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MEETI NG ABSTRACTS Open AccessProceedings of the International Conference on Human Genetics and 39th Annual Meeting of Indian Society of Human Genetics Ahmadabad, India. 23-25 January 2013 Published: 21 January 2014 These abstracts are available online at http://www.molecularcytogenetics.org/supplement/7/S1 I NTRODUCTI ON A1 Editorial Jayesh Sheth FRIGE’s Institute of Human Genetics, FRIGE House, Satellite, Ahmedabad, India Molecular Cytogenetics 2014, 7(Suppl 1):A1 The International Conference on Human Genetics and 39 th Annual Meeting of the Indian Society of Human Genetics organized from January 23-25, 2014 is an effort by the Foundation for Research in Genetics and Endocrinology (FRIGE), Ahmedabad and the Institute of Life Sciences, Ahmedabad University. With the emergence of newer technologies and expansion of scientific frontiers, the scope of genetics and allied sciences has crossed several boundaries and has opened several applications in medical diagnostics and treatment strategies. Human genetics and in particular molecular genetics has demonstrated a significant contribution in prediction and detection of genetic diseases (prenatal diagnostics) with advancement of techniques like Array- CGH, FISH and many others. For instance, microarray-based comparative genomic hybridization (array CGH) is a revolutionary platform that has been developed to screen entire genome for copy number variations (CNV) and can be used as the first line investigation modality in cases of non-syndromic mental retardation, unexplained developmental delay (DD), intellectual disability (ID), autism spectrum disorders (ASD) and multiple congenital anomalies (MCA). It can also be used for molecular characterization, to size the abnormality and study the gene content etc. In the field of cancer, several genes have been identified as the target for the treatment and cure. MicroRNAs (miRNAs) is yet another exciting advancement in the genetics. It may play an important role in regulation of expression of genes involved in cell competition at the post-transcriptional level. In silico screening of miRNAs involved in a cell competition is an effort to identify potential miRNAs and to reduce, economize and expedite experimental work. Under these circumstances identification of few novel and functional genes in different population can play an important role to define the future strategies for the diagnosis and treatment of various diseases. Further, many studies are emerging such that nutrition and certain micronutrients such as Vitamin B 12 , Vitamin D, folic acid etc. may have influence gene expression and genetic make-up. Therefore, the theme of the conference was chosen as ‘Healthy Genes - Healthy Life’. The presentations planned and research papers received are in conformity to this theme. There is a fair mix of papers with clinical and basic topics to be presented and included in this issue of the journal. It includes a vast area from basics of genetics to the complex of array CGH, SNP arrays and Next generation sequencing; prenatal diagnosis, pre-implantation genetics, epigenomics, pharmacogenomics, in- born errors of metabolic disorders, storage disorders, latest from the human variome project, point of care medicine and nanotechnology. We feel honoured and privileged to edit this special issue of Molecular Cytogenetics that highlights the genomic science presentations during this conference. The Indian Society of Human Genetics (ISHG) imparts knowledge related to Human genomics through annual meeting every year in January at different places of the country to encourage young students in biomedical science and invite learned scientists for the plenary talks. FRIGE is a nationally recognised organization with national and international alliances for research in human genetics. It is involved in carrying out basic and translation research in Human Genetics and Endocrinology with a motto of services to the society and imparting knowledge to the young students and researchers. Institute of Life Sciences is a part of the School of Science and Technology, Ahmedabad University with a motto of “Nurturing Science, Knowledge and Innovation”. The vision of the Institute is to undertake world class research in nano biotechnology leading to affordable health care to enrich human and environmental health. It has already forged alliances with national and international academic organisations as well as industries. We wish that the readers find these abstracts very interesting for their future research work. Jayesh Sheth Alok Dhawan Frenny Sheth Ramesh K. Goyal Sanjay Singh Acknowledgements: “The Organizers wish to put on record their sincere appreciation for the generous financial support provided by the Department of Science & Technology (Government of Gujarat), Gujarat State Biotech- nology Mission, Department of Science & Technology (Government of India), Department of Biotechnology (Government of India), Indian Council of Medical Research, Council of Scientific and Industrial Research” and other industries like Genzyme–Sanofi India, Zydus Cadila and many others as an exhibitors. International Conference on Human Genetics and 39 th Annual Meeting of the Indian Society of Human Genetics 23 rd – 25 th January, 2014 Ahmedabad, India Molecular Cytogenetics 2014, Volume 7 Suppl 1 http://www.molecularcytogenetics.org/supplement/7/S1 © 2014 various authors, licensee BioMed Central Ltd. All articles published in this supplement are distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. HUMAN GENOME / VARIOM PROJECT I1 Human variome project – current overview Richard Cotton 1,2 1 Human Variome Project International Limited, Melbourne, Australia; 2 Department of Pathology, The University of Melbourne, Australia E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):I1 The Human Variome Project (HVP) was initiated in 2006 to foster discussion around how patient outcomes could be improved by connecting and promoting the disparate work of genetic variation database curators. In the six years that have passed since that meeting, the concept of the Human Variome Project has evolved and matured. The challenges and complexities that face the field of human genetics, form a constantly changing technology landscape. The influx of raw data that those changes bring, has necessitated a shift away from the Project’s initial passive stance —a forum for the sharing of ideas and collaboration—to a more active initiative that actively engages with partners and stakeholders to establish and maintain the standards, systems and infrastructure that will enable the global collection and sharing of genetic variation information to be integrated into routine clinical practice. The Project is now well established, with over 900 HVP consortium members from 72 countries, 16 countries officially developing HVP country nodes, 6 major disease groups developing databases and over 140 databases having joined the Gene/ Disease Specific Advisory Council. With the Project Roadmap 2010-2012 nearing completion, the establishment of a new committee structure to govern the scientific aspects of the Project’s activities and the incorporation of a legal entity, Human Variome Project International Limited, to act as the Project’s International Coordinating Office outlined in the Roadmap are now in place. Most importantly, the Project Roadmap 2010-2012 proposed a strategy that would ultimately ensure the collection of all genetic variation information worldwide—the One Project, Two Channels, Multiple Locations Strategy. This strategy, the next Project Roadmap 2012-2016 and progress will be outlined in the presentation. I2 3D facial phenotyping Peter Hammond UCL Institute of Child Health, London, UK E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):I2 The recognition of facial dysmorphism remains an important skill for clinical geneticists to acquire despite the expanding use of next generation sequencing tools for the identification of gene mutations and related anomalies. Phenotype-genotype correlations, for example, still have an important role to play in diagnosis and prognosis. Facial morphology can now be quickly captured in 3D and analysed with the support of appropriate computer software. This talk will describe how: 3D images of affected individuals can be combined to delineate characteristic facial features of genetic and related conditions; static and dynamic visualisations can help a clinician to identify what is atypical in an individual child’s craniofacial development; quantitative analysis of face shape can assist with diagnosis and the study of phenotype-genotype correlations; links can be established between facial dysmorphism and neuro-cognitive disability arising from genetic anomaly and teratogen exposure. SESSION II: COMPLEX DISEASE: DIABETES & CARDIOVASCULAR DISORDERS I3 Prakruti genomics and prameha-proclivity: relevance to metabolic syndrome Ashok DB Vaidya ICMR-Advanced Centre of Reverse Pharmacology in Traditional Medicine, Kasturba Health Society, Mumbai, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):I3 Ayurveda, an art-science of a healthy way of life and a system of medicine, is being practiced in India for several millennia. Despite the remarkable description of diabetes (Madhumeha) and its precondition (Prameha), in Ayurveda, the current biomedical sciences stay generally uninformed of its rich concepts. The proclivity factors for Prameha are: Garbhakala Asatmyata (intrauterine influence), Shaithilya (poor muscle and adipose firmness), Meda asarata (dysfunctional adipocytes), Madhur Agraha (sweet tooth), Kapha chaya (accumulation of sero-mucoids), Kapha prakopa (aggravation of biofilms), Mansavahasrotadushti (hepatomuscular infiltration), Meda Vriddhi (adiposity), Avyayama (distaste for exertion), Divasvapna (daytime sleep), Medovahasrotadushti (adipose tissue inflammation), Kleda Dushti (excess of extracellular fluid) etc. The relative significance of each of these factors will depend on the individual’s Prakruti. Genetic determinants of these factors have to be investigated. The profiling of Prakruti in patients with diabetes has been studied by several groups. In a study from the Central Research Institute (Ayurveda), Jaipur, the frequency of kapha/ with vata/pitta was much more (64 %) than pure pitta/kapha/with vata (36 %). The response to the treatment with Nisha amalaki was also better in patients of kapha prakruti. The other study from the Banaras Hindu University showed a strong correlation of blood glucose response to exercise with prakruti. In another study from BHU, an association was found between the type 2 diabetes mellitus and 5, 10-methylene tetrafolate reductase MTHFR C677T. The CT genotype is protective. The study did not show any association of genotypes with the prakruti. MTHFR C677T gene polymorphism, common in the Chinese population, presents a genetic risk factor for diabetic nephropathy in type 2 diabetic patients. So there is also a need to consider Prakruti genomics for assessing the risk, onset and severity of complications in diabetes. Reverse Pharmacolgy coupled with Ayurgenomics can be applied to detect and measure the variation in drug response correlates vis-à-vis Prakruti. In the development of obesity, variants of more than three dozen genes are identified. For the type 2 diabetes, there are estimates of around hundred genes being involved; most of them are not identified at present. But it is being realized that it would be too simplistic to expect genomics to provide all the answers. Besides proteomics and metabolomics, we need a paradigm shift to consider the problem of proclivity to diabetes from the perspective of human biology at the bedside. Prakruti, Pathya and Ahar-Vihar of Ayurveda offer such bedside opportunities. These interactions, at present, are formidable challenges to the dominant reductionist paradigms in genomics and systems biology. However, personalized prevention and management of the metabolic syndrome can influence its resolution or progression to diabetes mellitus if one can identify the sets and subsets of prakruti with objective clusters of genomic, proteomic or metabolic markers; this would emerge by research at the interface of Ayurveda and Modern Biomedicine. I4 Next generation diagnostics on cardiomyopathy Jean-Louis Blouin 1,2* , Jeremy Bevillard 2 , Periklis Makrythanasis 2 , Michel Guipponi 1,2 , Federico Santoni 2 , Stylianos E. Antonarakis 1,2 , Siv Fokstuen 1 1 Genetic Medicine, University Hospitals of Geneva, Switzerland; 2 Genetic Medicine and Development, University of Geneva School of Medicine, Switzerland E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):I4 Cardiomyopathies are common, seemingly monogenic autosomal dominant cardiac disorders known as the primary cause of sudden cardiac death in young adults. These diseases are characterized by a remarkable genetic heterogeneity, which makes it difficult to unravel the causative mutation in a diagnostic laboratory that is very laborious and expensive by Sanger sequencing. To circumvent these limitations, we explored solutions of high throughput sequencing of targeted exomes with the aim to implement this approach in routine diagnostics. As a first test we designed a capture microarray with the total genomic length of 1 Mbp that includes all exons/splicing sites of 130 genes involved in cardiovascular mendelian disorders and analyzed simultaneously four samples by multiplexing patients with cardiomyopathies or Long-QT syndrome. Pathogenic mutations and variants of unknown significance were found thus resolving the genetic Molecular Cytogenetics 2014, Volume 7 Suppl 1 http://www.molecularcytogenetics.org/supplement/7/S1 Page 2 of 59 causes of the cardiopathy in three. In the fourth patient the mutation usually associated with hypertrophic cardiomyopathy was found with Long-QT. Further developments to next generation diagnostics are now in progress, and will be also discussed. In conclusion, high throughput sequencing holds considerable promises for molecular diagnosis of highly heterogeneous disorders in clinical practice and allows a better understanding of the complexity of mendelian disorders. I5 Genetics of cardiovascular disorders: influence of maternal nutrition Sukhinder K Cheema Department of Biochemistry, Memorial University of Newfoundland, St. John’s, Canada E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):I5 Cardiovascular disease (CVD) is the number one non-communicable disease of the world. According to the World Health Organization, 30% of global deaths in 2008 were caused by CVD, and it is estimated that by 2030, more than 23 million people will die annually from CVD. Unfortunately, India will be the leading country of the world to have the highest rate of CVD by 2030. Genetics, as well as diet and life style are the predominant factors predisposing the population to an increased risk of CVD. Although genetic makeup is generally considered as the culprit, recent phenomenon of “Developmental origins of health and diseases” or “in-utero programming” places significant importance on maternal diet in predisposing the next generation to metabolic disorders. According to this phenomenon, the diet of the mother during gestation and lactation affects the genetic makeup of the growing foetus, thereby causing perturbations in the metabolic regulations of the offspring, and later onset of diseases. Dietary fats are known to be associated with an increased risk of CVD; however the importance of maternal dietary fats in “in-utero programming” and the onset of CVD in the offspring in later life is not clear. We have shown that a maternal diet high in saturated fatty acids (SFA) increased the levels of low-density lipoprotein (LDL) cholesterol of the offspring by inhibiting hepatic LDL-receptor gene expression. Furthermore, maternal high fat diets caused aortic endothelial dysfunction, which is an additional factor associated with CVD. High fat diets during gestation and lactation also induce fatty liver and myocardium hypertrophy, due to inhibition of beta- oxidation through peroxisome proliferator activated receptors (PPARs). We found that maternal high fat diets altered the gene expression of PPARs, which likely involves epigenetic modifications. On the other hand, we found that maternal diets high in omega (n)-3 PUFA reduced plasma lipid levels of the offspring, thereby reducing the risk of CVD. Our findings have established the importance of maternal dietary fat intake during gestation and lactation to prevent the onset of CVD in the next generation. The Indian population is predisposed to an increased risk of CVD due to their genetic makeup. It is therefore recommended that the Indian population, especially women in their child bearing years, should manage the intake of dietary fat- both the quantity and the quality, to prevent the onset of CVD and other metabolic disorders in the next generation. SESSI ON I I I : CANCER GENETI CS I6 Affordable diagnosis and prevention of genetic disease John Burn Institute of Genetic Medicine, Newcastle University, International Centre for Life, Newcastle upon Tyne, UK E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):I6 Pathogenic variants in around 100 genes are responsible for a high risk of early onset solid tumours in close to 1% of the world’s population. As sequencing costs plummet and effective drugs are targeted at the molecular structure of cancers, many more of these gene carriers will be identified. They are a target population for low cost prevention strategies in their own right. An added benefit is that similar molecular pathways are disrupted in sporadic tumours, so effective chemoprevention strategies can be extrapolated to the general population. The Cancer Prevention Programme, CaPP, was developed 25 years ago to promote genetically targeted cancer prevention trials. CAPP2, published in 2011 (Burn et al Lancet) was the first trial with cancer as an endpoint to prove that regular aspirin significantly reduced the burden of colorectal and other cancers in people at risk of Lynch syndrome due to a defective mismatch repair gene. 600mg daily (2 tablets) for 2 years or more than halved the cancer rate after a lag of five years. Long term follow up of the other trial which tested cancer as a cancer preventive, the Women’s Health Study, has now shown a protective effect of very low dose aspirin taken on alternate days, but not until 10 years from the trial start (Cook et al 2013). CaPP3 starts in 2014 and will test the relative benefits of three different blinded doses of enteric coated aspirin -600mg, 300mg and 100mg in 3000 gene carriers. Failure of mismatch repair leads to the generation of frameshift peptides due to unrepaired slippage in repetitive DNA stretches (microsatellites). Antibodies to the resulting frame shift peptides will be tested as possible biomarkers. A vaccine raised against the important frame shift peptides affecting genes involved in carcinogenesis is also under investigation. Our novel nanowire technology is being investigated as a means of identifying microsatellite instability and BRAF mutations which help separate sporadic from hereditary tumours. This will make case identification much cheaper and easier in the context of countries with a developing economy. I7 An empirical assay for assessing genomic sensitivity and for improving cancer diagnostics Diana Anderson University of Bradford, Richmond Road, Bradford BD7 1DP, UK E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):I7 Detection tests have been developed for many cancers, but there is no single test to identify cancer in general. We have developed such an assay. In this modified patented Comet assay, we investigated lymphocytes of 208 individuals: 20 melanoma, 34 colon cancer, 4 lung cancer patients, 18 suspect melanoma, 28 polyposis, 10 COPD patients and 94 healthy volunteers. The natural logarithm of the Olive tail moment was plotted for exposure to UVA through different agar depths for each of the above groups and analyzed using a repeated measures regression model. Response patterns for cancer patients formed a plateau after treating with UVA where intensity varied with different agar depths. In comparison, response patterns for healthy individuals returned towards control values and for pre/suspected cancers, were intermediate with less of a plateau. All cancers tested exhibited comparable responses. Analyses of Receiver Operating Characteristic curves, of mean log Olive tail moments, for all cancers plus pre/suspected-cancer versus controls gave a value for the area under the curve of 0.87; for cancer versus pre/suspected-cancer plus controls the value was 0.89; and for cancer alone versus controls alone (excluding pre/suspected-cancer), the value was 0.93. By varying the threshold for test positivity, its sensitivity or specificity can approach 100% whilst maintaining acceptable complementary measures. Evidence presented indicates that this modified assay shows promise as both a stand-alone test and as a possible adjunct to other investigative procedures, as part of detection programs for a range of cancers. I8 Mutational landscape of gingivo-buccal oral cancer: new cancer genes and molecular subgroups identified Partha P Majumder National Institute of Biomedical Genomics, Kalyani, West-Bengal, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):I8 Gingivo-buccal oral cancer (GBOC), an anatomical and clinical sub-type of head and neck squamous cell carcinoma (HNSCC), is prevalent in regions where tobacco-chewing is common. Exome sequencing and other data on 50 GBOC tumor/normal DNA pairs revealed (a) significantly and recurrently mutated genes that are (i) specific (USP9X, MLL4, ARID2, UNC13C and TRPM3), and (ii) shared with HNSCC (e.g., TP53, CDKN2A, Molecular Cytogenetics 2014, Volume 7 Suppl 1 http://www.molecularcytogenetics.org/supplement/7/S1 Page 3 of 59 PIK3CA, HRAS, NOTCH1); (b) new genes with recurrent amplifications (e.g., DROSHA, YAP1) or homozygous deletions (e.g., DDX3X); (c) existence of molecular sub-types, with distinctive mutational profiles; (d) high proportion of C>G transversions, not noted earlier in HNSCC, among tobacco users with high numbers of mutations; and, (e) enrichment of alterations of pathways specific to GBOC, including Neurotrophin signaling, Wnt signaling, dorso-ventral axis formation and axon guidance. Recurrently mutated genes were validated on an independent set of 30 GBOC patients. These findings open new vistas for biological characterization and exploration of therapies. I9 Functional genomics of lung cancer progression reveals mechanism of metastasis suppressor function Ram Krishna Thakur 1† , Vinod Kumar Yadav 2† , Akinchan Kumar 1† , Richa Basundra 1 , Anirban Kar 1 , Rashi Halder 1 , Ankita Singh 1 , Pankaj Kumar 2 , Aradhita Baral 1 , MJ Mahesh Kumar 3 , Krishnendu Pal 4 , Rajkumar Banerjee 4 , Shantanu Chowdhury 1,2* 1 Proteomics and Structural Biology Unit; 2 G.N.R. Knowledge Centre for Genome Informatics, CSIRInstitute of Genomics and Integrative Biology, Delhi, India; 3 Animal House, CSIR-Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad, India; 4 Division of Lipid Science and Technology, CSIR-Indian Institute of Chemical Technology, Hyderabad, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):I9 The mechanism of action of NME2, a widely accepted metastasis- suppressor gene, is poorly understood. Recently we found that NME2 directly regulates transcription of the c-MYC proto-oncogene. This prompted a genome-wide study to ascertain whether NME2 exerts its anti-metastatic action through transcriptional regulation. Chromatin- immunoprecipitation followed by massively parallel sequencing (ChIPseq) along with transcriptome profiling uncovered a network of genes involved in intercellular contact, focal adhesion and actin assembly under direct transcriptional control of NME2. In line with this, NME2-depleted cells displayed increased focal adhesion points and altered actin stress fiber organization. Our findings demonstrate that NME2 regulates transcription of a key focal adhesion factor vinculin and its localization within adhesion foci. NME2-depleted A549 lung cancer cells showed higher invasiveness in vitro and seeded more metastases in vivo. Consistent with these findings, expression of several NME2-transcriptional target genes related closely to advanced tumor stages with metastatic proclivity, and NME2 levels predicted patient survival. I10 Non-coding rna based regulation of blood vessel development in zebrafish and relevance to humans Sridhar Sivasubbu CSIR-Institute of Genomics and Integrative Biology, Delhi, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):I10 Non-protein coding RNAs (ncRNA) represent a variety of transcripts having minimal or no protein coding capacity. The ncRNA have been studied with interest for their function as regulators of gene expression. Molecular studies on ncRNA have uncovered diverse interactions with protein coding genes. It has been suggested that ncRNAs are an additional layer of regulatory switches involved in gene regulation during development and disease. Detailed studies deciphering the function of ncRNAs have been limited to a few well-studied candidates. The function of ncRNAs during vascular development has attracted the interest of our laboratory. We designed reverse genetics screens to elucidate the role of miRNAs conserved between human and zebrafish, and potentially involved in regulating vascular development. In this screen we identified several miRNAs involved in the development of vasculature. We also undertook studies to understand transcription factor-miRNA interaction during vascular development. We extended the study to identify long non-coding RNAs (lncRNA) in diverse zebrafish tissues including vascular tissues. We identified several novel lncRNAs from zebrafish tissues. The functional importance of ncRNAs during zebrafish development will be presented and their relevance to humans will be discussed. SESSI ON I V: MOLECULAR CYTOGENETI CS I N DI SEASE DI AGNOSI S I11 Small supernumerary marker chromosomes – an update Thomas Liehr Jena University Hospital, Friedrich Schiller University, Institute of Human Genetics, Kollegiengasse 10, D-07743 Jena, Germany E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):I11 Genotype-phenotype correlations in patients with small supernumerary marker chromosomes (sSMC) are still difficult to asses. The presently known influence of chromosomal imbalance induced by sSMC size and origin, mosaicism of sSMC in different cells of the body and uniparental disomy (UPD) of sSMC’s sister chromosomes on the clinical outcome is summarized according to data on ~5,000 sSMC cases summarized on http://www.fish.uniklinikum-jena.de/sSMC.html. Two third of sSMC carriers are clinically normal. In the remainder 1/3 of sSMC patients, clinical symptoms may vary between slightly up to severely affected, including intrauterine death. Besides the known sSMC related syndromes Pallister-Killian-, isochromosome-15q12-, isochromosome-18p-, cat-eye- and Emanuel-syndrome there are numerous other yet unnamed and unidentified “sSMC-syndromes”. Recently, derivative-8- and derivative- 13/21 syndromes in complex sSMC were reported. The influence of chromosomal imbalance induced by sSMC size and its origin seems to have the largest impact on the phenotype of sSMC- patients. Besides UPD of sSMC’s sister chromosomes and mosaicism of sSMC may be important for the clinical outcome. The latter is especially important to be predicted in prenatal cases. Acknowledgments: Supported in parts by Deutsche Forschungsgemeins- chaft (DFG LI 820/22-1), Else Kröner-Fresenius-Stiftung (2011_A42) and the Deutscher Akademischer Austauschdienst (DAAD). I12 Molecular cytogenetic characterization of chromosomal rearrangements - utility in genetic counseling and research Ashwin Dalal Diagnostics Division, Centre for DNA Fingerprinting and Diagnostics, Hyderabad, Andhra Pradesh, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):I12 Chromosomal abnormalities can lead to a number of human genetic disorders. A large number of chromosomal rearrangements are known to be associated with a disease phenotype as a result of interrupting or modifying the expression of gene(s) localized in or close to the breakpoints. Structural changes in chromosomes can be classified according to cytological types and their effect on the phenotype. The main structural rearrangements are translocations, inversions, deletions, insertions, isochromosomes, dicentric chromosomes and ring chromosomes. Structural rearrangements alter the genome architechture and may result in human disease phenotypes. The identification of genes involved in human diseases resulting from chromosomal rearrangements is important to understand the pathophysiology of the disease, further it often provides new insights into normal human development and biology. Chromosomal analysis with routine methods gives a low resolution of about 5 Mb. Newer techniques like Fluorescent In Situ Hybridization (FISH), array Comparative Genomic Hybridization (CGH) have increased the resolution exponentially. These recent advancements in molecular cytogenetic techniques have made it possible to characterize the structural chromosomal rearrangements to very high resolution. Some of these techniques and their application to characterization of chromosomal rearrangements will be discussed using different case scenarios. Molecular Cytogenetics 2014, Volume 7 Suppl 1 http://www.molecularcytogenetics.org/supplement/7/S1 Page 4 of 59 I13 Microdeletion syndromes Chaitanya Datar Sahyadri Genetics, Unit of Sahyadri Hospitals, Barve Memorial Complex, J.M. Road, Pune, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):I13 Microdeletion syndromes are a group of disorders characterized by the deletion of a small chromosomal segment (usually <5 Mb in size) encompassing multiple disease genes, each potentially contributing to the disease phenotype independently. The mechanism of disease causation is usually due to haploinsufficiency of certain critical genes of that region. The genetic changes of these microdeletion syndromes are often not detected by the current band resolution using the routine or high resolution karyotyping (2-5 Mb) but require application of molecular cytogenetic techniques like Fluorescence in-situ Hybridization (FISH) or the latest array CGH technique. FISH is now the standard technique for the diagnosis of common microdeletion syndromes like Prader Willi syndrome, Angelman syndrome, Velocardiofacial (DiGeorge) syndrome, William syndrome etc. It is also possible to diagnose rare syndromes like Wolf Hirschhorn syndrome, Smith Magenis syndrome etc by FISH if the degree of clinical suspicion is high. With the advent of chromosomal microarrays, detection of newer microdeletion syndromes and better characterization of existing syndromes has become possible. SESSION V: CURRENT TRENDS IN PRENATAL SCREENING OF GENETIC DISORDERS I14 Non-invasive prenatal diagnosis using massively parallel sequencing - first experience in germany Rolf-Dieter Wegner 1,2* , Markus Stumm 1,2 , Wera Hofmann 3 1 Zentrum für Pränataldiagniostik & Humangenetik – Kudamm 199, Berlin, Germany; 2 BG Berlin Genetics GmbH, Berlin, Germany; 3 LifeCodexx AG, Konstanz, Germany E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):I14 Non-invasive prenatal diagnosis (NIPD) of aneuploidies by cell free fetal DNA (cff-DNA) from maternal plasma has reached the reliability to be applied in a clinical setting. It started with an observation that fetal DNA fragments are present in the blood of pregnant women. One main obstacle to be resolved had been the low concentration of cff-DNA among maternal DNA fragments, in general 2 – 10 % at 10 th week of pregnancy. This problem was resolved by the technical development of next generation sequencing applying massively parallel sequencing of millions of DNA fragments out of a single blood sample of 10 ml followed by bioinformatic processing of the data. Algorithms for calculations of z-scores were validated to allow a highly accurate distinction of pregnancies with an euploid fetus from those with a fetus carrying certain aneuploidies. With focus on the reliability of results it should be in mind that the cff-DNA is derived from the cytotrophoblast, e.g. from the placental tissue which is analyzed in CVS short term culture. Hitherto collected clinical data show sensitivity and specificity in agreement with a diagnostic test. In Germany, aneuploidy testing by NIPD started in 2012. Initially, the approach allowed the detection of trisomy 21. However, meanwhile probing for trisomy 13, trisomy 18 and the sex chromosome constitution is feasible. At the moment, the test is indicated for women with singleton pregnancies at an increased risk for aneuploidies. Blood samples should not be taken before 9+0 week of pregnancy. However, it is suggested to perform NIPD only in conjunction with a first trimester ultrasonographic examination for a profound judging of the fetal situation. For the time being, testing time is reduced to 10 working days or even less. In Germany, by now more than 4000 tests had been performed. In Berlin, data exceeding 250 cases had been collected by BG Berlin Genetics. The data of NIPD will be discussed in comparison to invasive prenatal diagnosis. I15 Basic principles of prenatal screening for aneuploidies Prakash Gambhir Birthright Genetic Clinic, Pune, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):I15 Prenatal screening processes are important public health interventions to counter common genetic disorders. For India prenatal screening against neural tube defects, thalassemia and Down syndrome is relevant to the health needs. However, there are many misconceptions regarding the principles of screening. Many clinicians wrongly consider it as substitute for prenatal diagnosis. Basically, prenatal screening protocols can be applied on a wide scale to identify women at risk of bearing a child with the common genetic disorder in that population. Prenatal screening for Down syndrome and other chromosomal abnormalities is a rapidly evolving science. It initially started with offering prenatal diagnosis to elderly mothers. The identification of low maternal serum alpha-fetoprotein levels as a marker in Down syndrome led to quest and recognition of many serum markers and maternal serum markers of AFP uE3 and hCG were used in combination as triple test and with addition of Inhibin as the quadruple test. The identification of Nuchal translucency as a marker in 1 st trimester led to much desired early risk prediction along with PaPPA and free beta hCG. It was also found that this test led to early risk determination and an early prenatal diagnosis with significantly better detection rate of 85 to 90%. This detection rate is head and shoulders better than the detection rate for the triple test rate of 65 to 70%. It has also been noted that combination of these markers can also lead to risk prediction of other chromosomal abnormalities. Presently risk prediction is possible with some remarkable softwares for Trisomy 18, Trisomy 13, Turner syndrome, triploidy, Cornela de Lange syndrome, Smith Lemlie Opitz syndrome and others. New markers like ADAM 12 with PaPPA will lead to early prediction of PIH and IUGR as well as prematurity. It is envisaged that in future maternal serum markers in combination with fetal biometry will give risk prediction for numerous chromosomal abnormalities many fetal disorders as well as pregnancy disturbances. Thus prenatal screening has an important place in antenatal care. I16 Prenatal screening for mendelian disorders in antenatal care Amar Verma Department of Paediatrics & Neonatology,Rajendra institute of Medical Sciences, Ranchi, Jharkhand, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):I16 Antenatal screening for fetal abnormality should be offered to all women, if available: In all cases of antenatal screening, the woman must be fully informed and understand the implications of the test, be promptly advised of their test result and be referred for further management and definitive diagnosis if their screening test is positive or suggestive of high risk. A positive prenatal diagnosis poses many ethical issues and challenging decisions for parents and clinicians. In those at increased risk of having a baby with a genetic condition, the risk should be identified and discussed fully before pregnancy and options for prenatal diagnosis discussed. Genetic counseling should be provided. At the present time around 5000 known disorders are inherited in a monogenetic mendelian fashion. Foremost among them are autosomal dominant, autosomal recessive and X-linked disorders, which carry a higher risk of illness than that conveyed by age-related risk. An autosomal dominant condition carries an a priori 50% inheritance risk where one parent is affected. An autosomal recessive disease carries a 25% inheritance risk for children of a healthy carrier couple. An X-linked recessive disorder carries a 50% risk for the son of a carrier mother. Specific, albeit non-screening genetic tests are currently available for more than 1000 of these diseases. Unlike cytogenetic, prenatal diagnosis based on maternal age, prenatal gene testing is not a screening test. Given the individuality of each case, prior planning is essential. Two differing strategies are possible: indirect and direct genetic testing. Molecular Cytogenetics 2014, Volume 7 Suppl 1 http://www.molecularcytogenetics.org/supplement/7/S1 Page 5 of 59 The following subsections cover the antenatal screening tests that are routinely offered like screening for potential for neonatal infection, Haemolytic disease of new born, Sickle cell disease and Thalassaemia, Down’s syndrome, Fetal anomaly, Measurement of fundal height etc. The different types of tests like Biochemical, Cytogenetic and Molecular genetic tests can be carried out by Chorionic villus sampling, Amniocentesis, Cordocentesis / percutaneous umbilical blood sampling Fetoscopy, Fetal radiology, Ultrasound-guided percutaneous skin and organ biopsy, Maternal blood tests, Ultrasound-guided percutaneous skin and organ biopsy and Preimplantation prenatal diagnosis. At present, in most cases, accurate prenatal diagnosis requires invasive testing. There is current research into noninvasive prenatal diagnosis using PCR and molecular genetic techniques to examine fetal DNA obtained from maternal blood. I17 Non-invasive prenatal testing (nipt): a better option for patients Ashish Fauzdar Quest Diagnostics India Private Limited, A-17 Info City, Sector 34 , Gurgaon, Haryana, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):I17 At present there are two methods to determine the chromosome health of unborn fetus in pregnant women. One is through maternal serum screening that is considered as simple but less sensitive method and the second options involves invasive methods like amniocentesis or chorionic villus sampling (CVS) for women who are found to be positive in maternal serum screening. Recent studies have demonstrated that non-invasive prenatal testing (NIPT) using cell-free fetal DNA (cfDNA) present in circulating maternal blood is considered as one the most effective method of screening for trisomy 21. The single nucleotide polymorphism (SNP) based Non-invasive Pre-natal test, being offered by Quest Diagnostics in technical collaboration with Natera Inc to determine chromosomal copy number by looking for specific patterns of (SNPs). This second generation technology analyzes cfDNA in a single reaction targeting 19,500 single nucleotide polymorphisms which are the most informative portions of an individual’s DNA selected across all the analyzed chromosomes. Two published study demonstrated in the women undergone both invasive chorionic villus samplings followed by karyotyping in conjunction with non- invasive prenatal testing the efficacy of test. Data from Nicolaides et al., 2013 paper on total of 242 cases that include thirty two cases aneuploidy included trisomy 21 (n=25) which were correctly identified with 100% sensitivity and 100% specificity with no false positive and false negative. Another study by Zimmerman et al., 2012 study on 166 samples from pregnant women, including 11 trisomy 21, three trisomy 18, two trisomy 13, two 45, X, and two 47,XXY samples including 146 low risk pregnancies. The study also correctly reported the chromosome copy number of all five chromosomes in 145 samples that passed a DNA quality test. The studies had demonstrated that SNP based NIPT is considered as an effective method of screening for common chromosomal abnormalities with detection rate of more than 99% and false positive rate of less than 0.1%. The initial finding shows NIPT approach has potential to avoid invasive procedure and can be provided an option to high risk pregnancies i.e. those with advanced maternal age, screens positive for biochemical screening and history of pregnancies with previous aneuploidies. SESSION VI: SINGLE GENE DISORDERS I18 Molecular diagnosis of genodermatoses in india Parag Tamhankar ICMR-Genetic Research Centre, National Institute of Research in Reproductive Health [NIRRH] Jehangir Merwanji Street, Parel, Mumbai, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):I18 Genodermatoses refer to inherited diseases of skin structure and function. Several genodermatoses present with multisystem involvement lead to increased morbidity and mortality. Genetic Research Centre focused on identifying molecular basis of such dreadful skin diseases with recessive inheritance. This would help us identify common mutations, founder effects etc that would reduce the cost of screening patients and their carrier parents. During the years 2011-13, 100 patients were referred to the centre with genodermatoses. The commonest group was ichthyosis followed by epidermolysis bullosa, ectodermal dysplasia, albinism, cutis laxa, progeroid conditions, precancerous conditions xeroderma pigmentosum, Rothmund Thomson syndrome, dyskeratosis congenita. Genetic heterogeneity is very common and molecular diagnosis requires an extensive effort. Recurrent mutations in unrelated families were seen in families with xeroderma, Griscelli. Prenatal diagnosis could be provided for ichthyosis, infantile hyalinosis and progeria. This is the largest cohort of mutation proven patients with genodermatoses from India. SESSION VII: GENETIC BASIS OF REPRODUCTIVE DISORDERS I19 Genetics of male infertility: Indian scenario K Thangaraj CSIR-Centre for Cellular and Molecular Biology, Hyderabad, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):I19 According to the recent epidemiological studies, nearly one out of every 10 couples face a problem in conceiving a child. Impaired fertility of male partner is causative in approximately 50% of all couples unable to conceive spontaneously. We have been studying the genetic factors associated with male infertility among Indian population. We have earlier shown that about 8.5% infertility among Indian men is due to the Y chromosome microdeletions. Further analysis of several autosomal (NR5A1, KLK3, CETN1, DEFB126, CAMK4, UBE2B, TNP1 & 2 and PRM1, 2 & 3); Y chromosomal (DAZ deletions, TSPY1 copy number) and mitochondrial genes accounted for additional 19.5% of the genetic factors responsible for male infertility. However, etiology of a large proportion (72%) of infertile men still remains unknown. Gene expression studies from our lab using microarray approach, showed several genes are many folds down regulated in the testicular tissue of infertile men, compared to the fertile men. Targeted resequencing of these differentially expressed genes and functional characterization of the observed mutations is in progress. In addition, we have recently initiated exome sequencing of idiopathic male infertile samples to identify additional genetic factors responsible for male infertility. The results of the findings would be discussed at the time of presentation. I20 Fertility options and challenges for patients with cytogenetic infertility & disorders of sex development Rama Ashok Vaidya * , Jaya Gogte Milan Polyclinic, 71-B Sarashwati Road, Santacruz (W), Mumbai, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):I20 Recent advances in assisted reproductive technology (ART), coupled with the emerging understanding in molecular mechanisms of disorders of sex development (DSD) and that of associated genetic infertility, have given hopes for fertility in groups of patients who till recently were denied biological parenthood. Heterologous fertility potentiation, in the cytogenetic infertility by donor oocytes, has been successfully used by us and others. However, homologous fertility preservation for this group of patients is evolving. Cryopreservation of either the mature oocytes following ovarian stimulation in post -pubertal Turner Syndrome (TS) girls or that of ovarian tissue in prepubertal TS patients are to be applied before actual ovarian failure occurs. Single embryo-transfer and strict selection criteria and judicious use of this advanced technology are advocated to minimize maternal morbidity and mortality. Microdissection testicular sperm extractions (Micro-TESE) in azoospermic Klinfelter syndrome patients have resulted not only in successful retrieval Molecular Cytogenetics 2014, Volume 7 Suppl 1 http://www.molecularcytogenetics.org/supplement/7/S1 Page 6 of 59 of spermatozoa for intracytoplasmic sperm injection (ICSI) but also in normal fertilization and clinical pregnancies. Swyer’s syndrome patients are reared as females and have female external and internal genitalia. However, with complete gonadal failure they need Oocyte donation with ICSI/IVFET for fertility. Those with partial androgen resistance (PAIS) due to genetic mutation of androgen receptor have a spectrum of abnormality like hypospadias, micropenis, undescended testes and male infertility. Though 46xy PAIS males have possibility of fatherhood complete AIS patients are female phenotype and are raised as females. This category of patients are encouraged and known to resort to fertilization of donated oocyte using husband’s sperms. Resultant embryos are transferred into surrogate uterus. Advances in reproductive medicine in general and those in assisted reproductive technology in particular have revolutionized diagnosis of disorders of sex development and the management of the associated infertility. These advances have certainly provided fertility potentiation but require judicious application coupled with expert genetic counseling. SESSI ON VI I I : EPI GENOMI CS I21 Uniparental disomy - clinical consequences due to imprinting and activation of recessive genes Thomas Liehr Jena University Hospital, Friedrich Schiller University, Institute of Human Genetics, Kollegiengasse 10, D-07743 Jena, Germany E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):I21 Uniparental disomy (UPD) is often considered as an event to be characterized exclusively by molecular genetic or epigenetic approaches. Still in at least one third of cases UPD emerge in connection with or due to a chromosomal rearrangement. Till date ~2,500 UPD cases detected in clinical, non-tumor cases are reported in the literature (http://www.fish.uniklinikum-jena.de/UPD.html). Based on this, the presently known imprinting syndromes, chromosomal contribution to UPD phenomenon, and cytogenetic subgroups of UPD and segmental UPD are reviewed. UPD may arise in clinical cases, as well as it may be exclusively tumor related. For clinical cases imprinting is quantitatively the most important problem. Still isodisomy may also be a problem, due to “activation” of recessive mutation-events, thus inducing rare autosomal recessive disorders. Overall, as UPD is more but an interesting rarity, the genetic background of each “UPD-patient” needs to be characterized besides by molecular methods, also by molecular cytogenetics in detail. I22 Epigenetic regulation of double c2 like domain beta (Doc2b) in cervical cancer K Satyamoorthy * , Samatha Bhat, Harish Rotti, K Shamaprasada Manipal Life Sciences Center, Manipal University,Manipal, Karnataka, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):I22 Associations of genetic changes and aneuploidy with tumor growth are traditionally attributed to alterations in DNA sequence manifested as mutations, deletions and amplifications. Inactive tumor suppressor genes could serve as drivers of tumor progression due to not only altered or lack of protein function but may also contribute to phenotypic changes that may provide distinct growth advantage in a hostile environment in the host. Human variation is also due to epigenetic alterations and heritable change that leads to altered gene expression; the functional consequence of which may contribute to definitive trait. A number of key regulatory genes associated with epigenetic silencing due to DNA methylation in cervical cancer have been reported. Elucidation of differentially methylated genes may identify new targets and further strengthen our understanding of molecular mechanism governing pathogenesis of cervical cancer. Thus, to identify DNA methylation regulated genes in cervical cancer, we have employed DMH based microarray experiments in pre-malignant and malignant cervical sample. Microarray data analysis and validation using bisulfite genomic sequencing lead to the identification of several CpG island as altered during cervical carcinogenesis and showed the potential for early screening of cervical cancer. One of the candidate gene identified was Double C2 like Domain beta (DOC2B), a key calcium regulator protein whose alteration has never been linked to cancer. We provide evidence that DOC2B is depressed in cervical cancer due to promoter hyper- methylation and act as a novel tumor suppressor gene by regulating multiple pathways in cervical cancer. I23 In search of epi-driver genes in head and neck cancer Sumana Bhattacharjya 1 , Kumar Singha Roy 1 , Nitai P Bhattacharyya 2 , Susanta Roychoudhury 1* 1 Cancer Biology and Inflammatory Disorder Division, CSIR-Indian Institute of Chemical Biology, Kolkata, India; 2 Crystallography and Molecular Biology Division, Saha Institute of Nuclear Physics, Kolkata, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):I23 Over the last decade, sequencing of a large number of tumour genomes has identified thousands of mutations in many genes. Among these mutations that confer selective growth advantage to the tumour cell are called ‘Mut-driver’ mutations. Furthermore, it has been proposed that ‘Epi-drivers’ are a class of driver genes that are not frequently mutated but aberrantly-expressed in tumours through epigenetic means. Intriguingly, it has been stated that the most obvious source of the proverbial ‘dark matter’ is in Epi-driver genes and human tumours contain large numbers of epigenetic changes affecting DNA or chromatin proteins. However, it is now clear that microRNAs (miRNAs) also have specific epigenetic functions whereby they recognize and bind to specific mRNA targets to repress their expressions. Using head and neck cancer tumour model we are trying to identify such miRNAs and their target Epi-driver genes important in this cancer. In this quest, we have carried out an in silico investigation of 53 miRNAs known to be deregulated in head and neck squamous cell carcinoma (HNSCC) and the expression and mutation status of their experimentally-validated target genes in the disease. Interestingly, our results have put forward 224 target genes as potential Epi-drivers specific to HNSCC. How miRNA regulation of mitotic genes could contribute to the HNSCC development and thus might be considered as potential Epi-drivers, will be discussed. I24 Birth defects: etiology to prevention Koumudi Godbole Deenanath Mangeshkar Hospital and Research Center, Erandawane, Pune, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):I24 Structural birth defects together of are a prominent cause of mortality and morbidity and are gaining importance with improving obstetric care and reduction in infective causes. Advances in understanding genetic (G) and environmental (E) factors and their interaction have added newer dimensions to the etiology of birth defects. Epigenetic is one such interesting field expected to provide mechanistic links between whether and how the environment interacts with maternal and fetal genomes. Some classical examples of environmental insults such as teratogens, maternal medical disorders and nutritional deficiencies as well as G-E interactions leading to birth defects will be discussed. It is important that the relevant scientific information should reach professionals and public for better awareness, appropriate policy decisions to prevent birth defects. I25 Genomic packaging and epigenetic regulation of genes Rakesh Mishra CSIR-Center for Cellular and Molecular Biology, Hyderabad, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):I25 Molecular Cytogenetics 2014, Volume 7 Suppl 1 http://www.molecularcytogenetics.org/supplement/7/S1 Page 7 of 59 Large proportion of the genome in higher eukaryotes does not code for proteins. This non-coding DNA is emerging as key to the packaging of the genome in form of chromatin, the functional form of genome, that is critical for chromosomal organization and gene regulation. It is clear that the packaging of the genome has regulatory consequences. We, however, do not know what the ‘packaging code’ of genome is, that allows as many packaging options as the number of cell types in the organism. What is clear is that packaging restricts enhancers/silencers that are capable of functioning over long distances, to interact with only appropriate promoters. Boundary elements that define topologically independent chromatin domains are expected to flank each gene or gene complex that is differentially regulated. Using comparative genomics, large scale mapping of epigenetic modification and genetic approaches we identified a large number of boundary elements by mapping epigenetic chromatin features across the hox complexes of vertebrates and use transgenic approaches to functionally analyze them. We find that the regulatory elements that are involved in the regulation genes by higher order chromatin structure are conserved across species - from flies to mouse. Finally, we use powerful genetic approaches in Drosophila melanogaster to explore the epigenetic mechanisms involved in the genomic packaging and regulation of genes. Our findings highlight long- range interactions involved in regulation of genes by means of genomic packaging in cell type specific manner. We propose that accumulation of non-coding DNA, including at least some of the repetitive elements, with the evolution of complexity is then consequence of the regulatory function embedded in this part of genome. SESSI ON I X: NEXT GENERATI ON SEQUENCI NG I26 Exome sequencing in unspecific intellectual disability and rare disorders Anita Rauch University of Zurich, Institute of Medical Genetics, Schlieren-Zurich, Switzerland E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):I26 Identification of disease causing mutations in genetically heterogeneous conditions such as intellectual disability by Sanger sequencing is time- consuming, costly and often unsuccessful. The advent of NGS techniques is paving the way for novel large scale approaches with an unforeseen diagnostic power. However, the plethora of variants of unknown significance detected by genome-wide approaches requires distinctive strategies to identify actually disease-related mutations. We recently showed that exome sequencing of patient-parent trios in sporadic cases of unspecific severe intellectual disability may unravel disease causing mutation in more than 50% of previously unsolved cases. Thereby it became also evident, that the current descriptions of phenotypes associated with mutations in a certain gene, are heavily biased towards certain recognizable patterns. However, while whole exome sequencing may currently provide theoretically the highest cost-efficient diagnostic power, it may miss mutations due to incomplete coverage of certain genes. Therefore in some phenotypes a “clinical exome” limited to a set of genes with currently known monogenic mutations may also be useful. I27 New paradigms in whole genome sequencing: from lab bench to cell phone Jonathan O’Halloran QUANTUMDx GROUP LIMITED, Newcastle, UK E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):I27 The DNA sequencing & MDx fields have seen dramatic advances since the first draft of the human genome was published, with companies reporting ever faster and cheaper methods. However, despite the race to attain the $1000 genome producing a plethora of exciting technologies, capillary electrophoresis (CE) is still being routinely used for targeted clinical sequencing and expensive real time PCR devices are still the work-horse of the MDx laboratory. QuantuMDx is developing a portable, handheld DNA sequencing device for PGx and infectious disease applications, to provide an alternative to slow and relatively expensive CE DNA sequencing & expensive slow and lab based MDx & CDx. I28 Personal genomes to precision medicine Vinod Scaria GN Ramachandran Knowledge Center for Genome Informatics, CSIR-Institute of Genomics and Integrative Biology, Mathura Road, Delhi, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):I28 The decade after the draft Human genome was published has seen tremendous developments in the technologies that can sequence genomes of individual Humans in a fast and affordable and accurate way. Reading 3 billion odd bases which comprise of the Human genome is today possible in realistic timelines and costs. This improvement was primarily brought about by the significant advances in the throughput and technology to sequence DNA and computational methods and resources to assemble and interpret the data. In addition to the improvements in technology, the miniaturization of technology that enables sequencing of human genomes on bench top sequencers has been thought to be one of the game-changers which could potentially accelerate the widespread application of genome sequencing. The availability of the reference human genome sequence has also in the last one decade accelerated the discovery of human genetic variations and their associations with traits which has provided a basis of annotating human genomes for predictive/ personalized medicine. The availability of effective tools, resources and datasets has poised genome sequencing in a unique position which could see its regular use in clinical settings. The application of genomics in clinical settings would primarily help in accurate and evidence based care, which encompass a new field of medicine called Precision Medicine. Precision medicine encompasses use of accurate clinical information and evidence to appropriately manage a patient at an individual level or at a community level. One of the major challenges which preclude the widespread application of genome sequences in clinical settings is the lack of know- how and expertise in analyzing and interpreting genome data in clinical settings. This would require a new breed of clinicians who have good clinical acumen, and are equally well versed with genomics and computational tools and methodologies. Towards accelerating the implementation of Precision medicine we have established a pipeline for human genome/exome sequencing and analysis in our laboratory and have developed a gamut of resources and tools for discovery, modeling and annotation of clinically actionable variants in genomes including Pharmacogenetic variations. In addition, we have implemented a pipeline for validation of functional variations in zebrafish, a popular vertebrate model system. Case studies and examples would be detailed during the lecture. SESSION X: DIAGNOSIS & THERAPEUTIC APPROACH OF LYSOSOMAL STORAGE DISORDERS I29 Enzyme replacement therapy for lysosomal storage disorders in India Mamta Muranjan Genetic Division, Depart of Pediatrics, KEM Hospital, Parel, Mumbai-400012, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):I29 Lysosomal storage disorders (LSD) are a heterogeneous group of inherited metabolic disorders with a combined frequency of 1 in 5000 affected live births. The commonest pathogenic mechanism is qualitative or quantitative deficiency of one of the 50 known lysosomal enzymes (acid hydrolases) Molecular Cytogenetics 2014, Volume 7 Suppl 1 http://www.molecularcytogenetics.org/supplement/7/S1 Page 8 of 59 involved in the catabolism of a variety of molecules. The outcome of this deficiency is progressive accumulation of partially degraded compounds which stealthily leads to multiorgan dysfunction. Unlike many inherited metabolic disorders, diet plays no role in the control of LSD. However treatment in the form of Enzyme replacement therapy (ERT) is available for a few disorders. The diseases for which ERT is currently the standard of care are Gaucher disease (Imiglucerase, Velaglucerase, Taliglucerase), MPS I (Laronidase), MPS II (Idursulfase), Pompe disease (Alglucosidase alpha), Fabry disease (Agalsidase beta, Agalsidase alfa) and MPS VI (Galsulfase). These drugs are human recombinant products manufactured in in-vitro tissue culture systems. ERT alters the natural history of these diseases and reverses many of the symptoms. However, as the drugs do not penetrate the blood-brain barrier, there is no impact on CNS disease. Gaucher disease was the first LSD to be treated with ERT in India since 1999-2000. Six centres in India located in Mumbai, Delhi, Lucknow and Chennai are treating patients with ERT. The major factor limiting widespread access to ERT in the country is the prohibitive cost, the need to import the drugs and lack of infrastructure for comprehensive evaluation and supportive care. Access has been facilitated for Indian patients through a charitable access program (INCAP). A board of seven National experts and a panel of International experts constitute the Indian Medical Advisory Board (IMAB) to screen patients for eligibility on the basis of pre-determined objective criteria. The experts also provide guidance for evaluation and management of LSD. To date approximately 60 individuals with Gaucher diseases are receiving ERT in India. Some of these have been receiving ERT for more than 10 years and are young adults pursuing graduate level academics. Additionally, 20 patients with MPS I, two with Fabry and 20 with classic infantile Pompe are receiving ERT. ERT is currently not available for MPS II and MPS VI in India. Twelve individuals with Gaucher disease (10 with type I) have been treated with Imiglucerase. Of these, one with severe disease resulting in hepato-pulmonary syndrome expired and one was transitioned to oral substrate reduction therapy. I30 Molecular study of lysosomal storage disorders in India Jayesh Sheth FRIGE’s Institute of Human Genetics, FRIGE House, Satellite, Ahmedabad, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):I30 Lysosomal storage disorders (LSDs) are gaining greater attention of the researcher and medical fraternity due to increasing awareness of occurrence, diagnostic facility, prenatal diagnosis and options for therapeutic intervention. Though the exact incidence is not known, they are likely to occur with a ratio ranging from 1:6000-1:8000 with the highest occurrence of Gaucher disease followed by GM2 gangliosidosis and mucopolysachharide disorder. However, our knowledge about the mutation spectrum for most of the LSDs and its phenotype consequences is limited. The present study was aimed to identify disease causing mutation for HEXA and GBA gene and its phenotypic consequences. Study was carried out in 37 biochemically confirmed subjects having Tay-Sachs disease and 32 subjects with Gaucher disease. Molecular analysis was carried out for all exons and exon-intron boundaries of HEXA and GBA gene by bidirectional sequencing method. In silico analysis was carried out using SIFT, Polyphen2 and Mutation T@ster softwares. In HEXA gene, 21 mutations were identified in 34 unrelated families in Tay-Sachs disease, 15 of which were novel, including 10 missense mutations [E114K, D175A, T263I, G269R, D322N, D322Y, Q374P, R393P, E462V, G478R] in 20 families, 4 nonsense mutations [Q237X, W474X, W485X, R510X] in 5 families, one 8 bp deletion [c.898_905TTCATGAG (p.F300HfsX21)] in one family. Earlier known 6 mutations were also observed that include 2 missense mutations [R170W and R178C] in 2 patients, one 4bp insertion in 5 patients [c.1277_1278insTATC (p.Y427IfsX5)], 3 splice site mutations [c.805+1 G>C, c.459+5 G>A and c.672+30 T>G] in four families. Mutation was not identified in 3 families. In GBA gene (Gaucher disease) we have identified 10 missense mutations in 32 unrelated families that include 9 known mutations [E326K, V352M, G355D, S356F, R359Q, R368C, R395C, R463C and L444P] in 31 families and one novel mutation [G289A] in one family. In silico analysis further confirmed the pathogenic effect of the novel mutations occurred at highly evolutionarily conserved and functionally active domain residues in the protein leading to conformational changes or mRNA producing truncated protein resulting in the diminish or absent activity of the protein. Present study demonstrated that E462V, D322Y and c.1277_1278insTATC (p.Y427IfsX5) mutations are the most common mutations observed in nearly 49% (18/37) of the children with TSD in India. Similarly, L444P missense mutation is commonly observed in 21/32 (65.62%) children with Gaucher disease. Additionally, the data also demonstrates that exon 5-12 in HEXA gene and exon 8-10 in GBA gene are the hot spot region where ~75% and 94 % mutations can be identified in Indian patients with the aforementioned diseases respectively. SESSI ON XI : ARRAY- CGH I31 The advantages of SNP arrays over CGH arrays Boris Keren Genetic Department, La Pitié-Salpêtrière Hospital, AP-HP, Paris, France E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):I31 In recent years, with the rapid development of Chromosomal Micro-array Analysis (CMA), the resolution limit of 5Mb which was imposed by conventional cytogenetics, has been significantly lowered. Currently, array CGH is the most widely used CMA technology. With the inclusion of thousands to millions of probes, it allows the detection of small copy number variations (CNVs) of a few kb. SNP (Single Nuleotide Polymorphism) arrays can also be used for CMA. They too enable the detection of CNVs, but unlike array CGH, each probe is located at an SNP and can determine the genotype of the corresponding SNP. Here, we report on our 6 years experience of the use of SNP arrays in cytogenetic diagnosis on more than 3000 patients and we will focus on the main benefits of SNP arrays over array CGH. Because of their ability to perform SNP genotyping, SNP arrays can detect long contiguous stretches of heterozygosity (LCSH). LCSH have 2 main interests: 1) they can detect uniparental isodisomies (UPD); 2) they can detect genetic identity by descent. UPD can be responsible for imprinting disorders and both UPD and identity by descent is associated withpromote the occurrence of autosomal recessive disorders. Moreover LCSH analysis allows performing homozygosity mapping and helping guide sequencing of candidate genes responsible for recessive conditions. Because of an abnormal number of different alleles, SNP arrays also enable the detection of polyploïdy and chimerism. Besides, SNP arrays also are of interest in quality control: they can detect DNA contamination and false paternity. Thus, while array CGH is still a very efficient technique to detect CNVs, the inclusion of SNP probes in arrays is desirable when possible. I32 Cytogenetic microarray in prenatal and postnatal diagnosis Shubha Phadke Department of Medical Genetics, SGPGIMS, Lucknow, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):I32 Due to high resolution cytogenetic microarray (CMA) has replaced traditional karyotyping for evaluation of individuals with intellectual disability, autism and congenital malformations. The diagnostic yield of CMA is 10 to 12 % and is more than any other investigation for evaluation of developmental disabilities. Due to its high resolution CMA is also being used as a prenatal test for chromosomal anomalies. The diagnostic yield is about 3% whatever may be the indication. The yield is higher in cases with fetal anomalies. The main concern with CMA is its ability detection of copy number variations of unknown significance. This is a major problem in prenatal diagnosis and is a challenge for the counselor and dilemma for the family in concern. With accumulation of more and more data of pathological and polymorphic variations in genome the variations of unknown significance will decrease. Molecular Cytogenetics 2014, Volume 7 Suppl 1 http://www.molecularcytogenetics.org/supplement/7/S1 Page 9 of 59 CMA is also useful in delineating abnormalities picked up by karyotyping. Some cases with double segment rearrangement may point towards familial chromosomal rearrangement and hence, CMA is indicated in familial cases with developmental disabilities and birth defects. Cost and availability of clinical cytogeneticists for appropriate interpretation of CMA results are important concerns for wider application of the technique. I33 Genomic copy number variations in glaucomatous neurodegeneration Arijit Mukhopadhyay CSIR-Institute of Genomic and Integrative Biology, New Delhi, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):I33 Copy number variation (CNV) is one of the major factors contributing to genomic diversity and diseases. It has been shown especially for neurodegenerative diseases that CNVs can play a very important role in genetic predisposition of the disease. Glaucoma is a major neurodegenerative disease causing irreversible vision loss across the globe. We wanted to analyze the impact of CNVs in a genome-wide scale in patients of primary open angle glaucoma (POAG) collected from the West Bengal state of India and reproduce our results in another cohort of Caucasian origin. Genome- wide data was generated on 347 POAG cases and 345 controls on Illumina 660W-Quad arrays and CNVs were called using PennCNV. The CNVs were classified as small (<100 kb) and large (>100 kb) and analyzed seprately for their involvement in the disease. A publicly available dataset of POAG cohort of 624 cases and 404 controls from Caucasian origin (GLAUGEN study) was used as a validation cohort and genome-wide CNV data of 208 HAPMAP samples was used as global control. We analyzed genome-wide CNV from 1928 samples. For the large CNVs distribution was significantly skewed toward larger size (>1 Mb) in cases compared to controls and this was replicated in the GLAUGEN data. We found that CNVs >1 Mb are enriched for gene rich regions in POAG patients with 125 genes while for controls a similar percentage of large CNVs overlapped with only 5 genes. In 208 HAPMAP samples CNV >1 Mb overlapped with 95 genes. Interestingly, genes found in the patients were unique and did not overlap with controls or HAPMAP samples. Within CNVs of >1 Mb gene-rich large deletions were ~2 fold enriched in patients compared to duplications irrespective of their ethnic background. Such a bias was not observed in the controls. In the smaller CNV range we performed association analysis and identifed novel regions to be under significantly higher CNV in patients’ comapred to controls. Particularly, a CNV encompassing the transcription factor FOXE3 was significantly enriched in patients of both Indian and Caucasian POAG patietns comapred to their respective controls. A sequence analysis of the gene revealed novel missense mutation in the patients. We have shown that genomic CNVs >1 Mb has significantly higher burden in POAG patient’s genome compared to controls irrespective of the population background. We have also identified candidate genes/regions which are uniquely present in POAG cases and absent in controls from all over the world. Our data provide new insights into role of CNV in pathogenesis of POAG. I34 Clinical utility and dilemmas of SNP microarray testing Virginia Kimonis Division of Genetics and Genomics, Department of Pediatrics, University of California, Irvine, CA 92868, USA E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):I34 The ability to diagnose patients with developmental delay, intellectual disability, congenital anomalies, and dysmorphic features has significantly improved with the introduction of SNP microarray technologies which includes more than 1.8 million markers on a single array. This high-density array will allow for sensitively detecting all known abnormalities with defined loci of interest as well as the discovery of ‘‘new syndromes’’ as a cause of mental retardation or autism. The most common micro deletion disorders include15q11-q13 Prader-Willi/Angelman, 22q11.3 velo-cardiofacial, 17pl3.3 William, 1p36and 16p11.2 microdeletion syndromes. SNP arrays are able to detect segmental uniparental disomy (UPD) as in Prader Willi syndrome and UPD14. SNP arrays identify parental consanguinity which may otherwise go undetected. Long stretches of homozygosity can be analyzed for recessive genes in patients born to consanguineous parents. (http://www.ccs.miami. edu/cgi-bin/ROH/ROH_analysis_tool.cgi) Arrays cannot however identify balanced chromosomal rearrangements, such as translocations or inversions, Marker chromosomes may also be missed, depending on the size, marker composition, and array coverage of the specific chromosomal region present on the marker. Dilemmas arise when ‘‘CNVs of uncertain clinical significance’’ are identified, or if a parent is not available for testing, thus making it difficult to identify if the rearrangement is inherited or arose de-novo. Further dilemmas arise if a parent is identified with the same rearrangement and is apparently asymptomatic, should one then consider the presence of a mutation in a recessive gene in the other allele to explain autism, mental retardation or other disorders. Overall SNP array technology has become the test of choice, permitting a 5.9% detection rate in patients with negative microarray-based CGH and an overall detection rate approaching 29%. I35 Detection and Inheritance Pattern of Copy Number Variations (CNVs) in Children with Multiple Congenital Anomalies Frenny Sheth FRIGE’s Institute of Human Genetics, FRIGE House, Satellite, Ahmedabad, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):I35 Microscopically visible chromosomal alterations or segmental aneusomy are one of the major causes for congenital anomalies and require cytogenetic investigations. Conventional G-banding analysis is limited to the detection of imbalances greater than 5-10 Mb. Expressivity of a phenotype generally correlates with the degree of genetic imbalance, therefore, patients with a greater genetic imbalance are more likely to be investigated, which creates a diagnostic bias. This does not reflect the full range of phenotypic presentation that may be associated with an imbalance of a particular chromosomal segment. Systematic clinical diagnosis of a rare syndrome can be carried out using FISH to characterize known deletion/duplication breakpoints or by screening for known micro-deletion syndromes and sub- telomeric imbalances where routine banding technique suggests “normal” karyotype. In the past decade, array- Comparative Genomic Hybridization (a-CGH) has made it feasible to detect cryptic imbalances in more number of cases with apparently normal chromosomal makeup in addition to characterize structurally rearranged chromosome. The study of inheritance pattern by a combination of conventional cytogenetic and a-CGH techniques has helped to determine the imbalance as either de novo or inherited. a-CGH could also provide information on whether an imbalance is pathogenic or polymorphic, which could aid in calculating the recurrent risk for future pregnancies. It provides several advantages over conventional cytogenetic techniques such as whole genome coverage in a single experiment with resolution of 20-150 kb, faster turnaround time, higher sensitivity and specificity and non-requirement of dividing cells. Having an extensive coverage of the genome and high resolution, this technique has been pinned as the “first line genetic test” to study cryptic genetic imbalances in cases with developmental delay and congenital anomalies, where G-banded karyotype is “normal”. SESSI ON XI I : GENETI CS OF NEUROBI OLOGY I36 Neuroferritinopathy: iron in the brain John Burn Institute of Genetic Medicine, Newcastle University, International Centre for Life, Newcastle upon Tyne, UK E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):I36 Molecular Cytogenetics 2014, Volume 7 Suppl 1 http://www.molecularcytogenetics.org/supplement/7/S1 Page 10 of 59 Careful attention to clinical phenotypes can identify new diseases which are now amenable to molecular genetic elucidation. In the late 1980’s I met a family labelled as having Huntington’s disease but with absence of dementia. Scanning revealed unusual cavitation of the basal ganglia. The pedigree was extended my connection to a second family using birth records. We mapped the gene to chromosome 19 and went on to identify an unusual mutation in the E helix of light chain ferritin. Staining for iron revealed huge accumulation of iron/ferritin complexes in the brain leading to neurodegeneration. We named the condition neuroferritinopathy and tested desferrioxamine without success. We have shown that accumulation of iron commences in childhood and are now preparing to test deferiprone as an iron chelator which crosses the blood brain barrier. I37 Clinical aspects of neuroregression: our experience on batten disease Mahesh Kamate KLES, Belgaum, Karnataka, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):I37 Neuroregression in a child is an important clinical problem faced by a pediatric neurologist. Depending on the initial clinical features they can be broadly divided into grey matter disorders, white matter disorders or combined. The age of onset and the progression of symptoms also help us in further characterization. Involvement of other systems and neuroimaging findings helps us in formulating the differential diagnosis and guides us in the laboratory evaluation and selection of appropriate confirmatory tests. After brief discussion on the clinical approach to neuroregression, here I would like to present our experience with one of the important poliodystrophies in children- Neuronal ceroid lipofuscinosis. Neuronal ceroid lipofuscinosis is a group of progressive neurodegenerative disorders characterized by accumulation of ceroid lipopigment in lysosomes in neurons and other cell types. Over a period of four years we have diagnosed 20 children with neuronal ceroid lipofucinosis. Of the 20 patients, 5 had infantile type and 15 had late-infantile neuronal ceroid lipofuscinosis. Diagnosis was confirmed by appropriate enzyme assay. Clinical presentation was quite varied. Common presenting features included refractory seizures, developmental delay/regression, and abnormal movements. Visual failure was not common in the present case series, and novel neuroimaging finding in the form of isolated dentate nucleus hyperintensities in PPT related neuronal ceroid lipofuscnioses was noted. During follow-up, all patients had a progressive downhill course and one patient died. Prenatal diagnosis could be offered to one family. Our experience suggests that infantile and late-infantile neuronal ceroid lipofuscinosis is not uncommon in this region of the country and the phenotype is different. I38 Early stress evokes age-dependent biphasic changes in hippocampal neurogenesis, epigenetic regulation of the bdnf gene, and cognitive behavior Vidita Vaidya Department of Biological Sciences, Tata Institute of Fundamental Research, Mumbai, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):I38 An experience of stress in early life is predominantly associated with negative consequences including increased anxiety and depressive behavior, as well as a failure to mount appropriate stress responses. It has remained a source of debate whether early stress also evokes potentially adaptive consequences that equip animals to cope better with their environment. We have shown that early stress exposure facilitates transient, adaptive changes in hippocampal neurogenesis, enhanced trophic factor expression and improved cognitive performance, thus providing possible competitive advantages in stressful environments. However, middle-aged animals with a history of early stress exhibit aladaptive effects on hippocampal neurogenesis, reduced trophic factor expression and impairments in cognitive performance. Our study provides novel insights into the short and long-term consequences of early stress, demonstrating biphasic, as well as unique, age-dependent changes at the molecular, epigenetic, neurogenic and behavioral level. These results compel a reappraisal of the traditional notion that early stress is deterministic for future negative outcomes. Our studies suggest that when observed across a life-span, early stress experience evokes both adaptive as well as maladaptive changes that emerge in a temporally regulated manner, with early adaptive outcomes that may eventually exert a high cost, evoking maladaptive consequences. SESSI ON XI I I : I NBORN ERRORS OF METABOLI C DI SORDERS I39 Dysmorphology of inborn errors of metabolism Virginia Kimonis Division of Genetics and Genomics, Department of Pediatrics, University of California, Irvine, CA 92868, USA E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):I39 As we discover the molecular mechanism of disorders, eventually all dysmorphic syndromes will ultimately be considered biochemical defects. An overview on the recognition and classification of dysmorphic features will be provided. Categories of inborn errors of metabolism associated with dysmorphic manifestations will be discussed. For e.g. abnormal eye findings are an important clue to the diagnosisin galactosemia, cystinosis, Lowe syndrome, and homocystinuria. Unusual kinky hair is seen in Menkes disease. Skin findings typically lead to the diagnosis in Fabry, Hunter and steroid sulphatase deficiency. Infants who have peroxisomal disorders (Zellweger) pyruvate dehydrogenase deficiency, cholesterol biosynthetic disorders (Smith-Lemli-Opitz), multipleacyl-CoA dehydrogenase deficiency (glutaric aciduria type II), infants of mothers with phenylketonuria have striking facial dysmorphism and structural anomalies at birth. In other disorders dysmorphic features may not be present at birth but may develop with age at varying rates such as in lysosomal storage disorders (mucopolysaccharidoses, oligosaccharidoses), congenital disorders of glycosylation and mitochondrial disorders. Salient clinical features, biochemical defects, molecular basis, and diagnostic strategies will be discussed to permit early treatment of these disorders. I40 New born screening program in India: ICMR multicentric experience Roli Mathur Indian Council of Medical Research, New Delhi – 110029, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):I40 Inborn Metabolic Disorders (IMD) are common genetic disorders imposing huge burden on health care infrastructure though many of these are treatable conditions. Indian Council of Medical Research (ICMR) recognized the need of initiating a multicentre Task Force (NTF-IMD) study to systematically collect data for congenital hypothyroidism and congenital adrenal hyperplasia to help in early diagnosis and management to prevent disability. This study was done to evaluate the feasibility of a newborn screening for different geo-ethnic regions of the Indian Population and attempt to define the incidence of the selected inborn metabolic errors i.e., CH and CAH in the population and also to develop the capability of treating and diagnosing inborn errors of metabolism. A series of brainstorming sessions were conducted between 2008-2013 at 5 regional centers in the country before the start of the study. The NTF- IMD Group developed a common protocol for implementation at all study sites for comprehensive screening, management, treatment of affected newborns, counseling, high risk screening in sick newborns, enrolment in quality assurance program, development of tools for advocacy, setting up of a dedicated website etc. A sample size of 100,000 newborns was set as a target and 1000 sick children admitted in ICU’s at study sites were also included. Molecular Cytogenetics 2014, Volume 7 Suppl 1 http://www.molecularcytogenetics.org/supplement/7/S1 Page 11 of 59 Coordinated effort of a dedicated team of investigators at 5 newborn screening centers, 3 high risk screening centers, data coordination centre, quality assurance centre and central coordination centers could lead to successful completion of newborn in more than 1,00,000 newborns from both urban and rural areas. The study involved the use of common protocols and involved data compilation and analysis to prepare comprehensive results from all centers and set a data representative for the country following thorough quality assurance procedures. The study helped to establish the incidence of CH and CAH in Indian population and prevented disability in affected newborns following early diagnosis and treatment. The study has far reaching implications as it helped to build regional capacity and in setting up an invaluable model for future newborn screening programs in India as well as other developing countries. I41 Newborn screening- the roadmap for India Seema Kapoor Department of Pediatrics, Maulana Azad Medical College, New Delhi, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):I41 India witnessed a major transition in 2013. It marked the completion of 50 years of the activities of the Indian Academy of Pediatrics, the completion of a taskforce study conducted by ICMR, organization of the Asia Pacific meeting on newborn screening and inclusion of congenital hypothyroidism as an important activity of a landmark program by the Government, the Rashtriya Bal Suraksha Kayakram. It has also created a platform for inclusion of many Delhi Hospitals in newborn screening. These activities have created 3 major thrust points in the country which include awareness about the need for screening, the feasibility of its execution in the metropolitan cities of our country and the commitment to make the special diets available. Both public and private players have developed deep commitments which are likely to change the face of newborn screening in the country. Screening for 5 nearly completely reversible and treatable disorders is possible. These include Congenital Hypothyroidism, Congenital Adrenal Hyperplasia, Glucose-6-phosphate dehydrogenase deficiency, Biotinidase deficiency and Galactosemia. These have been included in different pilot programs across India. Challenges which invite discussion are the feasibility of coverage in less well developed areas, hilly terrains and the home deliveries. MCTS (Mother child tracking system) an initiative launched by the Government of India once in full implementation may play a pivotal role in this program. Availability of good counselors and a well integrated follow up system needs to be developed so that all screen positive babies can be followed up. Generation of epidemiologic data for the currently untreatable conditions being the non availability of diets needs to be simultaneously addressed so that we can gear up for the expanded phase later. Generating ethnic cutoffs, ensuring quality compliance, improving availability of confirmatory tests needs to be addressed. The volumes are formidable but also suggest that with high rates of consanguinity and inbreeding one is likely to encounter a significant proportion of these in the country. The most positive aspect is the commitment to this noble cause which will help us cross and reach the horizon. I42 Treatment of inborn errors of metabolism Anil B Jalan Navi Mumbai Institute of Research in Mental and Neurological Handicap, C- 116, Om Rachna Society, Sector 17, Vashi, Navi Mumbai, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):I42 Inborn errors of metabolism (IEM), though individually rare are collectively common. Average incidence of 50+ common IEMs is considered to be approx 1 in 1,000 live births. With annual birth rate of approximately 25 million babies in India, we can expect at least 25,000 babies being born with IEM in India and hence it is a significant burden to the families and societies. Over the last 3 decades many new and effective therapies have emerged for the management and treatment of IEMs. As of today approximately 70 different forms of therapeutic agents are available to help people suffering from IEM. Besides these we have various forms of transplants–liver cell transplants, liver transplant (both ALT and OLT), bone marrow or HSCT and kidney transplant now easily available at various parts of India. Amongst hundreds of IEM, certain disorders are common and treatable with simple forms of therapeutic agents. Urea Cycle Disorders and Organic Acidemias are top on the list. Every pediatrician and neonatologist must be aware of emergency management of these two group of disorders as they may present at any age especially in the 1 st decade and more so in infancy. Both of these groups of disorders may present with hyperammonemia as their first manifestations and needs to be treated with easily available medications as oral form of Sodium Benzoate (250–500 mg/kg/day in 2-3 divided doses), low protein diet or temporary stoppage of protein intake till acute crisis is under control. For UCDs one can use arginine (granules or powder) hydrochloride or base–250 mg/kg/day in 2-3 divided doses except for Arginase deficiency. For disorders like CPS and NAGS deficiency Carbaglu of Carbamylglutamate or N Acetyl Glutamate can also be used in the dose of 100–300 mg/kg/day. Now a days it is also recommended for treatment of certain organic acidemias e.g. propionic acidemia. Other products like Sodium Phenyl butyrate or injectable forms of Arginine (10%) or Sodium Benzoate+Sodium Phenylbutyrate) are not available in India and are very costly for an average Indian patient. Once acute crisis is managed, special diets with low protein are successful in managing most of the UCDs. For Organic acidemias like Propionic acidemia, Methyl malonic Acidemia and Isovaleric acidemia, L-Carnitine in the dose of 100–300 mg/kg/day must be used. Injectable form of L-Carnitine is also available for emergency management. Correction of acidosis is very important along with supple- mentation of adequate amount of dextrose. One can use Dextrose-Insulin drip in emergency. Besides these, other medications like Betaine, NTBC, Dextromethrophane, Diazoxide, certain vitamins e.g. Biotin, Vit-B12, Thiamine, Riboflavin, Folic acid, Folinic acid, Pyridoxine, Pyridoxal–5- Phosphate, Vit C, certain aminoacids like Glycine, Ornithine, Citrulline etc are also available for the management of various types of IEMs. Of late many enzymes are available for enzyme replacement therapies of LSDs e.g. Gaucher, Pompe, Fabry’s Disease, MPS I, II and VI. SESSION XIV: INHERITED BLOOD DISORDERS: HEMOGLOBINOPATHIES I43 Thalassemias: can we reduce the national burden? Roshan Colah ICMR-National Institute of Immunohaematology, Parel, Mumbai, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):I43 The burden of inherited disorders of hemoglobin, the commonest group of single gene disorders in India is huge. With a population of 1.21 billion and an average prevalence of b-thalassemia carriers being around 3.5-4%, there would be 35-45 million carriers and the estimated number of births of affected babies annually would be 10,000-12,000. The carrier rates vary from 1-17% in different ethnic groups. Apart from b-thalassemia, Hb E is common in the north eastern region and in West Bengal (4 to > 50%) and Hb S is prevalent in parts of central, western and eastern India (5-40%). Thus interaction of the b-thalassemias with these Hb variants is not uncommon and can lead to a severe disorder. One way to combat the burden is by prenatal diagnosis but the only approach to reduce the national burden is by a comprehensive community control programme. Awareness is very limited in different states (<20% among pregnant women) and the entire public health infrastructure from medical colleges to district hospitals and down to the community health centres must be mobilized for education and generating awareness on these disorders. Experience shows that screening for carriers in India will have to be done at multiple levels – schools, colleges, antenatal clinics as well as cascade screening where extended family members of an affected child are screened. However, antenatal screening with subsequent testing of the husbands of carrier women would be the most cost effective way to identify couples at-risk and give them the option of prenatal diagnosis. For this, obstetricians must recognize the implications of hypochronic and microcytic red cell indices (MCV <80 fl, MCH < 27 pg and a high RBC count) and ask for a b-thalassemia screen by estimation of HbA 2 levels. Several laboratories in the country use automated HPLC for reliable HbA 2 estimation and identification of heterozygotes is not a problem. Late registration at antenatal clinics (only 15-20% in the first trimester in public Molecular Cytogenetics 2014, Volume 7 Suppl 1 http://www.molecularcytogenetics.org/supplement/7/S1 Page 12 of 59 hospitals) is an impediment resulting in many couples at-risk being identified late and requiring second trimester fetal diagnosis. Social stigmatization is an issue to be dealt with during premarital screening of marriage partners of carrier individuals. Only education can reduce this barrier. Many State Governments in India are now undertaking population screening and counselling programmes and Gujarat and West Bengal have taken the lead. There are 10-12 centres offering prenatal diagnosis by CVS and DNA analysis and recently the Indian Council of Medical Research has established 6 more centres in different regions. However, many more centres would be required once there is an increasing demand. The spectrum of mutations and their distribution are now known which would facilitate prenatal diagnosis. Thus, there are many challenges – a large and diverse population, limited awareness, late registration in antenatal clinics and inequality of available services (urban v/s rural areas) with around 70% of the population residing in rural areas. There is a need for the Central and State Governments to join hands and involve NGO groups to form networks in different regions which when backed by political will could gradually reduce the national burden of hemoglobinopathies in this vast country. I44 Haemophilia - diagnosis and management challenges Shrimati Shetty ICMR-National Institute of Immunohaematology Parel, Mumbai, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):I44 Haemophilia care in India is slowly progressing but the diagnostic and management challenges continue until optimum care for haemophilia patients become a reality in this country. Against an anticipated severe haemophilia population of more than 0.1 million, only 16000 haemophilia patients are registered in the 74 Haemophilia Chapters across the country. As per the recent WFH Survey, 43% of the World haemophilia population live in India, Bangladesh, Indonesia and China, out of which only 12% is diagnosed in these countries. Additionally these countries represent only 2% of the Worlds total factor usage. The per capita use of FVIII in India has been shown to be 0.0075 (1IU per capita is 20000 IU FVIII per haemophilia patient) which is approximately equal to mean consumption of 1654 IUs per PWH against 112508 IUs per PWH in United States. Haemophilia in India is thus a grossly under diagnosed disorder, mainly due to lack of awareness and diagnostic facilities in this country. Except a few Corporate Hospitals and a few Medical Colleges in India majority of the 630 District hospitals and Medical Colleges do not have even screening coagulation facilities. Haemophilia Federation (India) has taken wide initiatives in providing diagnostic facilities to the PWH in this country, through various measures such as supporting the cost of diagnosis of PWH, networking private laboratory facilities, setting up coagulation laboratories in close proximity to Haemophilia Chapters, involving faculty members of Medical Colleges in the activities of Haemophilia Chapters and so on. Indian Council of Medical Research has also initiated through its Translation Research Programme several workshops in the Laboratory diagnosis of bleeding disorders at Mumbai and several places in East and North Eastern parts of India. As quality control is an important exercise in a coagulation laboratory, many of the laboratories in India fail to establish factor and inhibitor screening assays in their respective laboratories. The prevalence of inhibitors in India is estimated to be 8.2–13%. Post operative inhibitors are another important problem which our PWH is facing in India. About 30% of PWH undergoing surgeries for various indications develop inhibitors postoperatively. Diagnosing inhibitors, demands specialized laboratories and expertise. The management of PWH and inhibitors comprises several approaches involving prompt treatment of bleeding episodes, managing its complications, preventing bleeds, and conserving and restoring joint function. The ultimate goal of treating PWH and inhibitors is to permanently eradicate inhibitors by immune tolerance therapy (ITT), or by use of alternative products like rFVIIa or FEIBA. However, because of prohibitive cost and logistics constraints many of our PWH are not able to utilize these products. Availability of free factors in some states of our country is a refreshing and welcome change for PWH. Significant progress has been made in awakening and alerting the State Governments for providing free factors to all our PWH. WFH has contributed to haemophilia care in India through a series of international twinning programme by initially twinning upcoming chapters in India or institutions of repute in India with some of the best centres in Europe and USA leading to the development of expertise in the area of genetic diagnosis and physical therapy. Despite this, there are several newer challenges that the hemophilia community will face in the coming years. Normalization of patients’ lives as a result of improved treatment has led to new problem areas. Malignancies specifically hepatocellular carcinoma (HCC) in HCV infected patients and non Hodgkin’s lymphoma in HIV infected patients are on the rise. About 25% of our patients is HCV infected. A less common albeit potentially severely morbid event is ICH. Osteoporosis in general has been considered to be an important cause of morbidity in patients with haemophilia and other bleeding disorders. I45 Current status of sickle cell disease in India: how can you attenuate? Dipika Mohanty Apollo Hospitals, Bhubaneswar- 751 005, Odisha, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):I45 Sickle cell disease is the most prevalent monogenic disorder worldwide resulting from single DNA mutation in beta globin gene. The mapping on the pattern of its distribution in India has been studied to a great extent. However paucity of adequate data throughout the country regarding the clinical manifestations, natural history of the disorder and correlation with genotype and phenotype of the SCD cases is observed. This communication will focus on following aspects: (i) prevention and control of the SCD in India. (ii) the good clinical management with particular reference to pain during the vaso-occlussive crisis. (iii) neonatal screening and genetic counseling in SCD in tribals of India. Between August 2009 and July 2010, 1668 newborns were enrolled and screened for SCD in Kalahandi district of Odisha. An average incidence of 17.62% of sickle cell trait (HbAS) was recorded for the area with the highest incidence observed in the tribal dominated part (19.03%). The data till date reveals a striking fact that more than 20 per thousand live births in the district are born with the disease. All the 34 cases of SCA detected were confirmed by parents testing, of which confirmation of 4 cases of compound heterozygosity for HbS and b-thalassaemia is an interesting finding. For pain management with an informed approved consent in 15 patients treated at Apollo Hospital, Bhubaneswar we have given nitric oxide inhalation at 80ppm for 6-12hrs and 90% of patients responded well, pain score being relieved significantly >60%. In 2 patients there was recurrence of pain on 2 nd day and they were given again the same therapy. Another 1 patient did not respond very well to pain. On investigation she was found to have superficial vein thrombosis of left hand. This NBS programme design provides scope for catering the benefit in rural areas and follow up for newborns. It also stresses on the acute need of such kind of extensive reach-out programme for early and confirmed detection of SCD in other places of the country. This will ultimately reduce the child mortality and morbidity in SCD. Nitric Oxide inhalation for pain relief in VOC of SCA is a very effective and less expensive method. I46 Community genetics approaches in the prevention of beta-thalassemia: towards achieving ‘Zero beta-thalassemia’ status in India V R Rao Department of Anthropology, University of Delhi, Delhi, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):I46 The estimates of prevalence of b-thalassemia in India are grossly underestimated, as it is based on 0-5% carrier frequency, while population surveys indicated communities with as high frequency as 24%. The overall carrier frequency distribution of b-thalassemia, is highly heterogeneous with wide geographical foci of high risk communities with 8% and above carrier frequency all over the country. As of now, there is no mechanism to evaluate whether the frequencies are increasing or decreasing in the populations. Prenatal diagnosis and prevention of births of b-thalassemia homozygotes is the most preferred approach adopted in India. However, the extent of distribution and the occurrence across various stratification in the society with large component of rural masses, it is difficult to assume Molecular Cytogenetics 2014, Volume 7 Suppl 1 http://www.molecularcytogenetics.org/supplement/7/S1 Page 13 of 59 that prenatal diagnosis alone can bring in ‘Zero’ b-thalassemia status in India. Countries like Cyprus could achieve such status where large scale population carrier screening programs involving education and premarital counselling were also effectively employed besides prenatal diagnosis. In India, there is urgent need to initiate large scale population carrier screening of high risk communities along with education and awareness. The extent of b-thalassemia distribution, identifying high risk communities and geographical regions presenting a population design for carrier screening program in India, will be presented. SESSI ON XV: HUMAN CHROMOSOMAL DI SORDERS I47 Chromosomal instability and molecular mutations in multi spectrum disease of Fanconi anemia Babu Rao Vundinti ICMR- National Institute of Immunohaematology, Parel, Mumbai, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):I47 Fanconi anemia (FA; MIM no. 227650), the most commonly inherited bone marrow disorder, has an overall prevalence of 1–5 per million and an estimated carrier frequency of 1:200-300 in most populations. Demonstrating either an autosomal or X-linked recessive mode of inheritance, FA is characterized by childhood progressive bone marrow failure and predisposition to acute myelogenous leukemia, older patients are at increased risk for squamous cell carcinomas of the head, neck, and genitourinary tract. Congenital abnormalities are present in approximately 70% of FA patients and may include radial ray defects, cafe´ au lait spots or hypopigmentation, short stature, microphthalmia, malformations of kidneys, gastrointestinal tract, and heart, mental retardation, and hearing defects. Because of the high degree of phenotypic variability exhibited by FA patients, diagnosis may be difficult on the basis of clinical manifestations alone. Because FA patient-derived lymphocytes and fibroblasts exhibit hypersensitivity to DNA crosslinking agents such as diepoxybutane (DEB) and mitomycin C (MMC), resulting in a high rate of chromosomal breakage and radial formation, analysis on the basis of this hypersensitivity has been routinely used to confirm clinical diagnosis. Molecular diagnosis of FA has been challenging because of the genetic heterogeneity associated with the disease. To date, 15 FA gene products (FANCA, B, C, D1, D2, E, F, G, I, J, L, M, N, O and P) have been identified and they constitute the FANC pathway, which is thought to function in preventing genome instability. The FA core complex comprises FAAP24, FAAP100, and 8 FA proteins (FANCA, B, C, E, F, G, L, and M) and mediates DNA-damage-induced or replication-stress induced monoubiquitylation of FANCD2 and FANCI. Monoubiquitinated FANCD2 and FANCI translocate to chromatin and function in DNA repair at least partially by recruitment of FAN1 nuclease. Cloning of the FANC genes has provided a means, via complementation group analysis, to routinely pinpoint the patient’s specific FA gene harboring the mutations, thus greatly simplifying subsequent molecular analysis. Although retroviral-mediated complementation analysis of FA patient-derived cells is commonly used to identify the patient’s genetic subtype as a prerequisite for mutation screening, such analysis has not been formally validated for clinical use. Previous studies have considered Fanconi complementation Group A (FA-A) assignments, but none have performed comprehensive sequencing to verify complementation assignment and/or evaluated complementation assignments performed using retroviral- mediated rescue as evidenced by correction of MMC and DEB-induced chromosome breakage and radial formation. Among all complementation groups, FAA accounts for the majority of FA patients (60–65%) followed by FAC (10%) and FAG (9%). Mutations in the gene for complementation group FA-A (FANCA; MIM no. 607139) confer autosomal recessive inheritance and account for approximately 66% of all FA cases. The FANCA gene is mapped to chromosome 16q24.3,11 spans approximately 80 kb of genomic DNA (gDNA), and consists of 43 exons. With more than 200 different mutations described thus far, the FANCA mutation spectrum is very heterogeneous. Because of the presence of a large number of Alu repeat sequences within the FANCA gene, large intragenic deletions involving multiple exons have been reported to account for more than 40% of all FANCA mutations. First time we have carried out a molecular study in the Indian population. We have studied FA by chromosomal breakage study using mitomycin C (MMC) and diepoxybutane (DEB) induction and FANCD2 monoubiquitination by western blotting to understand gene defects in FA pathway. The complementation analysis was done by retroviral transfection. The molecular study was carried out by Multiplex Ligation-dependent Probe Amplification (MLPA) and direct sequencing of FANCA, C, G, E, F, L, and M genes. The complementation analysis results showed different spectrum as compared to world literature. Our study revealed FA-A and E gene defects in 69% cases and followed by FANCE gene defect. The molecular analysis showed the large deletions in 11 patients and FANCA gene mutations were in 12 FA patients. Out of 12 mutations 5 mutations (c.3678 C>G, p.Ser 1226 X, c.3993G>A p.Leu 1331 Pro, c.1274 C>G p.Glu 425 His, c.2630 C>G p.Ser 877 X) found to be novel mutations. In our series the single nucleotide polymorphisms (SNP); Exon9,c.796A>G(p.Thr266Ala), Exon16,c.1501G>A(p. Gly501Ser), Exon26, c.2426G>A(p.Gly809Asp), of FANCA gene also observed in 23 patients, and these polymorphisms in disease association was reported in FA database, however, it needs to be established in Indian population. Interesting finding of our study is the existence of FANCE mutation in Indian population, and the same was reported rarely in other places of the world. The study also highlighted the uncharacterized FA patients, which may associated with new genes in Indian population and these patients should be studied molecularly and genotype-phenotype correlation need to be established, which helps in better understanding and management of the disease. SESSION XVI: NANOTECHNOLOGY IN BIOMEDICAL RESEARCH I48 Unlocking the chemistry of bile acids for cancer therapeutics Avinash Bajaj Laboratory of Nanotechnology and Chemical Biology, Regional Centre for Biotechnology, Gurgaon, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):I48 Breast cancer is the second leading cause of cancer deaths today after lung cancer and is the most common cancer among women. The primary drug tamoxifen is used treat breast cancer has several problems including poor oral bioavailability. Bile acids/salts are known to be components of endogenous molecular pool that solubilizes, absorbs dietary fat/lipid molecules in the form of micelles. Bile acids are interesting chemical scaffold for drug conjugation due to presence of different number of free hydroxyl groups and a free carboxylic acid. We have been exploring bile acids as drug carriers for cancer therapy. We used three bile acids: lithocholic acid (LCA), deoxycholic acid (DCA) and cholic acid (CA) to engineer bile acid tamoxifen conjugates with free amine and acid functionalities. In this talk, I would present the interactions of these new drug carriers and their therapeutic potential for breast cancer therapy. SESSION XVII: PRE-IMPLANTATION AND PRENATAL DIAGNOSIS OF GENETIC DISORDERS: INDIAN SCENARIO I49 Pre-implantation and polar body diagnosis in cases of parental chromosomal translocations applying array-CGH Rolf-Dieter Wegner 1,2* , Markus Stumm 1,2 , Heide Mundt 2,3 , Matthias Bloechle 4 1 Zentrum für Pränataldiagnostik und Humangenetik – Kudamm 199, Berlin, Germany; 2 BG Berlin Genetics GmbH, Berlin, Germany; 3 Freie Universität Berlin, Germany; 4 Kinderwunschzentrum an der Gedächtniskirche, Berlin, Germany E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):I49 Parents with a balanced chromosomal translocation show an increased risk of reduced fertility including spontaneous abortions and chromosomally Molecular Cytogenetics 2014, Volume 7 Suppl 1 http://www.molecularcytogenetics.org/supplement/7/S1 Page 14 of 59 unbalanced offspring. In the course of genetic counseling the parents frequently decide to seek artificial reproductive techniques. In vitro fertilization (IVF) offers the chance to perform Polar Body Diagnosis (PBD) or Preimplantation Diagnosis (PID). Female translocation carriers can opt for analysis of polar body, blastomeres or trophectoderm cells while male translocation carriers have only a choice between blastomere and trophectoderm diagnostic. In Germany, up to the year 2010 a restrictive “Embryo Protection Law” did allow only PBD excluding male translocation carriers from diagnostics. Thus, experience with PBD applying FISH or array-CGH was collected in cases of maternal translocations. For such cases, advantages and limits of array analysis as compared to the FISH approach will be presented. Furthermore, the resolution power of the BlueGnome 24sure and 24sure+ arrays will be shown. In particular we report here on the segregation pattern of maternal translocation chromosomes in 16 cases including 24 cycles and analyzing 97 first polar bodies. In addition to the distribution of the translocation chromosomes the number of segregation errors leading to aneuploidy in the 1 st . polar body will be presented. Since a German high court ruled that PID of trophectoderm cells should be legal under very stringent conditions, the Embryo Protection Law had been slightly modified in 2011. In future this will allow the application of array analysis also in cases of paternal translocation. I50 Challenges in prenatal and pre-implantation genetic diagnosis studies Prochi Madon Department of Assisted Reproduction and Genetics, Jaslok Hospital and Research Centre, Mumbai, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):I50 Prenatal diagnosis (PND) of chromosomal anomalies by karyotyping and fluorescence in situ hybridization (FISH) is a routine procedure in many cytogenetic laboratories. However, PND of single gene disorders is available in only a few centres specializing in molecular genetic testing in India. Preimplantation genetic diagnosis (PGD) is a state-of the art technique involving biopsy of a single cell from a cleavage stage embryo obtained after intra-cytoplasmic sperm injection (ICSI). This is an additional step during in-vitro fertilization (IVF). The biopsied cell is then fixed on a slide in a critical step where the cytoplasm is removed to expose the nucleus. Single cells from many embryos of the couple are fixed and then screened for common aneuploidies by FISH using multicolour DNA probes. A maximum of 5 chromosomes can be checked in 1 hybridization. Hence the probes are stripped and slides are rehybridized in 2-4 rounds to study more chromosomes within a limited time frame. Only the chromosomally normal embryos are transferred or cryopreserved for a subsequent transfer. Karyotyping of couples with recurrent early miscarriages helps to identify the cause if the husband or wife is a carrier of a balanced translocation. PGD can help such couples to have a healthy baby earlier than by normal chance. Specific FISH probes have to be ordered especially for reciprocal translocations and a pre-PGD work up is important. This helped to identify an additional cryptic translocation between chromosomes 9 and 12 in a case from a neighbouring country, where the husband had inversion 12. Hence PGD included testing of chromosome 9 in this case. The wife has an ongoing pregnancy in the first attempt. Currently, ours is the only centre in India which offers PGD even for reciprocal/ Robertsonian translocations and microdeletions. We have had success with the birth of healthy babies. PGD for single gene disorders is yet in its infancy in our country. It involves genetic testing from DNA extracted from a single cell. Pre-PGD work up is more demanding, especially in cases where a HLA matched normal savior sibling is desired to cure a child affected with a hematological disorder such as Thalassemia. I51 Consanguinity and perinatal medicine - the berlin perspective Rolf Becker Centre for Prenatal Diagnosis and Human Genetics, Berlin, Germany E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):I51 The present study was conducted to assess the impact of consanguinity on the prevalence of major anomalies in a prenatal study group of Berlin/ Germany. Over a time-span of 19 years (1993-2011), 34,900 fetuses were examined by prenatal sonography. In 659 cases (1.9%) the parents were consanguineous, with 45.2% related as first cousins (F = 0.0625) and 54.8% beyond first cousins (F< 0.0625). Detailed information on the pregnancy outcome of 31,145 fetuses was retrieved either through direct report by families or by active inquiry of patients or their physicians, 555 of these fetuses (1.8%) had consanguineous parentage. The prevalence of major anomalies among fetuses with non- consanguineous parents was 2.8% (863/30,590). By comparison, in the sub-group of fetuses with consanguineous parentage the prevalence was 11.0% (61/555 fetuses). Within the consanguineous sub-group a causal association between fetal anomaly and consanguinity was assessed as probable in 6.5% (36/555) of cases, as possible in a further 3.4% (19/555) of cases, and as improbable in 1.1% (6/555) of the diagnosed anomalies. The data indicate that the prevalence of major fetal anomalies associated with consanguinity was approximately eight percentage points higher than in non-consanguineous offspring. As a proportion of these anomalies result either in intrauterine death or medical termination of pregnancy, the prevalence of consanguinity-associated defects diagnosed post-birth is equivalently lower, thus under-estimating the overall adverse effect of intra-familial marriage on fetal and neonatal well-being. SESSI ON XVI I I : SI GNI FI CANCE OF PHARMACOGENOMI CS I N CLI NI CAL RESEARCH I52 Pharmacogenetics: polymorphism and genotype-phenotype correlation of drug response in indian population Harish Padh Sardar Patel University, Vallabh Vidyanagar, Gujarat, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):I52 Inter-individual genomic variations have recently become evident with advances in sequencing technologies. Polymorphism and human variants have been linked to disease susceptibility as well as drug efficacy and toxicity. Although polymorphism can occur at any loci and can affect any protein structure and function, it is largely studied in enzymes involved in drug metabolism where much correlation has been established with drug efficacy and toxicity. Among genetic variations SNPs are widely studied and better defined because of availability of large scale detection platforms. Besides SNP, insertion- deletions (INDELs), inversions, copy number variations (CNVs) and larger structural variations (> 3 Mb) have come to light in recent years, however, their link to health and disease remain ill-defined. CNVs are defined as the segment of DNA larger than 1 Kb in size, and compared to reference genome vary in copy number. All types of genomic variations are bound to play vital role in disease susceptibility and drug response. In this presentation, genetic variation in many DMEs like CYP2D6, CYP2C9, CYP2C19 will be discussed, and its effect on pharmacokinetic (PK) parameters like AUC, Cmax, Tmax and T1/2 will be presented. PK variation as phenotype will be compared and correlated with genotype variation in Indian population. Examples of CNV data in Indian population will be presented and compared with other populations. Available literature will pose significant policy issues about drug approval procedure. The issue of incorporation of local pharmacogenetic consideration in drug approval will be analysed. I53 Pharmacogenomics of cardiovascular drugs C Adithan Department of Clinical Pharmacology, JIPMER, Pondicherry, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):I53 Molecular Cytogenetics 2014, Volume 7 Suppl 1 http://www.molecularcytogenetics.org/supplement/7/S1 Page 15 of 59 Cardiovascular diseases account for the second largest number of non- communicable disease after mental illnesses. Coronary heart diseases, hypertension, atherosclerosis, congestive heart failure, arrhythmias are important cardiovascular diseases. Beta blockers, statins, antiplatelet drugs, anticoagulants, drugs modifying renin angiotensin systems and many others are being used for treating these ailments. Still, satisfactory treatment of these diseases is still elusive. Drug response in influenced by many environmental factors besides host factor. Besides the above, genetic factor is also important in modifying response to cardiovascular drugs. Now there is reasonable amount of evidence exists that Indian population is genetically distinct from other major ethnic groups. The allele and genotype frequencies of genes encoding important drug metabolizing enzymes, drug transporters and receptors of Indian population are different from Caucasians and Orientals. Studies done in our laboratory and other Indian laboratories suggested that pharmacogenomics of clopidogrel, warfarin, acenocoumarol, beta blockers etc. may have clinical significance in treating cardiovascular diseases of Indian population. There is a need for multi-institutional and multi-disciplinary approach for large scale implementation of pharmacogenomics and personalized medicines in cardiovascular diseases. I54 Point of care testing for improving risk- benefit ratio of aspirin and warfarin Harsh Sheth 1* , Emma Northwood 2 , Faye Elliott 2 , Michael Jackson 1 , Mauro Santibanez Koref 1 , John Tyson 3 , Ann Daly 4 , Jonathan O’Halloran 3 , Jayesh Sheth 5 , Frenny Sheth 5 , Keyur Parikh 6 , D Timothy Bishop 2 , John Burn 1 1 Institute of Genetic Medicine, Newcastle University, International Centre for Life, Newcastle upon Tyne, UK; 2 Leeds Institute of Molecular Medicine, St. James Hospital, Beckett Street, Leeds, UK; 3 QuantuMDx Ltd., International Centre for Life, Newcastle upon Tyne, UK; 4 Institute of Cellular Medicine, Medical School, Newcastle University, Newcastle upon Tyne, UK; 5 FRIGE’s Institute of Human Genetics, FRIGE House, Satellite, Ahmedabad, India; 6 CIMS Hospital, Nr. Shakun Mall, Sola, Ahmedabad, India Molecular Cytogenetics 2014, 7(Suppl 1):I54 The increase in identification of putative biomarkers and opportunities to develop tailored treatments are due to emergence of omics technologies. Application of pharmacogenetic knowledge with the help of quick and cheap companion diagnostics in the primary care setting is expected to deliver improved treatment and reduced heathcare costs. Warfarin and aspirin are the two most widely prescribed drugs for preventing cardiovascular diseases. Long term aspirin use has also been shown to reduce risk, recurrence and mortality from colorectal cancer. However, they both have narrow therapeutic windows and several genetic polymorphisms have been noted to influence their dose and efficacy. We therefore have launched two collaborative projects: first, to study the genetics of warfarin safety in the Gujarati Indian population and second, to identify further polymorphisms that modulates aspirin’s colorectal cancer chemopreventive efficacy. Understanding the impact of polymorphisms on dose and efficacy for these drugs would lead to development of a combined panel of markers that would predict accurate therapeutic dose with minimal risk for adverse reactions. These markers will be deployed at the point of care settings using a novel handheld genotyping device which will use disposable microfluidic cassettes and silicon nanowires currently developed by QuantuMDx. Results, future work, opportunities and barriers will be examined. SESSI ON XI X: GENE DI SCOVERY - TRANSLATI ONAL RESEARCH FROM BENCH TO BEDSI DE I55 Digital health, microfluidics, and bedside genetic testing Syed Hashsham Department of Civil and Environmental Engineering, Michigan State University, USA E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):I55 Decentralized, bedside, or point of care analysis of nucleic acids (DNA/RNA/ mRNA/microRNA)-based markers is expected to be a key component of digital healthcare. Nucleic acids-based approaches allow for screening of disease and pathogens, disease surveillance, selection of treatment, treatment effectiveness, differential diagnosis, risk assessment, staging, and prognosis. Hand-held genetic analysis systems have the potential to provide all these capabilities in a simple, affordable, field deployable, rapid, multiplexed, and robust manner without the need for electrical power or refrigerated reagents. Even the need for specific language can be eliminated by translatable or visual graphical user interface truly integrating the analytical system with the communication network. The presentation will introduce Gene-Z and iDx, two networkable platforms that are developed to provide simplified analysis of genetic markers. Gene-Z has Android/iPod based operation using Bluetooth TM and capable of carrying out quantitative isothermal amplification for 64 reactions in a disposable microfluidic chip. The device is battery operated and can be charged by solar panels integrated at the top of the device. A smaller version (iDx) working as an attachment to cell phones, also with real time amplification and quantification capabilities, allows 8 reactions in parallel. Both these devices are networkable and currently being validated for a number of key applications important to human and animal health, plant safety, industrial microbiology, and environmental protection. Simplification, reliability, and cost-effectiveness are key factors ensuring successful implementation of such approaches. Business model for adoption of such low cost approaches, however, is still evolving and major challenges need to be addressed about how to sustain a business that is built on small margins usually by small companies engaged in biomedical research. I56 G protein signaling in tumor cell growth and metastasis Danny Dhanasekaran Peggy and Stephenson Cancer Center, University of Oklahoma Health Sciences Center, 975 NE 10 th Street, BRC 1417, Oklahoma City, OK, 73012, USA E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):I56 G protein signaling has been implicated in different aspects cancer growth and progression. Our studies have identified that the G12-family of G proteins that defines the gep family of oncogenes are critically involved in tumor cell proliferation and metastasis. Defining these pathways has shown that the gep protooncogene GNA12 is specifically involved in the proliferation of ovarian cancer cells whereas GNA13 is involved in cancer cell metastasis. Consequently, the silencing of the gep proto-oncogenes potently inhibited tumor growth of ovarian cancer cells in a mouse xenograft model, thus suggesting the dominant role for the gep oncogenes in ovarian cancer growth and progression. In addition we demonstrate a similar role for GNA13 in the invasive migration of pancreatic cancer cells. Furthermore, we demonstrate that an eleven amino acid peptide derived from the gep oncogenes Ga 12/13 can effectively disrupt LPA-stimulated oncogenic pathways. Thus, in addition to unraveling the molecular mechanism underlying cancer progression and metastasis, our results provide evidence that the G protein signaling nodes can be targeted for cancer chemotherapy. This work was supported by grants from the National Institutes of Health (CA123233, CA 125752, CA 116984). I57 Vitiligo: a complex disease and a complex approach Rasheedunnisa Begum 1* , Yogesh S. Marfatia 2 , Naresh C Laddha 1 , Mitesh Dwivedi 1 , Mohmmad Shoab Mansuri 1 , Mala Singh 1 1 Department of Biochemistry, Faculty of Science, The Maharaja Sayajirao University of Baroda, Vadodara, India; 2 Department of Skin & V.D., Faculty of Medicine, The Maharaja Sayajirao University of Baroda, Vadodara, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):I57 Vitiligo is an acquired, circumscribed hypomelanotic skin disorder, characterized by milky white patches due to loss of functional melanocytes Molecular Cytogenetics 2014, Volume 7 Suppl 1 http://www.molecularcytogenetics.org/supplement/7/S1 Page 16 of 59 from the epidermis. Prevalence of vitiligo is found to be very high in Gujarat i.e., ~8.8%. Vitiligo is a multifactorial polygenic disorder with a complex pathogenesis, linked with both genetic and non-genetic factors. Several theories have been proposed to explain the etiopathogenesis of vitiligo, but none of the hypotheses explains the entire spectrum of this disorder. We are addressing this complex disease in our Gujarat population with various approaches. Our study mainly deals with the evaluation of oxidative stress, autoimmune, genetic and neurochemical hypotheses in Gujarat vitiligo patients. We have shown that our vitiligo patients exhibit significant oxidative stress and thus, systemic oxidative stress could play a pathophysiological role in precipitation of vitiligo in Gujarat population. Our studies revealed that presence of increased antimelanocyte antibodies and the imbalance of T-cell (CD4 + /CD8 + and Tregs) subsets along with their functional defects might result in melanocyte destruction in vitiligo patients. Our results on selected candidate genes in conferring oxidative stress and autoimmunity suggest that HLA-A*33:01, HLA-A*02:01,HLA- B*44:03, HLA-DRB1*07:01 and a few studied polymorphisms in IL4, CTLA4, SOD2, SOD3, GPX1, NALP1, MYG1, TNFA, TNFB, IFNG and IL10 genes are strongly associated with vitiligo susceptibility, whereas a few studied polymorphisms in PTPN22, MBL2, ACE, CAT, G6PD and SOD1 genes are not found to be significantly associated with Gujarat vitiligo patients. Gene expression studies of the IL4, CTLA4, SOD2, SOD3, NALP1, MYG1, TNFA, TNFB, IFNG, IL10 and ICAM1 genes suggest that these genes are strongly associated with vitiligo susceptibility. We are also addressing the role of immune-regulatory genes with respect to their expression in skin along with the effect of selected cytokines on in vitro cultured melanocytes derived from healthy and vitiliginous human skin to have an insight towards vitiligo pathogenesis. We are also exploring the potential microRNAs involved in pathogenesis of vitiligo. This integrated study will provide a better understanding of the role played by oxidative stress and autoimmunity in the pathogenesis of vitiligo in Gujarat population and also to develop selective therapy and the genetic marker/s for vitiligo. Various factors such as the antioxidant status, LPO (oxidative stress) levels and antimelanocyte antibody titer decide the selective therapy for our vitiligo patients. The pathogenesis of vitiligo though partially understood still remains complex and enigmatic to a greater extent. Though the condition may be precipitated by multiple etiologies, the interaction of oxidative stress and immune system clearly appears to be the key convergent pathway that initiates and/or amplifies the enigmatic loss of melanocytes in vitiligo. I58 STR Markers in clinics: a rapid prenatal diagnosis by quantitative fluorescent-pcr for aneuploidies Sarita Agarwal * , M Srinivasan, Shubha Phadke Department of Medical Genetics, SGPGIMS, Lucknow, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):I58 In the past few decades, prenatal diagnosis of fetal chromosomal abnormalities has relied on conventional cytogenetic analysis of cultured amniocytes, chorionic villi, or fetal blood. In recent years, the clinical validity of a newer technique, QF-PCR, to detect the common aneuploidies has been reported by a number of investigators. This technique has the advantage of providing rapid results for the diagnosis or exclusion of aneuploidy in chromosomes 13, 18, 21, X or Y. It is now possible to choose standard chromosome analysis or QF-PCR for the prenatal diagnosis of chromosomal abnormalities, or to perform both tests, depending on the clinical indication for testing. QF-PCR exploits the distribution of STR markers, 2-6bp tandem repeats, across the genome. This DNA marker is easily amplified by polymerase chain reaction (PCR) without the problem of differential amplification; that is, the PCR products for STRs are generally similar in amount, making analysis easier. An individual inherits one copy of an STR from each parent, which may or may not have similar repeat sizes. The number of repeats in STR markers can be highly variable among individuals, which make these STRs effective for diagnosis and human identification purposes. It has an added advantage of identifying maternal or paternal origin of nondysjunction. The establishment of QF-PCR has been initiated with the aim of providing rapid prenatal diagnostic service and to reduce anxiety of patients about their at risk pregnancies. For validation, we performed analysis on 100 confirmed cases of Down syndrome with the markers D21S1435, D21S11 and D21S1411, whose heterozygosity has been studied in North Indian population, with heterozygosity of 70.1%, 83.1% and 93.6% respectively. We observed 100% concordance with the clinical diagnosis as well as cytogenetic analysis. With these results we extended the methodology in prenatal services were chromosomal studies are very common for suspected Down syndrome pregnancies. However, in addition we included 2 more markers D21S1411 and D21S1413 for 21 chromosome, D13S238 and D13S631 for 13 chromosome, D18S391 for 18 chromosome and AMEL, XHPRT, X22 and SRY for sex chromosomes. Addition of 13, 18 and sex chromosomes marker were helpful at drawing conclusive reports for maternal contaminated samples and simultaneously to screen for rare aneuploides like 13, 18 trisomy and Klinefelter syndrome. The test was offered to the pregnancies with triple test positive as well as abnormal findings in ultrasonography. The source of DNA was 3 – 5 ml of amniotic fluid while in few cases chorionic villi sample. 190 pregnancies were studied to date, 3 pregnancies (1.6%) were turned to be Down syndrome positive while rest was negative for Down syndrome while 1 case was turned out to be homozygous for all 4 markers of 21chromosome in which we required additional markers to do reporting. However, karyotying showed this sample to be negative for Down syndrome. Rest all the finding was consistent with that of karyotyping results. The technique has its own advantage that can utilize the maternal blood contaminated samples, to add more, low volume of sample requirement and possibility to do reporting within 6 – 12 hours. Still we are working to record heterozygosity of rest of the 8 markers and to tune up the marker panel accordingly. SESSI ON XX: STEM CELL AND ERT THERAPEUTI CS I59 Mitochondrial donation by stem cells: potential for novel therapeutics Anurag Agrawal CSIR-Institute of Genomics & Integrative Biology, New Delhi, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):I59 There is increasing interest in whether mesenchymal stem cells (MSC) act as mitochondrial donors for rescuing injured cells. Islam et al (Nature Medicine, 2012) showed conclusively, using a mouse model of acute lung injury, that MSC mediated mitochondrial donation can lead to survival benefits (3). Contemporaneous work from Dr. Anurag Agrawal’s lab reveals the molecular regulation of mitochondrial movement during donation and shows how this can be engineered to increase therapeutic efficacy of MSC. They find that Miro1, a calcium sensitive mitochondrial Rho-GTPase that attaches mitochondria to KIF5 motor proteins on microtubules and regulates neuronal mitochondrial trafficking, is necessary for mitochondrial transfer by MSC. Overexpressing Miro1 in MSC (MSCmiro Hi ) led to increased mitochondrial transfer and therapeutic efficacy in mouse models of lung injury as well as asthma. This is a significant addition to the field of mitochondrial biology and stem cell therapeutics. It is also of general interest to physicians and members of public with interest in regenerative medicine, because this is highly translatable into more effective therapies. I60 Haematopoetic stem cell transplantation at apollo group hospitals Chirag Shah Apollo Hospitals International Limited, Gandhinagar, Gujarat, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):I60 Hematopoietic stem cell transplantation (HSCT) is the transplantation of multipotent hematopoietic stem cells, usually derived from bone marrow, peripheral blood, or umbilical cord blood. It is a medical procedure in the fields of hematology and oncology, most often performed for patients with certain cancers of the blood or bone marrow, such as multiple myeloma or leukemia. HSCT can be divided in two types, autologous and allogenic Molecular Cytogenetics 2014, Volume 7 Suppl 1 http://www.molecularcytogenetics.org/supplement/7/S1 Page 17 of 59 transplant based on the source of stem cells. Autologus is when stem cells are collected from patient’s own body. Allogeneic is when stem cells are collected from someone else. Apollo group hospitals perform largest number of HSCT in private sector. HSCT requires multidisciplinary effort with team of many experts including haematologist/oncologist, intensive care specialist, trained nursing staff, experts for venous access, infectious disease specialist, transfusion medicine expert, counsellors and many other experts. Over 700 patients have been treated by HSCT. The majority of these patients have haematological malignancies, consisting of acute and chronic leukaemias, lymphomas, plasma cell myeloma and other haematological neoplasms. Patients with other benign blood diseases including thalassemia, sickle cell disease, aplastic anemia, Fanconi anemia, and others have also been treated. The major facility for these patients is a specially designed haematology ward with HEPA-filtered air. The hemato-oncology department manages both adult and paediatric haematopoietic stem cell transplantation cases, where more than a hundred allogeneic and autologous transplantations are now performed every year. The full range of allogeneic transplantation is performed, with haematopoietic stem cells coming from HLA-identical siblings, matched unrelated donors, umbilical cord blood and haploidentical donors. I61 Perspective of stem cell research & therapy in diabetes Sarita Gupta Department of Biochemistry, Faculty of Science, The Maharaja Sayajirao University of Baroda, Vadodara, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):I61 In the area of regenerative medicine, researchers have been exploring the potential of Stem Cells in preclinical and clinical studies to improve therapies that can resolve injuries by enhancing endogenous repair, opening a new paradigm in cell therapy.Stem cells may it be embryonic stem cells (ESC), induced pluripotent stem cells (iPSC) or adult stem cells have been used as potential sources for beta cell replacement therapy across the globe. Numerous studies have shown the differentiation potential of ESCs and iPSCs towards producing pancreatic progenitors but after terminal differentiation into insulin producing cells these lacked various important pancreatic markers and have low index of glucose stimulated insulin secretion (GSIS). This along with other ethical conundrums makes it difficult to use ESCs as a source for cell therapy. Adult stem cells provide clinically and ethically accepted option over embryonic or induced pluripotent stem cells, as they can be used as autologous source for cell transplant in treatment of diabetes. Widely used option of cadaveric islet therapy has its own problems due to insufficient amount of islets available for transplantation. Hence, our research is focused on increasing islet mass from various adult stem cells using neutraceutic bioactive compounds isolated from a plant demonstrating efficient anti-diabetic activity and further explored them as potent differentiating agent under in-vitro and in-vivo condition. In-vitro differentiated islets from various adult stem cells sources were assessed at morphological, molecular, immunological and functional level and further evaluated for glucose lowering effect after transplantation in Streptozotocin (STZ) induced diabetic balb/c mice. ORAL PRESENTATI ON O1 A Poisson regression model for association mapping of count phenotypes Saurabh Ghosh 1* , Abhishek Chakrabortty 2 1 Human Genetics Unit, Indian Statistical Institute, Kolkata, India; 2 Department of Biostatistics, Harvard University, Cambridge, USA E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):O1 Background: Clinical end-point traits are often characterized in terms of quantitative precursors. For psychiatric disorders, traits such as symptom counts often serve as endophenotypes of interest for understanding the genetic basis of the clinical end-point trait. Since such traits are discrete in nature, it may not be optimal to use standard approaches such as Analysis of Variance (ANOVA) to detect association. Methodology: For population level data, we propose a Poisson regression approach that computes the likelihood of the count phenotype conditional on an additive allele count at a SNP. The test statistic is asymptotically distributed as chi-squares with one degree of freedom under no association between the SNP and the phenotype. For family-based data involving trios with at least one heterozygous parent at a SNP, we use a similar Poisson regression model conditional on two indicator variables: the marker allele transmitted by the heterozygous parent and the marker allele transmitted by the other parent. A one degree of freedom test based only on the coefficient of the first indicator is protected against population stratification as it tests for association in the presence of linkage. Two degrees of freedom test based on both the indicators is also a valid test for association, but is susceptible to population stratification. Results and conclusions: Based on extensive simulations under different genetic models, we find that for population level data, while the asymptotic tests for ANOVA yield an inflated rate of false positives, especially when there is heteroskedasticity in the distribution of the trait across the QTL genotypes, our proposed method maintains the correct size. Moreover, our method yields uniformly more power compared to ANOVA for the different genetic parameters in our simulations. For the trio design, we find that the two degrees of freedom test is more powerful than the one degree of freedom test. We applied our method to analyze externalizing symptoms, an endophenotype correlated with alcoholism using data generated in the Collaborative Study On the Genetics of Alcoholism (COGA) project. We found significant evidence of association in the class 1 alcohol dehydrogenase subunit ADH1C in the 4q22.3 region. O2 Therapeutic potential of histone deacetylase inhibitor for treatment of Niemann-Pick type C1 disease Nina H Pipalia * , Frederick R Maxfield Department of Biochemistry, Weill Cornell medical College, 1300 York Ave. New York, NY 10065, USA E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):O2 Background: Niemann-Pick type C (NPC) is a rare, autosomal recessive neurodegenerative lysosomal storage disorder (LSD) caused due to mutation in either npc1 or npc2 genes. However, 95% of the reported cases are caused due to mutation in npc1 gene and only 5% due to the mutation in npc2 gene. Mutations in either of these two genes results in similar phenotype such that there is an abnormal accumulation of unesterified cholesterol and glycosphingolipids in late endosomes/ lysosomes (LE/Ly) of many cell types. NPC1 is a multi-spanning transmembrane protein localized in limiting membrane of LE/Ly whereas NPC2 is soluble cholesterol binding protein localized in the lumen of LE/Ly. There are no therapeutic options to treat the disease and the children born with this defect die before the age of 20 years. Miglustat, a drug used to treat Gaucher disease has been reported to stabilize NPC patients and has also been approved for treating NPC patients in Europe. Infusion of very high dose of chemical chaperone like 2-hydroxypropyl-b-cyclodextrin (HPBCD) several times a week has also been shown to reduce the cholesterol load in peripheral tissues. Unfortunately, the blood brain barrier (BBB) cross over capability of HPCD is very poor and hence requires intrathecal CNS injections. Results: We have found that the treatment of several histone deacetylase inhibitors (HDACi), corrects the NPC defect specifically in human patient derived NPC1, but not in NPC2 fibroblast. Amongst the HDACi tested Vorinostat is a FDA approved drug that has been shown to cross BBB and is currently used in clinic for treatment of cutaneous T-cell lymphoma and many other type of cancer. Our initial study was conducted on most common mutation “I1061T” found in human patients but since then we have tested the effect of Vorinostat and other HDACi’s on several different patient derived mutant fibroblasts. Conclusions: Our data indicate that Vorinostat is effective in rescuing the phenotype in most cases but to a varying degree. This is a promising breakthrough example of repurposing existing FDA approved drug for the treatment of NPC1 and other LSDs. Molecular Cytogenetics 2014, Volume 7 Suppl 1 http://www.molecularcytogenetics.org/supplement/7/S1 Page 18 of 59 O3 Genetics of autism spectrum disorder & BDNF gene Usha P Dave Medical Geneticist & Neuroscientist, Mumbai, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):O3 Autism is a group of complex neurodevelopmental disorders which manifests problems with social interaction, language, communication and behavior deficits like stereotype and repetitive activities. Autism prevalence rate in last one decade has shown astonishing level of increase from 1 per 10,000 to 1 in 110 children in USA (CDC, 2007). Various environmental & genetic factors or combination are suggested as contributing factors in this clinically diagnosed neurobehaviour syndrome. However, similar data on prevalence are scarce in India. The etiology of autism still remains unknown, with many factors implicated in the development of autism phenotype. Autism Spectrum Disorder (ASD) is evaluated by a clinical psychologist using DSMR- IV criteria & may manifest mild to severe autistic features, clinical symptoms & low to high intellectual functioning. There are few well characterized genetic/ metabolic conditions (e.g. Rett Syndrome, Tuberous sclerosis, PKU) and chromosomal syndromes (e.g. Fragile-X, Angelman and Prader willi ) where autism has frequently associated features. Multiple genes are thought to be involved in the pathogenesis, but no evidence involving any one particular gene. The recent research focus is also on epigenetic mechanisms operating in complex autism. Brain Derived Neurotrophic Factor (BDNF) is a neurotophin in the mammalian Central Nervous System & important in neuronal survival, neurogenesis, and synaptic plasticity. BDNF gene is located on chromosome 11. In humans, Val66Met is probably the most investigated SNP of the BDNF gene. Since BDNF readily crosses the Blood-Brain-Barrier, the serum concentrations correlate directly to brain concentration, therefore plasma studies of BDNF are thought to accurately reflect CNS concentration. The significance of serum BDNF in ASD to explore its precise role in pathogenesis of ASD and therapeutic relevance is increased with the evidence of BDNF linked with autism. A genetically heterogeneous population of India where consanguinity and endogamous marriages are prevalent genetic risk factors, ASD is a challenge. An attempt is made to study BDNF level & mutations in correlation with the severity of neurobehavioral deficits in Indian patients with ASD & mental retardation. This will be discussed in the light of current scenario. O4 Identification and clinical evaluation of segments of homozygosity, uniparental disomy and complex chromosomal abnormalities revealed by copy-number SNP arrays Jia-Chi Wang, Leslie Ross, Loretta W. Mahon, Renius Owen, Morteza Hemmat, Boris T Wang, Mohammed El Naggar, Kimberly A Kopita, Mary Haddadin, Fatih Z Boyar, Arturo Anguiano, Charles M Strom, Trilochan Sahoo * Cytogenetics Laboratory, Quest Diagnostics Nichols Institute, 33608 Ortega Highway, San Juan Capistrano, CA, USA E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):O4 Background: Presence of such segments of homozygosity (SOH) may be due to parental relatedness, chromosomal recombination or rearrangements and provides important clues regarding ancestral homozygosity, parental consanguinity or uniparental disomy. We have determined the frequency and nature of copy neutral segments with allelic homozygosity identified in cases interrogated by oligonucleotide-SNP microarrays. Materials and methods: We collected cases from consecutive specimens sent to our clinical laboratory over the past two years. The cases were reported based on the presence of a contiguous SOH >10 Mb in a single region or >5 Mb in at least two regions. The percentage of the genome encompassed by SOH regions was calculated based on the total coverage of about 2,700 Mb. Results: Of 14,574 cases analyzed by SNP arrays, 872 (6%) cases harbored SOH, with 659 (76%) cases harboring multiple SOH and interpreted as arising due to identity by descent (IBD), 213 (24%) cases with SOH involving a single chromosomal segment and suspected or confirmed as resulting from UPD. For the cases with IBD, the coefficient of inbreeding was calculated: 5% cases due to first degree or closer parental relatedness, 9% second, 19% third, 16% fourth, and 51% fifth. Cases with UPD cases involved every single chromosome. In eight cases, identification of SOH was crucial to diagnosing autosomal recessive disorders. Conclusions: This study demonstrates that the identification of SOH, in addition to CNVs, is much more frequent than previously recognized, reflecting close parental relatedness, and often ascertains autosomal recessive diseases or unravels UPD in many cases. O5 Utility of CD-138 negative fraction for chromosome analysis in Plasma Cell Dyscrasias (PCD): a novel approach Gopalrao Velagaleti * , Christina Mendiola, Gihan Mohamed, William Ehman Jr., Vinaya Noronha, Veronica Ortega Department of Pathology, University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, TX, USA E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):O5 Background: FISH analysis is superior to chromosome analysis in detecting important prognostic genetic abnormalities in PCD. However, its sensitivity is hampered due to paucity of plasma cells in whole bone marrow and often shows false-negative results when frequency of abnormal cells is below the cut-off values. Studies have shown that the abnormality detection rate in enriched plasma cells (EPC) is greater than unselected plasma cells, but purification techniques are limiting to only FISH when bone marrow volumes are inadequate. The inability to perform chromosome analysis may compromise patient care since chromosome analysis is equally important for detecting non-plasma cell related abnormalities, such as secondary myelodysplastic syndrome. To resolve this critical issue and optimize limited quantity received, we designed a study where an immuno-magnetic CD138 enriched positive selection of plasma cells was used for FISH while the negative selection was used to retrieve the remaining cellular components (RCC) for chromosome analysis. After validating this approach in a pilot study, we implemented this strategy in 2012 for routine clinical diagnosis in patients with PCD. When there was adequate sample volume available, both whole bone marrow and RCC were used for chromosome analysis. Results: We found 100% success rate for chromosome analysis using RCC. Identical PCD related genetic abnormalities were observed by both chromosome analysis using whole bone marrow and EPC FISH in 4.2% (4/96) cases while 8.3% (8/96) cases showed discordant results. Population variants such as loss of Y chromosome were observed in 6.3% (6/96) cases. Karyotypic abnormalities of myeloid origin or population variants were found both in whole bone marrow and RCC cultures. Karyotypes with PCD related aberrations were seen only in whole bone marrow cultures in 37.5% of the cases (3/8) while the remaining 62.5% (5/8) of cases showed them in both whole bone marrow and RCC cultures. All PCD related aberrations showed concordant EPC FISH results. Conclusions: Our results confirm the feasibility of retrieving RCC from the CD138 negative fraction, and prove to be an innovative strategy for performing chromosome analysis on PCD patients with insufficient sample volumes. O6 Prenatal screening above aneuploidies that is Pre-Eclampsia Gurjit Kaur Department of Physiology, Consultant Incharge, Genetic Centre, GMCH-32, Chandigarh, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):O6 Background: Pre-Eclampsia is defined as high blood pressure and excess protein in the urine after 20 weeks of pregnancy in a normotensive woman. Even a slight increase in blood pressure may be a sign of preeclampsia. Despite extensive clinical trials no therapeutic approaches are available for either treatment or prevention of preeclampsia. Removal of placenta remains the only solution for resolution of preeclampsia which necessitates premature deliveries and can have adverse outcomes of low Molecular Cytogenetics 2014, Volume 7 Suppl 1 http://www.molecularcytogenetics.org/supplement/7/S1 Page 19 of 59 birth-weight babies, at the same time increasing the risk of maternal and neonatal mortality and morbidity worldwide. The concentrations of several placental proteins, inflammatory cytokines and growth factors are altered in the maternal circulation of women with preeclampsia. Nonetheless, early pregnancy screening for preeclampsia remains insufficient, and randomized controlled trials that used biomarkers to identify high risk women have been disappointing, perhaps because the sensitivity of most of these markers is high in the second or third trimester, long after the placental dysfunction is already established. At Genetic Centre, GMCH-32, Chandigarh we have tried to correlate first trimester biochemical and ultrasonographic markers (viz. Pregnancy Associated Plasma Protein-A, Placental Growth Factor, Doppler Pulsatility Index) with establishment of preeclampsia and other obstetrical complications (fetal growth restriction, pre-mature delivery, risk of miscarriage, still-birth) in pregnant women. This study will help in determining the possible diagnostic utility of PAPPA, PlGF, and Doppler Pulsatility Index as sensitive and specific biomarkers for screening early onset of pre-eclampsia. Materials and methods: All pregnant women visiting Government Medical College and Hospital, Chandigarh in first trimester of pregnancy are tested for PAPPA, PlGF, and Doppler Pulsatility Index after thorough counselling and written informed consent. PAPPA, PlGF and Doppler Pulsatility Index are quantitatively analyzed and multiple of medians are obtained of approximately 1000 pregnant ladies. Result and conclusion: In order to relate mode of delivery (NVD/LSCS) with PAPPA, PlGF, and Doppler Pulsatility Index an initial sample size of 222 was taken for analysis. The association of mode of delivery was carried out using chi square test and it has been observed that PAPPA risk has a significant association with mode of delivery however PlGF and Doppler Pulsatility Index do not have a significant association. However this is an ongoing study and statistical analysis of more samples is needed to get a significant association. O7 Suspected microdeletion syndromes and molecular cytogenetic techniques: an experience with 330 cases Ashutosh Halder * , Manish Jain, Isha Chaudhary Department of Reproductive Biology, AIIMS, New Delhi 110029, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):O7 Background: Microdeletion syndromes are characterized by small (< 5Mb) chromosomal deletion in which one or more genes are involved. They are frequently associated with multiple congenital anomalies. The phenotype is the result of haploin sufficiency of genes in the critical interval. Fluorescent In- Situ Hybridization (FISH), Multiplex Ligation-dependent Probe Amplification (MLPA), Quantitative Fluorescent Polymerase Chain Reaction (QFPCR) and Array (microarray) Comparative Genomic Hybridization (aCGH) techniques are commonly used for precise genetic diagnosis of microdeletion syndromes. Methods: This study comprised of 330 cases of suspected microdeletion syndromes. There were 184 cases of 22q11.2 microdeletion, 52 cases of William, 47 cases of Prader Willi/Angelman, 18 cases of Miller Dieker, 14 cases of Retinoblastoma (bilateral infantile), 5 cases of Trichorhinophalangeal (TRP) and 10 cases of other microdeletion syndromes. FISH was carried out on all 330 clinically suspected microdeletion syndrome cases using non- commercial FISH probes. Subsequently, we have performed aCGH in 100 cases (77 cases of 22q11.2; 9 cases of William Syndrome, 8 cases of Prader Willi Syndrome, 4 cases of Miller Dieker Syndrome and 2 cases of other microdeletion syndromes) including one monozygotic twin pairs with discordant phenotype. Another 50 cases, including 22q11.2 microdeletion, aCGH experiment and analysis are in progress. Results: FISH was confirmatory in 28 cases only (8.48%; 19 cases of 22q11.2 microdeletion, 5 cases of Prader Willi, 3 cases of William and 1 case of TRP syndrome). There were 8 cases with mosaicism and 20 cases with pure deletion. Microarray was picked up copy number variation (CNV) with or without copy neutral loss of heterozygosity (LOH) in approximately 70% of cases, mostly involving several chromosome loci. However, aCGH was failed to pick up mosaic cases (with even 45% deleted cell lines). Clinically suspected specific locus CNV was detectable in approximately 24% cases only by aCGH. Variation in deletion sizes and or break point differences (with genes involvement variations) as well as other CNVs with or without LOH was evident. Conclusions: We conclude that FISH in this format should not be the method of choice for clinically suspected microdeletion syndromes as cost, labor & time versus benefit is unjust. Microarray seems better technique, in clinically doubtful cases. However, microarray is likely going to miss mosaic cases, if deleted cell lines concentration is less than 50%. It seems time has come to follow strict clinical criteria for FISH testing or preferably to follow better methods viz., DNA microarray (array comparative genomic hybridization). We think that whole genome screening should be adopted as first line of investigation and FISH may be used for detecting mosaicism, screening family members and prenatal diagnosis. Furthermore, microdeletion syndrome best fitted with genomic disorder as several chromosomal loci are involved in CNV with or without LOH and alteration in deletion size or breakpoint. Our study has not found identical deletion profile in any cases, thus explaining reason for phenotypic variability between deletion positive cases. O8 Molecular characterization of mutations in galactosemia genes: structural and functional implications R Singh 1 , BR Thapa 2 , G Kaur 3 , R Prasad 1* 1 Department of Biochemistry, Postgraduate Institute of Medical Education and Research, Chandigarh, India; 2 Department of Gastroenterology, Postgraduate Institute of Medical Education and Research, Chandigarh, India; 3 Department of Physiology, GMCH, Chandigarh, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):O8 Background: Galactosemia is an autosomal recessive metabolic disorder caused by the deficiency of enzymes involved in galactose metabolism resulting in complications like cataracts, hepatocellular damage and developmental delay. Nonetheless, no report is available on mutations in galactosemia genes from our population. The objective of the present study was to determine blood GALT and GALK activity in infants with cholestasis and congenital cataracts and to establish a spectrum of mutations in GALT and GALK genes. Material and methods: 430 infants (2 days-11 months) with cholestasis admitted in Pediatric Gastroenterology over 3.5 years were evaluated for galactosemia. Basic investigations included hemogram, liver function tests, blood culture, urine culture, urine for non-glucose reducing substances and eye evaluation. Screening for GALT deficiency was done using Perkin–Elmer neonatal GALT kit. The levels of galactose-1-phosphate were also measured. Also, 115 patients with congenital cataracts were screened for the galacto- kinase (GALK) deficiency. Mutation analysis for most common Q188R and N314D mutations in GALT gene was performed by Restriction Fragment Length Polymorphism (RFLP). Single Stranded Conformational Polymorphism (SSCP) analysis and subsequently DNA sequencing were done for identification and characterization of unknown and novel mutations in GALT and GALK genes. Results: 55 (12.7%) infants were found to have reduced GALT activity with male: female: 37:18, jaundice in 55 (100%), hepatomegaly in 54 (98%), splenomegaly in 32 (58%), coagulopathy in 23 (42%), encephalopathy in 9 (16%), septicemia in 10 (18%) and cataracts in 12 (22%) were observed. Increased galactose-1 phosphate levels were fraternized with reduced activity of GALT. A total of 16 mutations and 4 polymorphisms were detected. 10 were novel mutations. Reduced blood galactokinase activity was found in 13 (10.2%) patients with congenital cataracts. 4 novel mutations were found in GALK gene. Conclusions: N314D mutation was found to be the most common mutation in our population. 10 and 4 novel mutations were also detected in GALT and GALK genes respectively. ARRAY- CGH P1 Use of array CGH for molecular characterization of genetic disorders Ashish Bahal * , Rajitha P , Ashwin Dalal Center for DNA Fingerprinting and Diagnostics, Nampally, Hyderabad – 500001, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):P1 Molecular Cytogenetics 2014, Volume 7 Suppl 1 http://www.molecularcytogenetics.org/supplement/7/S1 Page 20 of 59 Background: Microarray-based comparative genomic hybridization (array CGH) is a revolutionary platform that has been developed to screen entire genome for copy number variations (CNV) with resolution beyond the capacity of light microscope. ACMG has recommended that array CGH can be used as the first line investigation modality in cases of non- syndromic mental retardation. We present here three cases in which use of array CGH has provided an insight of genetic abnormality. Cases studies: Case 1 was a 2 month old child who presented with multiple dysmorphic features of face and extremities. The karyotype of the child showed presence of additional chromosomal material of unknown origin on chromosome 18. Array CGH was carried out which showed duplication of region 2q31.1-q37.3. Whole chromosome paint probe for chromosome 2 showed presence of chromosome 2 material on 18q. Case 2 was a 10 year old girl having intellectual disability and facial dysmorphism. The initial cytogenetic evaluation did not reveal any abnormality. Further, array CGH showed presence of gain in chromosome 1 from bases 1959715 to 2787707. The segment was analysed using computational software (Decipher) which showed it to be a pathogenic CNV for Intellectual disability. Case 3 was from two affected children in a consanguineous family with hypotonia and delayed motor milestones. Molecular analysis for Spinal Muscular Atrophy (SMA) was negative. Array CGH was done to look for regions of loss of heterozygosity which were detected in multiple regions. Further analysis of these regions was done to look for the genes associated with SMA and we found PLEKHG5 gene within homozygous regions. Conclusions: Our study demonstrates the usefulness of array-CGH in detailed characterization of chromosomal rearrangements, detection of submicroscopic copy number variations and detection of regions of homozygosity in single gene disorders in consanguineous population. P2 Diagnostic utility of array-based Comparative Genomic Hybridization in a clinical setting Frenny Sheth 1 , Chaitanya Datar 2 , Koumudi Godbole 3 , Joris Andrieux 4 , Manisha Desai 1* , Bhumi Patel 1 , Jayesh Sheth 1 1 FRIGE’s Institute of Human Genetics, FRIGE House, Jodhpur Gam Road, Satellite, Ahmedabad, India; 2 Sahyadri Genetics, Unit of Sahyadri Hospitals, Barve Memorial Complex, J.M. Road, Pune, India; 3 Deenanath Mangeshkar Hospital and Research Center, Erandawane, Pune, India; 4 Laboratory of Medical Genetics, Jeanne de Flandre Hospital CHRU de Lille, Lille Cedex, France Molecular Cytogenetics 2014, 7(Suppl 1):P2 Background: Submicroscopic genomic imbalances are a major cause of congenital and developmental abnormalities including unexplained Developmental Delay (DD), Intellectual Disability (ID), Autism Spectrum Disorders (ASD) and Multiple Congenital Anomalies (MCA). Submicroscopic imbalances are not visible at the resolution level offered by conventional cytogenetics techniques but could potentially be analysed with array based comparative genomic hybridization (aCGH) which offers high resolution scan of the genome. We present the motion backed by clinical evidence for the diagnostic utility of aCGH in a clinical settings for aforementioned cases. Material and methods: aCGH analysis was carried out in 37 non-syndromic individuals that clinically presented with either one or a combination of aforementioned phenotypes. Of which, 13 cases had congenital anomalies with or without mental retardation [Group-1]. Remaining 24 cases presented developmental and learning disabilities with seizure [Group-2]. Conventional cytogenetic [GTG-] banding analysis showed apparently normal chromosomal pattern at 550-band resolution. Hence, all 37 cases were further analyzed using Agilent 60k oligonucleotide array-Comparative Genomic Hybridization (aCGH). Sex matched genomic DNA (Promega Corporation, Madison, WI, USA) was used as reference. Relative fluorescence intensity data was analyzed with the aCGH analysis software v3.4 (Agilent Technologies Inc., Santa Clara, CA, USA) by applying Z-score segmentation algorithm with a window size of 10 points to identify chromosome aberrations. Parents in 12 out 16 families were analysed for genomic imbalances using q-PCR to confirm the mode of inheritance. Results: Cryptic quantitative genomic alteration was detected in a total of 16 cases that include 31% [4/13] from group-1 and 50% [12/24] from group-2. The chromosomes involved in submicroscopic alterations were 1p, 2q, 5q, 6q, 7q, 8q, 10q, 11q, 12q, 15q, 18q, 19q, 22q, Xp and Xq. Single copy number variation was detected in 14 cases whereas in the remaining two, multiple CNVs were detected. De novo alteration and parental inheritance was confirmed in 4 and 7 cases respectively. One case is currently under investigation and four cases did not provide consent for further investigation. Conclusions: aCGH is currently practiced as a front line diagnostic technique for patients with developmental delay and intellectual disability in various countries. This technique is also being applied to understand why patients get developmental disorders as a part of Deciphering Development Delay (DDD) study. Accurate information of etiology aids in genetic counselling and calculating risk for future pregnancy. Our clinical data indicate the application of aCGH in cases with complex phenotypes and apparently normal karyotype. P3 Combined classical cytogenetics and array Comparative Genomic Hybridisation for genomic copy number analysis Meena Lall * , Pushpa Saviour, Ratna Puri, Preeti Paliwal, Surbhi Mahajan, Ishwar Verma Center of Medical Genetics, Sir Gangaram Hospital, New Delhi 110060, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):P3 Background: There is a great need for reporting and cataloging of genotype phenotype correlation of clusters of individuals sharing similar genomic rearrangements and phenotypes. This will facilitate diagnosis and personalized genetic counseling and will also improve our understanding of gene function and disease. Karyotyping and FISH are microscopic classical cytogenetic methods of detecting gain, loss or rearrangement of genetic material. Array Comparative Genomic Hybridisation (CGH) can used for molecular characterization, to size the abnormality and study the gene content. Materials and methods: A pilot study was designed: Combined classical cytogenetic, Array CGH and phenotypic findings were correlated in 20 patients with autism, mental retardation or congenital malformations. Results: Ten out of 20 patients with autism, mental retardation or congenital malformations had a normal karyotype and ten had abnormal karyotype. Ten normal subjects were included as normal controls, of which two had balanced translocations, which were not recognized by ACGH. Conclusions: Copy number variations detected by ACGH can be novel or extremely rare such that uncertainty may remain as to whether the aberration is pathogenic or simply a benign variant. Copy number variant (CNV) loci have been listed in the database of genomic variants (DGV). Using this and the UCSC browser the analysis of the size of the aberration and gene content was done in all individual cases, with normal or abnormal karyotypes. These findings were correlated with the phenotype to recognize individual syndromes. Conclusions: This pilot study is just the beginning. Identification of more patients, who share a region of genomic duplication or deletion and have phenotypic features in common, will allow greater certainty to be given to the pathogenic nature of the rearrangement and delineation of new syndromes. Most data available is western. Therefore there is a need to document and register Indian data accumulated to recognize the more common syndromes affecting our population and the common benign CNVs which can be discounted in our population, during the CNV analysis. CANCER GENETI CS P4 A study of human Kallikrein-2 gene polymorphism with special reference to prostate cancer patients in India Rajesh Biswas 1* , Shiv Deep Bansal 2 , Kakoli Biswas 3 , Shrawan K. Singh 4 1 Department of Zoology, Government Home Science College, Sector-10, Chandigarh, India; 2 National Centre for Human Genome Studies and Research, Panjab University, Chandigarh, India; 3 Department of Biotechnology, DAV College, Sector-10, Chandigarh, India; 4 Department of Urology, Postgraduate Institute of Medical Education and Research, Chandigarh, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):P4 Molecular Cytogenetics 2014, Volume 7 Suppl 1 http://www.molecularcytogenetics.org/supplement/7/S1 Page 21 of 59 Background: Prostate cancer is the third most common cancer among men in the world. The human kallikrein-2 gene (KLK2 gene) contains a mutant (T) for wild-type (C) allele substitution, resulting in a Trp to Arg change on the 250 th amino acid at the 784 th nucleotide of KLK2 cDNA. The reports on polymorphism in the KLK2 gene in relation to prostate cancer are contradictory among American and Korean population. Reports are not available regarding polymorphism in KLK2 gene in relation to prostate cancer in Indian population. The relationship between mutants (T) for wild-type (C) allele substitution of the KLK2, circulating human kallikrein-2 (hK2) levels and prostate cancer risk in Indian population has been examined. Materials and methods: The study was limited to 15 subjects that include seven patients with prostate cancer and six controls who were persons with no cancer. Peripheral blood samples were collected from subject and control. DNA was extracted from these samples using standard protocol. DNA was dissolved in 1X TE buffer and used for PCR reactions for the amplification of 322bp KLK2 gene. Polymerase chain reaction- Restriction fragment length polymorphism (PCR-RFLP) procedure was employed using MspI to detect this polymorphism. The enzyme recognizes the 4bp sequence 5ʹCCGG3ʹ at codon 226 of KLK2 gene. PCR-RFLP products ware separated in 2% agarose gel and viewed under Gel-doc system. Results: The median age of the eight subjects diagnosed for prostate cancer were 60, out of which 6 are found to be homozygous for CC allele of KLK2 gene, 1 was heterozygous having CT allele and 1 was homozygous for TT allele. From 7 subjects diagnosed negative for prostate cancer 2 were having the CT genotype and 5 were found to have the TT genotype. Conclusions: The study indicates a possible correlation among the C/T polymorphism of the KLK2 gene and circulating levels of hK2 and, in combination, may be predictive for prostate cancer. Though significant association has been found with small subjects, uses of allele marker for prostate cancer need further extensive study with large number of subject. P5 Role of betel quid in changing oral pathology Aniket Adhikari 1* , Subhrajyoti Mukherjee 2 , Kaustav Roy 2 , Ranjan Roychowdhury 3 , Madhusnata De 1 1 Department of Genetics, Vivekananda Institute of Medical Sciences, Ramakrishna Mission Seva Pratisthan, 99- Sarat Bose Road, Kolkata 700 026, West Bengal, India; 2 Department of Oral and Maxillofacial surgery of Ramakrishna Mission Seva Pratisthan, 99- Sarat Bose Road, Kolkata 700 026, West Bengal, India; 3 Department of ENT of Ramakrishna Mission Seva Pratisthan, 99- Sarat Bose Road, Kolkata 700 026, West Bengal, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):P5 Background: Chewing of betel quid (BQ) and areca nut is an ancient custom in South East Asia. More than 600,000,000 people chew areca nut worldwide. In India there are 75,000 to 80,000 cases of oral cancer each year and incidence rates of cancers of the oral cavity in both male and female in all urban cancers are among the highest in the world. In India, 13 -50% of the student chew BQ and pan masala frequently and prevalence rate is 14 -15% among 11-15 year of children. Areca nuts, Catechu, Slaked lime are major components of Betel quid. Nitrosamines formed from alkaloids in betel nut and reactive oxygen species (ROS) are generated due to slaked lime during betel quid chewing may be implicated in the etiology of oral cancer. Micronuclei (MN) have been proposed as a good biomarker to assess cytogenetic damage. Among the xenobiotics metabolizing enzymes, the CYP2A family is characteristics of its catalytic properties to nitrosamines. Micronuclei (MN) and CYP2A6 genetic polymorphism were studied among the Eastern and North eastern population. Materials and methods: In this present study subjects were screened from different camps and Department of E.N.T. & Oral and Maxillofacial surgery of RKMSP hospital, Kolkata. Exfoliated cell from the buccal mucosa were examined for micronuclei (MN). Polymorphism of CYP2A6 gene was studied from EDTA blood. Results: Micronuclei percentage was higher in subjects who had betel quid chewing habit. Early metabolizers are susceptible to oral cancer whereas in case of poor metabolizers chances are less. Conclusions: Oral pathology seems to be altered due to chewing betel quid and its ingredients with increased susceptibility to cancer. P6 In silico screening of alleged miRNAs associated with cell competition: an emerging cellular event in cancer Manish S Patel 1* , Bhavesh V Antala 1 , Neeta Shrivastava 1,2 1 Department of Biotechnology, National Institute of Pharmaceutical Education and Research (NIPER)-Ahmedabad, Ahmedabad-380054, Gujarat, India; 2 Department of Pharmacognosy and Phytochemistry, B. V. Patel Pharmaceutical Education and Research Development (PERD) Centre, Ahmedabad-380054, Gujarat, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):P6 Background: Cell competition is identified as a crucial phenomenon for various conditions like cancer, aging and organ development. MicroRNAs (miRNAs) may play an important role in regulation of expression of genes involved in cell competition at the post-transcriptional level. In silico screening of miRNAs involved in a cell competition is an effort to identify potential miRNAs and to reduce, economize and expedite experimental work. In the present study we have identified miRNAs of Drosophila genome involved in a cell competition using in silico screening strategy. Material and methods: We used four steps of in silico (i) Selection of cell competition related genes of Drosophila genome through literature survey; (ii) Identification of miRNAs that target selected cell competition-related genes; (iii) Sequence conservation analysis of identified miRNAs with Human genome; (iv) Identification of genomic location of that Drosophila miRNAs and exploration of their expression profiles in tissues by use of host gene expression profile. Results and conclusions: In this study we have identified seven potential Drosophila miRNAs that are most probably involved in cell competition. Human homologs of these seven potential miRNAs may be very important in cell competition related diseases like cancer. Proof of this concept will be established with wet lab experimentation to reassert the role of miRNAs in cell competition. P7 Analyzing the expression of candidate microRNAs in primary tumors of oral squamous cell carcinoma Mayakannan Manikandan * , Deva Magendhra Rao A K, Arasambattu Kannan Munirajan Department of Genetics, Dr. ALM PG Institute of Basic Medical Sciences, University of Madras, Taramani campus, Chennai – 600113, Tamil Nadu, India Molecular Cytogenetics 2014, 7(Suppl 1):P7 Background: Accumulating evidences suggest that aberrant expression of microRNAs (miRNAs) contribute to the initiation, development, invasion and metastasis of human cancers including Oral Squamous Cell Carcinoma (OSCC). Materials and methods: In this study, we investigated the differential expression of six candidate miRNAs namely hsa-miR-21, hsa-miR-125b-2, hsa-miR-138, hsa-miR-155, hsa-miR-184 and hsa-miR-205 in thirty two OSCC primary tumors in comparison to five normal control tissue samples by employing TaqMan® MicroRNA Assays. Results: Amongst the studied candidates, miR-125b-2 and miR-205 were significantly down regulated while miR-21 and miR-155 were significantly upregulated in the primary tumors compared to controls. The other two miRNAs, miR-184 and miR-138, in general were down regulated in the cancer samples although not statistically significant. These observations suggest that microRNA dysfunction could be one among the major factors for initiation and progression of oral cancer with miR-125b-2 and miR-205 functioning as tumor suppressor miRs, and miR-155 and mir-21 acting as oncomiRs. Functional annotation of the experimentally validated targets of these miRNAs highlighted that miR-21, miR-125b-2 and miR-155 were predominantly targeting genes of the apoptotic pathway, while miR-205 was targeting genes involved in transcriptional and metabolic processes. Conclusions: It is concluded that microRNA dysfunction could be one among the major factors for initiation and progression of oral cancer. However, further experimental studies in more OSCC primary tumors are required to facilitate the use of these miRNAs as molecular biomarkers or as diagnostic/prognostic indicators. Molecular Cytogenetics 2014, Volume 7 Suppl 1 http://www.molecularcytogenetics.org/supplement/7/S1 Page 22 of 59 P8 The role of direct DNA repair gene O6-methylguanine-DNA methyltransferase (MGMT) in high grade malignant glioma M Jeru Manoj 1* , G K Chetan 1 , KVL Narasinga Rao 2 , HN Venkatesh 1 , MK Sibin 1 , Ch Lavanya 1 1 Department of Human Genetics NIMHANS, Bangalore-29, India; 2 Department of Neurosurgery, NIMHANS, Bangalore-29, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):P8 Background: High-grade [WHO, 2007- grade III-IV] gliomas are undifferen- tiated or anaplastic, are highly infiltrating and termed as malignant. The overall prognosis and survival rate is very low. Only few highly sensitive and specific biomarkers for Glioma have been identified, but are yet to be put for routine use due to challenges in their clinical validation for early disease detection, diagnosis and monitoring to improve long-term survival of patients. The inefficacy of currently available chemotherapeutic and radio- therapeutic agents largely depends on a number of resistance mechanisms among which DNA repair plays an important role. Hence bio-molecules involved in DNA repair mechanisms will be promising biomarkers. Current status in the validation of DNA repair genes include, studying the promoter methylation of the only direct DNA repair gene O6-methylguanine-DNA methyltransferase (MGMT). MGMT is epigenetically inactivated via hypermethylation of the 5′-CpG islands located in promoter region. It is associated with prediction of successful alkylating agent therapy with temozolomide in combination with radiotherapy and also longer overall survival in patients. The silenced MGMT gene possibly enhances radiation effects by inducing radiation-mediated double stranded DNA (dsDNA) breaks and suppression of dsDNA repair pathways after radiation exposure. Materials & methods: Post surgery excised tumor samples from 20 patients diagnosed histopathologically with high grade glioma were tested for MGMT promoter region methylation status. DNA extraction and bisulphate conversion using suitable commercially available kits (Qiagen) followed by Methylation specific PCR using specific methylated and unmethylated primers was carried out. Correlations are underway with clinical profile and treatment regimen. Results: Hypermethylation of the promoter region was observed in about 60%, (12) samples. The other samples only demonstrated unmethylated regions. Conclusions: Assessment of promoter methylation of the MGMT gene helps in the therapeutic choice of the patients. It has gained importance not only as a prognostic but also as a predictive biomarker in molecular profiling of high grade gliomas. P9 Correlation o P16 and Bmi1 gene expression in human high grade glioma M K Sibin 1* , GK Chetan 1 , Ch Lavanya 1 , Manoj M Jeru 1 , Dhananjaya I Bhat 2 1 Department of Human genetics, National Institute of Mental Health and neurosciences, Hosur road, Bangalore-29, India; 2 Department of Neurosurgery, National Institute of Mental Health and neurosciences, Hosur road, Bangalore-29, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):P9 Background: Gliomas are neoplasm of the central nervous system and make up approximately 80% of all malignant brain tumors. Even with the advancement in the field of surgery, chemotherapy and radiotherapy, the prognosis of glioma patients is dismal. Many molecular alterations of various oncogenes and tumor suppressor genes are seen in glioma and can be used for molecular characterization of tumor. The Bmi1 is a Polycomb group of protein which is associated with various cancers. The main alterations in Bmi1 gene in glioma includes copy number variation, over expression of mRNA and protein levels when compared to the non glioma brain samples. The CDKN2A (p16) is a tumor suppressor gene, mapped to 9p21. Alterations of the 9p21 locus have been implicated in many types of cancer, indicating the role of the tumor suppressor genes CDKN2A which encodes for p16 INK4a /p14 ARF . In this study we wanted to check the gene expression pattern of p16 and Bmi1 genes in glioma and their clinical correlations. Material and methods: 50 glioma tissues were collected from Neurosurgery department and histologically confirmed as glioma. Total RNA was isolated and 1µg was converted to cDNA using RT kit. Real time RT PCR was performed using Taqman probes specific for p16 and Bmi1 and GAPDH as internal control. Results: We showed that the p16 mRNA levels in glioma found to be decreased when compared to the non glioma tissues. Bmi1 gene was found to be over expressed in glioma. There is a correlation in the expression of both genes. Bmi1 is the main upstream regulator of p16 and we found that gene expressions of both are correlated in glioma. Conclusions: It is concluded that p16 and Bmi1 genes are important in glioma and can be used as independent biomarker in glioma characterisation. P10 Promoter methylation of PTEN gene in high grade gliomas C Lavanya 1* , GK Chetan 1 , MK Sibin 1 , Manoj M Jeru 1 , Bharath MM Srinivas 2 1 Department of Human genetics National Institute of Mental Health and Neurosciences, Bangalore, India; 2 Department of Neurochemistry, National Institute of Mental Health and Neurosciences, Bangalore, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):P10 Background: Gliomas are the most common primary malignant central nervous system tumors arising from glial cells accounting for 80% of all malignant brain tumors. Many mutations occur frequently in genes that control cell cycle and proliferation leading to tumor progression. Major genes that are associated with the malignant tumors are MGMT, hTERT, TP53, PTEN, P16. Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) is a tumor suppressor gene deleted or mutated in many cancers. It plays a significant role in cellular processes like cell cycle progression, apoptosis, translation, growth, proliferation and migration by negatively controlling PI3/ AKt pathway. Deregulation of PI3K signaling pathways proceed from genetic alterations in the PTEN gene on 10q23 at the level of mutation, LOH and methylation identified in 60% of glioblastoma. Recent reports show that absence of PTEN was associated with increased tumor malignancy in gliomas. This seems to have prognostic significance and accepted as independent prognostic factor. In this study 15 high grade glioma tissues were collected from neurosurgery department and confirmed as glioma histologically. Material and methods: DNA was isolated using Qiagen kit and bisulphite DNA conversion was done by Qiagen Epitect. PCR was performed to check the methylation status in the promoter region of PTEN gene. Results: We noticed that around 50% of patients DNA show methylation in promoter region of PTEN gene when compared with healthy control DNA. We are looking at the response of these patients to chemotherapy and radiotherapy. We are also looking in to changes in the PI3/AKt signaling pathway. Conclusions: There is a possibility of correlation between the methylation status and patients survival. P11 Role of p16 deletion and Bmi1 copy number variation in glioma GK Chetan 1* , M K Sibin 1 , Dhananjaya I Bhat 2 , Ch Lavanya 1 , Manoj M Jeru 1 , N Geethashree 1 1 Department of Human genetic, National Institute of Mental Health and neurosciences, Hosur road, Bangalore-29, India; 2 Department of Neurosurgery, National Institute of Mental Health and neurosciences, Hosur road, Bangalore-29, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):P11 Background: Malignant gliomas are the most common and lethal intracranial tumors. Glioma exhibit a relentless malignant progression characterized by widespread invasion throughout the brain, resistance to traditional and newer targeted therapeutic approaches. The classical genetic alterations in glioma target pathways governing cellular proliferation, cellular survival (apoptosis and necrosis), invasion, and angiogenesis. The p16 is the second most altered tumor suppressor gene and frequent mutations and Molecular Cytogenetics 2014, Volume 7 Suppl 1 http://www.molecularcytogenetics.org/supplement/7/S1 Page 23 of 59 deletions of p16 in human cancer cell lines first suggested an important role for p16 in carcinogenesis. The Bmi-1 is an important oncogene and its expression is found to be elevated in many types of cancers. Role of Bmi1 copy number variation in glioma is still under debate. In this study we analyzed the alterations in p16 and Bmi1 genes in glioma. Material and methods: 50 glioma samples from patients were collected from the neurosurgery OT. Tissues having >95% tumor cells were processed and DNA was isolated. For the analysis of p16 deletion multiplex PCR was done using primers specific for all 3 exons of p16. Copy number variation in Bmi1 gene was analyzed using real time PCR with Bmi1 specific primers. Results: Our results showed that there is 20% of p16 deletion in our samples and the deletion pattern vary with exons. There was no copy number variation found in Bmi1 gene in glioma. Conclusions: We concluded that the p16 deletion is a common alteration found in glioma and Bmi1 gene amplification is not the common mechanism to increase the expression of this protein in glioma. P12 Analysis of DNA damage in cells excreted in urine of cervical cancer patients using alkaline comet assay Avani Patel 1* , Mihir Shah 1 , Pinaki Patel 2 , Trupti Patel 1 1 School of Biosciences and Technology, Vellore Institute of Technology, Vellore – 632014, Tamil Nadu, India; 2 Sardar Patel University, Vallabh Vidyanagar, Anand, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):P12 Background: Cancer is one of the most unwanted menaces in the human body. Cancers of lower abdomen are not only life threatening but also painful and survival rate is low. Cervical cancer is second most common worldwide and fifth deadliest in women. It affects about 16 per 100,000 women per year and kills about 9 per 100,000 in a year. In developing countries occurrence rate is 80%. It is possible that there may be no symptom until an advance stage of the cancer is progressed. The single cell gel electrophoresis assay also called comet assay, which is versatile, reliable, powerful, uncomplicated and sensitive technique for detection of DNA damage at the level of individual eukaryotic cell. Understanding the extent of DNA damage in neoplastic cells discarded in the urine of cancer patients through comet assay. Usually normal urine sample have very rare cells. In case of cancer patient the number of cells increases drastically. The type of cells passed in the urine of cancer patient contains mainly leukocytes, some neoplastic cells and some infected tissue cells. Materials & methods: The analysis was carried out on 10 subjects having cervical cancer and different levels of DNA damages were seen in cells which were separated from urine. The choice of sample is so, as it is non-invasive. Results: The cells have damaged DNA as a result of cancer, which do not have proper binding with histone proteins giving a tail in comet assay which can be easily seen by ethidium bromide staining. Conclusions: Comet assay can be used to diagnose the cancer in the suspected patient as an alternative approach to detect the stages of the cancer instead of biopsy and cytology which are painful methods. Early stages of cancer will be detected by non-invasive technique. P13 Vitamin D receptor gene polymorphisms modulate the clinico- radiological response to vitamin D supplementation in knee osteoarthritis Divya Sanghi 1* , RN Srivastava 1 , Sarita Agarwal 2 1 King George’s Medical University, Near Daligunj Chauraha, Lucknow, India; 2 Post Graduate Institute of Medical sciences, Raibareily Road, Lucknow, India Molecular Cytogenetics 2014, 7(Suppl 1):P13 Background: Vitamin D receptor (VDR) gene plays an important role in bone mass regulation and is demonstrated in human articular chondrocytes of cartilage. We have previously shown a beneficial effect of vitamin D on symptomatic improvement in Osteoarthritis knee (KOA) patients. This study investigated whether the clinic-radiological response to vitamin D was modulated by VDR gene. Materials and methods: This randomized placebo-controlled trial recruited 103 KOA cases as per American College of Rheumatology (ACR) guideline having vitamin D insufficiency (25(OH)D≤50 nmol/L). Enrolled cases were randomly allocated in two groups to receive placebo (51) and vitamin D (52). Primary outcome measures: pain and functional disability which were recorded by knee specific WOMAC index and secondary outcome measure were radiological features (joint space width and osteophytes). The serum levels of vitamin D were assessed by ELISA method using IDS, UK kit. Detection of VDR polymorphisms (Taq1 & Apa I) were done by PCR-RFLP technique. 25(OH)D levels, clinical and radiological features were recorded at baseline and at one year follow up. Results: At one year, in vitamin D group, TT genotype of TaqI polymorphism showed increment in 25(OH)D levels in comparison to Tt and tt genotype whereas in placebo group it remained same. No such association was observed for ApaI polymorphism. In clinical features, pain significantly increased in Tt and tt genotype of placebo group whereas it decreased, although not significant in each genotype of vitamin D group. Total WOMAC scores significantly decreased in both Tt and TT(p<0.05) genotypes in vitamin D group, whereas functional disability only in Tt(p<0.05). In radiological features, decreased Medial-JSW was observed in tt genotype and increased Osteophyte was observed in TT and Tt genotype of placebo group whereas no significant changes were noted in vitamin D group. No such effect was observed with ApaI polymorphism. Conclusions: VDR gene polymorphisms influence the clinico-radiological response to vitamin D supplementation in KOA Bottom of Fo with low 25-OHD levels. P14 Anti cancer activity of colon specific osmotic drug delivery of acetone extract of Quercus Infectoria Olivier, Fagaceae in 1,2- dimethylhydrazine-induced colon cancer in rats Roshni Solanki 1* , Dhaval Madat 2 , Ramesh K. Goyal 3 1 Faculty of Pharmacy, Dharmsinh Desai University, Nadiad, India; 2 TBC; 3 Institute of Life Science, Ahmedabad University, Ahmedabad, India Molecular Cytogenetics 2014, 7(Suppl 1):P14 Background: Adenoma of colon and rectum is known as colorectal cancer. It is the fourth most common form of cancer in the United States. Various colon drug targeting systems are currently under development to minimize drug degradation, prevention of side-effects and increase drug bioavailability in the required zone. Galls of Q.infectoria contains about 50%- 70% tannin mainly gallotannic acid which is reported to be antimutagenic. In present investigation colon targeted AEQI formulation was prepared and evaluated by in vitro drug release study and in vivo study using 1,2- dimethylhydrazine(DMH) induced colon cancer in rats with or without diabetes. Materials and methods: Pellets of AEQI were prepared by extrusion- spheronization method using Chitosan as osmotic agent. Optimized formulation was tested further for in vitro study, stability study and release kinetic model fitting study. In vivo study was carried out using 1,2- dimethylhydrazine (DMH) 20 mg/kg s.c. in rats. Diabetes was induced by streptozotocin 40 mg/kg i.v. Colon targeted formulation of AEQI of 100mg/ kg and 200 mg/kg was administered for 16 weeks. At the end of study blood samples were collected for estimation of antioxidant and oxidant parameters and measurement of TNF a and TGF b levels. Colon was isolated at the end of study and development of aberrant crypt foci (ACF) was observed in histopathology and tissue VEGF levels were measured. Statistical analysis was carried out using analysis of variance followed by multiple comparison Tukey (p<0.05). Results: In vitro study showed that optimization gave sustained release of drug for 24 hr 98.37% release and stability with respect to release pattern. The release of drug follows Weibull model with minimum F value (22.40). In vivo treatment showed improvement in oxidant and antioxidant parameters significantly with improved histopathological characters in colon as compared to non treated model control group. Moreover significant reduction in TNF a, TGF b and VEGF levels was observed with treatment. Diabetes associated with colon cancer increases severity of colon cancer and it was prevented by treatment. Molecular Cytogenetics 2014, Volume 7 Suppl 1 http://www.molecularcytogenetics.org/supplement/7/S1 Page 24 of 59 Conclusions: Our data suggest that microbially triggered colon specific osmotic pump delivery have a potential to be used as targeted therapy for cancer. P15 Genetic variation in PSCA gene and bladder cancer susceptibility in North Indian population Praveen K Jaiswal * , Vibha Singh, Rakesh Kapoor, Rama D Mittal Department of Urology and Renal Transplantation, Sanjay Gandhi Post Graduate Institute of Medical Science, Raebareli Road, Lucknow 226014, Uttar Pradesh, India Molecular Cytogenetics 2014, 7(Suppl 1):P15 Background: Bladder cancer (BC) is a significant health problem worldwide. Prostate stem cell antigen (PSCA) gene has been reported earlier in Genome Wide Association Study (GWAS) for BC risk. It is highly expressed in bladder cancer and considered to be involved in the cell proliferation inhibition and/ or cell-death induction activity. It has been assumed as a useful marker for diagnosis and progression of bladder cancer. Materials and methods: We genotyped PSCA rs2978974G/A and PSCA rs2294008C/T gene by Real Time Taqman® probes to evaluate the risk of bladder cancer (BC) in histologically confirmed 225 BC patients and 240 healthy controls (age and gender matched) from North Indians in a hospital based case control study. Further statistical analysis for association studies was done by SPSSver16.0. Results: Significant increased BC risk was observed to be associated with heterozygous CT genotype of PSCA rs2294008C/T having 1.86 folds risk (p=0.004;OR=1.86). The variant allele T was also significantly associated with BC risk (p=0.027;OR=1.38) for PSCA rs2294008C/T. In case of PSCA rs2978974G/A, no significant association was observed with BC at genotypic level. Smoking significantly modulated the BC risk in patients with heterozygous CT genotype (p=0.025) for PSCA rs2294008C/T gene polymorphism. A significant BC risk was observed when risk was evaluated with tumor-grade-stage level for PSCA rs2294008C/T with heterozygous CT genotype (p=0.045;OR=1.02). Furthermore, BC patients receiving BCG treatment showed no significant association with any genotype of PSCA. Bioinformatics analysis (in-silico analysis) showed no significant association with BC risk. Conclusions: Our study has unveiled a complex intervention of PSCA rs2294008C/T conferring a higher risk of BC risk among North Indian population. Further studies in large sample size and different ethnic group are needed for validation. P16 Replicative study of GWAS reported TP63C/T rs710521, TERTC/T rs2736098 and SLC14A1C/T rs17674580 with susceptibility to bladder cancer in North Indians Vibha Singh * , Praveen Kumar Jaiswal, Rakesh Kapoor, Rama Devi Mittal Department of Urology, Sanjay Gandhi Post Graduate Institute of Medical Science, Raebareli Road, Lucknow 226014, India Molecular Cytogenetics 2014, 7(Suppl 1):P16 Background: Genome Wide Association Studies (GWAS) have confirmed association of rs710521, rs2736098 and rs17674580 gene variants with susceptibility to Bladder Cancer (BC) in European and Caucasian. However, the risk conferred for BC in north Indians is unknown. In present study, we replicate GWAS findings of TP63C/T(rs710521), TERTC/T(rs2736098) and SLC14A1C/T(rs17674580) gene polymorphisms for their association with BC susceptibility in North Indian population. Material and methods: Three SNPs were genotyped by real-time polymerase chain reaction in histologically confirmed 225 BC cases and 240 healthy controls (age and gender matched) from North Indians in a hospital based study. To evaluate SNP effects on BC susceptibility, odds ratio and confidence interval were calculated by SPSSver.16.0. Bioinformatics analysis was done by F-SNP free online web server. Results: In case of TP63C/T(rs710521), variant genotype (TT) showed significant reduced risk for BC (p=0.045;OR=0.53). On Combining heterozygous and variant genotype, it showed reduced risk for BC (p<0.001; OR=0.54). In case of TERTC/T(rs2736098) heterozygous genotype (CT) as well as variant genotype (TT) showed significant risk for BC susceptibility (p=0.031;OR=1.77,p=0.004;OR=2.78 respectively) along with T allelic level (p<0.001;OR=4.19). Further SLC14A1C/T(rs17674580), variant genotype (TT) also showed significant high risk for BC susceptibility (p=0.006;OR=3.01) along with variant T allele level (p=0.003;OR=1.52). Interestingly smoking was also found to modulate risks for BC in case of TERT and SLC14A1 variant genotype (TT). Further tumor-grade-stage level of cases supports the genotypic data with TERT and SLC14A1 for BC risk. Bioinformatics analysis supported our result at genotypic level for the BC risk for TERTC/T and SLC14A1C/T. Conclusions: Our results indicated that TERTC/T(rs2736098) and SLC14A1C/ T(rs17674580) showed high risk for BC in North Indian population. However, TP63C/T(rs710521) showed reduced risk of BC susceptibility. More study with large sample size and diverse ethnicity are required to validate our observations. P17 Association of miR-27a, miR-181a and miR-570 genetic variants with gallbladder cancer susceptibility on North indian population Annapurna Gupta 1* , Kiran L Sharma 1 , Annu Yadav 1 , Vijay Kumar 2 , Sanjeev Misra 2 , Ashok Kumar 3 , Balraj Mittal 1 1 Departments of Genetics, Sanjay Gandhi Post Graduate Institute of Medical Sciences (SGPGIMS), Lucknow, India; 2 Department of Surgical Oncology, KGMU, Lucknow, India; 3 Surgical Gastroenterology, SGPGIMS, Lucknow, India Molecular Cytogenetics 2014, 7(Suppl 1):P17 Background: MicroRNAs are small endogenously expressed short non- coding RNAs. They appear to be critical regulators of tumor biology as their aberrant expression is well characterized in cancer progression. The role of microRNA is not fully understood in gallbladder carcinoma, so in present study we investigated the role of miR-27a, miR-181a and miR-570 genetic variants with gallbladder cancer (GBC) susceptibility. Material and methods: In this case-control study, we evaluated the role of miR-27a, miR-181a and miR-570 genetic polymorphisms with GBC susceptibility in North Indian population. The present study included 515 GBC patients and 200 healthy controls from North India. Genotypes were determined by TaqMan probes. Statistical analysis was done by SPSS ver. 16. In silico analysis was performed using Bioinformatics tools (F-SNP, FAST-SNP). Results: Logistic regression analysis showed no significant association of miR-27a, miR-181a and miR-570 genetic polymorphism with GBC susceptibility (P> 0.05). On stratifying our data on the basis of gall stone status, the [AG+GG] genotypes of miRNA rs895819 (A>G) were significantly associated with increased risk of GBC in patients without stone (p=0.003 OR=1.83 [(95%CI) 1.23-2.72]. The genetic risk by miR-27a, rs895819 (A>G) was also modulated by tobacco consumption as the heterozygotes (AG) were at higher risk p=0.005 OR=1.94 [(95%CI) 1.22-3.08]. However, there was no association of miR-181a and miR-570 polymorphisms with disease risk in subgroup analysis. In-silico analysis showed change in transcriptional regulation of miR-27a and miR-570 variations. Conclusions: We found significant association of miRNA rs895819 A>G with gallbladder cancer risk through gallstone independent pathway and tobacco usage. P18 Selection of suitable housekeeping genes for gene expression analysis in glioma using quantitative real-time PCR Madhuri GS Aithal, Narayanappa Rajeswari * Department of Biotechnology, Dayananda Sagar College of Engineering, Bangalore, Karnataka, India- 560078 E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):P18 Background: Quantitative real-time PCR (qPCR) is the most reliable tool for gene expression measurements from tissue samples. Selection of housekeeping genes (HKGs) that are most stably expressed among tissues is vital to carry out accurate gene expression profiling of target genes. Expression of HKGs varies among tissues and experimental conditions. There is no ‘universal’ housekeeping gene having stable expression in all tissues under all experimental conditions. So, it is extremely important to Molecular Cytogenetics 2014, Volume 7 Suppl 1 http://www.molecularcytogenetics.org/supplement/7/S1 Page 25 of 59 identify most appropriate internal control genes for a particular tissue and experimental conditions. The aim of the present study is to identify most suitable HKGs for gene expression analysis in glioma tissue samples. Material and methods: Based on literature survey six most commonly used HKGs reported to be invariant in human gliomas were chosen for gene expression analysis. We performed qPCR using RNA from formalin fixed paraffin embedded glioma patient samples and normal brain samples to investigate the expression pattern of six HKGs [Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hypoxanthine phosphoribosyltransferase 1 (HPRT), b2 microglobulin (B2M), TATA-binding protein (TBP), 18S ribosomal RNA (RN18S1) and ribosomal protein L13a (RPL13A)] with different abundance. A simple ∆Ct approach was employed to calculate the fold change. Results and conclusions: Our data showed that out of the six genes studied, expression of GAPDH, B2M and RPL13A were found to have most constant expression across all the tumor samples in our experimental setup. Thus, these three genes proved to be the most suitable to be used as internal controls for gene expression analysis in human glioma. Except for GAPDH, none of the other conventionally used HKGs in glioma studies e.g., HPRT and TBP were found to be suitable as they showed large variation in RNA expression. Validation of housekeeping genes is therefore highly specific for a particular experimental setup and is important in assessing any new setup. P19 Development of allele specific hybridization assay and computer aided tool for detection of genetic variations in folate pathway genes Ravishankara 1* , Shreeshakala 1 , S Padmalatha Rai 1 , Kerthana Prasad 2 , C Deepthi 2 , PM Gopinath 1 , K Satyamoorthy 1 1 Division of Biotechnology, School of Life Sciences, Manipal University Manipal, India 576104; 2 School of Information Sciences, Manipal University Manipal, India 576104 Molecular Cytogenetics 2014, 7(Suppl 1):P19 Background: Most current single nucleotide polymorphism (SNP) genotyping methods are still too slow and expensive for routine use in large association studies that requires hundreds or more SNPs in a large number of DNA samples and for diagnostic purpose. Clinical implementation and ultimate use of nucleic acid biomarkers necessitate development of pragmatic detection assays that deliver adequate sensitivities, mutation selectivity and high throughput capabilities in a rapid, robust and cost effective manner. In the present study we have developed a sensitive and cost effective genotyping technique with computer aided tool for allele discrimination and implemented it to genetic epidemiology study. Material and methods: Modified allele specific oligonucleotide hybridization assay with elimination of DNA isolation step for PCR and novel allele discrimination method was used to screen folate metabolism gene polymorphisms in acute lymphoblastic leukemia patients and normal individuals. The plasma homocysteine and global DNA methylation was performed by HPLC method. Results: The developed novel allele discrimination method along with the newly developed image analysis software increases the sensitivity and elimination of DNA isolation step for PCR reduces the cost of the genotyping technique. The average kappa value of all designed probes was > 0.81. The technique was successfully designed for both diagnosis and research purpose. The evaluation of effect of gene polymorphism on plasma folate, homocysteine level and global DNA methylation explored that the only MTHFR C677T gene polymorphism is associated with elevated homocysteine level in healthy young adults. The genotyping of 22 folate metabolism genes explored that only RFC1 80GA and RFC1 80AA genotypes were significantly associated with increased risk of Acute Lymphoblastic Leukemia (n=203) when compare to healthy individuals (n=245) in south Indian population. Conclusions: The developed visual based allele specific oligonucleotide hybridization assay will be useful in clinical diagnostic system where large scale screening is often necessary without any sophisticated detection systems. RFC1 80GA and RFC1 80AA genotypes were significantly associated with increased risk of acute lymphoblastic leukemia. In young adults, only MTHFR C677T polymorphism is associated with elevated homocysteine level. P20 Cytogenetic and interphase Fluorescence in Situ Hybridization studies in patients with multiple myeloma G Perumal 1* , RS Chandra 2 , P Prabu 1 , N Indhumathi 1 , Anil Tarigopula 1 , Rama Mani 1 1 Department of Molecular Biology and Cytogenetics, Apollo Hospitals, No. 21, Greams Lane, Off. Greams Road, Chennai-600 006, Tamilnadu, India; 2 Department of Genetics, Dr. ALM PG. Institute of Basic Medical Sciences, University of Madras, Taramani, Chennai - 600113, Tamilnadu, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):P20 Background: Multiple myeloma (MM) is characterized by the clonal proliferation and accumulation of malignant plasma cells in the bone marrow, monoclonal protein in the blood or urine and associated organ dysfunction. Some patients may show a slow progressive evolution from monoclonal gammopathy of undetermined significance while others may be associated with features of high clonal aggressiveness such as plasma cell leukemia or extramedullary plasmacytomas. Chromosomal abnormalities (mainly IgH translocations and trisomies) have been shown to be of prognostic significance in MM. Interphase fluorescence in situ hybridization (i-FISH), in particular, has been much more effective in identifying these trisomies/ monosomies and specific translocations. Materials and methods: The bone marrow aspirates were processed for conventional cytogenetic and interphase FISH analyses using three probes to detect del(13)(q14.3), t(4;14)(p16.3:q32) and del(17)(p13.1). A total of 30 newly diagnosed patients were studied between May 2012 and March 2013. The affected were mostly elderly people with median age of 55 years (range: 32 to 80 years) at the time of diagnosis. There were 21 males and 9 females. Results and conclusions: Chromosomal abnormalities were detected in only 7 patients because of the low proliferation rate of plasma cells and the non-availability of analyzable metaphases. On the other hand, i-FISH revealed an abnormality in 14 patients and a normal pattern for the selected probes in the remaining 16 patients. The most frequent abnormality was found to be 13 monosomy (complete/ partial) in 13 cases followed by t (4;14) seen in 4 patients. However these abnormalities were not recognized in the karyograms. None of the cases showed a p53 deletion. Further studies employing the complete FISH panel are required for better diagnosis and prognosis. P21 APC is epigenetically down regulated in advance cases of gallbladder cancer Dinesh Tekcham Singh 1 , Satish Poojary 1 , Shushruta Bhunia 1 , Mustafa Ahmed Barbhuiya 1 , Manisha Kakkar 2 , Vishwajit Jalaj 2 , Sanjeev Gupta 2 , Braj Raj Shrivastav 2,3 , Pramod Kumar Tiwari 1* 1 Centre for Genomics, Molecular and Human Genetics, School of Studies in Zoology, Jiwaji University, Gwalior-474011, India; 2 Cancer Hospital and Research Institute, Gwalior-474007, India; 3 Gajra Raja Medical College, Gwalior-474007, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):P21 Background: The mortality rate of gallbladder cancer (GBC) is considerably high in India and world over. The Adenomatous Polyposis Coli (APC) gene is widely reported for its role in cancer. APC is known to have two promoter regions, 1A and 1B, that show differential role in various cancers. However, role of these promoters and their exons in the molecular pathogenesis of GBC is obscure. Our aim was to study the epigenetic control of APC promoters in GBC and to evaluate their utility as prognostic/diagnostic biomarker of GBC. Methods: We carried out methylation specific PCR of the modified genomic DNA from GBC and GSD tissues, followed by semi-quantitative and quantitative PCRs (qPCR). We compared the transcript levels of the two exons of APC in GBC and GSD as compared to their adjacent control tissues. We also performed Immunohistochemistry (IHC) on tissue micro array (TMA) of GBC and GSD. The scoring of different tissue cores was done by the expert pathologist and student’s t-test was performed to check the significance. Molecular Cytogenetics 2014, Volume 7 Suppl 1 http://www.molecularcytogenetics.org/supplement/7/S1 Page 26 of 59 Results: The APC 1A promoter was found significantly methylated in both GBC (96%; p=0.0155) and GSD (80%; p=0.015), but 1B was not. The down- regulation of APC exon 1 was observed in both GBC and GSD, whereas exon 2 appeared normally expressed. Immunohistochemistry of APC protein on Tissue Microarray (TMA) revealed down regulation with negative score of 34.48% in GBC (p=0.057), 1+ in 24.14% GBC (p=0.005), 2+ in 25.85% in early stage or GSD (p=0.091). Conclusion: We infer that APC is epigenetically down regulated in advance cases of GBC and might be a key step in the tumorigenesis of gallbladder. In future, APC may be considered for diagnostic, prognostic and therapeutic purposes in GBC. P22 Bladder-Exstrophy-Epispadias-Complex risk and C677T polymorphism in MTHFR gene: case-control study among Indian children Abid Ali * , Rajeev Kumar Pandey, Sukanya Gayan, Minu Bajpai Department of Pediatric Surgery, All India Institute of Medical Sciences, New Delhi-110029, India Molecular Cytogenetics 2014, 7(Suppl 1):P22 Background: Bladder exstrophy is a congenital anomaly in which part of the urinary bladder is present outside the body. It is rare in occurrence but the frequency is increasing very rapidly. 5, 10-methyltetrahydrofolate reductase (MTHFR) enzyme, which catalyzes the synthesis of 5-methylene- tetrahydrofolate and C677T polymorphism in MTHFR, shows a significant role with a thermolabile enzyme and decreased specific MTHFR activity. The objective of the present investigation was to study the case-control association between C677T polymorphism in relation to Bladder-Exstrophy- Epispadias-Complex. Materials and methods: For the present study, a total of 50 patients classified as Bladder-Exstrophy-Epispadias-Complex & cloacal exstrophy patient and 50 healthy school going children (as control) were recruited to participate in this study. Genomic DNA was extracted from peripheral blood lymphocytes by using commercially available kits. Genotypes of the MTHFR C677T polymorphisms were detected by polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP). Results: Frequency distributions of genotypes and combined genotypes were obtained. The overall distribution of the C677T genotype was found to be significantly associated with cloacal exstrophy but not with epispadias as compared to the controls. Conclusions: Genotyping of 50 patient cases and 50 controls revealed a significant association between C677T polymorphism and Bladder- Exstrophy. P23 MLPA analysis of transcriptional gene(s) variations in patients with congenital heart septation defects Rajasekhar Moka * , Neetha John, Yashvanthi Borkar, K Ranjan Shetty School of Life Sciences, Manipal University, Manipal-576104, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):P23 Background: Congenital heart diseases (CHD) occur in ~1% live births and are among the most common birth defects. It accounts nearly about 46% of all deaths from congenital malformations. Adults with repaired congenital heart disease represent a complex and heterogeneous group of patients that are increasingly surviving beyond childhood. Advances in our molecular understanding of normal heart development have led to the identification of numerous genes necessary for cardiac morphogenesis. The etiological factors of many genetic syndromes and familial CHD have been identified, but the genetic basis of majority of them remains unknown. Objective of this study was to screen the transcriptional genes for novel mutations since majority of these involved in the early stages of cardiogenesis. Materials and method: Multiplex Ligation-dependent Probe Amplification (MLPA) was employed in to detect copy number changes in 50 patients’ DNA using the P311-A1 CHD SALSA MLPA Kit for the genes NKX2-5, GATA4, TBX5, BMP4 and CRELD1 in 25 genomic regions. Results: Copy number changes were identified in four (8%) two heterozygous deletions in BMP4 and TBX5 were observed in common in two patients atrial septum defects. Conclusions: MLPA assay can be used for detection of copy number changes in patients with non-syndromic CHDs as a first line of screening test. P24 COL6A1 loss of function mutation underlie atrioventricular septal defects in down syndrome patients Priyanka Ghosh * , Pranami Bhaumik, Subrata K Dey West Bengal University of Technology, Department of Biotechnology & Biological Sciences, Kolkata, West Bengal, Pin-700064, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):P24 Background: The present investigation was undertaken to explore the role of COL6A1 variants on cardiovascular septal defects in Down syndrome. Materials and methods: We sequenced the entire reading frame of COL6A1 in the samples from Kolkata and adjoining areas. Four hundred participants were included in the genetic association study and they were stratified as Down syndrome (DS) with atrioventricular septal defect (AVSD), DS without AVSD, euploid with AVSD and euploid without AVSD. DNA of Down syndrome individual with atrioventricular septal defect and Down syndrome individuals without AVSD were sequenced for the SNP analysis of COL6A1 gene. Results: A missense mutation p.E887K in exon 35 of COL6A1 gene was identified in three DS patients with complete AVSD and was absent in control population. The causative potential of a sequence variation was evaluated by bioinformatics software like Mutationtaster, MutPred and SOPMA which predicted. The mutation was predicted to be deleterious and is predicted to affect normal protein function. Genetic analysis of the mutation carrier’s available family members showed that the substitution co-segregated with AVSD transmitted in an autosomal dominant pattern. The p.E887K variation was automatically predicted to be disease causing. Conclusions: Our data suggest that missense mutation p.E887K in exon 35 of COL6A1 mutation may serve as a potential biomarker for diagnosis of AVSD in individuals thus suggesting potential implication for early prophylaxis and personalised treatment of AVSD. P25 An intronic rare mutation in Presenilin-1 (PSEN-1) gene may be involved in the developement of Alzheimer’s disease Pranami Bhaumik * , Priyanka Ghosh, Subrata K. Dey Human Genetics Research Laboratory, Department of Biotechnology and Biological Sciences, West Bengal University of Technology, Kolkata 700064, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):P25 Background: The Presenilin-1 gene (PSEN-1) encodes a protein component of gamma-secretase complex which is involved in processing of amyloid precursor protein (APP). The PSEN-1 is involved in many cardinal mechanisms in several molecular pathway which when impaired leads to the manifestation of Alzheimer’s disease (AD). The aim of the study was to investigate the role of PSEN-1 gene in the developement of AD in Indian Bengali population. Materials and methods: Blood samples were collected from 96 AD patients and 173 age matched control individuals. DNA was isolated from each sample and then sequencing was performed for the exon 8 and its flanking introns of PSEN-1 gene. Results: A rare mutation rs201992645 was identified within intron 8 and several in. silico analyses (Bioinformatic tools like ‘Human Splicing Finder’, ‘SpliceAid’ and ‘mutation t@sting’) revealed the mutation as ‘potentially damaging’ at the transcript splicing level. The genotypic frequencies of mutant heterozygotes were 0.031 AD, but it was not found in the control population. Conclusions: We hypothesize that this rare mutation may be involved in the malfunctioning of Presenilin-1 protein and thus may play a role in the manifestation of Alzheimer’s disease. Further study with large population size may establish this mutation as a potential biomarker for diagnosis of AD. Molecular Cytogenetics 2014, Volume 7 Suppl 1 http://www.molecularcytogenetics.org/supplement/7/S1 Page 27 of 59 P26 Identification and characterization of disease causing genetic variant by conventional genotyping and whole genome sequencing in familial tooth agenesis Tanmoy Sarkar 1* , Rajesh Bansal 2 , Parimal Das 1 1 Centre for Genetic Disorders, Faculty of Science, Banaras Hindu University, Varanasi- 221 005, India; 2 Faculty of Dental Sciences, Institute of Medical Sciences, Banaras Hindu University, Varanasi- 221 005, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):P26 Background: Congenital tooth agenesis (CTA), a type of craniofacial disorders affects approximately 20% (including 3 rd molar or Wisdom teeth) and 2-10% (excluding 3 rd molar) of the world population. Five major candidate genes known to be associated with syndromic and non- syndromic CTA, these are PAX9, MSX1, AXIN2, EDA and WNT10A. The present investigation was undertaken to identify and characterize disease causing genetic variant by conventional genotyping and whole genome sequencing in familial tooth agenesis Material and methods: We have identified two Indian families (DEN11 and DEN12) segregating two distinct types of non-syndromic tooth agenesis with autosomal dominant (DEN11) and apparently indistinguishable either autosomal or X-linked dominant pattern of inheritance (DEN12) family. Eight affected and four unaffected members from DEN11 and four affected and two unaffected members from DEN12 family were enrolled for the present study to identify causative genetic defect associated with the disease. While direct DNA sequencing and microsatellite based genotyping were carried out to screen all but EDA genes in affected (II-5,-7, III-1,-7,-9, IV-2,-3,-4) and unaffected (III-2,-3,-5, IV-1) family members of DEN11, whole genome sequencing was carried out for two affected (III-2 and IV-4) family members from DEN12 along with four unaffected unrelated controls using Illumina 2500 Next Generation Sequencing platform for identification and subsequent characterization of causative variant/s. Results and conclusions: In the microsatellite and SNP based haplotype analysis we identified a haplotype block of a ~9.64 Mb region containing PAX9 gene located between D14S70 and D14S288 markers associated with CTA in DEN11. However, absence of any DNA sequence variant within the exons and exon-intron boundaries of the linked PAX9 was found and this indicates the involvement of other pathogenic mechanism. In the whole exome sequencing we identified 86 nonsynonymous novel nucleotide variations distributed among 84 different genes across the nuclear genome of two affected members of DEN12 subjected for investigation. Using bioinformatics, among those variations a specific variation in Ectodysplasin-A (EDA), c.956G>T transversion leading to p.Ser319Ile, at the Tnf homology domain, was considered as a potential pathogenic variation. This variation was not observed in any unaffected family member and in 100 unrelated control chromosomes as assayed through Sanger sequencing and/or RFLP. In silico analysis using SwisSPdb Viewer V4.1.0 reveals that this change destroys an H-bond between p.319 Ser and p.332 Cys establishing this as a plausible pathogenic variation for tooth agenesis in DEN12 family. P27 Identification of NKX2-5 and GATA4 sequence variations in patients with cardiac septation defects Yashvanthi Borkar 1* , K Ranjan Shetty 2 , K Krishnanand 2 , Rajasekhar Moka 1 1 Department of Biotechnology, School of Life Sciences, Manipal University, Manipal, India; 2 Department of Cardiology, KMC, Manipal University, Manipal, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):P27 Background: Cardiac septation defects (CHD) are the problems with the structure of the heart during the development and is one of the most common among congenital heart defect (CHD) which occurs ~1% in general population. The etiology for the majority of these defects may be multifactorial. However, advancement in understanding the molecular basis of normal heart development has revealed the necessity of numerous genes which are predominantly expressed during the heart morphogenesis. Many cohort studies have shown that cardiac transcription factors NKX2-5, GATA4 and TBX5 are the master regulators during heart development. The mutations in any of these genes results in the failure of normal development of heart leading to septal defects. The study was carried out to identify the nucleotide sequence variation in the patients with Atrial Septal Defects (ASD), Ventricular Septal Defects (VSD) and Atrioventricular Septal Defect (AVSD). Materials and methods: Fifty seven patients with ASD (n=51) and VSD (n=6) were included in the study after a thorough evaluation of Echocardiography and clinical phenotypes. All the coding regions and flanking introns of NKX2-5 and GATA4 which are predominantly expressed in the heart were amplified by polymerase chain reaction and sequenced using ABI PRISM 3130 automated sequencer. Results: No patient was found to have NKX2-5 and GATA4 mutations, however single nucleotide polymorphism (c.63A>G; rs2277923) in NKX2-5 was observed in 20 patients with Ostium secundum ASD. Conclusions: Since the mutation frequency is very low (NKX2-5 ~1-4%; GATA4 ~1%) in these cases, we need to study more number of patients to get expected frequency. P28 Factor V Leiden and MTHFR mutations as a combined risk factor for hypercoagulability in referred Patients population from Western India Priya Bansal Kokilaben Dhirubhai Ambani Hospital and Medical Research Institute, Four Bungalows, Andheri West, Mumbai, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):P28 Background: Factor V Leiden mutation is a recognized most prevalent genetic risk factor for venous thromboembolic disease. Factor V mutations, are known to potentiate the effect of MTHFR on deep vein thrombosis. The thermo labile variant of the MTHFR gene (C677T) increases the plasma homocysteine levels and hyperhomocysteneimia is a known risk factor of deep vein thrombosis. Results of studies concerning interaction of Hyperhomocysteneimia and thromobophilic risk factors like Factor V are contradictory. Some studies have shown an increased risk (10-50 times) of deep vein thrombosis because of MTHFR and FVL mutations combined, yet other studies fail to conclude similarly. We attempt to address this paradox in our study referred for DVT, Hyperhomocysteneimia and pulmonary embolism and assess the importance of the synergistic effects of FVL and MTHFR mutations. Material and methods: In this study we analyzed 190 (79 females and 111 males) cases referred to Kokilaben Dhirubhai Ambani Hospital and Medical Research Institute (Jan 2009 to Aug 2013) for DVT, Hyperhomocysteneimia and pulmonary embolism. MTHFR mutation was studied in 27 of the above subset. The detection of FVL mutation by PCR was studied by Restriction Fragment length polymorphism and for MTHFR (C677T & A1298C) mutations detection was done using commercially available kit. Results: FVL mutation was found to be present in 10% (19/190) in our study population. Of these, 18 patients were heterozygous and 1 was homozygous for this mutation. Of which 25% (19/76) patients with deep vein thrombosis were positive for variants of FVL. 74% (20/27) of the patients screened for MTHFR were found to be positive (5 for C677T, 4 were compound heterozygous & 11 for A1298C). 2 out of 4 patients who were positive for both FVL and C677T MTHFR mutations had poor prognosis and died. Conclusions: Our study reconfirms the Synergistic role of factor V Leiden and MTHFR (14.8%) in hypercoagulability disorders like DVT, throm- boembolism and others leading to poorer prognosis. P29 RAGE gene polymorphism and expression: risk factor for vascular complications in type 2 diabetes mellitus Diwesh Chawla, Savita Bansal, Pawan K Kare, Basu Basu Dev, Sri Venkata Madhu, Ashok K Tripathi * Department of Biochemistry and Department of Medicine, University College of Medical Sciences and G.T.B. Hospital, Delhi-110095, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):P29 Molecular Cytogenetics 2014, Volume 7 Suppl 1 http://www.molecularcytogenetics.org/supplement/7/S1 Page 28 of 59 Background: Advanced glycation end products (AGEs) are formed due to hyperglycemia in T2DM. Interaction of AGEs with its receptor RAGE induces signal transduction that culminates in vascular complications, the major cause of morbidity and mortality in diabetic subjects. Some functional polymorphism of this gene show differential activity of this receptor and therefore may be associated with the development of diabetic complications. In the present study we investigated the association of expression of RAGE gene and its polymorphism namely -374T/A and -429T/ C in the promoter region and Gly82Ser polymorphism in the exon 3 region with vascular complications in T2DM patients. Materials and methods: We screened 820 subjects which includes 200 healthy controls, 200 type 2 diabetes mellitus (T2DM) subjects without any vascular complications (DM), 220 T2DM subjects with microvascular complications (DM-Micro) and 200 T2DM subjects with macrovascular complications (DM-Macro) for -374 T/A, -429 T/C and Gly82Ser polymorphisms of RAGE gene. DNA isolated from the enrolled subjects was genotyped by PCR-RFLP. RAGE expression was determined by quantitative real-time PCR. Results: Mutant variant of -429T/C and Gly82Ser RAGE polymorphism was about three times more prone to develop macrovascular and microvascular complications respectively in T2DM subjects while -374A allele showed reduced risk towards the development of macrovascular complications (OR = 0.57, p = 0.006). Further, haplotype analysis revealed that CTG haplotype was significantly associated with the development of macrovascular complications while haplotype TAG was observed to be significantly protective towards development of macrovascular complications in T2DM subjects. The expression of RAGE correlated significantly with the genotypic variation of the RAGE gene. Conclusions: Mutant genotypes of RAGE gene and its enhanced expression may be considered as risk factor for vascular complications in North Indian T2DM patients. P30 Association of Matrix Metalloproteinase 1 and 3 (MMP1 and MMP3) gene polymorphisms with susceptibility to ESRD risk in North Indian population Archana Verma * , Aneesh Shrivastava, Rama D Mittal Department of Urology and Renal Transplantation, SGPGIMS, Lucknow, India Molecular Cytogenetics 2014, 7(Suppl 1):P30 Background: End Stage Renal Disease (ESRD) is influenced by genetic and epigenetic factors. Few studies have been reported earlier depicting the role of Matrix Metalloproteinase in different populations for ESRD risk, but we have reported few novel and functional genes in North Indian population. The prime objective of the present study was to find out the association of four variants viz. MMP1-519(A/G) and MMP1-1607(1G/2G) and MMP3+5356 (A/G) and MMP3 -1161(A/G) with ESRD risk. Materials and methods: 4 SNPs were genotyped by Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) in 200 ESRD patients and 200 healthy controls, that were age and gender matched. To evaluate the SNP effects on ESRD, odds ratio (OR) and confidence interval (CI) 95% were calculated by SPSSver16.0. Haplotype analysis was done for MMP1 by SNPanalyzer1.2 ver. Results: In case of MMP1-519(A/G) the variant genotype GG showed significant reduced risk of ESRD (p=0.005, OR=0.254). On combining the heterozygous and variant genotypes marginal reduced risk was observed (p=0.055, OR=0.677), although we found significance reduced risk of ESRD (p=0.007, OR=0.639) in G allele. In case of MMP1-1607(1G/2G) the variant genotype 2G2G showed significant reduced risk of ESRD (p=0.008, OR=0.476) whereas the combination of heterozygous and variant genotypes reduced risk was obtained (p=0.027, OR=0.614). At allelic level, 2G allele showed reduced risk (p=0.006, OR=0.675) of ESRD. Gene-gene interaction was also performed for above gene variants. We found a significant reduced risk in combination GG+AA (p=0.034, OR=0.185) on interacting MMP1-519(A/G) and MMP3-1161(A/G). We also found reduced risk for ESRD for MMP1-519(A/G) and MMP1-1607(1G/2G) while looking risk for ESRD at haplotype level. Bioinformatics analysis (in-silico) showed no association with ESRD. Conclusions: Our results indicated that polymorphism in MMP1-519(A/G) and MMP1-1607(1G/2G) showed reduced risk for ESRD in North-Indian population. More relevant studies with large sample size and diverse ethnicity are required to validate these observations. P31 Association of PKD1 sequence variants with pathophysiology of ADPKD in Indian patients Sonam Raj 1* , RG Singh 2 , Parimal Das 1 1 Centre for Genetic Disorders, Faculty of Science, Banaras Hindu University, Varanasi, India; 2 Dept. of Nephrology, Institute of Medical Sciences, Banaras Hindu University, Varanasi, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):P31 Background: Autosomal dominant polycystic kidney disease (ADPKD) is a systemic conditions with cardinal symptom of bilateral multiple renal cysts of variable sizes. ADPKD is genetically heterogeneous, Mendelian disorder, shows late onset and contributes 8-10% cases of end stage renal disease (ESRD). Linkage analysis in ADPKD familial cases revealed approximately 85% cases of PKD1, rest cases of PKD2 and an unidentified locus. Materials & methods: In the present study occurrence of mutation, amino acid change and three frequently observed SNPs from 3’single copy region (exon 44-46) and in 5’ duplicated region (exon 2-11) of were screened in 47 patients (24 sporadic and 23 familial) and in control individuals to search the responsible and/or susceptible spots for ADPKD in Indian patients using direct DNA sequencing of specified exons and RFLP for SNP assay. PKD1 exons 44-46 encoding intracellular and exons 2-11 encoding ligand binding domains, Ig-like PKD domains in extracellular compartment with their suggested role in cell signaling and cell adhesion were chosen for this study. Results: Screening of exons 44-46 in 47 patients identified five exonic (I4045V, 4059V, A4092A, L4137L, P4210P) and two intronic (IVS44+15G>A, IVS+22delG) variants. Assessment of the three SNPs viz. I4045V, A4059V, and A4092A screened in a multiplex family with four affected (II-1, II-7, II-9, III-12) and 30 unaffected members along with 100 ages and sex matched controls showed higher occurrence (2X) in patients compared to controls indicating their role for disease susceptibility. Screening of exons 2-7 in 19 patients uncovered six exonic (A92A, c.445delC, T191N, G340G, N416N, A443T) and two intronic (IVS4+21delC, IVS6+12delC) variants while screening of exons 8-11 in 15 patients detected twelve exonic (Q554X, T558M, Q562R, A637V, P676L, c.1987delC+2016_2017insG, D910D, L811L, L845S, N854S, V873A, N890N) and one intronic (IVS9+14_27del14) variants. Two exonic changes (c.445delC, c.1987delC+2016_2017insG) generate 288 amino acids long truncated protein and a stretch of nine altered amino acids sequence in LDL-A domain respectively. Conclusions: The spectrum of changes detected in various affected individuals indicates that the clinical as well as allelic heterogeneity compounding the pathophysiology of ADPKD in both sporadic and familial cases. Screening of remaining region of PKD1 and other candidate genes is therefore planned for future for better understanding. P32 Identification of mutations in bone morphogenetic protein 2, 4 and 7 in congenital heart disease patients from Indian population Ritu Bhardwaj 1* , Damyanti Agrawal 2 , Ashok Kumar 3 , Bhagyalaxmi Mohapatra 1 1 Cytogenetics Laboratory, Department of Zoology, Banaras Hindu University, Varanasi, India; 2 Department of Cardio Thoracic & Vascular Surgery, Banaras Hindu University, Varanasi, India; 3 Department of Pediatric Medicine, Institute of Medical Sciences, Banaras Hindu University, Varanasi, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):P32 Background: Congenital heart disease (CHD) is the most common congenital defect, affecting 1% of all livebirths. Initial cardiac development and the subsequent maturation of the heart are temporally and spatially regulated by several Bone Morphogenetic Proteins (BMPs). BMP signaling plays an important role in the specification and patterning of the early embryo. Animal model studies have established a strong link between BMPs and cardiac development. At least 6 BMPs (BMP2, BMP4, BMP5, BMP6, BMP7 & BMP10) are expressed in the heart where they have both independent and redundant functions. BMP2, 4 & 7 are expressed in the Molecular Cytogenetics 2014, Volume 7 Suppl 1 http://www.molecularcytogenetics.org/supplement/7/S1 Page 29 of 59 AtrioVentricular cushion and Out Flow Tract (OFT) and helps in their formation. However due to functional redundancy of BMP ligands, relatively little is known about the role of individual BMPs. The association of BMPs with CHD has not been studied so far in human subjects. Here we report the genetic variants in BMP2 (NM_001200.2), BMP4 (NM_001202.3) and BMP7 (NM_001719.2) in individuals with CHD. Materials & methods: We screened 190 unrelated probands with CHD and 100 healthy controls by PCR and DNA sequencing for 3 genes. Results & conclusion: We have identified 6 novel sequence variants in BMP2. Out of which 3 (H321L, E328K & S351C) are missense variants. One nonsense variants (K241X) has also been identified in one case having Patent Ductus Arteriosus and is not found in 100 controls. In BMP7, 10 novel sequence variants have been identified, out of which 3 (D85V, R175W & N372Y) are missense, 3 (c.366G>A, c.381G>A & c.384C>T) are in 5’UTR, 1 synonymous change (c.682G>T) and 3 (c.947-20G>C, c.1140+14G>A & c.1289+68G>A) are intronic variants. In BMP4, 3 sequence variants are found, out of which only one novel (c.1695A>C) in 3’UTR region has been found in one individual. Other than these some already reported single nucleotide polymorphisms have also been identified in these 3 genes and some of them are more frequently found in CHD cases. In-silico analyses based on evolutionary, biochemical and structural aspect of the gene revealed the disease causing effect of these variants. Further biochemical analysis would reveal the regulatory circuits involving these cardiac transcription factors and their possible role in pathogenesis of the disease. P33 Vitamin D Receptor (VDR) gene polymorphism and risk of ischemic stroke Puttachandra Prabhakar 1* , Rita Christopher 1 , Dindagur Nagaraja 2 1 Department of Neurochemistry, National Institute of Mental Health and Neuro Sciences, Bangalore-29, India; 2 Department of Neurology, National Institute of Mental Health and Neuro Sciences, Bangalore-29, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):P33 Background: Vitamin D deficiency is associated with various chronic disease conditions. The expression and nuclear activation of vitamin D receptor (VDR) are essential for the effect of Vitamin D, because vitamin D is involved in various signaling cascades. Few polymorphisms in VDR gene have been reported to be associated with cardiovascular disease and hypertension. We sought to examine the association between VDR gene variants and risk of ischemic stroke in Indian population. Methods: We recruited 250 patients diagnosed with ischemic stroke and 300 age and gender-matched healthy control subjects, after informed consent. 4 SNPs in VDR gene (ApaI, BsmI, FokI & TaqI), were genotyped by using PCR-RFLP method. Serum Vitamin D levels were determined by ELISA. The association of each SNP with the risk of stroke was analyzed by multiple logistic regressions by adjusting with age, gender, smoking, alcohol, hypertension and diabetes. Results: The VDR BsmI bb and FokI Ff genotypes were associated with increased risk for ischemic stroke (OR: 1.76; 95% CI; 1.01 – 3.08; P = 0.04) & (OR: 1.52; 95% CI; 0.99 – 2.31; P = 0.05) respectively. No association was observed between TaqI and ApaI polymorphisms and ischemic stroke. Vitamin D deficient (<20ng/ml) subjects with Bsm I Bb & bb genotypes had significantly higher risk for ischemic stroke (OR: 2.24; 95% CI; 1.11 – 4.56; P = 0.03) & (OR: 2.27; 95% CI; 1.05 – 4.99; P = 0.034), respectively. Similarly, increased risk for stroke was observed in Vitamin D deficient subjects with TaqI Tt genotype (OR: 2.77; 95% CI; 1.45 – 5.31; P = 0.002), and ApaI Aa genotype (OR: 2.456; 95% CI; 1.25 – 4.81; P = 0.009). Conclusions: In this first study to elucidate the role of VDR gene polymorphism in ischemic stroke patients, we found that genetic variants in VDR gene was associated with an increased risk for stroke especially in Vitamin D deficient subjects. Our findings could contribute to the development of strategies for the prevention of ischemic stroke. P34 Clinical Characterization of Idiopathic Restrictive Cardiomyopathy having rare variant (E949K) in b-cardiac myosin heavy chain gene Mitali Kapoor 1* , Amitabh Biswas 1 , Soumi Das 1 , Sandeep Seth 2 , Balram Bhargava 2 , Vadlamudi Rao 1 1 Department of Anthropology, University of Delhi, Delhi, India; 2 Department of Cardiology, AIIMS, India Molecular Cytogenetics 2014, 7(Suppl 1):P34 Background: Idiopathic Restrictive cardiomyopathy (IRCM) and hypertrophic cardiomyopathy (HCM) reflects the same or very similar disorders showing restrictive physiology with different names due to discretionary crosscuts in the LV wall thickness rather than two separate distinct diseases. The perspective of this study is to clinically evaluate the IRCM proband and comparison between the two distinct disease phenotype IRCM and HCM as an outcome of same genotype i.e.E949K in gene MYH7. Methods: Diagnosis was based on ECG, 2D-echocardiography, Cardiac Catheterisation and Endomyocardial biopsy. 5ml of Blood sample was collected and DNA was isolated using phenol-chloroform method. Sequencing of the hotspot region of MYH7 gene i.e. exon 23 by Sanger method (ABI 3730) was done. Screening of family members available was also done clinically as well as genetically. The study was ethically approved by Institutional committee and informed written consent was taken from all participants. Results: The proband, 11 year old male reported with chest discomfort, syncope, palpitations and arrthymias since last six months. Echocardiography showed marked biatrial enlargement (LA=46mm, RA=54mm), normal sized ventricles (IVS=8mm, LV=9mm) with mild MR and severe TR. Cardiac catheterization revealed grade III MR, severe TR severe PAH, mild RV systolic dysfunction. With these evidences a diagnosis of Idiopathic RCM was made. Sequencing of exon 23 of MYH7 gene led to the identification of rare variant E949K (c.2845G>A) in MYH7 gene. This variant was not found in our screening of 80 cardiomyopathies patients and 78 unaffected family members. This mutation was previously reported by Watkins et al.1992 in a case of familial HCM and suggested late onset of disease with severe LV hypertrophy and Rayment I et al.1995 also reported that this mutation shows loss of tensile strength of stiffness which represents the main phenotype of IRCM. We found this rare mutation in IRCM patient with early onset and no ventricular hypertrophy. Conclusion: This study represents the phenotypic heterogeneity of same rare variant in two ethnically different probands indicating environmental role or mutations in some other genes. This study reiterates the importance of whole genome sequencing approaches in clinical practice. P35 A novel donor site mutation in LMNA gene leading to severe form of Dilated Cardiomyopathy in a proband of a family from Bihar, India Soumi Das 1* , Amitabh Biswas 1 , Mitali Kapoor 1 , Sandeep Seth 2 , Balram Bhargava 2 , Vadlamudi Rao 1 1 Department of Anthropology, University of Delhi, Delhi, India; 2 Department of Cardiology, AIIMS, India Molecular Cytogenetics 2014, 7(Suppl 1):P35 Background: Dilated Cardiomyopathy (DCM) is poorly understood in terms of their mechanistic pathways. It may lead to sudden cardiac death with a prevalence rate of 0.04-0.2%. The cause of DCM is still unknown and referred to as Idiopathic Dilated Cardiomyopathy. There are many candidate genes associated with the DCM but most of the mutations are found in the LMNA and MYH7 genes. Methods: The proband underwent Echocardiography and ECG to confirm the diagnosis. 5ml blood was collected and DNA was extracted using Phenol-chloroform method. The hot spot regions exon 23 of MYH7 gene, exon3 and exon 4 along with the intron3 of LMNA gene were sequenced using Sanger sequencing (ABI 3730xl). ACE 287bpI/D and TNNT25bpI/D polymorphisms were also genotyped. In silico analysis of this novel mutation by using softwares, Human splicing finder (HSF) and MaxENT to understand the effect of mutation on splicing. The study was ethically approved by Institutional committee and informed written consent was taken from all participants. Results: The proband aged 50yrs diagnosed with DCM, age of onset 45yrs, showing severe symptoms such as dyspnea, palpitation, fatigue and pedal edema under NYHA III classification showing dilated LV with EF 30%. Proband’s mother died due to heart problem but was not clinically confirmed for DCM and his sister had a suspicious death. The son of the proband aged 24 years has the same LMNA mutation but is asymptomatic presently. A Novel donor splice site mutation G>C transversion in intron3 of LMNA gene and a synonymous mutation (C>T at codon 923) was found in MYH7 gene in proband. ACE and TNNT2 polymorphisms showed a heterozygous (ID) and homozygous (II) genotypes respectively. In silico analysis by HSF and MaxENT shows that the elimination of natural donor splice site leading Molecular Cytogenetics 2014, Volume 7 Suppl 1 http://www.molecularcytogenetics.org/supplement/7/S1 Page 30 of 59 to the use of a cryptic donor site which is located 7 nucleotide upstream of native splice site. Conclusion: As reported in previous studies, LMNA gene mutations are associated to the severe form of DCM. From the above results, it may be concluded that the severe form of the disease is due to the splice site mutation. COMPLEX TRAI T & POLYGENI C DI SORDERS P36 Association of Vitamin D 3 levels with Glycemic Control in Type 2 Diabetes Subjects from Gujarati population-India Avisek Majumder 1* , Bhavik Doshi 1 , Frenny Sheth 1 , Manan Patel 1 , Navneet Shah 2 , Thakor Premal 3 , Rama Vaidya 4 , Jayesh Sheth 1 1 FRIGE’s Institute of Human Genetics, FRIGE House, Jodhpur Gam Road, Satellite, Ahmedabad-380015, India; 2 Department of Diabetes and Endocrinology; Sterling Hospital, Ahmedabad- 380052, India; 3 Gujarat Diabetic Association, Ahmedabad-380007, India; 4 Kasturba Health Society, Medical Research Centre, Mumbai-400056, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):P36 Background: Considering the active role of Vitamin D 3 in the functional regulation of pancreatic b-cells, present study was carried out to know the occurrence of Vitamin D 3 deficiency in Type 2 diabetic [T2D] and non- diabetic subjects and demonstrate its influence on glycemic control. Materials and Methods: This prospective study comprises of 508 individuals (including 210 T2D & 298 controls). All subjects were categorized into 3 groups according to Vitamin D 3 levels. Group-I: included 171 individuals (61 T2D out of 171) with normal Vitamin D 3 concentration (≥25 nmol/l), group-II: included 264 subjects (118 T2D out of 264)with mild to moderately Vitamin D 3 deficiency (15-24.9 nmol/l) and group-III included 73 subjects (31 T2D out of 73) having severe Vitamin D 3 deficiency (≤14.9 nmol/l). Vitamin D 3 and glycosylated hemoglobin [HbA1C] level was analyzed for all the subjects. Results: Overall 66.34% subjects (both T2D & Controls) were found to have Vitamin D 3 deficiency. This was more common in T2D patients (71%)(mean ±SD Vitamin D 3 level was 17.77±7.19nmol/L) as compared to controls (63%) (mean±SD Vitamin D3 level was 24.38 ± 9.30nmol/L)(p<0.05).Female subjects were more prone for Vitamin D 3 deficiency as compared to male (71.09% vs 61.09%, p<0.03) subjects. Moreover, a gradual increase in mean HbA1C level was observed as Vitamin D 3 level when reduced from its normal level in T2D subjects (7.95- 9.08% in group-I through group-III) [bHbA1C, Vit. D3= -0.07, r = -0.28, p<2.73×10 -5 ]. No such changes in mean HbA1C level were observed in controls. Conclusion: Present study demonstrates the high prevalence (66.34%) of Vitamin D 3 deficiency in Gujarati population from India, more so for subjects with T2D. It is likely that Vitamin D 3 has a role in regulating insulin sensitization; resulting in poor glycemic control in subjects with low Vitamin D 3 levels. This study also indicates that females are likely to be at a higher risk for Vitamin D 3 deficiency compared to male in T2D and control subjects. P37 Effect of PPAR-g2 Gene Pro12Ala Polymorphism (Rs1801282) and Vitamin D 3 on Glucose Homeostasis in Type 2 diabetic Subjects from Gujarat-India Avisek Majumder 1* , Frenny Sheth 1 , Manan Patel 1 , Bhavik Doshi 1 , Navneet Shah 2 , Premal Thankor 3 , Rama Vaidya 4 , Jayesh Sheth 1 1 FRIGE’s Institute of Human Genetics, FRIGE House, Jodhpur Gam Road, Satellite, Ahmedabad-380015, India; 2 Department of Diabetes and Endocrinology; Sterling Hospital, Ahmedabad- 380052, India; 3 Gujarat Diabetic Association, Ahmedabad-380007, India; 4 Kasturba Health Society, Medical Research Centre, Mumbai-400056, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):P37 Background: Pro12Ala polymorphism in PPAR-g2 gene is known to be involved in insulin sensitization and metabolic deregulation in Type 2 Diabetes (T2D) subject. Considering beneficial effects of Vitamin D 3 in functional regulation of pancreatic-b cells, present population based study was designed to determine the association of Pro12Ala polymorphism with serum Vitamin D 3 level and its effects on glucose homeostasis in T2D and non-diabetic subjects. Materials & methods: Total 508 subjects (including 210 T2D & 298 controls) were divided into two groups according to serum Vitamin D 3 level. GrouPI: included 338 subjects (150 T2D out of 338) having Vitamin D 3 deficiency (≤25.0 nmol/l) and group-II: included 170 subjects (60 T2D out of 170) with normal vitamin D 3 level (>25.0 nmol/l). All cases were investigated for Vitamin D 3 , glycosylated hemoglobin (HbA1C) level and Pro12Ala variant of PPAR-g2 gene. Results: It was observed there is 12Ala allele frequency of PPARg2 gene in 10.19% of T2D and 9.46% in control subjects (p>0.36). The mean HbA1C was better controlled in group-II T2D subjects with 12Ala allele compared to group-I T2D subjects having same allele (7.26±0.44% vs. 8.35±0.43%, p>0.014). In contrary to the above, patients who were homozygous for 12Pro allele, the mean HbA1c remains high irrespective of the normal Vitamin D 3 levels (8.59±0.18% vs. 8.29±0.28% in group-I and group-II respectively). Conclusions: Significant decrease in mean HbA1C level was observed in T2D patients with 12Ala allele and normal Vitamin D 3 level compared to the patients having same allele with Vitamin D3 deficiency. No such effect was observed in T2D patients with homozygous status for 12Pro allele. This indicate that biologically active form of Vitamin D 3 (125(OH)D 3 ) together with 12Ala allele may affect glucose homeostasis by some gene-nutrition interactions. P38 Transferrin (rs3811647) gene polymorphism in iron deficiency anemia K Sri Manjari 1* , KSPS Teja 1 , M Sujatha 1 , A Jyothy 1 , Pratibha Nallari 2 , A Venkateshwari 1 1 Institute of Genetics and Hospital for Genetic Diseases, Osmania University, Hyderabad; 2 Department of Genetics, Osmania University, Hyderabad E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):P38 Background: Iron-deficiency anemia (IDA) is the most common type of anemia, caused by inadequate iron availability for hemoglobin production due to the lack of dietary iron or insufficient uptake of iron. Transferrin (TF) exerts a crucial function in the maintenance of systematic iron homeostasis. The expression of the TF gene is controlled by transcriptional mechanism, although little is known about its influence on IDA. Hence, the aim of the current investigation was to determine the functional polymorphism (rs3811647) of TF gene in iron deficiency anemia. Material and Methods: A total of 207 school children of age from 12-16 years were selected from Government High School Seetaphalmandi, Secunderabad, Andhra Pradesh and screened for iron deficiency. Out of which 70 school children had iron deficiency anemia with hemoglobin levels less than 11.5 g/dl and 137 were normal. Demographic details and blood samples were obtained from all the subjects. Genotyping was carried out by tetra-primer ARMS PCR followed by agarose gel electrophoresis, and appropriate statistical analysis. Results: The genotype distribution of TF (rs3811647) region were 4.3% (AA), 81.4% (AG) and 14.3% (GG) in iron deficient children compared to 8% (AA), 72.3% (AG), and 19.7% (GG) in normal children. No significant variation was observed with respect to the allelic distribution [OD = 1.035 (0.67 – 1.59), p = 0.917] in the IDA group when compared to normal group. Conclusions: There was no significant association of the TF (rs3811647) gene polymorphism with iron deficiency anemia. Thus, our study highlights the importance of other genetic variants influencing the outcome of iron deficiency anemia. However, larger samples have to be analyzed to confirm the same. P39 CD36 gene variants and their potential in determining T2DM susceptibility Monisha Banerjee 1* , Sunaina Gautam 1 , CG Agrawal 2 1 Molecular & Human Genetics Laboratory, Department of Zoology, University of Lucknow, India; 2 Department of Medicine, King George’s Medical University, Lucknow, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):P39 Molecular Cytogenetics 2014, Volume 7 Suppl 1 http://www.molecularcytogenetics.org/supplement/7/S1 Page 31 of 59 Background: Type 2 diabetes (T2DM) is a non-communicable disease affecting huge populations in India. Several groups all over the world are in search of prognostic genetic markers for the early detection of T2DM. Single nucleotide polymorphisms (SNPs) in CD36, a macrophage scavenger receptor were implicated in the pathogenesis of T2DM and its complications. This molecule is responsible for the uptake of free fatty acids specially oxidized low density lipoprotein (Ox-LDL) during diseased conditions. Aim of the present study was to find out the allelic and genotypic frequencies of 11 SNPs spanning the entire CD36 gene in north Indian population and find their association with T2DM. In addition, haplotypic analysis was undertaken to find out the risk haplotype in individuals of families with a history of T2DM. Material and Methods: All the 11 SNPs were genotyped in at least 100 each of controls and T2DM subjects using polymerase chain reaction- restriction fragment length polymorphism (PCR-RFLP) and single-strand conformation polymorphism (SSCP) methods. Haplotype analysis was done by SHEsis software. Ten families with diabetic history were identified and blood samples were collected from available family members. DNA was extracted by salting out method and three SNPs viz. rs1761667 (G>A) in exon 1A, rs3211938 (T>G) in exon 10 and rs3212018 (16 bp del) in exon 14 were genotyped in all individuals. Anthropometric characteristics viz. systolic/diastolic blood pressures, body mass index (BMI), waist hip ratio (WHR) and biochemical parameters were measured in all individuals. Results: Out of 11 SNPs, six were polymorphic (rs1984112, A>G; rs1761667, G>A; rs1527479, C>T; rs3211938, T>G; rs1527483, C>T; rs3212018, 16bp del) in the study population. Individuals having a haplotypic combination ‘AACGC1’ showed highest significant association with T2DM (P<0.001). Family studies showed that individuals with risk genotype ‘GA’ of rs1761667, ‘G’ allele of rs3211938 (T>G) or ‘G’ allele of rs1984112 (T>G) had an increased BMI and relatively higher glucose levels. Conclusions: Therefore, we conclude that haplotypes/genotypes/alleles of the CD36 gene variants can be potential markers in determining diabetes risk and such analyses may be useful for early identification of individuals susceptible to T2DM and its complications in the north Indian population. P40 Mitochondrial-nuclear epistasis contributes to phenotypic variation in wild yeasts Swati Paliwal * , Anthony C. Fiumera, Heather L. Fiumera Department of Biological Sciences, State University of New York at Binghamton, 4400 Vestal Pkwy E, Binghamton, NY 13902, USA E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):P40 Background: Mitochondria are ubiquitous organelles and are the main source of cellular energy. Mitochondria affect nearly every cellular process, including energy production, metabolite biosynthesis, ion homoeostasis, growth, cellular differentiation and apoptosis. In human and mice models, mitochondrial DNA variation is associated with various metabolic diseases. Mitochondrial functions require intricate interactions between mitochondrial (mt) and nuclear (n) genomes. The extent to which mitochondrial-nuclear (mt-n) interactions contribute to phenotypic variation in a population is unknown. We have made an attempt to find out if different combinations between the different mt and n genomes are found in natural populations would may lead to the expression of complex traits through epistatic interactions. Materials and methods: We created a novel population of 100 novel yeast strains with all possible combination of 10 naturally occurring and polymorphic, nuclear and mitochondrial genomes. These strains were phenotyped for metabolic growth under a variety of conditions, including high temperature, carbon source, and oxidative stress using high- throughput micro-cultivation plate readers. The results were analysed statistically for the growth rates using ANOVA. Results: It was found that mt-n epistasis significantly contributes to phenotypic differences among these strains, explaining up to 40% of phenotypic variation. A large number of strains were found to contribute, indicating the mt-n epistasis is a wide-spread phenomenon. The patterns of genome interactions vary across environments, indicating that multiple interactions affect fitness. Interestingly, we found that certain strains harboring their native mitochondrial genomes are more fit than when harboring non-native mtDNA, suggesting nuclear-mitochondrial co-evolution. Conclusions: It is suggested that mt-n epistasis may provide a fitness landscape upon which selection can operate. This work highlights the need to consider mt-n epistasis when characterizing the genetic basis behind complex traits and “missing heritability” and in developing treatments for mitochondrial disorders in humans. P41 Association of Pro-inflammatory Cytokine Gene Polymorphisms with Schizophrenia in South Indian Population Lekshmy Srinivas 1* , NV Neetha 1 , Chandrasekharan Nair 2 , Priya M. Allencherry 3 , Moinak Banerjee 1 1 Human Molecular Genetics Laboratory, Rajiv Gandhi Centre for Biotechnology, Trivandrum, Kerala, India; 2 Nair’s Hospital, Ernakulam, Kerala, India; 3 Mental Health Centre, Trivandrum, Kerala, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):P41 Background: Schizophrenia is a severe and debilitating mental illness. Around 0.26% of people in South India suffer from schizophrenia. It is a complex disorder which may involve multiple genes with mild to moderate effect and non-genetic risk factors like environmental and psychological assaults. Cytokines, regulators of immune/inflammatory reactions and brain development, emerge as part of a common pathway of genetic and environmental components of schizophrenia. Our study explored the association of polymorphisms in cytokine genes with schizophrenia, the interaction of these genes in the causation of the disease and the population genetics of cytokine gene polymorphisms. Materials and methods: We performed a case-control association study using 248 patients from Kerala, South India and 244 ethnically matched normal healthy controls. We screened polymorphisms in 10 cytokine genes (IL1A, IL1B, IL1RN, IL3, IL4, IL6, IL10, IFNG, TNFA and TGFB1). Genomic DNA was isolated from blood and genotyping was carried out by PCR-RFLP, TaqMan allelic discrimination and KASPar assays. Allelic, genotypic, haplotypic and diplotypic frequencies were calculated and compared. Gene- gene interactions among cytokine genes were also assessed. Results: We found significant association of SNPs in pro-inflammatory cytokine genes IL1A, IL6, TNFA and IFNG with schizophrenia in the Kerala population. No significant association was observed with any of the anti- inflammatory cytokine genes IL4, IL10 and TGFB1. Our study provides significant evidence for strong gene-gene interactions among pro- inflammatory cytokine genes in the development of schizophrenia. Our findings support the immune hypothesis in the predisposition to schizophrenia. Conclusions: The phylogenetic analysis of Kerala population with HapMap populations show proximity to HapMap Gujarati Indian population (GIH), Mexican population (MEX) and the African populations ASW, MKK and YRI. This similarity can be attributed similar selection pressures that lie in the same latitudinal belt indicating similar environmental conditions with respect to temperature, rainfall, pathogens, sunlight etc. This information might facilitate the identification of clinical subgroups of patients with strong immunological basis for the outcome of the disease. P42 Glutamate Transporter Genes Are Associated With Schizophrenia in South Indian Population Ajay Jajodia 1* , Harpreet Kaur 1 , Ruchi Baghel 1 , Gurpreet Kaur 1 , Sanjeev Jain 2 , Ritushree Kukreti 1 1 CSIR-Institute of Genomics and Integrative Biology, Mall Road Delhi, India; 2 NIMHANS, Honsur Road, Bengaluru, India Molecular Cytogenetics 2014, 7(Suppl 1):P42 Background: Schizophrenia is a neurodevelopmental disorder and is manifested by disruption in cognitive ability along with positive and negative symptoms. Neurodevelopment abruptions involve pathologic processes caused due to genetic and environmental factors. Study aims to evaluate the association between the genetic polymorphisms of the neurodevelopmental gene and the risk of schizophrenia. Molecular Cytogenetics 2014, Volume 7 Suppl 1 http://www.molecularcytogenetics.org/supplement/7/S1 Page 32 of 59 Method: The study includes 482 schizophrenia cases and 401 age, sex matched controls. Genotyping was performed for single nucleotide polymorphisms (SNPs) of genes by primer extension reaction followed by the MALDI-TOF mass spectrometry (Sequenom TM ). Polymorphisms involved in neurodevelopment processes like synaptic plasticity, synaptogenesis, signal transduction and activity of receptors/transporters. Genotypic tests were applied to examine the association of SNPs with disease. SNPs with p value < 0.1 were investigated by haplotypes analyses. Further, p values were adjusted with clinical variables using multivariate logistic regression. To evaluate interaction between the loci multifactor-dimensionality reduction (MDR) test was performed. Test for multiple corrections was applied using 10000 MaxT permutations. Results: Single locus association analysis showed association of rs2033267 (SLC1A3) OR=3.08, 95%-CI=1.98-4.77, rs10430590(PIP4K2A) OR= 1.71, 95%- CI=1.29-2.26. We identified a significant three marker haplotype of SLC1A3 significantly associated with disease with OR=3.81, 95%-CI=2.03-7.16. MDR analysis reveal interaction between rs2494750 and rs3803300 of v-AKT murine thymona viral oncogene homolog 1(AKT1) and rs16917204, rs56164415 of Brain Derived Neurotrophic Factor (BDNF) with cross validation 8/10 and Pvalue 0.0001,OR=13.07,95%-CI=8.97-19.03. Conclusions: We report a association of SLC1A3(5p13) with risk of schizophrenia development, which codes for glutamate transporter found in glial cells that functions to regulate neurotransmitter concentration at excitatory synapse. In addition interaction study revealed AKT1 and BDNF genes both having neuroprotective role. P43 Association of HLA-G 14bp INS/DEL Polymorphism with brain morphology in Schizophrenia Ashwini Rajasekaran 1,2* , Venkataram Shivakumar 2 , Deepthi Venugopal 1,2 , Sunil V. Kalmady 2 , Anekal C. Amaresha 2 , Mahavir Agarwal 2 , Janardhanan C. Narayanaswamy 2 , Ganesan Venkatasubramanian 2 , Monojit Debnath 1 1 Department of Human Genetics, NIMHANS, Hosur Road, Bangalore-560029, India; 2 Translational Psychiatry Laboratory, Department of Psychiatry, Neurobiology Research Centre, NIMHANS, Hosur Road, Bangalore-560029, India Molecular Cytogenetics 2014, 7(Suppl 1):P43 Background: Multiple lines of evidence have implicated dysregulated immune processes in the pathogenesis of schizophrenia. Being an important immuno-modulatory molecule, Human Leukocyte Antigen (HLA)-G plays a pivotal role in successful pregnancy. Altered expression of HLA-G due to environmental and genetic variations not only lead to pregnancy complications but also a range of immunopathologies, some of which are being considered to confer risk for schizophrenia. One of the polymorphic marker, 14bp insertion/deletion (INDEL) located within the 3´ UTR region of the HLA-G locus in Chr.6p21.3 is associated with HLA-G expression and function. The current study is aimed at analysing the role of 14bp polymorphism and the impact of feto-maternal compatibility/ incompatibility at this locus on the risk of schizophrenia. In addition, the effect of 14bp INDEL on brain structure alterations in schizophrenia patients was also investigated. Methods: A total of 151 (male-75 and female-76) schizophrenia patients and 113 (male-68 and female-45) ethnicity matched healthy controls (HC) were considered. In addition, mothers of 64 schizophrenia patients were also recruited. Genotyping of 14bp INDEL was determined by PCR. Structural brain images were acquired using a 3-Tesla MRI in 108 HC and 76 schizophrenia patients. Voxel-Based Morphometry toolbox in SPM8 was utilized for brain imaging analysis using bilateral masks of the following brain regions: dorsolateral prefrontal cortex (DLPFC), hippocampus, parahippocampal gyrus (PHG) and posterior cingulate gyrus (PCG) [uncorrected p<0.01; small-volume-correction for the respective mask (family-wise-error) p<0.05; 10-voxel threshold]. Results: There were no significant allele and genotype differences between patients and controls. However, a significant increase of heterozygous (+14bp/-14bp) genotype was observed in the female patients (p≤0.05). Interestingly, mother and the female patients also shared increased +14bp/- 14bp compatibility. Imaging analysis indicated that patients exhibiting +14bp/-14bp genotypes had significantly deficient volume in the right hippocampus [30, -13, -12] and right PHG [41, -38, -11]. Conclusion: Our results demonstrate a possible role of HLA-G polymorphism and feto-maternal matching of HLA-G in conferring the risk of schizophrenia. Importantly, this genetic variant also influences brain morphometric measures. Taken together, these findings suggest HLA-G could be an important biomarker for schizophrenia. P44 Stress and 5-HTT (SLC6A4) as an indicator of suicidal behavior: a population study among the Dubla tribe of Daman Sweta Saha * , Masan Kambo Newmei, Shivani Pasi, Piyoosh Kumar Singh, Vadlamudi Rao Department of Anthropology, University of Delhi, Delhi, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):P44 Background: Suicide is considered to be characteristic of modern societies and thought to be an alien feature in tribal societies. Few earlier studies have reported cases of suicide in tribal communities but there is a dearth of evidences from India and around the globe investigating suicide among the tribal communities. The stress diathesis model is the best framework to understand the complex mechanisms interacting throughout the relationship between stress and suicide. The present study aims to understand the genetically determined vulnerability of suicide under stressful life events among the Dubla tribe of Daman. Method: 84 unrelated individuals were recruited for the study. Face to face interview was conducted to collect information. Columbia Suicide severity Rating Scale (C-SSRS) and Presumptive Stressful Life Event Scale (PSLES) was administered to measure suicidal behavior and stressful life events respectively. Alcohol abuse was assessed using DSM-IV based questionnaire. Sociodemographic variables were also recorded. Buccal cell samples were collected for genetic analysis. Genotyping of 5HTT-VNTR (Stin2) was done with 55 samples. Result: Bivariant analyses showed significant difference in stressful life events among suicide ideators and non-ideators (OR=8.625, p= 0.00007). Also, stressful life events were higher in suicide attempters both in males and females [OR=8.438, p= 0.02 (males); OR=9.454, p= 0.04 (females)]. The frequency of the 5HTT VNTR 10 repeat allele and12repeat allele was 27% and 73% respectively in the studied population. Significant difference was observed in 10 repeat allele (Stin 2.10) for suicide attempters and non attempters among females (odds ratio, OR = 4.263; p = 0.000003). Conclusion: Stress was found to be a major predictor of suicide among the Dubla tribe. 5HTT VNTR 10 repeat allele was associated with suicide attempt among females. This finding of 5-HTT genes associated with female suicide is justified by the dimorphic nature of the serotonergic system. CYTOGENETI CS P45 Significance of Gain and Loss of Chromosomal Abnormalities in AML: An Indian Experience Pina J Trivedi 1* , Manisha M Brahmbhatt 1 , Dharmesh M Patel 1 , Esha N Dalal 1 , Geeta Joshi 2 , Prabhudas S Patel 1 1 Cell Biology Division, Gujarat Cancer Research Institute, Ahmedabad, India; 2 Department of Cancer Biology, Deputy Director, The Gujarat Cancer & Research Institute, Ahmedabad, Gujarat, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):P45 Background: Acute myeloid leukemia (AML) is a heterogeneous group of disorder. Recurrent translocations are generally recognized to be a major parameter for prognostication in AML. Recurrent chromosomal translocation t(15;17),t(8;21) and inv(16) have good prognosis, whereas, loss and gain of different chromosomes and/or chromosome segments play a vital role by different mechanism in leukemogenesis. Cytogenetics is one of the most powerful independent prognostic indicators in AML. It serves to identify biologically distinct subsets of disease and has been widely adopted to provide the framework for risk-adapted treatment approaches. The aim of the present study was to appraise the clinical significance of numerical and structural chromosomal abnormalities in AML patients in terms of loss and gain of chromosomal material. Molecular Cytogenetics 2014, Volume 7 Suppl 1 http://www.molecularcytogenetics.org/supplement/7/S1 Page 33 of 59 Materials & Methods: Bone marrow and peripheral blood lymphocytes of 321 AML patients were carried out by cytogenetics and FISH studies. Short term cultures and GTG banding were done for karyotyping. FISH and Multicolour FISH; were carried out as and when required as per standard protocols. Results: Out of all 321 patients, trisomy 8 showed the highest prevalence (n=14) and found as sole, complex and secondary change. Apart from most commonly observed recurrent chromosomal abnormalities, there were loss and gain of different chromosomes also observed. The loss of sex chromosome was observed in the highest frequency (n=20). Gain of whole chromosomes were; 8 (X23), 10 (X5), 19 (X7), 21 (X10), 22 (X6) and loss of X (X6), Y (X17). Mainly involved breakpoints in structural abnormalities were gain or loss of different chromosomes i.e. 1q (X8), 5q (X5), 8q (X4), 9q (X5), 11p (X5), 11q (X8), 17q (X9), and 22q (X 6). Conclusions: Study revealed that the, loss of chromosomal material was observed much more often than gain in AML with aberrant karyotypes. Hence, loss of tumor-suppressor genes may occur which may involved in mechanism of leukemogenesis. Numerical abnormalities in karyotype may affect gene-dosage and may play a significant role in the pathogenesis of AML. This study also highlights the importance of diagnostic cytogenetics as an independent prognostic factor in AML, providing the allocation for a stratified treatment approach of the disease. P46 Chromosomal Abnormalities and Hormonal Imbalance in Patients with Amenorrhea in Tamilnadu G Bhavani 1* , RS Chandra 1 , D Anuradha 2 , ST Santhiya 1 1 Dr ALM Post Graduate Institute of Basic Medical Sciences, University of Madras, Taramani, Chennai-600113, India; 2 Department of Medical Genetics, Institute of Obstetrics and Gynecology, Madras Medical College, Government Hospital for Women and Children, Egmore, Chennai-600008, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):P46 Background: Amenorrhea is the absence of menstrual bleeding in women of reproductive age. Multifarious causes such as pregnancy, absence of uterus and vagina, hormonal imbalance, excess of male testosterone, endometritis and improper functioning of ovaries could be attributed. It is the sixth major cause of female infertility. The present study was undertaken to determine the prevalence of chromosomal anomalies in patients with amenorrhea and to correlate the karyotype with the clinical condition. Materials & Methods: The study comprised of cases with provisional diagnosis of Primary Amenorrhea [n=74], Secondary Amenorrhea [n=13], Turner Syndrome [n=8], Gonadal dysgenesis [n=4] and a case of Androgen insensitivity syndrome. Results: In the present study, Eighty-one cases revealed normal karyotype (46,XX) while, the remaining 19 cases showed abnormal chromosomal pattern. The most frequent abnormal karyotype in these patients was of 46,XY females [n=6] followed by 45,X [n=5]. Two individuals showed 45,X/ 46,X,i(X)(q10) and a single case each of 45,X/46,XX; 45,X/46,XY; 45,X/46,X,r (X); 46,X,i(Xq); 46,X,del(X)(q21.2) and 46,X,del(X)(p11) respectively were also observed. The percentage of chromosomal abnormalities in patients with PA and SA were 18% and 7.6% respectively. Hormonal profiling wherever possible revealed an elevated level of FSH in 47 individuals [52.8%] and that of LH in 34 [43.2%] of them. Altered levels of other hormones such as PRL [3.09%], T3 [11%], T4 [9%] and TSH [12%] were seen. Most of the patients had hormone values appropriate to the post-menopausal range. Detailed molecular analysis of possible candidate genes in cases of 46,XY females has been proposed to resolve genetic etiology. Conclusion: This study has shown the spectrum of chromosomal abnormalities underlying amenorrhea. DYSMORPHOLOGY P47 Partial Deletion of Distal Long Arm Encompassing Jacobsen Syndrome Manisha Desai 1* , Bhumi Patel 1 , Chaitanya Datar 2,3 , Anand Pandit 3 , Prakash Ghambhir 4 , Darshana Nayak 5 , Jayesh Sheth 1 , Frenny Sheth 1 1 FRIGE’s Institute of Human Genetics, FRIGE House, Jodhpur Gam Road, Satellite, Ahmedaba, India; 2 Sahyadri Genetics, Unit of Sahyadri Hospitals, Barve Memorial Complex, J.M. Road, Pune, India; 3 KEM Hospital, Department of Paediatrics, Rasta Peth, Pune, India; 4 Birth Right Genetic Clinic, Erandawane, Pune, India; 5 Asian Child Neuro Clinics, Park Avenue, Ellisbridge, Ahmedabad, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):P47 Background: The terminal 11q deletion syndrome also known as Jacobsen syndrome (JS) is a rare genetic disorder associated with multiple dysmorphic features and occurs in 1 in 100,000 live births. The etiology behind the disorder is loss of contiguous set of genes due to 7-20Mb deletion that has proximal breakpoint at 11q23. Materials and Methods: Segmental aneuploidy or alteration involving #11q arm was detected in 2 cases during conventional cytogenetic analysis carried out in children having multiple congenital anomalies (MCA). Chromosome preparations were obtained from PHA stimulated lymphocyte cultures according to the standard procedure at 500-band level in both patient and their parents. Evaluation of the break point region was performed by 60K oligonucleotide array-Comparative Genomic Hybridization (aCGH) using Agilent platform. Female genomic DNA (Promega Corporation, Madison, WI, USA) was used as a sex-matched reference, which was analyzed with the aCGH analysis software v3.4 (Agilent Technologies Inc., Santa Clara, CA, USA) by applying Z-score segmentation algorithm with a window size of 10 points to identify chromosome aberrations. Results: Chromosomal study demonstrated structural rearrangements on #11q arm in both the cases i.e. 46,XX,der(11)del(11)(q24). Oligonucelotide aCGH analysis was performed using 3-points filter and 0.2 variation which lead to the confirmation of partial deletion of 11.8-11.9Mb at 11q24.1q25 [arr 11q24.1q25(123,045,174-134,868,407)x1] in case-1 where presence of deletion was suggested by banding technique. Whereas, a 13.9- 14Mb deletion at 11q23.3q25 together with 7.3-7.6Mb duplication at 12q24.32q24.33 was detected in the second case [arr 11q23.3q25 (121,000,318-134,868,407)x1 and 12q24.32q24.33(126,482,698-133,767,986) x3]. The complex structural rearrangement was missed by the conventional cytogenetic analysis in case 2. Paternal inheritance was confirmed in the latter case. Conclusion: Conventional cytogenetic analysis provided an overview of the deleted segment whereas aCGH analysis added detailed information about the breakpoint region. The complex structural rearrangement detected only by aCGH indicates its utility in diagnosis of rare genetic diseases especially in cases with multiple congenital anomalies. GENETI C TOXI COLOGY P48 In Vitro Fluoride Induced Genotoxic Effect on Human Blood Lymphocyte Cells and its Amelioration by Emblica Officinalis Extract Swati B Thakur * , Mandava V Rao Human Genetic Unit, Department of Zoology, School of Sciences, Gujarat University, Ahmedabad, Gujarat, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):P48 Background: Fluoride is a widespread industrial pollutant. Although, acute and chronic exposure of fluoride results in adverse health effects, in vitro studies demands for further evidences to conclude on the role of F as genotoxic agent. We have investigated the genotoxic properties of fluoride on peripheral blood lymphocyte cells and evaluated the protective effect of Emblica officinalis (Amla) against fluoride toxicity. Materials and Methods: Peripheral blood lymphocytes were cultured and treated with different concentrations of fluoride (17 µM, 34 µM, and 51µM) and supplement with amla extract(20 µg) for the study of various genotoxic parameters such as sister chromatid exchanges (SCEs) and cytokinesis block micronucleus (CBMN) assay. To rule out the antioxidant properties of amla, indices like 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay and High performance thin layer chromatography (HPTLC) were done. Results: Fluoride exhibited a significant increase in SCEs per metaphase plate (p<0.001) and SCEs per chromosome (p<0.05). Similarly, cell cycle proliferative index significantly decrease (p<0.001) in a dose-dependent manner in the three fluoride dose groups. Genotoxic indices such as nuclear deformities and frequency of micronucleus significantly (p<0.001) Molecular Cytogenetics 2014, Volume 7 Suppl 1 http://www.molecularcytogenetics.org/supplement/7/S1 Page 34 of 59 elevated with increased fluoride concentration. Furthermore, nuclear division index (NDI) and cell viability also noticed to be declined in fluoride treated cultures. Cultures with high dose of fluoride co-supplement with amla extract indicated a remarkable recovery in these genotoxic indices as comparable to control cultures. Antioxidant analysis of amla extract showed high free radical scavenging activity with EC 50 value of 55.44 ± 0.12µg/ml. Conclusion: Amla has a strong antioxidant system to scavenge the free radicals generated through toxic effect. Amla showed an antigenotoxic effect against fluoride and thus has a great potential for the application in medicinal products. P49 Genetic Damage Biomarkers in Buccal Epithelial Cells of Healthy Individuals Staying Near Three Mobile Phone Base Stations Naresh Mahajan * , Akbar Bhat, Gursatej Gandhi Department of Human Genetics, Guru Nanak Dev University, Amritsar, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):P49 Background: Massive surge in mobile phone usage throughout the world has increased the installation of mobile phone base stations to meet the subscribers demand. However, as mobile phone towers and handset use microwave radiation for signal transmission, their arise health concerns from exposures to these radiations especially for people living in areas where the mobile phone base stations have been installed. Materials and Methods: In cross sectional case control study, effort was made to assess genetic damage in some individuals (n=50; exposed group) residing in an area with three base stations close by (150 meters) with power density ranging from 172.6 to 550.3µw/m 2 at the threshold of residences from where sampling was done. Healthy controls (n=50) with no exposure were contacted from areas with no base stations (power density 0.0023µw/m 2 ).The standard buccal cytome assay was performed to assess DNA (genetic) damage, all proliferation and cell death biomarkers. Results and Conclusion: Genetic damage (micronuclei and nuclear buds) was significantly (p<0.05) higher in the exposed group while cell proliferation markers (basal cells and binucleated cells) were not significantly raised. However, among the cell death markers (condensed chromatin, karyorrhectic cells, pyknotic cells and karyolitic cells), the condensed chromatin (p<0.05) and karyolitic (P<0.05) cells also showed significant increase in the exposed group. The observed significant increase in genetic damage assessed in the buccal epithelial cells in individuals residing near mobile phone base stations is of concern as all neoplasia initiate from genetic damaging events. P50 Role of δ-Aminolevulinic Acid Dehydratase (ALAD) Gene Polymorphism in Lead Induced Nephrotoxicity Mugdha Tiwari 1* , LJ Bhagia 2 , I Shaikh 2 , D Rohila 1 1 Environmental Carcinogen Unit, National Institute of Occupational Health, Ahmedabad, India; 2 Hygiene Department, National Institute of Occupational Health, Meghani Nagar, Ahmedabad, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):P50 Background: Lead is an important environmental and occupational pollutant and it can cause nephrotoxicity even at low doses. Early detection of renal disease from occupational and environmental exposure to nephrotoxic chemicals is currently limited by the lack of sensitive or chemical specific tests. There are growing body of evidence that supports Kidney injury molecule-1 (KIM-1) as a specific marker for nephrotoxicity, specifically- ischemic renal injury. δ- aminolevulinic acid dehydratase (ALAD) gene polymorphism is known as an important factor affecting workers susceptibility to lead toxicity, further role of ALAD-1-1 and ALAD2-2 genotype is not clear yet. Correlation among the blood lead level, ALAD genotype present, urinary KIM-1 level of workers can provide better understanding about lead intoxication pattern and the role of genetic factor in susceptibility towards lead intoxication among workers. Methods: A total 200 biological samples (blood and urine) collected from workers working in lead-acid battery manufacturing unit, were analysed for blood lead levels, urinary KIM-1 and presence of ALAD genotype. Results: In the present study significant correlation between blood lead levels and kidney toxicity were found. In individuals having ALAD 1-1 genotype, blood lead levels were observed significantly higher. Conclusion: Higher KIM-1 level in urine in lead exposed workers denotes that KIM-1 can be considered as early kidney injury for lead induced nephrotoxicity. GWAS STUDY P51 Genome Wide Association Study to Identify SNPs Associated with Homocysteine, Vitamin B 12 and Holotranscobalamin in Indian Population Vinay Singh Tanwar 1* , Gaurav Garg 1 , Ankur Mukherjee 2 , Ganesan Karthikeyan 3 , Sandeep Seth 3 , Analabha Basu 2 , Shantanu Sengupta 1 1 CSIR-Institute of Genomics and Integrative Biology, Delhi, India; 2 National Institute of Biomedical Genomics, Kalyani, West Bengal, India; 3 Department of Cardiology, All India Institute of Medical Sciences, New Delhi, India Molecular Cytogenetics 2014, 7(Suppl 1):P51 Background: Vitamin B 12 , a cofactor for the enzyme methionine synthase, catalyzes the remethylation of homocysteine to methionine. About 50-60% of the Indian population are deficient in vitamin B 12 , a micronutrient that is only synthesized by microorganisms while mammals have evolved ways for its absorption from diet. Of the various factors involved in Vitamin B 12 absorption, Transcobalamin II (TC II) is most important as vitamin B 12 bound to TCII is bioavailable. Thus, the objective of our study was to identify the genetic variants that are associated with homocysteine, vitamin B 12 and holotranscobalamin levels. Methods: A total of 3024 healthy individuals of Indo-European ethnicity were included in the study. Biochemical parameters like homocysteine, vitamin B 12 , holotranscobalamin etc were determined for each individual. In the first phase (discovery phase) Genome wide association studies were performed using Illumina Omni express chip and 524 individuals were genotyped for 731,442 single nucleotide polymorphisms (SNPs). Statistical analysis was performed using PLINK software (v 1.07) after stringent quality control. In the second phase SNPs that were found to be significantly associated were genotyped in 2500 healthy individuals. Results: Several genetic variants, some of which are novel, were found to be significantly associated with homocysteine, vitamin B 12 and holotransco- balamin. This is the first GWAS for holoTC which we found is a better predic- tor of vitamin B12 status. Conclusion: Although we found several SNPs earlier reported to be significantly associated with the biochemical traits measured in our population also, many of the SNPs were previously not reported to be associated with the biochemical traits measured. METABOLI C DI SORDERS P52 Identification of Novel Mutations in Glucocerebrosidase (GBA) Gene in Indian Patients with Gaucher Disease (GD) Chitra Ankleshwaria 1* , Jayesh Sheth 1 , Mehul Mistri 1 , Ashish Bavdekar 2 , Sheela Nampoothiri 3 , Sarita Gupta 4 , Frenny Sheth 1 1 Institute of Human Genetics, FRIGE House, Jodhpur Gam Road, Satellite, Ahmedabad, India; 2 KEM Hospital, 489, Rasta peth, Sardar Mudliar Road, Pune, India; 3 Amrita Institute of Medical Sciences and Research Centre, AIMS Ponekkara, Kochi, India; 4 Department of Biochemistry, The MS University, Baroda, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):P52 Background: Gaucher disease (GD) is the most common glycolipid storage disorder due to inherited deficiency of lysosomal enzyme acid b-glucosidase (glucocerebrosidase, E.C.3.2.1.45) occurring due to mutations in the GBA gene. More than 300 mutations have been reported with higher frequency of most common mutant allele N370S (c.1226A>G), Molecular Cytogenetics 2014, Volume 7 Suppl 1 http://www.molecularcytogenetics.org/supplement/7/S1 Page 35 of 59 Leu29AlafsX18 (c.84dupG), L444P (c.1448T>C), IVS2+1G > A (c.115+1G>A) in Jewish population. The objective of the investigation was to identify mutations in Indian patients with GD and to understand genotype/ phenotype correlation. Materials and Methods: Our study comprises of forty five patients with GD and we have reported two novel mutations observed in two of the patients. Genomic DNA was extracted from whole blood by salting-out method and was screened for the common N370S (c.1226A>G), L444P (c.1448T>C), R463C (c.1504C>T), and IVS2 (+1) G>A (c.115+1G>A) mutations by RFLP PCR. Bidirectional sequencing was carried out using automated sequencer in absence of above common mutations. In silico analysis was carried out using Polyphen2, SIFT and Mutation t@sting softwares. Results: Our study has identified two novel missense mutations G289A (c.866G>A) in homozygous state in Exon-7 and I466S (c.1397T>G) in heterozygous state in Exon-10 in two patients respectively. Experimental program Polyphen 2 showed G289A (c.866G>A) and I466S (c.1397T>G) as probably damaging with a score of 0.963 (sensitivity: 0.78, specificity: 0.95) and 1.000 (sensitivity: 0.00, specificity: 1.00) respectively. SIFT/ PROVEAN Human program showed G289A (c.866G>A) and I466S (c.1397T>G) mutation as deleterious with score of -3.233 and -5.045 respectively and Mutation testing showed G289A (c.866G>A) as disease causing mutation and I466S (c.1397T>G) mutation as polymorphism. Conclusion: In this study, we have observed two novel GD mutations G289A (c.866G>A) and I466S (c.1397T>G) and effect of this genotype was studied using protein modeling. Identification of the genotype helps in predicting phenotypic expression, therapeutic response, and carrier screening for genetic counselling. P53 Identification of Novel Mutations in HEXA Gene in Children Affected with Tay-Sachs Disease from India Mehul Mistri 1* , Chaitanya Datar 2 , Frenny Sheth 1 , Sarita Gupta 3 , Jayesh Sheth 1 1 FRIGE’s Institute of Human genetics, Ahmedabad, Gujarat, India; 2 Sahyadri Medical Genetics and tissue engineering facility (SMGTEF), Pune, Maharashtra, India; 3 The M. S. University, Vadodara, Gujarat, India Molecular Cytogenetics 2014, 7(Suppl 1):P53 Background: Tay-Sachs disease (TSD) is an autosomal recessive storage disorder due to impaired activity of the lysosomal enzyme b-Hexosaminidase-A (EC 3.2.1.52) due to the mutation in HEXA gene. As per HGMD database, 134 mutations have been reported from different ethnic groups, while in India only few mutations have been reported till date. Here we reported three new novel mutations in HEXA gene causing TSD in children from Maharashtra. The objective of the investigation was to determine the disease causing mutations in HEXA gene in children affected with Tay-Sachs disease confirmed by deficient enzyme activity of b-Hexosaminidase-A. Materials and Methods: Seven children in the age range of 1 month to 1.5 years were enrolled in this study. Enzyme study was carried out using 4-MU substrate (MUGS) specific for b-Hexosaminidase-A. The exons and exon-intron boundaries of HEXA gene were bidirectionally sequenced using automated sequencer. In silico analysis was carried out using SIFT, Polyphen2 and Mutation T@ster softwares. Written consent was obtained from guardian of the study subjects. Results: Overall, we have identified 8 mutations in seven unrelated families, three of which are novel, including combined heterozygous missense mutations c.524A>C (p.D175A) and c.805G>C (p.G269R) was observed in one case and one homozygous nonsense mutations c.1528C>T (p.R510X) in one case. A previously known missence mutations c.532C>T (R178C), c.964G>T (p.D322Y), c.1385A>T (p.E462V), 4bp insertion c.1277_1278insTATC (p.Y427Ifs5) and splice site mutations c.459+5 G>A were observed in 5 children. In silico analysis further confirmed the pathogenic effect of the novel mutations occurred at highly evolutionarily conserved and functionally active domain residues in the protein leading to conformational changes or mRNA producing truncated protein resulting in the diminish or absent activity of the protein. Conclusion: Mutations responsible for TSD in Indian population are unique. We have found 3/8 (37.5%) novel mutations [D175A, G269R and R510X] in present study along with 5/8 (62.5 %) previously reported mutations [E462V, D322Y, R178C, c.1277_1278insTATC (p.Y427IfsX5) and c.459+5 G>A]. This study further confirms that nearly 37.5 % of TSD children harbor novel mutations in India and 62.5 % have common or earlier reported mutations. Overall study provides the new insights into the molecular basis of the disease that can be utilized for the molecular screening of TSD. P54 Live-Cell Imaging of Compartment-Specific Redox Changes in Menkes Disease Fibroblasts Ashima Bhattacharjee 1* , Martina Ralle 2 , Svetlana Lutsenko 1 1 Johns Hopkins School of Medicine, Baltimore, Maryland, USA; 2 Oregon Health and Science University, Portland, USA E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):P54 Background: Copper is an essential micronutrient and its misbalance in the body is associated with severe neurodegenerative disorders. While the importance of copper in the body is evident, mechanisms by which copper misbalance induces pathologic changes and disease symptoms are poorly understood. In this study, using fibroblasts from Menkes disease patient, as a cellular model of copper accumulation, we examined whether excess copper triggers specific and distinct changes in the redox environment of different cellular compartments. Subjects and Methods: Skin fibroblasts from Menkes disease patient (YS cells, ATP7A -/- ) and his heterozygous mother (ATP7A +/- ) were used as an experimental system. Glutathione mediated redox environment and levels of H 2 O 2 were investigated in nuclei, cytosol, and mitochondria of live cells by tagging the respective ratiometric sensors (GRX-roGFP and HyPer) with a compartment-specific localization signal. Results: Under basal conditions, the YS and XS cells show similar glutathione mediated redox environment in the nucleus and cytosol. However, the mitochondria are oxidizing in YS cells. YS cells were observed to accumulate higher level of peroxide in the cytosol and mitochondria. We also found that copper accumulation in cytosol and nuclei of YS cells sensitize cells to glutathione depletion suggesting importance of glutathione in protection of cells against copper overload Conclusion: Our experiments revealed differential response of cellular compartments to excess copper in cells. Nuclei, in spite of being a site of copper accumulation, do not show marked redox changes, suggesting presence of robust protective mechanisms operating in this compartment. H 2 O 2 accumulation in cytosol in YS cells does not change glutathione balance, whereas mitochondria appear most affected, since both H 2 O 2 levels and glutathione balance are altered. We propose that mitochondria could be a primary site of copper toxicity in Menkes fibroblasts. Molecular mechanisms underlying differential redox responses are presently being investigated. MOLECULAR CYTOGENETI CS P55 Characterization of prenatally detected small Supernumerary Marker Chromosomes (sSMC) by molecular cytogenetic technique: FISH Bhumi Patel 1* , Thomas Liehr 2 , Manisha Desai 1 , Bindu Parikh 3 , Jayesh Sheth 1 , Frenny Sheth 1 1 FRIGE’s Institute of Human Genetics, FRIGE House, Satellite, Ahmedabad-380 015, India; 2 Jena University Hospital, Institute of Human Genetics, Kollegiengasse 10, D-07743 Jena, Germany; 3 Satyam Hospital, 6/65 Nilam Park, Bapunagar, Ahmedabad India Molecular Cytogenetics 2014, 7(Suppl 1):P55 Background: Microscopically recognized chromosomal trisomies and monosomies are clinically well described. However, clinical effects in the unborn baby having imbalances of small chromosomal regions resulting from karyotypes containing small supernumerary marker chromosomes (SMCs) in addition to normal chromosomal count are less predictable. Moreover, due to extreme heterogeneity of sSMCs in size, structure and chromosomal origin, full characterization of sSMCs by molecular techniques – FISH has become imperative. Materials and methods: Two out of 1600 cases investigated showed sSMC during amniotic fluid (AF) analysis. In case-1, single sSMC (47,XN,+mar1) Molecular Cytogenetics 2014, Volume 7 Suppl 1 http://www.molecularcytogenetics.org/supplement/7/S1 Page 36 of 59 [100%] was detected during third gravida in a young couple having previous child with Down syndrome and second pregnancy ending in first trimester miscarriage. In case-2, two sSMCs were detected in the foetus of an elderly couple during primi gravida (i.e. 48,XN,+mar1,+mar2)[100%]. In both cases, fetal anomaly scan and triple marker study were normal. Parental chromosomal analysis at 500 band resolution was apparently normal at the time of prenatal study confirming de novo origin of the SMCs. Various FISH probes were applied such as (acro-)cenM-FISH, SRY, and subtel X/Ypter; besides immunohistochemistry using antiboies CENPB (all centromeres apart from Y) and CENPC (all active centromere) was done. Microdissection and reverse painting FISH was also performed for complete characterization. Results: Microdissection and reverse FISH was carried out in case-1 and showed signal only on the SMC. cenM-FISH did not yield any further information. Since the fetus had two X chromosomes, reverse FISH was carried out on a normal male control which gave signal on the #Yp. Additional probes specific to the SRY and subtel X/Y pter gave two signals confirming a neocentric inv dup(Y) i.e. 47,XN,+mar.ish inv dup(Y) (pter/Yp11.2::Yp11.2/pter)(SRY++)(subtelX/Y++)[100%]. The sSMC thus consisted of euchromatin exclusively. In case-2, sSMC were characterized as inv dup(13 or 21)(q10) and del(13 or 21)(q10) i.e. 48,XN,+inv dup(13 or 21)(q10)(CENPC++)(D13/21Z1+),+del(13 or 21)(q10)(CENPC+)(D13/21Z1+) [100%]. Marker chromosomes in case 2 solely consisted of heterochromatic material according to FISH. Conclusions: This shows that molecular technique FISH is one of the most powerful tools for precise identification and detail characterizations of SMCs and thereby providing valuable information to the families regarding genotype-phenotype correlation during prenatal diagnosis. P56 Assessment of 1p/19q deletion by Flourescence Insitu Hybridization (FISH) in Glioma Patients from Andhrapradesh Eppa Kavitha 1* , Iravathy Goud 1 , Swarna Latha 2 , Meenakshi Swain 2 , Michelle De Paude 2 , Tejal Modi 2 , Anuradha 2 , Ravi V 1 , Sakina Aneeb 1 , Adi Maha Lakshmi M 1 , Vijayanand Reddy P 3 1 Molecular Biology and Cytogenetics Department, Apollo Health City Building, Apollo Hospitals, Jubilee Hills, Hyderabad, India; 2 Department of Histopathology, Apollo Hospitals, Jubilee Hills, Hyderabad, India; 3 Department of Oncology, Apollo Hospitals, Jubilee Hills, Hyderabad, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):P56 Background: Oligodendroglial tumors represent approximately 4-7% of all gliomas, however, in some series the incidence has been reported to be as high as 10-20% due to improved histological appreciation and recently recognized molecular signatures. The discovery of 1p and 19q chromosomal arms deletion in glial tumors influences both more objective diagnosis and more accurate prediction of chemotherapy response. As a result an attempt has been made to detect deletion using fluorescence in-situ hybridization (FISH) and to determine its prognostic value in a cohort of glial tumor patients from Andhra pradesh. Materials and Methods: FISH was performed on 66 FFPE tissue sections by using Vyis LSI 1p36/LSI 1q25 and LSI 19p13/LSI 19q13 dual coloured FISH probe sets. Signals were scored from at least 150-250 non- overlapping, intact nuclei. Results: Simultaneous occurrence of both 1p and 19q deletions was observed in (21/35) 60% of oligodendroglyomas which included (8/21) 38% of grade II and (13/21) 61.9% of grade III. Isolated 19q deletion was seen in (1/21) 4.76% & lone 1p loss was not observed in oligodendroglyomas. In Mixed Oligoastrocytomas combined 1p/19q loss was observed in (7/16) 43.75% cases, including one grade II and 6 grade III tumors and 1/16 (6.25%) showed isolated 1p loss & 19q deletion. This disorder was not observed in astrocytomas. The oligodendroglial phenotype was found to be significantly associated with a loss of 1p (P<0.05), a loss of 19q (P<0.05) and a combined loss of 1p and 19q (P< 0.05). Frontal location of a tumor occurred to be a statistically significant factor unfavourable for prognosis, p<0.05. Conclusion: In the work presented the FISH was successfully applied to identify deletion 1p/19q. Its incidence depends on the type of diagnosed glioma. Deletions also have prognostic significance in the test group what constitutes the basis for inclusion of determining deletion 1p/19q into diagnostic and treatment algorithm in gliomas. P57 Split Hand/Foot Malformation Type 1 Associated with 7q21.3 Deletion - A Case Report S Aswini 1* , S Ambika 2 , KS Pooja 2 , D Anuradha 3 , JS Kadandale 1 , CR Samuel 1 1 Department of Genetics, Dr. ALM PG. Institute of Basic Medical Sciences, University of Madras, Taramani, Chennai, Tamil Nadu, India; 2 Centre for Human Genetics, Bangalore, Karnataka, India; 3 Department of Medical Genetics, Institute of Obstetrics and Gynecology, Madras Medical College, Government Hospital for Women and Children, Egmore, Chennai , India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):P57 Split hand/split foot malformation (SHFM) is a rare congenital deformity involving limb development. SHFM, also known as ectrodactyly, is characterized by absence of digits, fusion of remaining digits, and a deep median cleft in the hands and feet. It has been observed to occur at a prevalence of approximately 1:18,000 newborns. This malformation is genetically heterogeneous involving several loci including 7q21-q22.1, Xq26, 10q24-q25, 2q31 and 3q27. New loci requiring further validation have also been suggested as valuable candidate regions. Chromosomal rearrangements involving7q21-q22 is most commonly associated with isolated or syndromic ectrodactyly, referred to as SHFM type 1. We report a case of SHFM1 in an eight-month-old female baby with developmental delay. Follow up after two years revealed bilateral hearing loss also. Chromosome analysis using high resolution banding technique showed an interstitial deletion of the sub-band 7q21.3. FISH using BAC clones resolved the break to have occurred within the band 7q21.3. Cytogenetic evaluation of her parents showed the deletion to be of a de novo origin. SFHM1 is expressed as an autosomal dominant trait with reduced penetrance and variable expression and is accompanied by deafness in 35% of the patients as observed in the proposita. Several studies have pointed out the probable role of three genes present in this region – DLX5, DLX6 and DSS1 - in limb development. This report reiterates the importance of high resolution banding and molecular cytogenetic techniques such as FISH in the detection and delineation of subtle abnormalities. Array CGH will help in further refining the deleted region and thus in the discovery of candidate genes for the phenotypic characteristics. MOLECULAR STUDY P58 Autosomal Dominant Mutation in COL7A1 Gene causing Epidermolysis Bullosa Dystrophica Jayesh Sheth, Mehul Mistri, Harsh Patel, Chitra Ankleshwaria, Aradhana Parikh * FRIGE’s Institute of Human Genetics, FRIGE House, Ahmedabad, Gujarat, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):P58 Background: Epidermolysis bullosa dystrophica is a rare genetic disease involving skin fragility, resulting in blistering of the skin either spontaneously or following minor skin contact, or trauma. Mutations in COL7A1 gene are associated with all forms of dystrophic epidermolysis bullosa. The gene which is present on the short arm of chromosome 3, codes for a protein called Collagen alpha-1(VII) chain which functions as an anchoring fibril between the external epithelia and the underlying stroma. The aim was to examine a de novo Mutation in Gene causing Epidermolysis bullosa dystrophica in a thirty year old female patient. Materials and Methods: The case of a thirty-year-old female patient was analyzed. The patient had vesiculobullous lesions, peeling of skin on pressure, milia formation and dorsa of both hands. Lichenified lesions with few fresh bullae were present over the knees and elbows. The histopathological examination was carried out for skin biopsy samples. For the molecular analysis, a sequence study of exons 73 to 75 of the COL7A1 gene was conducted. Molecular Cytogenetics 2014, Volume 7 Suppl 1 http://www.molecularcytogenetics.org/supplement/7/S1 Page 37 of 59 Results: The histopathological examination of the skin biopsy revealed separation of epidermis at dermoepidermal junction along with acanthosis, hyperkeratosis and focal parakeratosis of the overlying epidermal layer. Dermis revealed moderate inflammatory cell infiltration. The immunofluorescence study was negative for IgG, IgM, IgA and C3. These findings were consistent with clinical diagnosis of EB Dystrophica. Molecular Analysis showed that the patient was heterozygous for c.6712 G>A (p.G2043R) mutation in exon 73 of the COL7A1 gene. Interestingly, the patient had no family history of the disease indicating that the mutation was de novo. Conclusion: It is one of the rare examples of a patient who was heterozygous for the mutation with dominant type of EB dystrophy. P59 A Balanced Reciprocal Translocation T (X;20) in A Girl with Seizures and Intellectual Disability Disrupting ARHGEF9 Usha R Dutta 1* , Vijaya Kumar Pidugu 1 , Vera M Kalscheuer 2 , Ashwin B Dalal 1 1 Centre for DNA Fingerprinting and Diagnostics, Hyderabad, India; 2 Max Planck Institute for Molecular Genetics, Berlin, Germany E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):P59 Background: Chromosomal aberrations are a significant cause of human disorders. The purpose of the present study is to characterize a balanced reciprocal translocation identified in a girl who presented with seizures, disturbed sleep, intellectual disability and focal hypopigmentation on the skin and identify the gene(s) involved. Materials and Methods: Several methods like GTG banding, array CGH, X- inactivation studies by methylation specific PCR for the human androgen- receptor gene (HUMARA), FISH (Fluorescence-in situ-hybridization) with whole chromosome paint probes (WCP) and with Bacterial Artificial Chromosome (BAC) clones from the regions of interest, RT-PCR expression analysis for ARHGEF9 gene were used. Results: The chromosomal analyses revealed a translocation between the long arm of chromosome X and the short arm of chromosome 20 [46,X,t (X;20)(q12;p13)]. This result was confirmed by WCP FISH. Additionally, array CGH ruled out any gains or losses at the breakpoints or elsewhere in the genome. Also, X-inactivation studies by methylation specific PCR for HUMARA indicated skewed X-inactivation of the normal X chromosome. Breakpoint mapping of both derivative chromosomes was performed by serial FISH using BAC clones and RP11-943J20 from chromosome X showed split signals on patient derivative translocation chromosomes, indicating that this clone spanned the breakpoint. The breakpoint on 20p13 was mapped to a region of about 28 kb. Subsequent in silico analysis of the fine mapped breakpoint regions showed that on chromosome X, ARHGEF9 was likely disrupted by the chromosome rearrangement, whereas on chromosome 20 the breakpoint region does not seem to harbor a known gene. RT-PCR expression analysis of ARHGEF9 using RNA isolated from the patient’s lymphoblastoid cell line and a control suggested that in the patient the breakpoint maps between exons 1 and 2 of this gene. Further, the rearrangement has potentially resulted in fusion genes, suggested by the low expression of ARHGEF9 exons 2 to 10 in the patient. Conclusion: We have previously reported another chromosome rearrangement that truncated ARHGEF9 in a patient with epilepsy, anxiety, aggression, insomnia and learning and memory loss. Given the similar clinical phenotypes of both patients we propose that in the patient reported here ARHGEF9 loss of-function is likely to be the cause of disease. P60 Molecular analysis of mucopolysaccharidoses: identification and characterization of pathogenic mutations in Indian population Anusha Uttarilli * , S Jamal Md Nurul Jain, Ashwin B Dalal, Prajnya Ranganath, Shubha R Phadke, Girisha Kumar, Sankar, SJ Patil, Madhulika Kabra, Sumita Danda Diagnostics Division, CDFD, Nampally, Hyderabad, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):P60 Background: Mucopolysaccharidosis (MPS) are a group of rare inherited metabolic disorders which are caused due to the deficiency of a specific lysosomal enzyme involved in the catabolism of glycosaminoglycans. These disorders show a wide clinical spectrum ranging from severe, intermediate and mild phenotypes. Most of them show overlapping clinical features such as corneal clouding, coarse facies, hepatosplenomegaly, skeletal dysplasia, short stature, dysostosis multiplex, joint stiffness, joint contractures, cardiovascular and respiratory difficulties. Certain MPS disorders also show impaired neurological functions leading to mental retardation. They are inherited as an autosomal recessive with exception of MPS II, which is X- linked recessive disorder. They exhibit clinical, genetic as well as molecular heterogeneity. To date more than 200 mutations in IDUA, ~ 500 in IDS, ~ 200 in GALNS and ~ 200 in ARSB have been reported worldwide. The mutation spectrum of MPS disorders in Indian population is not characterized and established yet. This study was done to establish mutation spectrum in the Indian population that can be useful for the design of cost-effective strategies towards the molecular diagnosis of MPS disorders. Materials and Methods: We have carried out mutation analysis of a total of 90 different MPS disorder patients (MPS I, II, IV and VI) and identified a total of 64 different mutations comprising majorly missense, nonsense, small deletions and splice site mutations. The mutations were further characterized using mutation prediction software and protein structure analysis for pathogenicity prediction. Conclusion: The characterization of mutational spectrum of Indian patients is likely to be useful in provision of better carrier diagnosis and prenatal diagnosis for patients with MPS disorders and also aids in designing of newer therapeutics for efficient treatment. It also helps in establishment of genotype-phenotype correlation and provision of better genetic counselling to the families with MPS affected members. P61 Association of ESR and FOXP3 Gene Polymorphisms with Outcome of Ovarian Stimulation in Infertile Females Undergoing IVF Arun Kiran Patnam 1* , R Vinu 2 , J Vijayalakshmi 2,1 , P Venkatachalam 1 , G Usha Rani 3 1 Department of Human Genetics, Sri Ramachandra University, Porur, Chennai, India; 2 Department of Biomedical Sciences, Sri Ramachandra University, Porur, Chennai, India; 3 Department of Obsteritics and Gynecology, Sri Ramachandra University, Porur, Chennai, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):P61 Background: Estrogen receptor (ER) gene plays a major role in folliculogenesis, maturation of oocytes and fertilization of embryo. FOXP3 gene is a crucial regulatory factor for the development and function of Treg cells; it takes part as a central role in the induction and maintenance of fetal- maternal immunologic tolerance. Therefore, the present study was aimed to investigate the association of ESR and FOXP3 gene polymorphisms in infertile women who are undergoing in vitro fertilization (IVF) with poor ovarian reserve upon controlled ovarian hyperstimulation (COH) induced by follicle-stimulating hormone (FSH). Materials and Methods: Genomic DNA was extracted from EDTA- peripheral blood by using high salting-out method, polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis was done followed by DNA sequencing to confirm the results for ESR1 gene on intron 1, PvuII T/C (rs2234693) and XbaI A/G (rs9340799); ESR2: RsaI G/A (rs1256049) on exon 5; and FOXP3 gene: HaeIII T/C (rs2294021) single-nucleotide polymorphisms (SNPs). Genotyping was carried out in infertile female (n=25) undergoing in-vitro fertilization (IVF) with poor ovarian reserve and healthy fertile controls (n=25). Results: Statistical analysis of ESR 1 (rs2234693) gene polymorphism showed a significant association (p<0.05) between cases and controls. Whereas, ESR 1 (rs9340799), ESR2 (rs1256049) and FOXP3 (rs2294021) genotypes showed no significant association (P>0.05) in cases and controls. Conslusion: As polymorphism rs2234693 (PvuII) showed a significant association in dominant model (Odds ratio: 3.19 (95%CI: 1.00-10.17); P: 0.04), genetic variability of the Estrogen receptor gene may exert an indirect effect on the pregnancy outcome of IVF patients by affecting the Molecular Cytogenetics 2014, Volume 7 Suppl 1 http://www.molecularcytogenetics.org/supplement/7/S1 Page 38 of 59 development of the follicles, oocytes and embryos. Still, further studies are necessary to confirm our findings with larger sample size that might improve our understanding of ESR1 gene polymorphisms and its importance for advancing infertility diagnoses, treatment and genetic counselling. P62 Protein structure prediction for novel mutations in Arylsulfatase-A gene M Divya * , S Jamal Md Nurul Jain, SR Phadke, Ratna Kishore, Mahesh Kamate, Neerja Gupta, Ashwin Dalal Diagnostics Division, Centre for DNA Fingerprinting and Diagnostics, Hyderabad, Andhra Pradesh, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):P62 Background: Protein structure prediction is the prediction of three- dimensional structure of a protein from its amino acid sequence. It is useful in determining the effect of a mutation on protein structure and associated function in detail. Along with the use of mutation prediction servers (Mutation taster, Polyphen etc.) protein structure prediction is an additional approach to functionally annotate genetic variants detected from different individuals. Methods: The X-ray structure of the P15289 protein was used for protein structure prediction of effect of mutations (PDB code 1AUK). TRANSEQ pro- gram of EMBOSS was used to obtain translated products of the deletion and insertion mutants. PyMOL and Swiss PdbViewer were used for performing structural analysis. Potential changes in overall hydrophobi- city due to non-synonymous mutations were calculated using CLC workbench. Results: We performed protein structure prediction for six novel non- synonymous missense mutations (P180Q, Y33S, Q139K, R299P, G34E and R311P) and four frameshift mutations (c.188-189insA, c.752-753insT, c.576delC and c.445-446insT) obtained from sequence analysis of ARSA gene. The residue Q139 is in 3 10 helix and R311 is in a b-sheet. Missense mutations at these positions affect the secondary structure of the protein. R299P mutation predicted to destabilize surrounding helical structure by hydrogen bond disruption. G34E, P180Q, R299P, and R311P showed a wide range of alterations in the overall hydrophobicity at the sites of mutation. Y33S and R311P were involved in active site modification and P180Q affecting the catalytic ability. All frameshift mutations were predicted to be leading to nonsense mediated decay. Conclusion: Protein structure prediction helps to provide a means of generating a plausible protein structure resulting after the mutation and understanding the effect of mutations on a protein whose effects have not been experimentally determined. P63 A Family Based Study on T-C Transition Polymorphism in Cyp17a1 Gene in Indian Children Sukanya Gayan * , Abid Ali, Rajeev Kumar Pandey, Minu Bajpai Department of Pediatric Surgery, All India Institute of Medical Sciences, New Delhi, India Molecular Cytogenetics 2014, 7(Suppl 1):P63 Background: Congenital adrenal hyperplasia (CAH) or 46, XX DSD is a result of a defect in the P450 adrenal enzymes responsible for the conversion of progesterones to glucocorticoids and mineralocorticoids. This syndrome affects both males and females but causes ambiguous genitalia only in females. Mutations in the CYP21 cause 90% of cases of CAH. The remainder of CAH cases is distributed among deficiencies of P450c11, CYP17 or steroid acute regulatory protein, depending on the ethnic origin of patients. Rarely, 46, XX, DSD can result from exposure to exogenous androgens such as those given in the past to prevent loss of pregnancy. The present study was conducted to replicate a family based correlation of C to T transition polymorphism in Congenital adrenal hyperplasia (CAH). Materials and Methods: A total of 60 samples (20 families) within a period of one year (October 2012 – 2013) associated with CAH, were collected from patient visiting the Out Patient Door (OPD) facility of the department of Paediatric Surgery, All India Institute of Medical Sciences (AIIMS). Genomic DNA was isolated from peripheral blood leukocytes of twenty patients and their family members using the phenol - chloroform method. Polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) was used to detect the polymorphism in CYP17A1 using restriction enzyme, MSPA1. Results: This study revealed no association between CAH risk and CYP17A1 gene in Indian children. Conclusion: Our results do not suggest a role of CYP17A1 as a high susceptibility gene for Congenital adrenal hyperplasia in Indian family. P64 Frequency analysis of Spinocerebellar ataxia types 1, 2, 3 & 6 in patients with ataxia from Gujarat Harsh Patel * , Mehul Mistri, Chitra Ankleshwaria, Frenny Sheth, Jayesh Sheth Institute of Human Genetics, FRIGE House, Ahmedabad, Gujarat, India Molecular Cytogenetics 2014, 7(Suppl 1):P64 Background: The autosomal dominant spinocerebellar ataxias (ADCA) are a clinically and genetically heterogeneous group of neurodegenerative disorder characterized by progressive deterioration in balance and coordination as well as cerebellar ocular disturbance. There is a lack of information about the frequency of SCAs in Gujarat (western part of India), which can be used as a common screening test in our population. The study was conducted to analyze the frequencies of SCA1, SCA2, SCA3 and SCA6 in patients with ataxia from Gujarat. Materials and Methods: Prospective analyses of 64 unrelated patients were included after an informed written consent. They were presented with progressive cerebellar ataxia with other features like gait and speech disturbance, saccadic eye movements and tremors. The study included 35 males and 29 females in the age range of 20 years to 85 years. All patients were investigated for CAG repeat expansion in SCA1, SCA2, SCA3 and SCA6 gene by PCR. Genomic DNA was used for the molecular study. Results: CAG repeat expansion was detected in 27 patients (42.1%) of 64 unrelated individuals. Among these, 7.41% (female-2) subjects were found to have SCA1 with CAG repeat expansion copy in the range of 49-56 in ATXN1 gene, 59.3% (female-9, male-7) were found to have SCA2 with CAG repeat expansion copy in the range of 37-47 in ATXN2 gene, 33.33% (female-3, male-9) were found to have SCA3 with CAG repeat expansion copy in the range of 63-82 in ATXN3 gene. While none of the subject was found to have SCA6 as has been shown by normal CAG repeats in the range of 10-16 in CACNA1A gene. Age range of onset of the affected subjects was 3 rd - 5 th decade in SCA1, 3 rd - 6 th decade in SCA2 and 3 rd - 8 th decade in SCA3. Conclusion: Our study demonstrates that SCA2 is the commonest dominant spinocerebellar ataxia in Gujarati population followed by SCA3 affecting in the 3 rd - 6 th decade of life. P65 Genetic susceptibility of Henoch-Schönlein purpura in children Ritu Aggarwal * , Anju Gupta, Jasmine Naru, Neha Nanda, Manila Salaria, Deepti Suri, Surjit Singh Department of Immunopathology and Allergy and Immunology Unit, Deptartment of Pediatrics, Post Graduate Institute of Medical Education and Research, Chandigarh, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):P65 Background: Henoch-Schönlein purpura (HSP) is a small vessel vasculitis typically observed in children, 3-10 years old. The aetiology is unclear. Interaction of several environmental factors, including infections and multiple genes has been proposed to play a role in pathogenesis. An increased familial occurrence is an indicator of genetic predisposition; association with a major histocompatibility complex is plausible. The aim of the study was to investigate the association of HLA-DRB1 (HLA class II antigen) with HSP. Subjects and methods: The study was prospective and hospital based. Patients up to the age of 14 years, who fulfilled the diagnostic criteria of HSP, laid by the ‘European League Against Rheumatism’ were enrolled. Age matched healthy controls were included as well. One ml blood in EDTA was collected from patients as well as controls. DNA extraction was Molecular Cytogenetics 2014, Volume 7 Suppl 1 http://www.molecularcytogenetics.org/supplement/7/S1 Page 39 of 59 performed using commercially available kit. The quantity and quality of DNA was estimated by spectrophotometer and PCR for housekeeping gene beta actin, respectively. PCR with 24 sequence specific primers for HLA-DRB1 antigen was performed. Commercially available HLA-DR tissue typing kit (Inno-train, Kronberg im Taunus, Hesse, Germany) was utilized. Frequency of HLA-DRB1 was correlated with gastrointestinal and renal involvement. Results: The study included 43 patients and 53 controls. The mean age of the patients and controls was 8.5 years (range: 3-14) and 7.4 years (range: 1-14), respectively. Frequency of HLA-DRB1*11 was significantly increased in patients (p=0.006). A greater gastrointestinal (p=0.03) as well as renal (p=0.004) involvement was observed in patients with HLA- DRB1*11. Conclusion: This is the first study from India to report the HLA susceptibility genes in children with HSP. Presence of HLA-DRB1*11 was observed to predispose to HSP in children, as well as for greater likelihood of gastrointestinal and renal involvement. P66 Evaluation of galectin-3 genetic variants and lipid profile in RA patients in North Indian population Tarnjeet Kaur 1* , Manpreet Kaur 1 , Jatinder Singh 2 , Sukhdev Singh 2 , Sumeet Arora 3 1 Department of Human Genetics, Guru Nanak Dev University, Amritsar, India; 2 Department of Molecular Biology and Biochemistry, Guru Nanak Dev University, Amritsar, India; 3 Rheumatology Clinic, Amritsar, India Molecular Cytogenetics 2014, 7(Suppl 1):P66 Background: Rheumatoid arthritis (RA) is a chronic, inflammatory, systemic disease characterized by inflammation and destruction of peripheral joints which leading to deformity and disability. Galectin- 3 is emerging as one of key molecules in pathogenesis of RA. The aim of the present study was to evaluate association of two genetic variants rs4644 and rs4652 of galectin-3 with susceptibility towards RA in North Indian population. The study further involved evaluation of lipid profile variables in cases and controls. Methods: The present case-control study involved 200 RA patients diagnosed according to 1987 revised criteria of American college of Rheumatology and 200 unrelated age, sex and ethnicity matched controls. Genomic DNA was isolated from blood samples and genotyping was done with PCR-RFLP. Sample size for genetic association was calculated by CaTS Power calculator (http://www.sph.umich.edu). Serum was analyzed for lipid profile biomarkers using standard reagents and kits. Genotypic distribution of control and RA was compared by odds ratio statistics using medcal software. Differences in lipid profile were analyzed by independent `t’ test using SPSS version 18.0 (IL, USA, and Chicago) Results: The genotypic distribution of +191(A/C) showed significant differences between patients and controls (odds ratio = 1.9552, 95% CI = 1.0461-3.6542, p<0.05). AA genotype was found to be more prevalent in patients in comparison to controls. However, genotypic distribution for +292 (C/A) showed no significant difference between controls and cases (odds ratio = 0.2768, 95% CI = 0.0541-1.4149, p>0.05 ). RA patients were found to be dyslipidemic as indicated by the significantly higher atherogenic index as compared to controls (p<0.01). Conclusion: Galectin-3 may play an important role in pathogenesis of RA. P67 Possible involvement of altered expression of BDNF exon II gene and specific dopamine receptors in simvastatin induced beneficial effects in depression Digvijay G Rana 1* , Amrut Patel 2 , CG Joshi 2 , Ramesh K Goyal 3 1 Department of Pharmacology, Sigma Institute of Pharmacy, Vadodara, India; 2 Department of Biotechnology, Anand Veterinary College, Anand, India; 3 Institute of Life Sciences, Ahmedabad University, Ahmedabad, Gujarat, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):P67 Background: The up regulation of central BDNF gene expression has been suggested in the treatment of major depression. Chronic administration of dopaminergic agents activates the function of CREB which results into the up regulation of the BDNF gene expression. Statin therapy is associated with a reduced risk of depression and could be of therapeutic potential for major depression. Materials and methods: We have examined a possible link amongst simvastatin, bromocriptine, haloperidol and levodopa in accordance with BDNF exon II gene expression using RT-PCR method in mice treated with standard paradigm of chronic mild stress procedure for 14 days. We specifically determined if the oral administration of simvastatin would affect the efficacy of bromocriptine, haloperidol or levodopa in mediating the regulation of the BDNF exon II gene expression. Results: The results of RT-PCR method revealed the differential expression patterns for the expression of BDNF exon II gene in brain of mouse by indicating the three different bands, as evidenced previously to be the three different exon II transcript variants in mouse namely BDNF IIA, BDNF IIB and BDNF IIC. Mice treated with bromocriptine or levodopa in combination with simvastatin for 14 days could synergize the up regulation of for the expression of specific BDNF exon II transcript as compared to simvastatin alone whereas the mice treated with haloperidol in combination with simvastatin for 14 days could abolish the for the expression of up regulation of specific BDNF exon II transcript compared to simvastatin alone. Conclusion: The results of the above study suggests linkage between the function of dopamine or dopamine D 2 like receptor and differential expression patterns for the expression of BDNF exon II gene in brain of mouse which further strengthen the emerging hypothesis, suggesting the ability of neuronal systems to exhibit the appropriate adaptive plasticity could contribute to the treatment of depression. Further, the dopaminergic agents in accordance with the cholesterol lowering drug as adjuncts may reduce the depressive like behavior more significantly and facilitation of antidepressant action of dopaminergic agents may be correlated with HMGR or cholesterol or mevalonate pathway. P68 Genetic, metabolic and cellular factors influencing intracellular localization of the Wilson disease protein, ATP7B Arnab Gupta 1* , Ashima Bhattacharjee 2 , Nesrin Hasan 2 , Lita Braiterman 1 , Svetlana Lutsenko 2 , Ann L Hubbard 1 1 Department of Cell Biology, Johns Hopkins University, Baltimore, USA; 2 Department of Physiology, Johns Hopkins University, Baltimore, USA Molecular Cytogenetics 2014, 7(Suppl 1):P68 Background: Wilson disease (WD) is a disorder of copper accumulation in liver and brain caused by mutations in the copper-transporting ATPase ATP7B that affects 1 in 5000 live births. Under basal conditions, ATP7B protein localizes to the trans-Golgi network (TGN) but traffics to vesicles in response to high copper. The purpose of the study is to identify genetic, metabolic and regulatory factors that regulate ATP7B function and localization to maintain normal copper homeostasis in cells. The study is divided into three parts, (a) Role of copper in normal protein folding and its ER exit, (b) Role of regulatory phosphorylation of ATP7B in its trafficking from TGN to vesicles (c) Interaction of ATP7B with regulatory proteins in its trafficking route. Material and methods: Recombinant ATP7B (wt and variants), Confocal microscopy, NMR, Mass Spectroscopy, targeted knockdowns and other basic molecular biology techniques were utilized in this study. Results: We demonstrate that the polymorphism (producing the Gly 875 >Arg substitution) of ATP7B drastically alters the intracellular properties of ATP7B, while copper reverses the effects. Unlike the wtATP7B, the Arg 875 variant is located in the endoplasmic reticulum (ER) and does not deliver copper to the TGN. Elevated copper rectifies the ATP7B-Arg 875 phenotype. Analysis by NMR suggests that the ER retention of ATP7B-Arg 875 is due to increased unfolding of the Arg 875 -containing. conserved domain (b) I characterized the sites of copper dependent regulatory phosphorylation of ATP7B critical in normal ATP7B trafficking. I discovered that a stretch of Ser (S 340-343 ) at the N-terminal of ATP7B are the sites of phosphorylation and that phosphorylation alters the interaction between two conserved domains of ATP7B, causing the protein to attain a TGN exit conformation (c) Finally, while identifying the cellular machinery that is involved in trafficking of ATP7B vesicles, I found that MyosinVb is a major regulator of ATP7B trafficking. Presently, the molecular mechanism of the role of MyoVb in ATP7B trafficking is being investigated. Molecular Cytogenetics 2014, Volume 7 Suppl 1 http://www.molecularcytogenetics.org/supplement/7/S1 Page 40 of 59 Conclusion: We characterized an ATP7B genetic variant (c.2623A/G) that is causal to the disease in Indians and is a non-disease causing SNP in Chinese population. P69 Association of microRNA-146a and their target gene IRAK-1 polymorphism with enthesitis related arthritis category of juvenile idiopathic arthritis Sushma Singh 1* , Geeta Rai 2 , Amita Aggarwal 1 1 Department of Clinical Immunology, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow, India; 2 Department of Molecular and Human Genetics, Faculty of Science, Banaras Hindu University, Varanasi, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):P69 Background: MicroRNAs (miRNAs) are non-coding RNA molecules that play pivotal role in modulating the expression of multiple target genes at the post-transcriptional level. Single Nucleotide Polymorphisms (SNP) in pre-miRNAs can alter miRNA expression, and polymorphism in target molecules can affect binding to target mRNA. Studies have shown an association between miR-146a polymorphisms and autoimmune disease. Taking into account that interleukin-1 receptor-associated kinase-1 (IRAK-1) is a target of miR-146a, we studied the association between SNPs of miRNA-146 and its target IRAK-1 with susceptibility to Juvenile Idiopathic Arthritis-Enthesitis Related Arthritis (JIA-ERA). Methods: One hundred and fifty patients with JIA-ERA (ILAR criteria) were included in the study. 216 blood donors (201 male) with a mean age of 30.5 years served as controls. miR-146a (C/G) (rs2910164) and it’s target IRAK-1(C/T) (rs1059703) at Exon 12 region and IRAK-1 (A/C) (rs3027898) at 3’UTR polymorphisms were analyzed using PCR-RFLP method. Results: Among 150 patients, 134 were males and the mean age at onset of disease was 11 (4-16) years, mean disease duration was 4.5 (0.3-12) years. 22 had uveitis and 21 had positive family history. 73 had enthesitis and 75 had inflammatory back pain and all had arthritis. 116 were HLA B27 positive. Genotype frequency of miR-146a gene was in Hardy Weinberg equilibrium in healthy controls whereas in IRAK-1 genotype the frequency was contrary owing to its presence on chromosome X. The genotype frequency for miR-146a were different in controls and patients [GG (51.85% vs 50.0%), GC (42.13% vs 37.29%) and CC (6.02% vs 12.71%), OR = 2.18; 95% CI 1.02 – 4.68; p value = 0.0418] IRAK-1 (rs1059703) allelic frequencies in controls and patients were similar [CC (33.8% vs 32.9%), and TT (66.2% vs 67.0%)]. IRAK-1 (rs3027898) allelic frequencies were also similar among control and patients [CC (70.1% vs 76.1%) and AA (29.8% vs 23.9%)]. Conclusion: The CC genotype of the miR-146a rs2910164 polymorphism was significantly associated with the susceptibility to JIA-ERA. P70 TMC1 may be a common gene for nonsyndromic hereditary hearing loss in Indian population Pawan Kumar Singh * , Shipra Sharma, Manju Ghosh, Shivaram S Shastri, Neerja Gupta, Madhumita Roy Chowdhury, Madhulika Kabra Division of Genetics, Department of Pediatrics, All India Institute of Medical Sciences, New Delhi, India Molecular Cytogenetics 2014, 7(Suppl 1):P70 Background: Hearing impairment is very heterogeneous and most common sensory disorder. The prevalence of prelingual hearing loss is 1:500, with both environmental and genetic factors being equally responsible. To date, more than 128 loci and 74 genes responsible for nonsyndromic hereditary hearing loss have been identified, of which GJB2 gene is the most common across populations. Transmembrane channel like 1 or transmembrane cochlear- expressed gene 1 (TMC1) at DFNB7/11 locus at 9q13–q21 is another gene that is responsible for prelingual, severe to profound hearing impairment. It contains 24 exons and encodes 760 amino acids long 87.8 kDa multipass transmembrane protein, which is required in maintaining electrochemical homeostasis, structure and function of neurosensory hair cells in the inner ear. More than 29 different mutations have been reported in 48 families. Materials and methods: DNA was extracted from 47 multiplex families. Sanger sequencing identified connexin 26 mutations in six families and in the rest of the 41 families, homozygosity mapping was done using 35 fluorescent markers for eight common loci. DNA sequencing of TMC1 gene was done in all individuals of two families, in which linkage to DFNB7/11 locus was seen. Results and conclusion: Linkage to DFNB7/11 by four markers was found in two families. DNA sequencing of TMC1 gene in this locus identified a reported homozygous mutation, c.100C>T (p.R34X) in one family. In the other family, a novel homozygous change, c.1283C>A (p.Ala428Asp) was found in all the affected children. The mutation segregated with the hearing loss in both the families. Online protein prediction software SIFT and PolyPhen 2, predicted this novel change as damaging and probably damaging respectively. As TMC1 gene mutations have also been reported earlier in Indian and Pakistani families, it appears that TMC1 may be a common gene after GJB2 in the Indian subcontinent. c.100C>T is a common mutation in this gene. TMC1 gene should be considered in routine diagnosis if GJB2 is negative. P71 Significance of nucleophosmin1 (NPM1) gene mutation status on acute myeloid leukaemia patients with normal karyotype in South India R Sureshkumar 1* , S Santhi 1 , V Sangeetha 1 , N Geetha 2 , S Hariharan 1 1 Division of Cancer Research, Regional Cancer Centre, Thiruvananthapuram, Kerala, India; 2 Division of Medical Oncology, Regional Cancer Centre, Thiruvananthapuram, Kerala, India Molecular Cytogenetics 2014, 7(Suppl 1):P71 Background: Acute myeloid leukaemia (AML) is a heterogeneous group of haematological malignancy. In spite of recurrent chromosomal abnormalities present in a significant proportion of AML patients, more than 50% of the patients have a normal karyotype (NK-AML) and lack reliable molecular markers. NPM1 has been recently characterized as the most frequently mutated gene in AML Patients. The objective of the present study was to profile the karyotype and to assess the types of NPM1 gene mutations in AML patients in south India. Subjects and methods: A total of 200 de novo AML patients were investigated in this study. Cytogenetic G- banding analysis, Fluorescence in situ hybridization was performed with standard methods. Mutation screening of NPM1 mutation hotspot exon12 performed in DNA by PCR- SSCP and suspected samples were sequenced. Results: Among 200 de novo AML patients 119 showed normal karyotype. Fourty out of two hundred patients showed mutations in NPM1 exon12. Most of the NPM1 mutation were detected in NK-AML (37/40) (92.5%).Three of them showed abnormal karyotype. The highest incidence of mutation was detected in AML-M1 with 47.5% followed by M5, M4, M2 and M3. Predominant type of mutation was a type A mutation (c.860-863dupTCTG) (75%). Other type of mutations includes tetranucleotide insertion of TGCA (10%), CTGC (10%), GTCA (2.5%) and GTAG (2.5%). All the mutated cases were heterozygous in nature retaining wild type allele and have a distinct sequence in the protein c-terminal. Conclusion: Presence of 31% mutation in the NK-AML in the present study indicates that it is the most prevalent mutation in NK-AML patients in south India. NPM1 mutations are associated with good prognosis in AML. So predominance of NPM1 mutation in the AML especially in NK- AML patients suggests that it could be used as a reliable molecular marker for those patients. P72 Role of TNF-A, IL-6 and IL-4 with the susceptibility to chronic periodontitis in North Indian population: a multi-analytic approach Garima Prakash 1* , Sadhna Ajay 2 , KK Gupta 2 , Balraj Mittal 1 1 Dept. of Genetics, SGPGIMS, Lucknow, India; 2 Dept. of Periodontics, SPPIDMS, Lucknow, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):P72 Background: Periodontitis is a chronic inflammatory disease causing destruction of tooth supporting tissue and loss of teeth. Inter-individual variation in genetic factors and one or more variations in genes could play critical role in susceptibility to Chronic Periodontitis and its pathogenesis. Therefore, in present study, we aimed to perform combined risk genotype analysis and Multifactor dimensionality reduction (MDR) to identify combination of alleles in modifying the risk of chronic periodontitis. Molecular Cytogenetics 2014, Volume 7 Suppl 1 http://www.molecularcytogenetics.org/supplement/7/S1 Page 41 of 59 Materials and methods: A total of clinically diagnosed 200 periodontitis cases and 200 age and gender matched controls were genotyped for TNF-a, IL-6 and IL-4 gene polymorphisms using ARMS PCR and PCR-RFLP methods. Multi-analytic approach (combine risk genotype analysis and MDR) were used to find out the combination of allele contributing to risk of chronic periodontitis. All statistical analyses were performed using SPSS ver.15.0 and MDR analysis by MDR 2.0 Beta 8.4. Results: Single locus analysis showed association of TNFa-308GA (rs1800629) with risk of CP whereas IL-6-174GC (rs1800795) and IL-4-590CT (rs224325) polymorphisms did not show any significant differences. However, combined risk genotypes of all three polymorphism depict the increased risk of CP (P trend = 0.001) where MDR analysis revealed TNF-a-308GA and IL-6-174GC as a best polymorphic model for the risk of CP. When subjects were stratified on the basis of gender, only TNFa-308GA polymorphism showed association with risk of CP in North Indian population. Conclusion: TNF- a -308G>A polymorphism is associated with chronic periodontitis. However, multi-analytical approach suggests TNF-a-308GA and IL-6-174GC genetic variants interact to confer significant risk for chronic periodontitis. Acknowledgment: ICMR, New Delhi, India. P73 Bio-chemical and molecular analysis in cardiomyopathy patients Rutvik J Raval 1* , Tapan A Patel 1 , PL Sachapara 2 , Mandava V Rao 1 1 Gujarat Genetic Diagnostic Center (GenDiCe), Department of Zoology, University School of Sciences, Gujarat University, Ahmedabad, Gujarat, India; 2 Samarpan Hospital, Madhavjyot, Kalubha road, Bhavanagar, Gujarat, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):P73 Introduction: Cardiomyopathy refers to diseases of the heart muscle. Symptoms of cardiomyopathy includes: Shortness of breath, Chest pain, Dizziness, Lightheadedness or fainting, Palpitations. Cardiomyopathies are divided in two types that are Primary (intrinsic) and Secondary (extrinsic) cardiomyopathies. Primary cardiomyopathies are divided in three types i. e. genetic, mixed and acquired. Present study was planned to evaluate the biochemical alterations in patients with cardiomyopathy or myocardial Infarction using bio-chemical and molecular indices. Methods: Blood samples were collected from patients (n=12) with cardiomyopathy/myocardial infarction and same age and sex matched healthy controls (n=12). Serum myoglobin and serum creatine kinase-MB (CK-MB) levels were evaluated. Genomic DNA was isolated from peripheral blood and its quality and quantity were checked. PCR amplification of the various exons of genes MYH7 (exon 8 and 9) and TNNT2 (exon 8) was carried out. PCR products were checked by 2% agarose gel electrophoresis. Further, mutations were screened by Polymerase Chain Reaction-Single Strand Conformation Polymorphism (PCR-SSCP) technique. Results: In cardiomyopathy patients the ratio of Male: Female was 1:1. Out of twelve patients four patients showed high levels of CK-MB and two patients with high Myoglobin levels and only one patient reported high levels of both myoglobin & CK-MB. None of the patient showed mutation in exon8 and 9 of MYH7 gene & exon 8 of TNNT2 gene. Conclusion: In Gujarat population, cardiomyopathy patients exhibited alterations in biochemical parameters, but did not reveal any mutation in exon 8 and 9 of MYH7 gene &exon 8 of TNNT2. P74 Cytogenetic and Yq microdeletion screening studies in infertile males J Suganya 1* , Kamala Selvaraj 2 , SS Muthaiah 3 , RS Chandra 1 1 Department of Genetics, Dr. ALM PG. Institute of Basic Medical Sciences, University of Madras, Taramani, Chennai, TamilNadu, India; 2 G.G. Hospital, 6-e, Thirumoorthy Nagar, Nungambakkam High Road, Nungambakkam, Chennai-, TamilNadu, India; 3 Kanmani Fertility Centre, 43, South Usman Road, T Nagar, Chennai, TamilNadu, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):P74 Infertility is defined as the inability of a sexually active couple to achieve pregnancy despite unprotected intercourse for a period of greater than 12 months. Infertility affects 15% of the infertile couples and in about half of them, male factor is responsible. It is due to low number, abnormal or immobile sperm production, or blockages that prevent the delivery of sperm. Illnesses, injuries, chronic health problems, lifestyle choices and other factors can also play a role. A total of 130 infertile males (43 azoospermic, 31 asthenospermic, 7 cryptozoospermic, 20 oligoasthenozoospermic, 29 normozoospermic) were analyzed for the presence of chromosomal abnormalities and Y chromosome classical microdeletions in the azoospermia factor (AZF) regions. Multiplex PCR amplification of genomic DNA was performed using two STS markers for each of the three non-overlapping regions, AZFa (sY84, sY86), AZFb (sY127, sY134) and AZFc (sY254, sY255) following standard guidelines. The markers ZFX/ZFY and sY14 (SRY) were included as internal controls. Two individuals showed Klinefelter syndrome (47,XXY) and three cases exhibited reciprocal translocations involving autosomes - t(3;17)(q26.2; p12), t(1;11)(p22.3;p13) and t(1;10)(q25;q24). Polymorphisms involving the heterochromatic regions of Y and the acrocentric chromosomes were seen in seven patients. The rest of the individuals exhibited a normal chromosomal pattern. The occurrence of chromosomal abnormalities was confined to only the azoospermia category which could reflect the small sample size. A deletion involving all the three AZF regions was detected in one individual while another showed a deletion in AZFb (partial) and AZFc regions. These results indicate a distinctly lower frequency of Y chromosomal microdeletions in the selected group of men with idiopathic infertility. The study needs to be extended using more primer sets defining these loci which play a major role in spermatogenesis. P75 Molecular studies on ARX gene in syndromic and non-syndromic mental retardation Gayatri Kulkarni * , Suvidya Ranade Department of Chemistry, University of Pune, Pune, Maharashtra, India Molecular Cytogenetics 2014, 7(Suppl 1):P75 Background: Mental retardation (MR) is frequently the result of genetic mutations. Syndromic mental retardation is intellectual deficits associated with other medical and behavioral signs and symptoms. Non-syndromic mental retardation refers to intellectual deficits that appear without other abnormalities. The newly identified ARX (Aristaless related homeobox) gene consists of five exons and encodes 562 amino acid proteins and is thought to regulate brain development. Mutations in the ARX gene are associated with a diverse spectrum of phenotypes ranging from severe developmental abnormalities of the brain to syndromic and nonsyndromic forms of X-linked mental retardation (XLMR) syndromes that can be associated with normal or abnormal brain morphology. Mutations in the human ARX gene are the major cause of developmental and neurological disorders. Material and methods: The present study was focused on screening of exon 2 of ARX gene in Indian families with mental retardation in order to obtain the relative prevalence of ARX mutations. Method adapted in present study was amplification of target exon by using polymerase chain reaction, qualitative conformation of amplicons by agarose gel electrophoresis and their use for conformation sensitive gel electrophoresis to find heteroduplex formation which is followed by sequencing. Conclusion: In present study we have found insertion mutation at genomic position 25031668, this change leads to loss of homeobox, OAR domains of ARX protein. The other mutation obtained was substitution of C to A/G is observed and does not affect protein functioning. P76 Squared nasal root, nasal voice -indicators of 22 q11.2 deletion in patients with psychiatric illness Jyothilakshmi Annavarapu * , Prabhavathi Halappa, Niby J Elackatt, Mitesh Shetty, Sridevi Hegde Dept. of Medical Genetics, Manipal Hospital, Bangalore, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):P76 Background: 22q11.2 Deletion Syndrome DGS (22q11) is a micro deletion syndrome caused by the deletion on chromosome 22. It is a multi system disorder which affects Cardiovascular system, immune system, facial Molecular Cytogenetics 2014, Volume 7 Suppl 1 http://www.molecularcytogenetics.org/supplement/7/S1 Page 42 of 59 features covered by acronym CATCH22 (Cardiac defects aortic arch anomalies, conotruncal anomalies, ventricular septal defect, patent ductus arteriosis and tetra logy of fallot; Abnormal facies ; Thymic hypoplasia; Hypocalcemia). Few children will not present with all of the above clinical features but only delayed motor mile stones, learning disability and mild behavioral issues which may progress onto psychiatric illness in adulthood. In this study, we aim to study the prevalence of DGS in patients with psychiatric illness. Materials and methods: To further explore physical, behavioral and psychiatric findings associated with 22q11 deletion in adults with psychiatric illness, we assessed 12 patients. All were confirmed psychiatric cases referred from a well-known Institute for mental health studies and also had mild facial dysmorphism. All patients were screened using the clinical checklist for DGS and Fluorescence In Situ Hybridization(FISH) studies was conducted using the probe specific for TUPLE1 gene located on chromosome 22 q11.2 in cultured blood samples. Results: Out of 12 cases, six cases (50 %) tested positive for 22q11 deletion indicating a strong association between 22q11.2 deletion syndrome and psychiatric illness in adult population. All six patients presented with squared nasal root, and nasal voice in addition to psychosis and one also had cardiac abnormality (VSD). Recent studies report that Digeorge critical region (DGCR) spanning up to 2 Mb on chromosome 22q11 region contains several genes like TBX1, GNB1L, PRODH and ZDHHC8 which are strong candidate genes for schizophrenia susceptibility. In conclusion, all patients with psychiatric illness, squared nasal root and nasal voice should be investigated for DGS. NANOTECHNOLOGY P77 TiO2 nanoparticles induce cytotoxicity and genotoxicity in human alveolar cells Krupa Kansara * , Pal Patel, Darshini Shah, NV Srikanth Vallabani, Ritesh K Shukla, Sanjay Singh, Ashutosh Kumar, Alok Dhawan Institute of Life Sciences, School of Science and Technology, Ahmedabad University, Ahmedabad –Gujarat, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):P77 Background: Engineered nanoparticles (ENPs) such as TiO 2 are widely used in products such as cosmetics, clothing, food packaging, drug delivery systems, etc. due to their unique physicochemical properties. This has increased the liklihood of ENP exposure in humans. As the ENPs are having small size and high diffusion coefficient, they can migrate rapidly in the air. Therefore, inhalation is considered to be the primary route of exposure to such ENPs. Hence, in the present study an attempt was made to assess the potential toxicological effects of TiO 2 NPs in human alveolar cell line (A549). Materials and methods: The average hydrodynamic size, size distribution, zeta potential and stability of TiO 2 NPs in DMEM-F12 media were determined by dynamic light scattering (DLS). Internalisaiton of ENPs in cells was detected using flow cytometry. Cytotoxicity was assessed using the MTT and neutral red uptake (NRU) assays. The genotoxic potential of TiO 2 NPs was assessed by cytokinesis block micronucleus (CBMN) assay and flow cytometry based assays. Results: The mean hydrodynamic diameter of TiO 2 NPs in DMEM-F12 media, as measured by DLS was 23.27 ± 2.1nm and the zeta potential was -10.1 ± 1 mV. The particles were also found to be stable in the media for upto 72 hr. A significant (p<0.05) concentration dependent uptake of TiO 2 NPs was obseverd as evident by an increase in the side scatter (SSC) intensity in flow cytomtery after 6 hr of exposure. A reduction in cell viability was observed as evident by the results of MTT and NRU both as a function of NP concentration as well as time of exposure. Moreover, significant (p<0.05) induction in the micronucleus formation was observed by conventional and flow cytometry based methods at non cytotoxic concentrations. Conclusion: Our data demonstrate that TiO2 ENPs are internalised in the human alveolar cells and induce cyto- and geno- toxicity. This warrant minimizing the unwanted exposure to the nanotechnology based products and suggests ensuring its safe use both by consumers and industry. Acknowledgements: The financial assistance for the Centre for Nanotechnology Research and Applications (CENTRA) by The Gujarat Institute for Chemical Technology (Grant no. ILS/GICT/2013/003) is acknowledged. P78 PEGylated nanoceria protect human epidermal cells from reactive oxygen species Ragini Singh * , Ritesh K. Shukla, Ashutosh Kumar, Alok Dhawan, Sanjay Singh Institute of Life Sciences, Ahmedabad University, Ahmedabad, Gujarat, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):P78 Background: Cerium oxide nanoparticles (CeNPs) have shown promise as catalytic antioxidants in cell culture and animal models due to its superoxide dismutase and catalase mimetic activities. CeNPs can exist in +3 and +4 oxidation states, which have been suggested as the mechanism behind the free radical scavenging activity. It has also been shown that unique crystal structure of CeNPs with surface oxygen defects promote the shuffling between the +3 and +4 oxidation states that help in eliminating the free radicals. This activity of CeNPs has been suggested as the mechanism, at least in part, behind increase in cellular longevity and decrease in the toxic insults in mammalian cells/tissues. Further, the uniform distribution of CeNPs, upon cellular uptake, within the cells could prevent the accumulation of reactive oxygen species in the cells. Material and method: PEG coated CeNPs were synthesized by methods described by Karakoti et al. CeNPs were characterized by UV-visible spectra and dynamic light scattering. Cytotoxicity of CeNPs was done by MTT and Neutral Red Uptake (NRU) assay in a time and dose dependent manner. The level of intracellular ROS generation was estimated by using 2,7- dichlorofluorescein diacetate (DCFDA) dye by time and dose dependent approach. Superoxide dismutase (SOD) mimetic activity of ceria nanoparticles was determined by a ferricytochrome C based assay. Results: The mean hydrodynamic diameter of PEG CeNPs in MillQ and culture medium were 250.6 nm and 153.3 nm respectively whereas Zeta potential were found to be -7.86 mV and -10.4 mV respectively. CeNPs did not exhibit cytotoixicity at the tested doses and time period in human epidermal cells (A431) as evident by MTT- and NR uptake- assays. The co- incubation of CeNPs with A431 cells showed significant (p<0.05) decrease in ROS generation as evident by a decrease in the DCFDA fluorescence. Conclusion: The present study demonstrates that CeNPs are stable in cell culture media conditions, non cytotoxic to A431 cells up to a relatively high concentration. CeNPs show free radical scavenging activity when co-incubated with A431 cells. Acknowledgements: The financial assistance for the Centre for Nanotechnology Research and Applications (CENTRA) by The Gujarat Institute for Chemical Technology (Grant no. ILS/GICT/2013/003) is acknowledged. P79 TiO2 nanoparticles induced micronucleus formation in human liver (HepG2) cells: comparison of conventional and flow cytometry based methods NV Srikanth Vallabani * , Ritesh K Shukla, Dinesh Konka, Ashutosh Kumar, Sanjay Singh, Alok Dhawan Institute of Life Sciences, School of Science and Technology, Ahmedabad University, Ahmedabad –Gujarat, India Molecular Cytogenetics 2014, 7(Suppl 1):P79 Background: TiO 2 nanoparticles (TiO 2 NPs) are extensively used metal oxide NPs in cosmetics, as an additive in pharmaceuticals, food colorant etc. Being widely used NPs, their toxicity assessment studies help in understanding the adverse effects to the humans. It is likely that NPs exposure to the humans can be through different routes but will finally reach the liver. Therefore, an attempt was made to explore the genotoxicity of TiO 2 NPs on human liver cells (HepG2). Materials and methods: TiO 2 NPs were characterized by transmission electron microscopy (TEM) for their primary size, shape and dynamic light scattering for their size, size distribution and zeta potential in culture medium. Cellular uptake of TiO 2 NPs in HepG2 cells was detected using Molecular Cytogenetics 2014, Volume 7 Suppl 1 http://www.molecularcytogenetics.org/supplement/7/S1 Page 43 of 59 flow cytometry method. Moreover, ultrathin sections of cells were analysed using TEM to visualise the internalisation of TiO 2 NPs. The genotoxic potential of TiO 2 NPs was assessed by micronucleus assay using the conventional and flow cytometry methods. Results: TEM analysis of TiO 2 NPs revealed a mean diameter size of 30-70 nm and DLS measurements showed a mean hydrodynamic diameter and zeta potential of 192.5 ± 10 nm and -11.4 ± 1.2 mV, respectively. The electron microscopy and flow cytometry studies for particle internalisation showed a significant cellular uptake of TiO 2 NPs in the human liver cells (HepG2). A significant (p < 0.05) induction in micronucleus formation was observed at 20 µg/ml of TiO 2 NPs exposure when compared to control cells. However further treatment to HepG2 with higher concentrations (40 and 80 µg/ml) showed a decrease in micronucleus formation than 20 µg/ml. In contrary, the flow cytometric results exhibited a significant concentration dependent induction of micronucleus in HepG2 cells exposed to all concentrations (20, 40 and 80 µg/ml) of TiO 2 NPs than control. Conclusion: The difference in the micronucleus frequency obtained from conventional and flow cytometry methods in HepG2 may be due to the accumulation of TiO 2 NPs on prepared slides, which hinders the counting of micronucleus (in conventional method). However, in the flow cytometry analysis, these nanoparticles do not interfere with the optics. Hence, it is proposed that in the case of NPs treatment, flow cytometry based micronucleus assay should be used instead of the conventional method. Acknowledgements: The financial assistance for the Centre for Nanotechnology Research and Applications (CENTRA) by The Gujarat Institute for Chemical Technology (Grant no. ILS/GICT/2013/003) is acknowledged. P80 Host-guest mediated sensing of biologically relevant small molecules using supramolecular nanoassembly Alok Pandya 1* , Pinkesh G Sutaria 2 , Anand Lodha 2 , Heena Goswami 2 , Shobhana K Menon 2 1 Institute of life Sciences, School of Science and Technology, Ahmedabad University, Ahmedabad, -380 009, Gujarat, India; 2 Department of Chemistry, School of Sciences, Gujarat University, Ahmedabad, 380 009, Gujarat, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):P80 Background: A high concern for human health and safety has motivated dynamic research on the potential impact of transition metal ions or molecule and their toxic effects. Thus, selective detection of biologically relevant molecule have enormously gained its attention due to involvement in a variety of fundamental environmental and biological process in organism because its deficiency and excess can induce a variety of diseases. Therefore, biomolecule detection have received a great deal of study. Here, we have designed an efficient strategy using supramolecular nanoassembly to detect biologically relevant small molecules with high specificity and selectivity and applicable to the biological milieus. Method: In our method, we designed a microwave assisted new promising approach using Silver (Ag) and Gold (Au) nanoparticles (NPs) based colorimetric sensing system (ANCSS) which form calix[4]arene- functionalized Ag and Au nanoprobe complex (CX-AgNPs/AuNPs) for the detection of biologically relevant small molecules such as ferric ion and glucose in water. Result: Driven by the need to detect trace amounts of Fe 3+ and Glucose from blood samples, a molecular receptor based on calix[4]arene functionalized AuNPs/AgNPs was designed which proficiently and selectively recognizes glucose and Fe 3+ in nanomolar level from aqueous solution with excellent discrimination against other heavy metals and biomolecule. The assembly was characterized by, DLS, UV-Vis, FT-IR, ESI-MS and 1 H NMR spectrometry which demonstrates the higher binding affinity for Fe 3+ and Glucose via weak forces. It is easy to operate and, most importantly, it exhibits fast response time (<80 s) and has long shelf-life (>4 weeks). Conclusion: The calix[4]arene functionalized Ag or AuNPs are able to selectively detect Fe +3 and glucose from aqueous medium and even from human blood. The key to the successful formation of this strategy is multi binding site of functionalized calix[4]arene which sensitively and selectively target to small molecules. The developed functionalized calixarene-Ag or AuNPs based human heme and gluocse biosensor has been proved to be a simple, reliable and accurate which can also indirectly assist to medeco-legal system for the routine investigation process. P81 Cytotoxicity assessment of ZnO nanoparticles on human epidermal cells Pal Patel * , Krupa Kansara, Darshini Shah, NV Srikanth Vallabani, Ritesh K Shukla, Sanjay Singh, Alok Dhawan, Ashutosh Kumar Institute of Life Sciences, School of Science and Technology, Ahmedabad University, Ahmedabad, Gujarat, India Molecular Cytogenetics 2014, 7(Suppl 1):P81 Background: Nanotechnology is growing rapidly worldwide and engineered nanoparticles have found tremendous applications in consumer and industrial products. Metal oxide nanoparticles (NPs), especially zinc oxide (ZnO), are widely used in cosmetics, catalysis, electronics, biosensors, medicine, paints, food packaging and imaging. As, the ZnO NPs have major application in cosmetics, their exposure will mainly be through the skin, which is the largest organ of the body and could serve as an important portal route for entry. Therefore, the present study was carried out to assess the cytotoxicity of ZnO NPs on human epidermal cells (A431). Materials and methods: The average hydrodynamic size, size distribution, zeta potential and stability of ZnO NPs were determined by dynamic light scattering (DLS). The detection for the internalization of ZnO NPs was carried out using flow cytometry. Cytotoxicity response was assessed by neutral red uptake (NRU) and MTT [3-(4, 5-dimethylthiazoyl-2-yl)-2, 5-diphenyltetrazolium bromide] assays. Further, to analyze the ability of ZnO NPs to induce the reactive oxygen species (ROS) generation and oxidative damage, 2, 7- dichlorofluorescein diacetate (DCFDA) dye was used. The effect of ZnO NPs in progression of cell cycle was also assessed using propidium iodide dye by flow cytometer. Results: The mean hydrodynamic diameter and zeta potential of ZnO NPs in DMEM with 10% FBS was 30.95 ± 3.72 nm and -12.8 ± 0.6 mV respectively. In flow cytometry, a concentration dependent significant (p<0.05) increase in the side scatter (SSC) intensity of treated cells was observed after 6 hr exposure. A significant (p<0.05) dose and time dependent cytotoxicity as evident from MTT and NRU assays was also observed. Additionally, a significant (p<0.05) induction in ROS generation was also observed in a dose dependent manner. Conclusion: Our study demonstrates that ZnO NPs induce cytotoxicity in human epidermal cells. Hence, proper precautions need to be taken while handling such NPs. Acknowledgements: The financial assistance for the Centre for Nanotechnology Research and Applications (CENTRA) by The Gujarat Institute for Chemical Technology (Grant no. ILS/GICT/2013/003) is acknowledged. P82 TiO2 NPs induced hepatic injury in mammals: a mechanistic approach Ritesh K Shukla * , Ashutosh Kumar, NV Srikanth Vallabani, Sanjay Singh, Alok Dhawan Institute of Life Sciences, Ahmedabad University, Opposite University Bus Stand, University Road, Navrangpura, Ahmedabad, Gujarat, India Molecular Cytogenetics 2014, 7(Suppl 1):P82 Background: The rapid advancement in nanotechnology has increased the production of metal oxide nanoparticles (NPs) especially TiO 2 for consumer and industrial products. This has also increased the likelihood for their exposure to human. TiO 2 NPs exposure to humans can occur through different routes, but will finally reach to liver through the circulatory system. Hence, the present study was planned to assess the effects of TiO 2 NPs in mammalian liver and their possible mechanism. Materials and methods: TiO 2 NPs were characterized by transmission electron microscopy (TEM) and dynamic light scattering (DLS). Genotoxicity assessment of TiO 2 NPs was carried out by fpg-modified Comet assay both in in vitro (HepG2 cells) and in vivo (mice liver). Additionally, to understand Molecular Cytogenetics 2014, Volume 7 Suppl 1 http://www.molecularcytogenetics.org/supplement/7/S1 Page 44 of 59 the mechanism of hepatotoxicity, biochemical parameters, oxidative stress markers, reactive oxygen species (ROS), and expression profile of different stress proteins, tumour suppressor apoptotic/antiapoptotic proteins were investigated. Results: TEM measurements and DLS analysis showed that TiO 2 NPs were in nano size regime, stable and mono-dispersed in different exposure vehicles, making them suitable for in vitro and in vivo toxicity studies. Our data from in vitro and in vivo study exhibited that TiO 2 NPs induced significant (p<0.05) oxidative DNA damage assessed by the fpg-Comet assay. This could be attributed to a concentration-dependent significant (p<0.05) increase of ROS generation as evident from the enhanced fluorescence intensity of DCFDA dye. A significant alteration in the level of different hepatic enzymes in TiO 2 NPs treated mice was also observed. Furthermore, immunoblot analysis revealed a significant increase in the expression profile of Hsp60, Hsp70, p53, BAX, Cyto-c, Apaf-1, caspase-9 and caspase-3 protein and a concomitant decrease in the level of antiapoptotic protein Bcl-2. Our data demonstrate the role of mitochondrial intrinsic pathway for TiO 2 NP induced apoptosis in liver cells. Conclusion: The present study using fpg-modified Comet assay, blood biochemical parameters, oxidative stress markers and immunoblot analysis confirmed that oxidative stress induced by TiO 2 NPs trigger the DNA damage, which consequently initiates the expression of apoptotic proteins resulting in hepatic injury. Hence the use of such nanoparticles should be carefully monitored. Acknowledgements: The financial assistance for the Centre for Nanotechnology Research and Applications (CENTRA) by The Gujarat Institute for Chemical Technology (Grant no. ILS/GICT/2013/003) is acknowledged. P83 BSA coated gold nanoparticles exhibit size dependent interaction with lung cancer (A549) cells Rahul Purohit * , NV Srikanth Vallabani, Ritesh K Shukla, Ashutosh Kumar, Alok Dhawan, Sanjay Singh Institute of Life Sciences, School of Science and Technology, Ahmedabad University, Ahmedabad – 380009, Gujarat, India Molecular Cytogenetics 2014, 7(Suppl 1):P83 Background: Due to high surface to volume ratio and other unique properties, nanomaterials interact within living system (cells, tissues, organisms) significantly different. Therefore, developing a rational basis for investigation and understanding about how these nanomaterials interact with living system is still a fundamental challenge. Further, it has been well documented that nanomaterials get coated by the proteins present in their surrounding thus constructing a “protein corona”. This event also plays a major role in determining the interaction of nanomaterials with cells/tissues. Therefore, to probe this we have chosen BSA coated gold nanoparticles (AuNPs) as a model system to study the interaction with a mammalian lung cell culture model system. Materials and methods: AuNPs of different sizes (15, 30, 50 and 70 nm) were synthesized using sodium citrate as a reducing agent. Size distribution, zeta potential and stability of coated (AuNPSBSA) as well as uncoated AuNPs were determined by dynamic light scattering (DLS) and UV-vis spectroscopy. Cytotoxicity response was assessed by neutral red uptake (NRU) and MTT [3-(4, 5-dimethylthiazoyl-2-yl)-2, 5-diphenyltetrazolium bromide] assays. Results: Zeta potential values for AuNPs of different sizes were found negative, which was decreased significantly after BSA coating. Further, the stability of AuNPSBSA in saline solution (1M and 500 mM NaCl) and cell culture media (DMEM-F12), which showed that BSA coated AuNPs were stable in both saline solution and serum containing culture media, as compared to uncoated AuNPs. Cytotoxicity studies revealed that BSA coated and bare AuNPs were non-toxic up to 50 µM, however, at higher concentration (above 50 µM) bare AuNPs were found to be more toxic as compared to AuNPSBSA. Further, the cytotoxicity of AuNPs on A549 cells was also found to be size dependent. Conclusion: This study demonstrates the size and BSA coating on AuNPs significantly control the cytotoxicity on lung cancer cells. BSA coating imparts the biocompatibility thus non toxic nature of these particles even at higher concentration. However, the relation between cytotoxicity and internalization of AuNPs (bare and BSA coated) in A549 cells remains to be seen. Acknowledgements: The financial assistance for the Centre for Nanotechnology Research and Applications (CENTRA) by The Gujarat Institute for Chemical Technology (Grant no. ILS/GICT/2013/003) is acknowledged. P84 A data mining approach for identifying novel target specific small molecules Varun Khanna * , Alok Dhawan Institute of Life Sciences, Ahmedabad University, Opp. University Bus Stand, Navrangpura, Ahmedabad - 380009, Gujarat, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):P84 Background: There has been a paradigm shift in drug discovery from being a single-target approach to a multi-target comparative analysis. This has shifted the emphasis from designing lead candidates with desirable pharmacokinetic properties against individual targets to synthesizing small molecules active at family and subfamily levels. Therefore, in the present study, protein targets and small molecule ligands available in PubChem BioAssay and DrugBank databases were comparatively analyzed to identify novel target specific small molecules. Materials and methods: The data obtained from public databases was mined using shell scripts and R-statistical computing software (2.15.3) installed under Linux environment. Results: Our data from small molecule analysis showed that 210 FDA approved drugs bind to a single protein target whereas 157 bind to two targets, 82 bind to three targets and rest bind to four or more targets. This shows that out of 1541 FDA approved drugs only 14% are specific binders whereas majority of the drugs are promiscuous binders. Similarly in PubChem BioAssay dataset 20% of the compounds bind to a single target whereas 11% compounds bind to two targets while the rest of the compounds bind to three or more targets. Further, our results from the comparison of target datasets showed that there are 2097 and 1271 unique domains in DrugBank and PubChem BioAssay target datasets respectively. There are 1190 domains common to protein targets in DrugBank and PubChem BioAssay datasets. Further, we note that of the top 10 domains 8 domains are shared between both the datasets. Conclusions: The above observations have implications in drug design approaches where the goal is to find target specific small molecules. Our analysis shows that promiscuity plays a major role in drug discovery therefore, should not be overlooked while designing novel drugs. From domain analysis of target proteins we conclude that protein target space is fairly narrow and the majority of the drug design efforts are concentrated only on few target classes containing limited domains. Acknowledgements: The financial assistance for the Centre for Nanotechnology Research and Applications (CENTRA) by The Gujarat Institute for Chemical Technology (Grant no. ILS/GICT/2013/003) is acknowledged. NEXT GENERATION SEQUENCING P85 Next generation sequence analysis of the transcriptional response to neonatal hyperoxia Soumyaroop Bhattacharya * , Zhongyang Zhou, Min Yee, Ashley Lopez, Valarie Lunger, Bradley Buczynski, Gloria Pryhuber, Thomas Mariani, Michael OReilly Division of Neonatology, Department of Pediatrics, University of Rochester Medical Center, Rochester NY, USA E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):P85 Background: Bronchopulmonary Dysplasia (BPD) is a major complication of preterm birth associated with significant morbidity. BPD is a debilitating condition characterized by inflammation, enlarged airspaces, vascular dysmorphia and aberrant extracellular matrix accumulation that is typically described as arrested lung development. Rodent models involving Molecular Cytogenetics 2014, Volume 7 Suppl 1 http://www.molecularcytogenetics.org/supplement/7/S1 Page 45 of 59 neonatal exposure to excessive oxygen concentrations (hyperoxia) have been used to study the mechanisms contributing to BPD pathology. Transcriptomic assessment of the effects of hyperoxia in neonatal mouse lungs using RNASeq will help to identify genes and pathways associated with BPD. Materials and methods: Whole lung tissue from newborn C57BL/6 mice exposed to 100% oxygen for 10 days (n=8) and room air-exposed age matched controls (n=6) were compared. Total RNA was isolated from individual whole lung tissues (n=14) and pooled in duplicates to perform transcriptome Sequencing (RNA-seq). Alignments were generated using multiple algorithms (CASAVA; TopHat; and SHRiMP). Raw counts obtained from each alignment algorithm (using HT-Seq) were further and filtered to remove undetected genes. Differentially expressed genes were detected using Significance Analysis of Microarrays (SAM) and CuffDiff2, on each version of mapped and normalized data. Ingenuity Pathway Analysis (IPA) was used for pathway and network analyses. Expression patterns for selected genes were examined by quantitative polymerase chain reaction (qPCR). Results: 248 genes were identified as differentially expressed between hyperoxia and control samples by both SAM (median FDR = 0) and CuffDiff2 (p<0.05) and had a fold-change ≤ 2. We successfully validated 17 of 24 genes by qPCR. Canonical pathways significantly dysregulated in hyperoxia lungs included Nrf2-mediated oxidative stress signaling, p53 signaling, hepatic fibrosis and sildenafil pathways. Interestingly most genes significantly affected following hyperoxia exposure (~70%) showed a pattern of expression consistent with an arrest in lung development. A subset of the genes dysregulated in hyperoxic neonatal mouse lungs, were also differentially expressed in human BPD lung tissue. Conclusions: We have identified genes dysregulated in mouse of BPD-like pathology. Further analysis of these data will enhance our current knowledge of BPD, and may be useful for developing novel therapeutic strategies. P86 Comparative analysis of human mitochondrial methylome show distinct patterns of epigenetic regulation in mitochondria Sourav Ghosh 1* , Shantanu Sengupta 1 , Vinod Scaria 2 1 Genomics and Molecular Medicine, CSIR Institute of Genomics and Integrative Biology, Mall Road, Delhi, India; 2 GN Ramachandran Knowledge Center for Genome Informatics, CSIR Institute of Genomics and Integrative Biology, Mall Road, Delhi, India Molecular Cytogenetics 2014, 7(Suppl 1):P86 Background: Understanding the epigenetic regulation of genes and their functional implications in physiological and pathophysiological states has been one of the emerging areas of genomics. DNA methylation and histone modifications across the nuclear genome have been recently extensively analyzed in this perspective. However, the mitochondrial epigenome, though has been discussed widely has not been analyzed at genome-scale resolutions. The recent availability of genome-scale epigenome datasets allowed us to analyse profiles of methyl cytosine across mitochondrial genomes. Method: We analyzed methyl-cytosine profiles as evident from Methylated DNA immunoprecipitation across 39 tissues and cell lines that were available as part of the NIH RoadMap Epigenomics project. The mitochondrial reference genome used in the current study was derived from the UCSC human genome build hg19. We used custom scripts to retrieve reads mapping to the mitochondrial genome. Results: Our analysis suggests that the general profile of methylated cytosines across the samples show a distinct pattern. This pattern was generally conserved across the datasets considered, with exceptions of a few regions which showed variability in methylation amongst datasets analyzed. We show that certain regions of the mitochondria could be differentially methylated in datasets which show distinct temporal and functional characteristics, like Brain and Blood. One such region harbors the loci associated with mitochondrial encoded NADH dehydrogenase (MT-ND6), variations in which are associated with neurological disorders. To date this is the first and comprehensive analyses of genome-scale methylation data for Human mitochondria. Conclusion: This study describes the first comprehensive map of methyl- cytosines across the Human mitochondrial genome. P87 Defining the effects of prematurity on the lymphocyte transcriptome Soumyaroop Bhattacharya 1* , Ravi Misra 1 , Heidi Hyuck 1 , Christopher Slaunwhite 1 , Shannon Castiglione 2 , Deanna Maffett 1 , Anne Marie Reynolds 2 , Gloria Pryhuber 1 , Thomas Mariani 1 1 Division of Neonatology, Department of Pediatrics, University of Rochester Medical Center, Rochester NY, USA; 2 Division of Neonatology, Department of Pediatrics, Women’s and Childrens Hospital, University at Buffalo, Buffalo NY, USA E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):P87 Background: We hypothesize that intrinsic and extrinsic factors associated with oxidative stress drive lymphocyte dysfunction contributing to lung disease in premature infants. Comprehensive transcriptomic assessment of CD8 + lymphocytes may identify biomarkers, and define disease-related mechanisms of chronic lung disease in premature infants. Methods: Peripheral blood samples were collected from premature infants at the time of hospital discharge at multiple sites and shipped to a central laboratory. Freshly purified PBMCs were isolated by Ficoll gradient centrifugation, sorted into individual lymphocyte cell types, and processed for total RNA. RNA isolated from CD8 + lymphocytes (n=79) was used for RNA-Seq analysis using the Illumina HiSeq2500. Sequences were aligned using the SHRiMP algorithm and expression values were summarized using HTSeq. Normalized gene expression data were analyzed for significant changes in expression using various statistical approaches. Ingenuity Pathway Analysis software was used for gene set interpretation. Results: Sufficient blood was collected to complete sorting from 137 of the 183 subjects recruited and discharged. The total lymphocyte frequency from the PBMC fraction was highly variable, with an average of 29.6±9.3%. Lymphocyte sub-type frequencies were also highly variable across subjects, with CD8 + cell averaging 5±2%. Total RNA yield from sorted cells varied according to cell type with CD8 + RNA averaging 177±140ng. RNA-Seq analysis using 1ng total RNA was completed for CD8 + cells from 79 subjects, generating an average of 11±5 million reads/sample with detection of 67 ±4% of the transcriptome. One-way ANOVA identified 147 genes that were differentially expressed among the 79 samples. A total of 47 genes were identified as differentially expressed between subjects born before 29-weeks and those born after 29-weeks of gestation. Pathways analysis of these genes identified mechanisms related to B and T cell signaling. Conclusion: These data demonstrate both the fidelity of our methodology and purity of the samples. Although we assessed a homogeneous cell type, our data includes substantial gene expression variability across subjects, and with respect to gestational age at birth. Our results support the feasibility of using these data and methods to identify biomarkers of, and mechanisms for, chronic respiratory morbidity following premature birth. OTHERS P88 Oxidant–antioxidant imbalance in the serum of Myotonic Dystrophy type 1 (DM1) patients correlates with the progression of disease Ashok Kumar 1* , Sarita Agarwal 1 , Sunil Pradhan 2 , Shubha Phadke 1 1 Department of Medical Genetics, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow, 226014, India; 2 Department of Neurology, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow, 226014, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):P88 Background: Myotonic dystrophy type 1 (DM1) is the most common form of muscular dystrophy affecting adults and is due to trinucleotide sequence (CTG) in the 3′ UTR region of DMPK gene located at 19q13.3 chromosome. The increased levels of ROS/free radicals and lipid peroxides and decreased antioxidant levels play an important role in the pathogenesis of DM1. Aim: The intention of the present study is to assess the lipid peroxidation and antioxidant status and its association with clinical phenotypes in DM1 patients of Indian origin. Material and methods: Clinically diagnosed 20 DM1 patients (16 men and 4 women; median age 32.8 years±9.3, range 17–52) and 40 age and sex matched controls (32 men and 8 women; median age 31.0 years±8.6, range 16–54) were included in the study. The collected blood samples were processed for serum separation used in measurement of MDA Molecular Cytogenetics 2014, Volume 7 Suppl 1 http://www.molecularcytogenetics.org/supplement/7/S1 Page 46 of 59 (Cat. No. 10009055-96), SOD (Cat. No. 706002-96), GPX (Cat. No. 703102- 96), GST (Cat. No. 703302-96), GSH (Tietze et al. 1969 method) and total antioxidant status or TAS (Koracevic et al. 2001 method) levels and its association with clinical phenotype were evaluated. Results: Analysis revealed significantly higher levels of MDA (p=0.002), SOD (p=0.006) and TAS p= 0.004) and lower level of GPX (p=0.003), GST (P<0.001) and GSH (P=0.016) in DM1 patients. A significant negative correlation of MDA level with dyspepsia and CK-MB and GST level with serum SCK, CK-MB and diabetes were observed. However, a significant positive correlation of SOD level with serum CK-MB, CK-MM and diabetes and negative correlation with facial weakness were noted. Though, GSH level had significant positive correlation with learning and writing disability, speech and languages disability yet found negative correlation with duration of the disease. The GPX and TAS showed no correlation with any clinical findings. Conclusion: Our data supports the pathogenic role of oxidative stress in DM1 of Indian origin and supports the opportunity to undertake clinical trials with antioxidants in this disorder. P89 Mental retardation in younger children Grishma Shukla 1* , Swati B Thakur 1 , GT Swamy 2 , MV Rao 1 1 Gujarat Genetic Diagnostic Center (GenDiCe), Department of Zoology, University School of Sciences, Gujarat University, Ahmedabad, Gujarat, India; 2 B M Institute of mental health, Ahmedabad-09, Gujarat, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):P89 Background: It is a generalized disorder appearing before adulthood, characterized by significantly impaired cognitive functioning and deficits in two or more adaptive behaviours. The present study was aimed to investigate the delayed development and psychological aspects in symdromic and non syndromic mental retarded individuals. Material and methods: This study was conducted at B.M. Institute, Ahmedabad. The aetiology was ascertained after proforma studying and number of patients observed. The spectrum of causative conditions and contribution of genetic disorders was established. A total of 40 patients were observed in which 30 were male & 10 were female between the ages of 3 -11 years. The various aetiological categories were: chromosomal disorders in 17, non chromosomal syndromes in 23. In which chromosomal disorders include Down syndrome, non-chromosomal disorders include delayed development and cerebral palsy. Results: Among non syndromic mental retardation (NSMR) children; the walking is delayed by 1 yr in 22% of children, 2 yrs in 57 % of children, 3 yrs in 4% and 13 % children were not able to walk. In syndromic mental retardation (SMR) walking is delayed by 1 yr in 41 % of children, 2 yrs in 23 % of children, 3 yrs in 24 %, 6 % were not able to walk. Other developmental milestone such as sitting and speech in both syndromic and non syndromic mental retardation also showed the same pattern of delayed development as seen in the walking. In IQ, level the highest is SMR which is 44% severe & 45% moderate while in NSMR 31% severe & 38% moderate level were observed. Conclusions: The present investigation concluded that there was a delayed milestone in mental retarded children which was supported by IQ levels in this study. P91 Role of miRNA binding site SNPs in candidate genes in a North Indian schizophrenia cohort Jibin John 1* , Smita N Deshpande 2 , Vishwajit L Nimgaonkar 3 , BK Thelma 1 1 Department of Genetics, University of Delhi South campus, Benito Juarez Road, New Delhi, India; 2 Department of Psychiatry, Dr. RML Hospital, New Delhi, India; 3 Department of Psychiatry, Western Psychiatric Institute and Clinic, University of Pittsburgh School of Medicine, PA 15213, USA Molecular Cytogenetics 2014, 7(Suppl 1):P91 Schizophrenia (SZ) is a debilitating neuropsychiatric disorder with ~80% heritability. Despite several genetic studies including linkage and candidate gene association and more recently GWAS, which have identified several risk variants, the total heritability of SZ remains elusive. In addition, a number of gene expression studies have reported dysregulation of candidate genes both in brain and blood of SZ cases compared to controls. Although, the role of coding, promoter, intergenic and UTR SNPs, have been demonstrated, very little is known about the role of miRNA binding site SNPs. In this study, we undertook to investigate the association, if any, of this important class of regulatory variants with SZ. Using in silico prediction tools, 27 functionally relevant SNPs from around 150 candidate genes were prioritized and genotyped in a north Indian SZ cohort (n=507 cases; n=522 controls). Test of association of these SNPs showed only one variant rs7430 in PPP3CC to be associated (p=0.01) with SZ. Analysis of genotype data in a subset of patients (TD positive n=89; TD negative n=160) with Tardive dyskinesia (TD), an iatrogenic disorder of SZ, showed association of rs4846049 in MTHFR (p=0.04) & rs17881908 in GCLM (p= 0.05 ) with this condition. Further regression analysis of the genotype data with neurocognitive measures in a subset (cases n=152; controls n=290) of the study cohort, showed significant association of nine SNPs (p< 0.05) with different domains of cognition. Based on this moderately powered study, the contribution of miRNA binding site SNPs in candidate genes to SZ and to TD seems negligible. However, their promising contribution to cognitive parameters warrants additional investigations. P92 Understanding insulin resistance pathophysiology in PCOS: a genetic approach Srabani Mukherjee 1* , Nuzhat Shaikh 1 , Roshan Dadachanji 1 , Nalini Shah 2 , Anushree Patil 1 1 National Institute for Research in Reproductive Health, J.M. Street, Parel, Mumbai- 400012, India; 2 Department of Endocrinology, Seth GS Medical College, Mumbai- 400012, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):P92 Polycystic ovary syndrome (PCOS) is a common endocrine disorder which affects women in their childbearing years, characterized by chronic anovulation, hyperandrogenemia, hyperinsulinemia and obesity. It is frequently associated with an increased risk for insulin resistance with long term health implications like type 2 diabetes mellitus and atherosclerosis disease. However, the aetiopathogenesis of this syndrome still remains elusive. The familial clustering of women with PCOS suggests that heredity is implicated in the origin of this syndrome. PCOS is now considered a polygenic trait that might result from the interaction of various susceptible and protective genomic variants along with the influence of environmental factors. A candidate gene based approach for investigating association of genes involved in insulin resistance such as INSR, PPARg, and PON1 with PCOS and its related phenotypes in Indian women has been undertaken. Clinical and biochemical parameters to evaluate PCOS associated traits related to metabolic and hyperandrogenemia were also assessed in all the participants. A polymorphism in insulin receptor gene (INSR), C/T His1058 showed association with PCOS only in lean women and also with hyperandrogenemia and hyperinsulinemia traits. Peroxisome proliferator activated receptor gamma (PPARg) is a transcription factor that regulates glucose, lipid homeostasis and intracellular insulin signaling. The most common variant Pro12Ala of PPARg which has a role in preserving insulin sensitivity showed significant association with decreased PCOS susceptibility while the other His447His polymorphism along with former was significantly associated with lower insulin related traits in women with PCOS. Paraoxonase 1 (PON1) gene encodes an antioxidant enzyme which protects against LDL oxidation and thus prevents atherosclerosis. Serum paraoxonase levels were significantly reduced in our PCOS group. A coding region L55M SNP showed association with PCOS. The study establishes that in Indian women, variations in genes related to insulin resistance do have an influence on the pathophysiology of PCOS. P93 Do variations in Insulin-like Factor 3 (INSL3) gene affect PCOS susceptibility? Nuzhat Shaikh 1* , Nalini Shah 2 , Srabani Mukherjee 1 1 National Institute for Research in Reproductive Health, J.M. Street, Parel, Mumbai- 400012, India; 2 Department of Endocrinology, Seth GS Medical College, Mumbai- 400012, India Molecular Cytogenetics 2014, 7(Suppl 1):P93 Polycystic ovary syndrome (PCOS) is a multigenic complex disorder causing metabolic and gynecologic dysfunctions in women of reproductive age. Its Molecular Cytogenetics 2014, Volume 7 Suppl 1 http://www.molecularcytogenetics.org/supplement/7/S1 Page 47 of 59 cardinal features include hyperandrogenemia, chronic anovulation, insulin resistance and hyperinsulinemia. Women with PCOS are at increased risk to develop long term health implications like endometrial cancer, T2DM and cardiovascular diseases. Insulin-like factor 3 (INSL3), also known as relaxin- like factor (RLF), is a member of the relaxin-like hormone family. Relaxin and INSL3 are peptide hormones with a number of important physiological roles in reproduction, regulation of extracellular matrix turnover, and cardiovascular function. INSL3 is a theca cell-secreted paracrine factor which regulates androgen production and has been implicated a role in follicle selection and resulting ovulation. In women, it is produced in lower amounts by ovarian theca and luteal cells, and circulating levels are increased in women with polycystic ovarian syndrome. This suggests that INSL3 may have a modulatory role in ovarian function. The INSL3 gene is localized on chromosome 19 and comprises of two exons and an intron. Novel and known variations in both exonic regions were investigated to understand their influence in PCOS pathophysiology. Genotyping of these polymorphisms were carried out by direct sequencing in 150 women with PCOS and 150 normal menstruating women. Clinical, biochemical and hormonal parameters were also assessed in these women. Analysis revealed association of db6523 SNP in exon 1 and rs1003887 polymorphism of exon 2 with susceptibility to PCOS. Other exonic region polymorphisms failed to show any association with PCOS pathophysiology. P94 Role of TNF a in the etiopathogenesis of PCOS: a clinical, biochemical and molecular genetic study Sujatha Thathapudi 1* , Vijayalakshmi Kodati 1 , Ahuja Yog Raj 1 , Uma Addepally 2 , Anuradha Katragadda 3 , Qurratulain Hasan 4,5 1 Vasavi Medical Research Centre, Hyderabad, India; 2 Jawaharlal Nehru Technological University, Hyderabad, India; 3 Anu Test tube baby centre, Hyderabad, India; 4 Kamineni Academy of Medical Sciences and Research Centre, Hyderabad, India; 5 Hyderabad Science Society, Hyderabad, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):P94 Background: Polycystic ovarian syndrome (PCOS) is one of the most common endocrine conditions affecting women of reproductive age with a prevalence of approximately 5-10 % worldwide. PCOS is characterized by hyperandrogenism, irregular menstrual cycles, polycystic ovaries, insulin resistance, obesity particularly abdominal phenotype. We evaluated the role of TNF a in PCOS patients and compared to age matched healthy controls. Methods: 204 women clinically diagnosed with PCOS and 204 healthy women controls in the age group of 17 to 35 years were evaluated. PCOS were subdiveded into Obese and lean based on BMI (< 25 or ≤ >≥ 25 kg/m 2 ). Clinical, biochemical characteristics of PCOS and molecular study of TNF alpha gene C850T polymorphism were studied. Results: Significant differences were observed in PCOS phenotypes and controls. All the PCOS including subtypes had elevated WC, W/H, Fasting Insulin, HOMA-IR, LH, LH/FSH, TG, and decreased HDL than Controls (P<0.0001). Patients had elevated serum TNF a, Free and Total testosterone, Androstenedione and DHEA when compared to controls (P <0.05). We have demonstrated an association between TNF a –C850T polymorphism and PCOS. Frequency of T allele was 93.5% in PCOS and 73.5% in controls (OR 5.1863, CI 3.2893 to 8.1773 and P value <0.0001). TT genotype confers a fivefold high risk for PCOS in our population (OR 5.7, CI 3.4673 to 9.3724 and P value <0.0001). Conclusion: TNF a contributes to the clinical , biochemical manifestations of PCOS, and –C850T TNF a gene polymorphism is associated with PCOS and could be used as a relevant molecular marker to identify women with risk of developing PCOS in our population. P95 Assessment of MBL2 gene polymorphism and lipid peroxidation in Chronic Obstructive Pulmonary Disease (COPD) Aarti Sharma 1 , Gursatej Gandhi 1 , Jatinder Singh 2 , Sukhdev Singh 2 , Manpreet Kaur 1* 1 Department of Human Genetics, Guru Nanak Dev University, Amritsar, India; 2 Department of Molecular Biology & Biochemistry, Guru Nanak Dev University, Amritsar, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):P95 Background: Chronic Obstructive Pulmonary Disease (COPD) is fourth leading cause of death worldover. It has been defined as a state characterized by airflow obstruction due to inflammatory reaction. Various innate and adaptive immune system molecules are involved in pathogenesis of COPD. Mannose-binding lectin (MBL) is a Ca 2+ dependent collagenous lectin which intervene immune response by inhibiting pathogen activity. Point mutations, in codon 54 and 57 of exon 1 of the MBL2 have been reported to affect the serum levels of MBL. Aim of the present study was to investigate the association of MBL2 gene polymorphism with severity and susceptibility towards COPD. Methodology: 129 COPD patients and 90 age- and sex-matched controls were recruited for the study. Genomic DNA was isolated from blood samples. PCR-RFLP of codon 54 and 57 of the MBL2 were studied using enzymes BanI and MboII respectively. In addition to this, serum MDA concentrations were evaluated by TBA-TCA-HCl method. Genotypic distribution was compared by odds ratio statistics using medcal software. Differences in MDA concentrations were analyzed by student’s‘t’ test using SPSS version 18.0 (IL, USA, and Chicago). Results: The genotypic frequencies of codon 54 in COPD patients were significantly higher (p< 0.05) than that of controls. GG genotype was found to be more prevalent in cases (OR= 3.402; CI= 1.14-10.10; p< 0.05). However, there was no significant difference in genotypic distribution for codon 57 of MBL2 gene. Serum MDA concentrations were significantly (p< 0.001) higher in patients (9.00±2.906) as compare to controls (6.31±2.361). Conclusion: The results of the present study revealed that MBL2 polymorphism may be involved in pathogenesis of COPD. P96 Role of IL-6/JAK/STAT pathway in inducing vascular insulin resistance Aswath Balakrishnan * , Kapaettu Satyamoorthy, Manjunath B Joshi Division of Biotechnology, School of Life Sciences, Manipal University, Manipal, India Molecular Cytogenetics 2014, 7(Suppl 1):P96 Introduction: Insulin resistance is a hall mark of metabolic disorders. Studies have demonstrated that inflammatory regulator interleukin-6 (IL-6) plays an important role in disruption of IR/Akt/eNOS signaling pathway resulting vascular insulin resistance. Accumulating evidences suggests a significant role of epigenetic mechanisms such as DNA methylation in progression of metabolic disorders. Hence the present study aimed to understand the role of epigenetic mechanisms involved during IL-6 induced vascular insulin resistance and its consequences in cardiovascular diseases. Materials and methods: Human umbilical vein endothelial cells (HUVEC) and Human dermal microvascular endothelial cells (HDMEC) were used for this study. Endothelial cells were treated in presence or absence IL-6 (20ng/ml) for 36 hours and followed by insulin (100nM) stimulation for 15 minutes. Levels of phosphorylated- and total Akt served as readout for insulin resistance. To investigate changes in DNA methylation, cells were treated with or without neutrophil conditioned medium (NCM) as a physiological source of inflammation or IL-6 for 36 hours. Genomic DNA was processed for HPLC analysis for methyl cytosine content and cell lysates were analyzed for DNMT1 (DNA (cytosine-5)-methyltransferase 1) and DNMT3A (DNA (cytosine-5)-methyltransferase 3A) levels using immunoblotting. Results: Endothelial cells stimulated with insulin exhibited an increase in phosphorylation of Akt ser 473 in serum free conditions but such insulin response was not observed in cells treated with IL-6, suggesting chronic exposure of endothelial cells to IL-6 leads to insulin resistance. HPLC analysis for global DNA methylation resulted in decreased levels of methyl cytosine in cells treated with pro-inflammatory molecules (both by NCM and IL6) as compared to 3.2% in untreated control to 2% in treated. Kinetic studies depicted a transient increase DNA methylation at 24 hours which was followed by steep decrease at 36 hours. Subsequently, analysis in cells treated with IL-6 showed a significant decrease in DNMT1 levels but not in DNMT3A. Other pro-inflammatory marker such as TNF-a did not exhibit such changes. Interestingly we also observed Akt phsophorylation refelcetd DNMT1 changes suggesting plausible role of PI3K/Akt signaling axis in regulation of DNMT1 expression. Molecular Cytogenetics 2014, Volume 7 Suppl 1 http://www.molecularcytogenetics.org/supplement/7/S1 Page 48 of 59 Conclusion: Taken together our study suggests that IL-6 induces vascular insulin resistance and involvement of epigenetic changes. P97 In silico docking studies for designing potent anti-diabetic derivatives of swertiamarin with enzyme HMG COA reductase Jayshil Bhatt 2* , Hitesh Vaidya 1 , Varun Khanna 2 , Naisargee Patel 2 , Ramesh Goyal 2 1 Memorial University of Newfoundland, St. John’s, Canada; 2 Institute of Life Sciences, Ahmedabad University, Ahmedabad, India Molecular Cytogenetics 2014, 7(Suppl 1):P97 Background: Swertiamarin, a secoiridoid glycoside is found in abundant quantity in Enicostemma Littorale herb and is the main constituent responsible for anti-diabetic and anti-obesity effects of the plant extract. It has been reported to act on various enzymes and transcription factors involved in glucose and lipid metabolism, including inhibition of the enzyme HMG-CoA reductase which might be one of the mechanisms responsible for the antihyperlipidaemic activity. However, owing to its high water solubility, it has a low plasma half life; and thus we have designed its derivatives which bind more efficiently with HMG CoA Reductase. Materials and methods: Docking was carried out using software namely Autodock and Vina, and repeated at least thrice to minimise the error and to confirm the repeatability. The obtained results were scored and sorted on the basis of the binding energy. The Autodock docking was only done on the inhibitor ligands that were taken from the crystal structure to cross check the results of Molecular Operated Environment [MOE] in terms of binding orientation. Attempts were also made to evaluate in silico toxicity and identification of other possible targets for the lead molecule swertiamarin. Results: We could design 23 compounds which were docked into the active site of the crystal structure of HMGR. The interactions of these molecules were compared with the presently known inhibitors such as atorvastatin and simvastatin. Based on the results of docking score a number of potent ligands for the enzyme HMGR as compared to swertiamarin were established. It was observed that the designed molecules SWL18, SWL21, SWL22 showed better docking score (-60.74; -61.43 and –55.68 respectively) and tight binding in pockets. Docking score of these molecules were very similar to the atorvastatin and simvastatin. Conclusions: SWL18, SWL21, SWL22 possess tight binding with HMGR as compared to swertiamarin, which suggest that these molecules could have better HMGR inhibition. PHARMACOGENETI CS P98 Influence of CYP3A5 polymorphism on tacrolimus drug dosing in Indian renal allograft recipients: initial experience Mohan Patel 1* , Manoj Gumber 1 , Vivek Kute 1 , Pankaj Shah 1 , Himanushu Patel 1 , Hargovind Trivedi 1 , Aruna Vanikar 2 , Jayesh Sheth 3 1 Department of Nephrology and Clinical Transplantation, Institute of Kidney Diseases and Research Center, Dr HL Trivedi Institute of Transplantation Sciences, Ahmedabad, India; 2 Department of Pathology and Immunohematology, IKDRC-ITS, Ahmedabad, India; 3 Foundation for Research in Genetics and Endocrinology (FRIGE) Institute Of Human Genetics, Ahmedabad, India Molecular Cytogenetics 2014, 7(Suppl 1):P98 Background: Tacrolimus (Tac) is the mainstay of standard immuno- suppressive regime in renal transplantation today. However, it needs regular drug level monitoring in view of narrow therapeutic window to keep blood level in therapeutic range. The objective of the study was to assess the potential influence of a functional polymorphism in CYP3A5*3 gene on dose requirements and trough blood levels of tacrolimus in renal transplant patients Materials & methods: This prospective observational study included 20 patients of end stage renal disease who underwent renal transplantion and a follow up of 1 year at our hospital. All the patients were started on standard immunosuppressive regime of Tacrolimus-Mycophenolate mofetil along with steroids with a starting dose of Tac 0.08 mg/kg/day. Genotype of CYP3A5 was studied in 20 patients requiring low dose of Tac. At 7 th and 30 th day, and 3 monthly after transplant, Tac dosages (mg/kg/d), were adjusted based on blood levels and complications. Polymerase chain reaction, followed by restriction fragment length polymorphism analysis was used for genotyping CYP3A5*3 gene (A6986G) in intron 3. Results: Out of 20 patients (17 males and 3 females) included in the study, 16 had live related renal transplantation and 4 had diseased donor renal transplantation. Out of 20 patients 16 were found to have CYP3A5 homozygous status (A3986G polymorphism in CYP3A5*3 gene-wild allele) and 4 with heterozygous status. Patients of CYP3A5 homozygous status had mean Tac level of 16.84 ng/dL, median of 15.5 ng/dL (range- 13.2-25 ng/dL) on 7 th day of transplant. So Tac dose was reduced to achieve TDL and mean Tac level at 30 th day of transplant was 6.6 ng/dL and median of 7.4 ng/dL (range- 5.5-12.5 ng/dL). At 6 th month and 12 th month of transplant, mean Tac levels were 6.1 ng/dL and 5.9 ng/dL. In patients having CYP3A5 homozygous status, mean and median Tac dose after 4 weeks was 0.03 mg/kg/day (range- 0.008 to 0.05 mg/kg/day) and mean creatinine was 1.17 mg/dL (range-0.8 to 1.4 mg/dL) after 30 th day. Remaining 4 patients having CYP3A5 heterozygous status were maintaining TDL at Tac dose of 0.06 mg/kg/day. Five patients had graft biopsy during first 4 weeks of renal transplant showing acute tubular necrosis (ATN) in three patients, acute cellular rejection in one patient and cellular rejection with evidence of calcineurine inhibitor (CNI) toxicity in one patient. Conclusion: This study demonstrates the usefulness of CYP3A5 genotype in transplant patients taking Tacrolimus-Mycophenolate mofetil and also shows that majority of our patients carry mutant allele A3986G in CYP3A5*3 gene. P99 Multi- analytical approach: better predictor of pharmacogenetic based clinical outcomes in breast cancer therapies Sonam Tulsyan Department of Genetics, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):P99 Background: Chemotherapeutic drug clinical outcomes are genetically determined as there is a large heterogeneity in the response to, and toxicity of, chemotherapeutic agents in breast cancer patients. Polymorphisms in genes encoding Phase I – Cytochrome P450 (CYP450) and NAD(P)H dehydrogenase quinine (NQO1); phase II - glutathione-Stransferase (GST) and methylene tetra hydrofolate reductase (MTHFR); phase III- p-glycoprotein (ABCB1) and solute transporter (SLC22A16) drug metabolizing enzymes can possibly predict clinical outcomes, and can be of prognostic significance in breast cancer patients. The aim of this study was to determine the role of genetic variations in drug metabolizing enzymes in predicting response and toxicity in breast cancer patients, using multi-analytical approaches. Materials & methods: Two hundred and four North Indian cases of histology proven invasive breast carcinoma treated at the our institute were genotyped for 17 polymorphisms by Polymerase Chain Reaction (PCR) or PCR-Restriction Fragment Length Polymorphism (RFLP) or Taqman allelic discrimination assay. All patients were treated with combination chemotherapy containing an anthracycline drug—epirubicin (62.70 %) or doxorubicin (37.25%) along with cyclophosphamide and 5-fluorouracil. Tumor response to NACT was recorded in 96 patients according to Response Evaluation Criteria in Solid Tumors (RECIST). Toxicological data was recorded according to National Cancer Institute- Common Terminology Criteria for Adverse Events (NCI-CTCAE). The highest grade toxicity that occurred in an individual patient during the course of treatment was used for the analysis. Genetic variations were correlated with response to NACT and chemo-toxicity using logistic regression through SPSS software, version 17.0. Furthermore, higher-order gene-gene interactions were determined through multifactor dimensionality reduction (MDR) using software version 2.0 beta8. Results: Heterozygous (CT) and variant (TT) genotype of ABCB1 1236C>T polymorphism was found to be significant with breast cancer response to NACT. Similarly, heterozygous (CT) genotype of ABCB1 1236C>T Molecular Cytogenetics 2014, Volume 7 Suppl 1 http://www.molecularcytogenetics.org/supplement/7/S1 Page 49 of 59 polymorphism was significantly associated with grade 2-4 anemia. Furthermore, we found significant association of heterozygous genotype (*1/*3) of CYP2C9*3 with grade 2-4 leucopenia and CT genotype of NQO1 polymorphism with dose delay/ reduction. However, none of the polymorphisms were found to be statistically significant with grade 2-4 toxicity. On MDR analysis, ABCB1 1236C>T polymorphism yielded the highest testing accuracy for response to NACT (CVT=0.61, CVC= 10/10, p =0.0067). However, ABCB1 1236C>T, ABCB1 2677G>T/A, CYP2C19*2 combination of polymorphisms yielded the highest testing accuracy for grade 2-4 anemia (CVT=0.66, CVC= 10/10, p < 0.0001). Similarly, the above combination of polymorphisms provided the best interaction model for overall toxicity as well (CVT=0.65, CVC= 10/10, p < 0.0001). For dose delay/ reduction, NQO1 609 C>T polymorphism yielded the highest testing accuracy (CVT=0.60, CVC= 9/10, p =0.0048). However, CYP2B6*9 polymorphism with the testing accuracy (CVT=0.43, CVC= 5/10) for grade 2-4 leucopenia did not achieve statistical significance (p= 0.1088). Conclusions: Multi-analytical approaches may provide a better prediction of pharmacogenetic based clinical outcomes in breast cancer patients. P100 Impact of KCNJ11, TCF7L2, SLC30A8, IGF2BP2, PPARG, SLC47A1, STK11, HHEX, KCNQ1, CDKAL1, FTO, CYP2C9, ADIPOQ, CAPN10 gene polymorphisms on risk of type 2 diabetes and therapeutic response to sulfonylurea and metformin therapy Nagaraja Phani 1* , Padmalatha Rai 1 , Prabha Adhikari 2 , Shivashankara Nagri 3 , Sydney D’Souza 2 , Mundyat Gopinath 1 , Kapaettu Satyamoorthy 1 1 Division of Biotechnology, School of Life Sciences, Manipal University, Manipal, India; 2 Department of Medicine, Kasturba Medical College, Manipal University, Mangalore, India; 3 Department of Medicine, Kasturba Medical College, Manipal University, Manipal, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):P100 Type 2 Diabetes (T2D) represents a spectrum of metabolic disorders is genetically heterogeneous disease with multiple genes located on different chromosomes contributing to its susceptibility. Prevalence of T2D has increased sharply in recent years with more than 300 million people worldwide and 70 million in India. The application of molecular, genomic knowledge will provide new opportunities to explore the heterogeneity which patients clearly exhibit and provide a more accurate understanding of individual patients. Single nucleotide polymorphisms (SNPs) represent the most profuse form of genetic variation in humans which are gaining increased popularity and emerging as a new generation genetic markers. With several classes of drugs available to treat T2D its clinical response exhibits significant variation among individuals which exerts a huge impact on healthcare system and economic burden due to diabetes treatment. Pharmacogenetic research which assesses the role of genetic variants can be used in elucidating the nature of these variants on differential drug response. In the current study, we evaluated the association of KCNJ11, TCF7L2, SLC30A8, IGF2BP2, PPARG, SLC47A1, STK11, HHEX, KCNQ1, CDKAL1, FTO, CYP2C9, ADIPOQ, CAPN10 gene polymorphisms with T2D and response to sulfonylurea and metformin using PCR-RFLP, TETRA-ARMS and DNA sequencing techniques. Study subjects were 330 T2D patients who are on sulfonylurea (178) and metformin (152) treatment with age and sex matched normal healthy controls of south Indian origin. Allele frequencies, genotype and haplotype distribution were analyzed by chi-squared test. Logistic regression analysis was used to predict the effect of gene variants on treatment. Gene–gene interactions were analyzed by generalized multifactor dimensionality reduction method. Linkage disequilibrium between each pair of SNP loci, were estimated and plotted with JLIN. We have also performed a systematic review and Meta-Analysis of KCNJ11 rs5219 and PPARG rs1801282 SNPs on South Asian populations to clarify inconsistency in association results. Our analysis showed KCNJ11 rs5215, SLC30A8 rs1326634, TCF7L2 rs7903146 were associated with susceptibility to T2D. Multivariate regression analysis showed that TCF7L2 rs12243326 TT genotype, KCNJ11 rs5219 TT genotype were associated with response rate to sulfonylurea treatment and STK11 rs741765 GG genotype, SLC30A8 rs1326634 TT genotype were associated with response rate to metformin treatment. POPULATI ON GENETI CS P101 Effect of PPAR-g2 gene Pro12Ala and ADR-b3 gene Trp64AArg polymorphism on glucose homeostasis in Type 2 diabetes subjects from Western India Ankna Shah 1* , Frenny Sheth 1 , Avisek Majumder 1 , Bhavik Doshi 1 , Navneet Shah 2 , Premal Thakor 3 , Rama Vaidya 4 , Jayesh Sheth 1 1 FRIGE’s Institute of Human Genetics, FRIGE House, Jodhpur Gam Road, Satellite, Ahmedabad-300015, India; 2 Department of Diabetes and Endocrinology; Sterling Hospital, Ahmedabad- 380052, India; 3 Gujarat Diabetic Association, Ahmedabad-380007. India; 4 Kasturba Health Society, Medical Research Centre, Mumbai-400056. India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):P101 Background: Several studies have shown the effect of Pro12Ala polymorphism of PPAR-g2 on insulin sensitivity and Trp64Arg polymorphism in ADR-b3 gene on obesity and insulin resistance in Type 2 Diabetic (T2D) subjects. The present study was carried out to find the interaction of these two gene polymorphisms and their combined effect on glucose homeostasis (HbA1C) in T2D subjects. Materials and methods: The present study comprises of 535 subjects (including 235 T2D & 300 controls). Genotyping was carried out for the above mentioned polymorphisms and glycosylated hemoglobin [HbA1C] levels were analyzed for each subject. All T2D subjects were divided into four groups according to their genotype. Group-I: 31 patients with Pro/ Pro and Trp/Arg genotype; Group-II: 159 patients with Pro/Pro and Trp/ Trp genotype; Group-III: 6 patients with Pro/Ala and Trp/Arg genotype; and Group-IV: 39 patients with Pro/Ala and Trp/Trp phenotype. Results: It was observed that 12Ala allele frequency was nearly equal in T2D patients and controls (9.0% vs. 9.1%, p>0.05). 64Arg allele frequency was 8.3% in T2D patients and 6.7% in controls (p>0.05). The mean HbA1C level was lower in T2D patients with 12Ala allele compared to patients homozygous for 12Pro allele (7.73±1.42% vs. 8.47±1.92%, p<0.02). However, no significant difference in mean HbA1C levels was observed in T2D patients with 64Arg allele compared to patients homozygous for 64Trp allele (8.50±1.81% vs. 8.31±1.88%, p>0.05). The mean HbA1C levels were higher in Group-I (8.62±1.84%, p<0.0092), Group-II (8.47±1.94%, p<0.001) and Group-III (8.26±1.71%, p>0.05) compared to grouPIV having a mean HbA1C of 7.65±1.37%. Conclusions: The protective effect of 12Ala allele is likely to be diminished in Group-III T2D patients in the presence of 64Arg allele. Polymorphisms of 64Arg and 12Pro alleles that is likely to play a role in controlling glucose homeostasis by gene-gene interactions. P102 Alarming findings about genomics of sudden cardiac arrest in India Pankaj Mankad * , Srisanth Balan, Saleem Mohammed Xcode Life Sciences, Chennai, 600 034, Tamil Nadu, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):P102 Background: Over 10% of total deaths and 50% of cardiac deaths in India are Sudden Cardiac Deaths (SCA) and occur at a young age of 5-8 years. We analysed risk allele plus genotype frequencies and polymorphism associations of 38 SNPs, confirmed in GWAS studies that increasing the risk of SCA in 124 random people in the general population. The data were compared with Caucasians reported in HapMap (n = 113). Material and methods: Following DNA extraction from saliva, genotyping was performed using Illumina golden gate genotyping assay. Allele and genotype frequencies were determined by direct gene count method. Results: Population Genomics indicated Odds Ratio of risk alleles as 1.62 (1.31 -1.92). The carrier rate of risk allele frequency was significantly higher (p = 0.037 to p = 9.18E-37) in Indians compared to Caucasians in 56% of SNPs in H-W equilibrium (15 of 27).6 SNPs showed statistically insignificant comparatively lower risk allele frequency in Indians. Mean increase in higher risk allele frequency SNPs was 2.4 fold (1.1 – 6.7). Frequency in Indians, for the two SNPs with the strongest SCD risk correlate in the gene BAZ2B was 2 times (rs4665058) and 6 times (rs 174230) greater than in Caucasians. Molecular Cytogenetics 2014, Volume 7 Suppl 1 http://www.molecularcytogenetics.org/supplement/7/S1 Page 50 of 59 Personalized Genomics indicated that even the frequency of risk homozygous genotype, resulting in the SCA threat in affected individuals being the square of the risk allele hazard, was markedly different with 4.7% Caucasians carrying homozygous risk genotype versus 10.6% of Indians (p<.0001). In 18 (out of 38) SNPs studied, the homozygous genotype difference in Indians was more than double). Although interplay of gene-gene interaction in individualized risk prediction is still undefined, 59% (73 of 124) people carried between 10 to 19 risk alleles and 41% (51 of 124) had between 20 to 30 risk alleles (median 19, range 10 -25), of the total maximum of 76. Conclusions: This is the first report, highlighting extremely high genomic propensity for Sudden Cardiac Arrest in Indians compared to the Caucasians. In India, there is also a high propensity for risk towards homozygous genotype and people carrying several risk alleles for SCA. At the population level, this data reinforces an urgent need to train community in different locations in basic resuscitation skills and at a personal level, it raises an important point to test the vulnerable group for assessing the risk alleles for better individualized preventive action plans could be advocated. P103 Transmission Disequilibrium Test for quantitative traits based on multiple sibs Hemant S Kulkarni * , Saurabh Ghosh Human Genetics Unit, Indian Statistical Institute, Kolkata, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):P103 Background: The Transmission Disequilibrium Test (TDT) is a well established family-based test for genetic association. The advantage of TDT over population-based association studies test, which is protected against population stratification, and hence, an association finding can be attributed to the presence of linkage. Quantitative traits (QT) are more informative on within-genotype variability and hence, statistical tests for identifying genes based on such traits tend to be more powerful compared to binary or qualitative traits. The TDT is based on a trio design (two parents and one offspring per family). However, if multiple sibs are present, TDT is only a valid test for linkage. In our study, we have extended the test for quantitative traits based on families with multiple offspring using a logistic regression framework. Materials and methods: We selected one offspring at random from each family to compute the usual TDT statistic. We then repeated the process and carry out separate permutation tests based on the mean and the maximum values of the TDT statistics obtained over different replications. We performed extensive simulations to evaluate the power of our proposed tests under a wide spectrum of genetic parameters (allele frequencies, QTL means and variances, extent of linkage disequilibrium between the QTL and the marker locus) and probability models (normal and chi-squares). We also compared the performance of the tests with a correction strategy using the Benjamini-Hochberg threshold. Results: We found that the test based on the mean TDT value is more powerful than that based on the maximum value. However, in either case, the power is higher compared to that obtained using the Benjamini- Hochberg threshold. In general, the power decreases with increase in heteroskedasticity at the QTL as well as the difference between the minor allele frequencies at the two loci. The powers are marginally higher for the chi-squares distribution compared to the normal distribution. P104 Evaluation of MC4R [RS17782313, RS17700633], AGRP [RS3412352] and POMC [RS1042571] polymorphisms with obesity in Northern India Apurva Srivastava 1* , Balraj Mittal 3 , Jai Prakash 2 , Neena Srivastava 1 1 Department of Physiology, King George’s Medical University, Lucknow, (U.P), India; 2 Department of Pediatrics, King George’s Medical University, Lucknow, (U.P), India; 3 Department of Medical Genetics, SGPGIMS, Lucknow, (U.P), India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):P104 Background: Genetic variants of the melanocortin-4 receptor gene (MC4R), agouti related protein (AGRP) and proopiomelanocortin (POMC) are reported to be associated with obesity. Therefore, we examined MC4R rs17782313, MC4R rs17700633, AGRP rs3412352 and POMC rs1042571 for association with obesity in North Indian individuals. Material and methods: The variants were investigated for association in 300 individuals with BMI≥30kg/m 2 and 300 healthy non-obese individuals with BMI<30kg/m 2 . The genotyping were analyzed by Taqman probes. The statistical analysis was performed by means of the software SPSS, ver.19 and P≤ 0.05 was considered statistically significant. Results: The genotypes of MC4R rs17782313 and POMC rs1042571 were significantly associated with obesity (BMI≥30kg/m 2 ) (p=0.02; OR=1.7 and p=0.01; OR=1.6 respectively). However, MC4R rs17700633 (p=0.001; OR=0.55) was associated with low risk. AGRP rs3412352 (p=0.93; OR= 0.96) showed no association with obesity (BMI≥30kg/m 2 ) in North Indian individuals. Conclusions: The study provides the first report of association of MC4R rs17782313 and POMC rs1042571 that these may have an effect on obesity BMI≥30kg/m 2 but MC4R rs17700633 and AGRP rs34123523 may not have any influence on obesity BMI≥30kg/m 2 in North Indian individuals. P105 Population allele frequencies of disease associated SNPs in India: a paradigm shift from HapMap Pankaj Mankad, Srisanth Balan * , Saleem Mohammed Xcode Life Sciences, 6B, Eldorado, Nungambakkam High Road, Nungambakkam, Chennai, 600 034, India Molecular Cytogenetics 2014, 7(Suppl 1):P105 Background: One of the challenges facing us in India, translating recent discoveries of chronic disease associated SNPs into clinical domain for prevention, is the lack of knowledge about the frequency of the polymorphism in our population. Hence, using available HapMap data has become a norm. However, it is anticipated that population genomics in India could be different from the HapMap Caucasian population. Relative disease risk prediction based on relevant SNPs, both for personalized medicine and for population genetics, has little value without accurate information of population allele frequencies. We report allele frequency of 384 SNPs directly related to chronic disease risk and metabolic traits in the Indian population. Materials and methods: We report the allele in a random sample of 146 individuals and compare them with the data reported in HapMap Caucasian population (n = 112). Genotyping was performed using Illumina golden gate genotyping assay following DNA extraction from saliva. Allele frequencies were determined by direct gene count method. Results: GWAS studies confirmed 384 SNPs to be associated with disease risk (364) of Diabetes Type 1 and 2 (54 & 118 respectively), Coronary artery disease(71), myocardial infarction(9), cardiac failure(24), sudden cardiac arrest (38), atrial fibrillation(9), hypertension(18), obesity(10), metabolic syndrome(2) and stroke(11); or were associated with metabolic traits (20). The master table of their ‘rs’ id, chromosomes, location and association is presented. Of the 384 SNPs, 44 were not in H-W equilibrium and were omitted. HapMap data were not available for 13 SNPs. We are reporting their allele frequencyon the Indian population for the first time. Of the remaining 307 disease association SNPs, statistically significant difference (p<.05) from HapMap Caucasian population was observed in 53% of them (164 of 307) and the difference of >10% (considered major in population genetics) was found in 42% (130 of 307). Of the 20 metabolic association SNPs, 50% (10 of 20) had statistically significant difference and in all of them it was >10%. Conclusions: We are reporting the largest repository, documenting disease and related SNP and allele frequencies in Indian population. We have also highlighted clear differences with HapMap data and would caution against indiscriminate use of HapMap for bench-to-bedside application of genetic knowledge in our population. P106 Genetic Variation in Intercellular Adhesion Molecule-1 (ICAM-1): candidate gene in susceptibility to malaria in the Indian population Anuroopa Gupta * , Harish Padh Department of Cell and Molecular Biology, B.V. Patel Pharmaceutical Education and Research Development (PERD) Centre, Ahmedabad-380 054, Gujarat, India Molecular Cytogenetics 2014, 7(Suppl 1):P106 Molecular Cytogenetics 2014, Volume 7 Suppl 1 http://www.molecularcytogenetics.org/supplement/7/S1 Page 51 of 59 Background: Plasmodium falciparum malaria is the most severe form of malaria causing morbidity and mortality worldwide. The key event in P. falciparum pathogenesis is the sequestration of the infected erythrocytes to the capillary endothelium, mediated by the receptor, Plasmodium falciparum erythrocyte membrane protein-1 (PfEMP1). PfEMP1 has distinct characteristics of binding to varied host ligands including Inter Cellular Adhesion Molecule (ICAM-1). Polymorphisms in the gene encoding ICAM-1 influence the rate of progression or confer protection from or susceptibility to malaria parasite infection. The objective of our study was to evaluate the frequency of ICAM-1 SNP in exon 6 (rs5498, K469E) in the Indian population and compare it with the global population influenced by the parasitic infection. Materials and methods: The genotyping for ICAM-1 SNP rs5498 was performed by Restriction Fragment Length Polymorphism (RFLP) PCR. Analysis of ICAM-1 variants was performed in 200 healthy subjects. Results: The allelic frequency for the variant ICAM1-Aallele was 59% in healthy control subjects. Chi square analysis reveals that it does not follow the Hardy Weinberg Equilibrium. Comparison with literature data indicates that frequency distribution in Indian population is similar to German, Swedish and Danish populations while it is significantly different from Finnish and Japanese populations. The genotypic frequency of AA was 35%, AG was 49% and GG was 16% in healthy control subjects. Conclusions: The frequency data from the above study will help in understanding the role of genetic variations in ICAM-1 adhesion molecule in the falciparum malaria pathogenesis in the Indian population. Upon comparison, a noteworthy difference in the genotype frequency of Nigerian children (p<0.001) was evinced. This data can be useful for predicting the potential risk of malaria in world population. P107 Genetic affinities of six populations of Manipur using a microsatellite (STR) marker Ahsana Shah * , Ruqaiya Hussain, Mohammad Afzal Aligarh Muslim University, Aligarh, U.P., India Molecular Cytogenetics 2014, 7(Suppl 1):P107 Background: The development of molecular genetic technology has led to the discovery of large number of polymorphic loci in the human genome. This has renewed the interest in investigating genomic diversity (and affinity) between human populations with much more depth and clarity than that was possible with the erstwhile traditional serological and biochemical genetic markers. DNA markers are presently widely used in gene mapping, forensic studies and information on population structure and population genetics of regional and global populations. The study attempts to understand the genetic structure and affinity among six different populations of Manipur. Materials and methods: The diversity of the studied populations was examined by investigating the polymorphism for a STR locus (TPOX). Unrelated individuals belonging to Muslims with different caste viz. Sheikh, Syed, Pathan & Moghul; Hindu (Meitei) and tribal (Naga) were randomly selected. Blood samples were collected after obtaining an informed consent. The DNA was extracted using standard phenol – chloroform extraction method and then amplified with PCR using previously published primers. The amplified PCR products were electrophoreses followed by Silver staining. Results: Different population containing allele with repeated number varying from 5 -11 were detected. Discriminating Power Analysis (PD) lie in the range from 0.444 to 0.889 and Matching Probability (MP) was observed to vary from 0.111 to 0.556. Based on genetic distance, dendrogram was constructed to depict the genetic affinities among the six populations. The tree shows that the ethnic background of six populations is different. P108 Microsatellite variation and allele frequency distribution for (TPOX) STRS locus in North Indian Muslim populations Ruqaiya Hussain * , Mohammad Afzal Human Genetics and Toxicology Lab., Section of Genetics, Department of Zoology, Aligarh Muslim University, Aligarh, Uttar Pradesh, India Molecular Cytogenetics 2014, 7(Suppl 1):P108 Background: Autosomal short tandem repeats (STRs) have become the most informative molecular markers because they are highly polymorphic and multiallelic. The objective of the present work is to study the genotypic polymorphisms, allele frequencies and forensic parameters at highly polymorphic STR loci (TPOX) among four Muslim Populations of North India: Pathan, Ansari, Saifi and Mansoori. Materials and methods: The DNA was extracted from blood samples by using the standard phenol-chloroform extraction method and then amplified by PCR using specific primers for TPOX locus. The PCR products were separated by electrophoresis on denaturing 6% polyacrylamide gel and silver staining was done to resolve and observe different alleles. The observed and expected heterozygosity (H obs and H exp ) as well as forensic and paternity indices including matching probability (MP), power of discrimination (PD), power of exclusion (PE) and polymorphism information content (PIC) were calculated using the Power Stats v.1.2 software for TPOX locus of the studied population. The POPGENE (v.32) statistical package was used for analyzing allele frequency. Results: The 6 to 10 different alleles were detected in the studied populations with 8 allele repeats are most common in all four groups of sub-population. The maximum expected heterozygosity was found in Mansoori population (H exp = 0.867). The polymorphism information content (PIC), has shown that this marker is highly informative for Mansoori (0.67) and Ansari (0.63) population group. Conclusion: In conclusion the interpopulation differentiation has been found significantly to differ from each other (F ST = 16.37%). A dendrogram was constructed using the Neighbor-joining (NJ) clustering method. The dendrogram shows the low genetic distance between Pathan and Ansari population groups. According to the statistical parameters, the combined analysis of this TPOX STRs locus is a powerful tool for forensic personal identification, paternity testing and population genetic studies in North Indian Muslim Populations. P109 Genetic variation of ITGB3 is associated with Autism Spectrum Disorders (ASD) in South Indian children Femina KMB Nair 1* , PA Suresh 2 , Moinak Banerjee 1 1 Human Molecular Genetics laboratory, Rajiv Gandhi centre for Biotechnology, Thiruvananthapura, India; 2 ICCONS, Shoranur, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):P109 Background: Autism spectrum disorders (ASDs) are a group of develop- mental disabilities that can cause significant social, communication and behavioral challenges in children. Genetic factors contribute significantly to ASD. ITGB3 encodes integrin b3. This cell adhesion molecule has been implicated as a modulator of serotonergic systems as well as in regulation of synaptic plasticity and maturation. In the brain, integrin b3 couples to integrin av to form a functional receptor, making integrin avb3 an interesting target for regulation of neural 5-HT systems. The aim of this study was to investigate the potential associations of single-nucleotide polymorphisms (SNPs) of the integrin gene with Autism Spectrum Disorder (ASD). Material and methods: Hundred and twenty five patients with ASD and 210 healthy volunteers were recruited. Four SNPs of Integrin genes were analyzed by direct sequencing and polymerase chain reaction–restriction fragment length polymorphism genotyping. Results: We detected significant allelic and genotypic associations with rs3809865 (Allelic and genotypic p value = 0.0089, 0.0044). Haplotypic association involving risk allele was observed in two, three and four locus. This 3’UTR SNP would decrease/break or enhance/create miRNA-mRNA binding sites and thus affect the expression of host genes in the brain. Conclusions: Our finding identified the possible function of this SNP locus, and provides the basis for subsequent functional research. P110 Association of Genetic Polymorphisms in STAT 3, STAT 5b and GWAS Identified PTPN22 Gene with Rheumatic Heart Disease Usha Gupta 1* , Avshesh Mishra 1 , Saurabh S. Rathore 1 , Snober S Mir 2 , SK Agarwal 3 , Naveen Garg 4 , Balraj Mittal 1 1 Department of Genetics, Sanjay Gandhi Postgraduate Institute of Medical Sciences (SGPGIMS), Lucknow, UP, India; 2 Department of Biotechnology, Molecular Cytogenetics 2014, Volume 7 Suppl 1 http://www.molecularcytogenetics.org/supplement/7/S1 Page 52 of 59 Integral University, Lucknow, UP, India; 3 Department of Cardiovascular and Thoracic Surgery, Sanjay Gandhi Postgraduate Institute of Medical Sciences (SGPGIMS), Lucknow, UP, India; 4 Department of Cardiology, Sanjay Gandhi Postgraduate Institute of Medical Sciences (SGPGIMS), Lucknow, UP, India Molecular Cytogenetics 2014, 7(Suppl 1):P110 Background: Rheumatic heart disease (RHD) is an inflammatory, autoimmune disease, occurring as a consequence of group A streptococcal infection complicated by rheumatic fever (RF). Cytokines are important mediators of inflammatory and immune responses. JAK-STATs have been demonstrated to be critical elements in signaling by certain families of cytokines. GWAS has identified PTPN22 SNPs as non-HLA genetic variants to be associated with susceptibility to autoimmune diseases. Based on these, we looked for association of genetic variants of STAT 3, STAT 5B and GWAs identified PTPN22 with RHD in North Indian population. Methods and results: This case-control study included 400 RHD patients and 200 controls. The polymorphisms were identified using RFLP/Taqman probes. Statistical analysis was performed by using SPSS. We observed that STAT3 CG and GG genotypes were significantly associated with RHD (p=0.024 & p=0.027 respectively), STAT5b CT&TT genotypes were significantly associated with RHD (p=0.001 & p=0.002 respectively) while both the SNPs of PTPN22 gene did not show any association with RHD. Further categorization of RHD patients into mitral valve disease (MVD) and combined valve disease (CVD) subgroups revealed that STAT3 CG&GG genotypes were associated with MVD and STAT5b CT&TT genotypes were also associated with both MVD&CVD. Conclusions: STAT3 & STAT5b gene polymorphisms may play an important role in the pathogenesis of RHD but GWAS identified PTPN22 SNPs may not be associated with susceptibility of RHD. P111 Role of cholecystokinin receptor-A gene polymorphism in development of functional dyspepsia Rajan Singh * , Balraj Mittal, Uday C Ghoshal Departments of Gastroenterology and Genetics, Sanjay Gandhi Postgraduate Institute of Medical Sciences (SGPGIMS), Lucknow-226014, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):P111 Background: Functional dyspepsia (FD) is characterized by epigastric pain, burning, early satiety and post-prandial fullness in absence of organic or metabolic causes. Cholecystokinin receptor-A (CCK-AR) is known to modulate satiety signal and delay gastric emptying, which are associated with FD. CCK-AR (rs1800857, T/C) polymorphism is associated with a defective splicing of the primary transcript of CCK-AR mRNA, which may result in the lower expression of the CCK-AR. Therefore, we evaluated the role of genetic polymorphism of CCK-AR gene (rs1800857, T/C) in FD. Material and methods: 237 consecutive patients with FD (Rome III) and 250 healthy controls (HC) were genotyped for CCK-AR gene polymorphism (PCR-RFLP). Patients with FD were sub-classified into epigastric pain syndrome (EPS), post-prandial distress syndrome (PDS) and EPSPDS overlap. Results: Patients with FD [173 (73%) male, age 38±12-y] were comparable with HC [195 (78%) male, age 37±12-y] with respect to age and gender. 26/237 (11%) had EPS, 55 (23.2%) PDS and 156 (65.8%) EPSPDS overlap. Among 237 patients with FD, CC (variant) genotype of CCK-AR (rs1800857) was infrequent among patients than HC [19 (8%) vs. 46 (18.4%) p=0.001, odds ratio (OR) =0.36, 95% confidence interval (CI) =0.19-0.66]. However, genotypes distribution was comparable among patients with different subtypes of FD (p=0.44). Conclusions: CC genotype of CCK-AR polymorphism is protective for FD. EPSPDS overlap was common among patients with FD. P112 Role of sarcomeric gene polymorphisms on left ventricular dysfunction in coronary artery disease patients Surendra Kumar 1* , Avshesh Mishra 1 , Anshika Srivastava 1 , Naveen Garg 2 , Surendra Kumar Agarwal 3 , Shantanu Pande 3 , Balraj Mittal 1 1 Department of Genetics, Sanjay Gandhi Post Graduate Institute of Medical Sciences (SGPGIMS), Lucknow-UP, India; 2 Department of Cardiology, Sanjay Gandhi Post Graduate Institute of Medical Sciences (SGPGIMS), Lucknow-UP, India; 3 Department of CVTS, Sanjay Gandhi Post Graduate Institute of Medical Sciences (SGPGIMS), Lucknow-UP, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):P112 Background: Coronary artery disease (CAD) is a major cardiac disease in humans. Many CAD patients develop left ventricle dysfunction (LVD), leading to congestive heart failure. Mutations in several genes including those encoding sarcomeric proteins such as MYBPC3, TNNT2, and TTN are common genetic cause of hereditary cardiac myopathies. An intronic 25- bp deletion in MYBPC3 at 3’ region is associated with dilated (DCM) and hypertrophic (HCM) cardiomyopathies in Southeast Asia. We sought to determine the role of MYBPC3 25bp, TNNT2 5bp and TNN 18bp ins/del polymorphisms on LVD in CAD patients. Methods and results: The study included 200 healthy controls and 988 consecutive patients with angiographically confirmed CAD. Among them, 253 with reduced ejection fraction (LVEF <45%) were categorized as having LVD. MYBPC3 25bp, TNNT2 5bp and TNN 18bp ins/del polymorphisms were determined by polymerase chain reaction. Our results showed that MYBPC3 25bp deletion was significantly associated with CAD as well as LVD (healthy controls v/s CAD; p value = 0.003; OR=4.08, healthy controls v/s LVD; p value < 0.0001; OR=6.67 and Non-LVD v/s LVD; p value = 0.031; OR=1.67). The TNNT2 5bp and TNN 18bp polymorphisms were not found to be associated with CAD (Pvalue=0.580, OR=0.88; Pvalue=0.795, OR=0.91; respectively) or LVD (Pvalue=0.146, OR=1.35; Pvalue=0.935, OR=0.97 respectively) when compared to controls. Conclusions: The frequency of MYBPC3 DW genotype and D allele was associated with LVD implying that genetic variants of MYBPC3 encoding mutant structural sarcomeric protein could increase susceptibility to left ventricular dysfunction. Therefore, 25bp deletion in MYBPC3 may represent a genetic marker for cardiac failure in CAD patients. P113 A novel mutation in 3’UTR of GJB2 gene in autosomal recessive nonsyndromic sensorineural hearing loss in South Indian population Maria sebastian 1* , Praveena Davis 2 , Padmaja Ramdas 2 , Moinak Banerjee 1 1 Human Molecular Genetics Laboratory, Rajiv Gandhi Centre for Biotechnology, Kerala, India; 2 National Institute of Speech & Hearing, Kerala, India Molecular Cytogenetics 2014, 7(Suppl 1):P113 Background: Autosomal recessive nonsyndromic sensorineyral hearingloss also known as “DFNB” causes 20% of all childhood deafness and may have carrier rate as high as 2.8%. Fifty to eighty percent of DFNB cases causing severe to profound hearing impairment, results from mutations in a single gene, GJB2(DFNB1), that encodes the protein connexin 26(Cx26) located on chromosome 13q11-12. Aim of this study was to explore the status of reported pathogenic mutation as well as novel variants in South Indian population. Material and methods: Promoter region, exons and 3’UTR of GJB2 gene were screened to find mutations associated with autosomal recessive nonsyndromic deafness from 50 families speaking Dravidian Malayalam language. Primers were designed using primer Z software for sequencing promoter, exons as well as 3’UTR of GJB2 gene. Mutation surveyor software was used to find changes in the sequence. Results: Pathogenic mutations in the coding exon like W24X, R127H, M163V were found in our study population. W24X mutation was the most commonly found mutation causing Stop codon. Screening of 3’UTR region of GJB2 lead us to find a novel mutation in this region located 1031 bases downstream of the gene causing a change from G to A. Bioinformatics analysis using different miRNA prediction tool like Microsniper, mirSNP suggest, this change causing a differential binding of miRna including hsa- miR-924, hsa-mir-501-5p,hsa-mir-1225-3p,hsa-mir-558 and hsa-mir-615-3p. Further it was revealed that this mutation indeed causes changes in expression of this gene. Conclusions: The present study could find a novel mutation in the 3’UTR region 1031bp downstream in GJB2. Bioinformatic analysis provides evidence for functional implication of this mutation which might have a role in pathogenicity of the disease. This study could also validate the importance of reported mutations in our population with W24X to be the common pathogenic mutation found in this population. Molecular Cytogenetics 2014, Volume 7 Suppl 1 http://www.molecularcytogenetics.org/supplement/7/S1 Page 53 of 59 P114 IL 10 gene -1082 G/A shows an associatoin with rheumatoid arthritis patients of South Indian Population EK Krishna Priya 1* , Moinak Banerjee 2 , S Rajesh 3 , K Sasikala 1 1 Department of Zoology, Bharathiar University, Coimbatore; 2 Rajiv Gandhi Center for Biotechnology, Trivandrum, India; 3 Department of Rheumatology, KIMS Hospital, Trivandrum, India Molecular Cytogenetics 2014, 7(Suppl 1):P114 Background: Rheumatoid arthritis (RA) is a systemic, chronic and inflamma- tory joint disease of unknown etiology with genetic predisposition. This disease is three times more frequent in women than men and is strongly associated with the circulating auto-antibodies like rheumatoid factor (RF) and anti cyclic peptide antibody (ACPA). The cytokine network dysfunction plays a vital role in the pathogenesis of rheumatoid arthritis. The purpose of the study was to understand the autoantibody specificities and its association with Disease activity score Interleukin 10(-819 T/C, -592 A/C & -1082 G/A) promoter polymorphisms in rheumatoid arthritis patients of South Indian population. Materials and methods: According to ACR criteria 1987, 168 rheumatoid arthritis patients and 244 controls groups were selected as study population. Genotyping of IL-10 promoter regions -819T/C and -592 A/C were performed by PCR- Direct sequencing and -1082 G/A promoter polymorphism by PCR- RFLP method. Results: The results show that -1082 G/A promoter region shows a significant association with rheumatoid arthritis patients in South Indian population (genotypic p = 2 x 10 -4 & allelic p = 2 x 10 -5 ). The disease activity score were positively correlated with rheumatoid factor of RA patients (p = 4 x 10 -2 ). Conclusions: The study concludes that the allele G of -1082 promoter region predisposes susceptibility to Rheumatoid Arthritis. We also found a strong association of positive rheumatoid factor with the disease phenotype in RA patients. P115 A case-control association study of K121Q and G/T Variants in ENPP1 and TCF7L2 gene with type 2 diabetes mellitus in North Indian Punjabi Population Basanti Barna * , Badaruddoza, Kawaljit Matharoo, Amarjeet Singh Bhanwer Department of Human Genetics, Guru Nanak Dev University, Amritsar- 143005, Punjab, India Molecular Cytogenetics 2014, 7(Suppl 1):P115 Background: The K121Q and G/T polymorphism of the ENPP1 and TCF7L2 genes respectively plays a significant role and found to be associated with increased risk of T2DM in many worldwide populations. However, a very few studies have been done for this association in North Indian Punjabi populations. In the present cross section study K121Q polymorphism of ENPP1 gene and G/T polymorphism of TCF7L2 gene were analysed to determine the association of these genes with type 2 diabetic North Indian Punjabi subjects. Materials and methods: A total of 450 participants consisting of 239 T2DM and 211 healthy subjects were recruited for this study. Genomic DNA was amplified by PCR-RFLP method. Anthropometric and physiometric variables such as height, weight, Body Mass Index (BMI), Waist hip ratio (WHR), Waist circumference (WC), Hip circumference (HC), biceps skinfold, triceps skinfold, SBP, DBP, MBP, Pulse rate and pulse pressure were measured using standard protocol. The Chi-square analyses were used to test the significance difference in genotype, allele frequencies and Hardy- Weinberg equilibrium deviation. Associations of genotypes with T2DM and its corresponding Pvalues were calculated using Web-Assotest program. Results: The results revealed that anthropometric and clinical characteristics such as WHR, fasting and random glucose levels, SBP, DBP, pulse rate and pulse pressure have significant (p<0.001) differences between T2DM and control subjects. However, none of the anthropometric and clinical characteristics have found significant difference with respect to their genotypic distributions for both of genes except DBP for ENPP1 K121Q polymorphism. Conclusions: The association analysis of the present results showed K121Q variant of ENPP1 gene G/T variant of TCF7L2 gene have significant association with type 2 diabetes with dominant and recessive model of action respectively in North Indian Punjabi populations. P116 A multifactorial dimensionality reduction model for gene polymorphisms and environmental interaction analysis for the detection of susceptibility for type 2 diabetic and cardiovascular diseases Badaruddoza * , Basanti Barna, Kawaljit Matharoo, Amarjeet Singh Bhanwer Department of Human Genetics; Guru Nanak Dev University; Amritsar- 143005, Punjab, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):P116 Background: The present study has presented a comparative gene- environment interactions such as three genes (ENPP1-K121Q, TCF7L2-G>T and GYS1 A1>A2) and six environmental factors (obesity and cardiovascular related risk factors) for the detection of susceptibility for T2DM and CVD and for the interpretation of epistasis involved in genetic studies of disease susceptibility. Materials and methods: The final sample size included 250 cases and 250 controls to focus on better methodological quality and a higher statistical power analysis. All the interactions and approaches were carried out using the methods of MDR. Results: The MDR analysis showed the interaction between environmental factor (SBP) and the genetic factor (ENPP1) for pooled and female T2DM patients which indicated that SBP and TCF7L2 had significant contribution on susceptibility to T2DM. The analysis showed that environmental factors (BMI, WHR, WC, SBP, DBP and PR) and genetic factors (ENPP1-K121Q, TCF7L2 -G>T and GYS1 A1>A2) have identified risk factors and their interaction. All these interactions were observed to be significant. The MDR method showed all interaction models first to ninth order interactions for pooled and male T2DM patients as significant for susceptibility of obesity. Whereas in female T2DM patients, a first order (WHR) and third order (WHR * SBP * ENPP1) have found a significant interaction for obesity. Both the genes ENPP1 and TCF7L2 interacting with WHR and WC increase the susceptibility of obesity many folds among T2DM patients and non-diabetic controls. These results were also supported by dendrogram and interaction entropy model. The three factor interaction model (BMI * SBP * ENPP1) for pooled T2DM patients have been found significant for predicting hypertension in T2DM patients whereas, in female T2DM patients all the interaction models have been found as significant. However, third order model (SBP * TCF7L2 * GYS1) have been found as a strong predictor for hypertension. In T2DM male patients there has been no significant interaction observed for gene- environment interaction although a seven factor model (BMI * WHR * WC * SBP * PR * ENPP1 * TCF7L2) seems to be comparatively a good predictor for hypertension. Conclusions: The results showed that both the genes ENPP1 and TCF7L2 interacting with WHR and WC increase the susceptibility of obesity many folds among T2DM patients and non-diabetic controls. P117 Association of clock gene variants with Autism Spectrum Disorder in South Indian population Ann Mary Alex 1* , PA Suresh 2 , Moinak Banerjee 1 1 Human Molecular Genetics Laboratory, Rajiv Gandhi Centre for Biotechnology, Kerala, India; 2 Institute for Communicative and Cognitive Neuro Sciences, Kerala, India Molecular Cytogenetics 2014, 7(Suppl 1):P117 Background: Autism Spectrum disorder is a group of neurodevelopmental disorders that manifests in the first three years of life. Social impairments, communication difficulties and repetitive/stereotyped behaviour are the common symptoms of the spectrum. One of the major endophenotype associated with the disease is circadian and sensory dysfunction. Circadian dysfunction is mainly observed by difficulties in sleeping. Around 56-83% of patients with ASD suffer from sleep problems. There is an endogenous circadian clock that regulates the sleep and wakefulness, cognitive function, systematic hormonal release and body temperature. We hypothesize that Molecular Cytogenetics 2014, Volume 7 Suppl 1 http://www.molecularcytogenetics.org/supplement/7/S1 Page 54 of 59 the genes functioning to maintain this molecular clock may be associated with ASD either directly or by its transcriptional regulation of other genes. Materials and methods: The study population was from the Malayalam speaking population of Kerala. The patients were diagnosed and characterized based on DSM-IV criteria. We selected 2 genes which are core clock components, hCLOCK and PER3 due to its functional relevance. The CLOCK gene is the first essential component of the mammalian clock and was found to be associated with circadian rhythm sleep disorders. PER3 gene is implicated in delayed sleep phase syndrome and extreme diurnal preference. Single Nucleotide Polymorphisms in both genes were studied for association with the disease. Genotyping was done by sequencing and PCR RFLP. Results and conclusion: Genotypic and allelic frequencies of the SNPs studied were analyzed to understand if there exists an association with the disease. We could not find any association with the 8 polymorphisms screened in hCLOCK gene in our population. However we were able to get an association with a VNTR in PER3 gene, with the 5 repeat allele being the risk allele. This is also the first report of PER3 gene being associated with ASD. P118 Skin miRNA profiling reveals differentially expressed miRNA signatures from non-segmental vitiligo patients Mohmmad Shoab Mansuri 1* , Mala Singh 1 , Naresh C. Laddha 1 , Mitesh Dwivedi 1 , Yogesh S. Marfatia 2 , Rasheedunnisa Begum 1 1 Department of Biochemistry, Faculty of Science, The M.S. University of Baroda, Vadodara, Gujarat, India-390002; 2 Department of Skin and V.D., Faculty of Medicine, The M.S. University of Baroda, Vadodara, Gujarat, India- 390002 Molecular Cytogenetics 2014, 7(Suppl 1):P118 miRNAs are small conserved non-coding RNA molecules that post- transcriptionally regulate gene expression by targeting the 3’ UTR of specific mRNA for degradation or translational repression. miRNAs have been shown to be promising biomarkers for different diseases. At present, the expression and function of miRNAs in human skin is largely unknown. The aim of the present study was to detect the differentially expressed miRNAs in non-segmental vitiligo (NSV) patients and to explore the potential role of these miRNAs in vitiligo pathogenesis. We performed the whole miRNA profiling of lesional as well as non-lesional skin from four NSV patients and four healthy skin samples from controls using TaqMan® Low Density Array. Our results suggest that 38 miRNAs were differentially expressed in the skin of patients compared to controls. We identified 13 miRNAs which were significantly differentially expressed in lesional skin of patients compared to healthy control skin. Further, 29 miRNAs were found to be significantly differentially expressed between non-lesional skin of patients and healthy control skin. Interestingly, three miRNAs were specifically down-regulated in the lesional skin compared to non-lesional skin from patients with NSV. In conclusion, for the first time the present study suggests the crucial role of differentially expressed miRNAs in NSV patients from Gujarat. P119 Gene copy number variation in Indian population and its implication in health Suhani Almal, Harish Padh * Cellular and Molecular Biology, B. V. Patel Pharmaceutical Education & Research Development (PERD) Centre, Ahmedabad-380054, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):P119 Objectives: Copy number variations (CNV) are important source of human genetic variation, found to be widely prevalent than what was initially predicted. The involvement of CNVs in disease susceptibility and drug response is well reported in other populations but has not been studied to a larger extent in Indian population. The aim of the present study was to evaluate the distribution of copy number variable genes in Indian population and their link, if any, to health, disease and drug response along with development of a comprehensive resource of CNV frequency distribution among different populations. Methods: A total of 100 – 280 healthy controls and in variable number of patients from Indian population were genotyped. Genotyping of the copy number (CN) variable genes was carried by PCR-based methodologies (long range PCR, real-time PCR and PRT assay). Results: An indicative correlation with disease susceptibility and significant (p < 0.05) difference in the frequency distribution of the CNV variants was observed in our study. MTUS1 deletion variant was found to be significantly (p = 0.0207) associated with a decreased risk to breast cancer. A significant association between CCL3L1 copy number and risk to HIV-1 was also observed. The inter-ethnic comparison of CCL3L1 gene copy number frequency demonstrated significant difference worldwide, being highest in Africans. The common deletion frequency comprising LCE3B and LCE3C genes was found to be highest in European and American populations. The frequency of FCGR3B CN < 2 was found comparatively higher in Indians, Americans and Africans as compared to European Caucasians. Thus, inter- ethnic differences were observed among populations, highlighting varied frequency distribution pattern based on their distinct geographical location. Conclusion: This is our first attempt to create a comprehensive map of the frequency distribution of CNVs and its association to disease susceptibility in Indian population. This varied worldwide distribution can thus be relevant from clinical or evolutionary point of view in future. P120 Maternal gene polymorphisms of folate metabolism as genetic risk factor for Down syndrome in North Indian population Sushil Kumar Jaiswal 1* , Ashok Kumar 2 , Vineeta Gupta 2 , Anjali Rani 3 , Amit Kumar Rai 1 1 Centre for Genetic Disorders, Institute of Medical Sciences, Banaras Hindu University, Varanasi-221005, India; 2 Department of Pediatric, Institute of Medical Sciences, Banaras Hindu University, Varanasi-221005, India; 3 Department of Gynaecology, Institute of Medical Sciences, Banaras Hindu University, Varanasi-221005, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):P120 Down syndrome (DS), a chromosomal disorder has higher prevalence in population occurring 1 in 700 live births. Recent reports have shown that almost 92% of the DS children are born from young mothers, suggesting that along with advanced maternal age some other risk factors are involved for predisposition of mother to Down child. Polymorphism in genes involved in folate metabolism as well as insufficient folic acid intake could result in genomic instability, DNA hypomethylation and non-disjunction even resulting in trisomy 21. In present study we compared the frequency of Thymidylate Synthase (TYMS) 28 bp repeat polymorphism in 5’UTR region, Cystathionine- Beta-Synthase (CBS) 844ins68bp polymorphism and Solute Carrier (SLC) 19A1 G80A single nucleotide polymorphisms in 80 triads (mother, father and child) and 77 matched control mothers in order to observe whether these variants act as risk factors for DS. A significant association was observed for TYMS 5’UTR 28 bp repeat with odds ratio 2.9 (95% CI 1.2-7.1, p=0.027). An association which is very close to be significant was observed for SLC19A1 G80A with odds 2.01(95% CI 1.04-4.24, p=0.055). Heterozygosity for 68 bp insertion at 844 in CBS showed significant association with odds ratio 10.5 (95% CI 1.29-85.1, p=0.019). Transmission disequilibrium test (TDT) for 28 bp repeat polymorphism in TYMS gene presented more than four times greater preferential transmission of maternal two repeats allele whereas paternal three repeats allele had about 1.5 times higher rate of transmission. TDT for SLC19 A1 G80A SNPs revealed preferential transmission of maternal A allele more than two times greater as compared to G allele whereas paternal alleles transmission didn’t show much difference. The result shows that above three polymorphism are significantly associated as a risk factor for predisposition of mother to DS children in the North Indian population. P121 Molecular basis of DYT1 and DYT6 primary dystonia in Indian patients Subhajit Giri 1* , Arindam Biswas 1 , Shyamal Kumar Das 2 , Kunal Ray 3 , Jharna Ray 1 1 S. N. Pradhan Centre for Neurosciences, University of Calcutta, Kolkata, India; 2 Bangur Institute of Neurosciences & Psychiatry, Kolkata, India; 3 CSIR-Indian Institute of Chemical Biology, Kolkata, India Molecular Cytogenetics 2014, 7(Suppl 1):P121 Molecular Cytogenetics 2014, Volume 7 Suppl 1 http://www.molecularcytogenetics.org/supplement/7/S1 Page 55 of 59 Background: Dystonia is the third most common movement disorder. It is manifested by involuntary and sustained muscle contractions with frequent twists and repetitive movement, abnormal posture and functional impairment. Genetic factors play significant role for causing dystonia. Till date twenty five loci (DYT1-DYT25) and sixteen genes have been reported for dystonia. The present study reports the screening of TOR1A (DYT 1) and THAP1 (DYT 6) among primary torsion dystonia patients of India. Materials and methods: First, the most common ∆GAG mutation (c. 904- 906/907-909 ∆GAG; p. Glu302/303del) and the rs1801968 (p. Asp216His) in TOR1A gene were screened following the published method (Naiya et al., 2006). THAP1 was screened in 214 patients to identify mutations (mean age of onset, 30.8 ± 16.42 years) and 254 controls (mean age, 41.9 ±11.59 years). All three exons and their flanking sequences including exon-intron boundaries were screened by PCR , sequencing and/or RFLP analysis. Results: A total of 321 patients were screened and ∆GAG mutation was identified in two brothers in a family suffering from primary generalized dystonia. The analysis of rs1801968 (Asp216His) demonstrated that the minor allele (216His) is significantly over represented in patients (p = 0.027; OR = 1.915; 95% CI = 1.058-3.451), suggesting a risk for the disease. On screening THAP1 in 214 patients, a total of five nucleotide variants were identified including a reported missense mutation (c.427 A>G; p.Met143Val), a novel heterozygous deletion mutation (c. 208-209 del AA; p. K70VfsX15) in two juvenile onset primary dystonia patients and a rare variant in 3ʹ UTR region (c. 1030 T>C) in a patient having Blepharospasm. In addition two SNPs, (rs71521601 and rs111989331), were also found both in patients and controls. The association study using rs111989331 (IVS2-87 A>G) demonstrate that the major allele (A) can play as a risk factor (p = 0.001; OR = 1.977; 95% CI = 1.302-3.008) for primary dystonia. Conclusion: Our preliminary results suggest that the TOR1A and THAP1 genes play significant role in the pathogenesis of Indian dystonia patients. This is the first report on mutation detection in TOR1A and THAP1 among Indian dystonia patients. Acknowledgements: This study is funded by the Department of Science & Technology (DST), Govt. of India, University Grant Commission (UGC), Government of India. P122 Role of Dopamine b Hydroxylase (DBH) in Parkinson’s disease patients of Indian population Arunibha Ghosh 1 , Arindam Biswas 1 , Tamal Sadhukhan 1 , Shyamal Kumar Das 2 , Jharna Ray 1* 1 S. N. Pradhan Centre for Neurosciences, University of Calcutta, Kolkata, India; 2 Burdwan Medical College & Hospital, Burdwan, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):P122 Background: Parkinson’s disease is a neurodegenerative disease affecting at least 1% of the population over age of 55. It is characterized by selective loss of dopaminergic neurons in substantia nigra pars compacta and the appearance of intracellular inclusions termed Lewy bodies. As the age progresses, neurons in the other regions of brain are also degenerated. Depletion of brain dopamine initiates aberrant motor activities including rest tremor, rigidity, bradykinesia, postural instability. Apart from motor symptoms, cognitive impairments, like depression, psychiatric illness, and decreased mental ability are also observed in the patients. Genetic and non- genetic components are believed to govern the pathogenesis of PD. Genes in dopaminergic pathway & also dopamine synthesis, storage, binding, metabolism are needed to be studied which seem to be major determinants conferring differential risk of developing PD. Methods: In this study, three reported SNPs, rs1611115 (T>C), rs1108580 (A>G), rs129882 (C>T) of DBH have been analyzed. For rs1611115 (T>C), 229 patients & 252 controls have been screened. A total of 298 patients & 276 control samples have been analyzed for rs129882 (C>T). A total of 378 patients & 253 control samples have been analyzed for rs 1108580 (A>G). All these SNPs were screened by PCR , sequencing and/or RFLP analysis. Also, DBH activity has been measured in plasma isolated from patients’ blood. Results: For rs1611115 (T>C), the minor allele T is over represented in control samples & pose a protection (p=0.043, OR=0.731, 95% CI=0.731 (0.538-0.847) for this disease. No significant association has been found for rs1108580 (A>G). For rs129882 (C>T), the minor allele T is over represented in patient pool & confers a risk (p=0.036, OR=1.322, 95% CI=1.012-1.727) for this disease. DBH activity has been measured in plasma isolated from patients’ blood. A correlation has been found between plasma DBH activity & rs1611115. Conclusion: This study suggests that DBH might have a role in susceptibility of developing Parkinson’s disease. Acknowledgements: This study is funded by the Department of Science & Technology (DST), Goverment of India, University Grant Commission (UGC), Goverment of India. P123 Beta thalassemia prevention in India: evaluation of socio- cultural factors Swati Chawla * , Rajnish Singh, VR Rao Department of Anthropology , University of Delhi, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):P123 Aim and background: There are approximately 240 million people across the world who are heterozygous for Beta thalassemia and 200,000 affected homozygous are born annually. Prevention is a big challenge. But the situation in India is different. Social, cultural, and religious issues are found to be closely intertwined with Beta thalassemia prevention. In spite of 10,000 annual Beta thalassemic births, the situation here is not well combated. Social stigma and negative attitude are largely understood to be the bounding factors. The present study examines the prevention issue in the high risk community of India, analyzing their knowledge and perceptions in the light of available health models. Methods: A semi structured interview schedule was framed and imple- mented in the rural and urban (Delhi) population of a high risk community i.e. ARORAS and compared with the cosmopolitans (not at high risk). Results: Correct knowledge of carriers is a very important criteria, which is a major factor for the prevention of Beta thalassemia in a multi ethnic country like in India. Participants were found to carry positive attitude towards the public perception of Beta thalassemia. In general, social discomfort were not a serious issue, but acceptance of life partner with Beta thalassemia trait was unacceptable among all the three populations but more among the rural Aroras which showed, lack of knowledge among them. The acceptability for prevention strategies and its implication was high among all the three populations but rural Arora was found to be more proficient towards premarital screening(80.8%) and prenatal diagnosis (98.0%). Conclusion: A lack of knowledge about the disorder, its manifestations, survival rate, treatment availability, and psychosocial and cultural issues have created barriers to optimal health care including disclosure of Beta thalassemia status as well as to carrier testing. To overcome the problem there is an urgent need to have knowledge about people’s perception to find a common platform to harmonize various approaches for prevention. PRENATAL DI AGNOSI S P124 Prenatal diagnosis of Tay-Sachs disease: our institutional experience Jayesh Sheth 1 , Mehul Mistri 1* , Frenny Sheth 1 , Sarita Gupta 2 1 FRIGE’s Institute of Human Genetics, FRIGE House, Jodhpur Gam road, Satellite, Ahmedabad-380015, Gujarat, India; 2 M.S.University, Vadodara, India Molecular Cytogenetics 2014, 7(Suppl 1):P124 Introduction: Tay-Sachs disease (TSD) is the second most common storage disease after Guacher disease in the group of lipid storage disorders in India. In absence of any therapeutic option, prenatal diagnosis is the only way to prevent the disease burden. Aims and objectives: The present investigation was undertaken to provide cost effective prenatal diagnosis of TSD by enzyme based study from cultured chorionic trophoblast (CT)/uncultured CV or cultured amniotic fluid cells (AF) and further confirmation by molecular analysis of HEXA gene. Molecular Cytogenetics 2014, Volume 7 Suppl 1 http://www.molecularcytogenetics.org/supplement/7/S1 Page 56 of 59 Material and methods: Prenatal diagnosis was carried out in six women having confirmed index case with TSD was enrolled in this study. Enzyme study and molecular analysis was carried from CT in 3 women at 11-13 weeks of gestational age while in remaining 3 women cultured AF cells were used at 16 weeks of gestation. The b-Hexosaminidase-A activity was carried out with synthetic substrates, 4-methylumbelliferyl-6-sulfo-N- acetyl-beta-glucosaminide (4-MUGS) and DNA-based analysis was carried out by the bidirectional sequencing of HEXA gene. Written informed consent was obtained from all patients. Results and discussion: Deficiency of b-Hexosaminidase-A enzyme activity [21.7 nmol/hr/mg protein (NR: 271–1621 nmol/hr/mg protein)] from CT cells was detected in one case and mutation study observed homozygous mutation of 4bp insertion at c.1277_1278insTATC in the CT same as index case. Whereas, remaining 2 enzymatically normal fetuses were normal for earlier identified index case mutations [Homozygous mutation D322N and compound heterozygous mutations (D322N/c.1277_1278insTATC) respectively]. The enzyme activity was carried out from cultured AF cells in three women and deficiency of b-Hexosaminidase-A enzyme activity of 23.4 nmol/hr/mg protein (NR:271-2213.2 nmol/hr/mg protein) with homozygous mutations E114K in one case; intermediate enzyme activity of 247.2 nmol/ hr/mg protein (~50% of mean) having heterozygous mutation D322N in one case and normal enzyme activity (670.5 nmol/hr/mg protein) was detected in one subject also showed absence of W485X mutation which was present in the index case. Conclusion: This study clearly demonstrates that for storage disorders like Tay-Sachs enzyme analysis from cultured/ uncultured CV and cultured AF provide highly reliable information for prenatal study and can be used as with high sensitivity and specificity for prenatal diagnosis of the disease where index case has confirmed diagnosis proven by enzyme activity or by molecular analysis. P125 Prenatal diagnosis of autosomal recessive osteopetrosis: a case report Mehul Mistri 1 , Harsh Patel 1 , Tanmay Tanna 2* , Chitra Ankleshwaria 1 , Frenny Sheth 1 , Jayesh Sheth 1 1 Institute of Human Genetics, FRIGE House, Ahmedabad-15, Gujarat, India; 2 National Institute of Technology Warangal, Andhra Pradesh 506004, India Molecular Cytogenetics 2014, 7(Suppl 1):P125 Background: Autosomal Recessive Osteopetrosis (ARO) or Malignant Infantile Osteopetrosis (MIOP) is a congenital disorder characterized by increased bone density on radiographs. It is known to be caused by mutations that cause loss of function in the TCIRG1, CLCN7 or OSTM1 genes. Classic symptoms include bone marrow failure, visual and hearing impairment, pancytopenia and extramedullary hematopoiesis leading to hepatosplenomegaly. The disease is lethal in infancy and the only curative treatment known is Hematopoietic Stem Cell Transplantation (HSCT). Prenatal diagnosis is done either by autoradiography or by identifying disease causing mutation. Case presentation: We present a family with consanguineous marriage with a male child born at full term with an uneventful incident and normal Apgar score. The child had normal milestones of development till two years of age. After this, he developed symptoms such as low platelet, low hemoglobin, splenomegaly and hydrocephalus. The patient was started with fortnightly blood transfusions. Clinically he was suspected to have osteopetrosis and investigated for mutations in TCIRG1 gene. Targeted exon sequencing identified the patient to be homozygous for a novel nonsense pathological mutation p.R426X (c.1276 C>T) in TCIRGI gene. He expired in the course of treatment at the age of 2.5 years. Subsequently, both parents were found to be heterozygous carriers for the same mutation. Prenatal counseling was advised for any future pregnancies. In the subsequent pregnancy, prenatal testing was done at 11 weeks of gestation using uncultured chorionic villus sample (CVS) to investigate for the aforementioned mutation. Analysis of genomic DNA obtained from uncultured CV has shown heterozygous status for TCIRGI p.R426X (c.1276 C>T) mutation. The family was informed of the carrier status of the fetus and their psychological trauma and stress was alleviated. Conclusions: Prenatal Diagnosis of Malignant Infantile Osteopetrosis (MIOP) through targeted sequencing of genomic DNA obtained from uncultured CV can be carried out once the mutation has been identified in an index case. This novel mutation also provides a new insight into the molecular basis of the disease which can be utilized for molecular diagnosis. P126 Prenatal diagnosis of lysosomal storage disorders: our experience in 120 cases Mehul Mistri, Nrupesh Oza * , Frenny Sheth, Jayesh Sheth FRIGE’s Institute of Human Genetics, FRIGE House, Ahmedabad-380015, Gujarat, India Molecular Cytogenetics 2014, 7(Suppl 1):P126 Background: Molecular study is considered to be the gold standard for single gene disorders. For Lysosomal storage disorders (LSDs), plethora of literature report indicates the use of lysosomal enzyme activity in uncultured chorionic villus sample (CV), cultured chorionic villus (CT) or cultured amniotic fluid cells (AF) as a reliable tool for prenatal diagnosis (PD) followed by mutation study as a confirmation where mutation is known and/or unequivocal enzyme activity is observed. Very few studies are available from India with anecdotal reports using either molecular methods or enzyme study in PD of LSDs. Nonetheless considering the reported prevalence of 1:5,000-7,000 live birth and limited availability of therapeutic option PD remains the only preventable cure for storage disorders. Aim: To establish prenatal diagnosis of LSDs and demonstrate the reliable use of lysosomal enzyme study for prenatal diagnosis. Material and method: One hundred twenty pregnancies with confirmed index case of LSD were selected for lysosomal enzymes study from CV, CT and AF. Written consent was obtained from guardian of the study subjects. Results: Of 120 pregnancies, 57 (47.5%) were normal, 8 (6.66%) had an intermediate enzyme values and rest 55 (45.8%) were found to be affected with specific LSD. From the affected fetuses, 11 (9.1%) were affected with MPS-I, 2 (1.7%) with MPS-II, 1 (0.8%) with MPS-IIIA, 1 (0.8%) with MPS-IIIB, 4 (3.4%) with MPS-IVA, 2 (1.7%) with MPS-VI, 7 (5.8%) with GM-1 gangliosidosis, 1 (0.8%) with Gaucher, 3 (2.5%) with Tay-sachs, 2 (1.7%) with Sandhoff, 1 (0.8%) with NPD-A/B, 1 (0.8%) with MLD, 9 (7.5%) with Krabbe, 4 (3.4%) with Pompe, 2 (1.7%) with Batten and 4 (3.4%) with Mucolipidosis- II/III. All affected fetuses have shown very low enzyme activity (~0-15% of normal mean). We have also identified 8 (6.66%) pregnancies with intermediate enzyme activity (~50% of mean) this includes 3 (2.5%) fetuses were carrier with Sandhoff, 2 (1.7%) with Mucolipidosis-II/III, each one (0.8% for each) with MLD, NPD-A/B and Tay-sachs disease. Except one case of MPS-IVA who was found to have carrier (~30% enzyme activity) during prenatal diagnosis was found to be affected with MPS-IVA after delivery. Conclusions: Prenatal diagnosis of LSDs can be made with equal sensitivity and specificity of the molecular study using CV, CT and AF. Nonetheless carrier identification needs to be confirmed by molecular analysis. P127 Application of Chromosomal Microarray and Multiplex Ligation- dependent Probe Amplification in prenatal diagnosis Pankaj Sharma 1* , Madhumita Roy Chowdhury 1 , Neerja Gupta 1 , Rashmi Shukla 1 , Shruthi Sudarshan 1 , Manju Ghosh 1 , Deepika Deka 2 , Madhulika Kabra 2 1 Genetics Unit, Department of Pediatrics, All India Institute of Medical Sciences, New Delhi, India; 2 Department of Obstetrics & Gynecology, All India Institute of Medical Sciences, New Delhi, India Molecular Cytogenetics 2014, 7(Suppl 1):P127 Background: Chromosomal Microarray (CMA) and Multiplex Ligation- dependent Probe Amplification (MLPA) are relatively newer techniques for detecting cryptic copy number variations (CNVs). Here we are presenting the data of eight families where CMA and MLPA were used for prenatal diagnosis (PND) in view of their previous child with Intellectual disability/ developmental delay (ID/DD) and normal karyotype. Methods: Families with ID/DD children were referred for genetic counseling in Genetics Clinic, Department of Pediatrics, AIIMS. CNVs were studied in the probands using Illumina Cyto-SNP12 chips and MLPA kits for subtelomeric screening and microdeletion syndrome (P036 and P064) and PND by chorionic villus biopsy or amniotic fluid was performed in cases with pathogenic CNVs. Molecular Cytogenetics 2014, Volume 7 Suppl 1 http://www.molecularcytogenetics.org/supplement/7/S1 Page 57 of 59 Results: In eight families, CNVs of clear pathogenic significance were identified using CMA and confirmed by MLPA in seven cases. The probands had 2q32.3q33.3, 22q11.2, deletion in interstitial region while subtelomeric CNVs were 9q34 deletion, 8p deletion and 12p duplication, 5p deletion and 8q duplication in two affected sibs, 7q deletion and 10q duplication, 5p deletion and 6q duplication, 7q deletion and 20p duplication respectively. PND by MLPA (six cases) and CMA(two cases) were done. In six cases, fetus was found to be normal. In one case fetus had 8p deletion and 12p duplication while in another, the fetus had 6q deletion and 5p duplication. Discussion: CMA and MLPA have clearly demonstrated an increased resolution and improved detection rate of CNVs. CMA detects CNVs across the genome at high resolution enabling precise breakpoints of the CNVs. MLPA is a cost effective technique for developing countries but only limited (upto 45) nucleic acid targets in the genome can be investigated. In our settings it is an efficient technique for confirmation of CNVs and PND. STEM CELLS P128 Exposure to ionizing radiations can cause hazardous effects on differentiation of human CD34+ hematopoietic stem cells Angshuman Biswas 1* , Asiti Sarma 2 , Subrata Kumar Dey 1 1 Stem Cell Research Laboratory, West Bengal University of Technology, Kolkata – 700 064, India; 2 Radiation Biology Lab, Inter University Accelerator Centre, New Delhi- 110 067, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):P128 Statement of purpose: The objective of this study is to investigate effects of ionizing radiation on human hematopoietic stem cell differentiation. Background: Despite being a strong mutagen, causing several genetic and chromosomal aberrations, ionizing radiation in the form of gamma rays and heavy ion radiation is becoming increasingly important in medical therapies and cancer. Heavy ions especially the particle beam of carbon ion are high energy radiations that is considered to be extremely hazardous during occupational radiological emergencies, manned space missions, high altitude flights, accidents casualties etc. Healthy blood cells in human body arise by the differentiation of a very small population of pluripotent hematopoietic stem cells that have the capacity of self renewal throughout their lifespan. Effects of radiation whatsoever minimum on these unique cells thus have greater impact on the human system in both long term and short term scenario which might currently lead to an idea of a new concept leading to cancer stem cells. Methods: Umbilical cord blood collected in-utero was subjected to immuno-magnetic enrichment, followed by flowcytometric estimation for stem cell content. Isolated hematopoietic stem cells were then cultured into specialized cytokine based medium and exposed to various doses of ionizing radiation for investigations on CD34+ marker based flowcytometric survival assay, differentiation & clonogenic potential by CFC assay, genotoxicity and cytotoxicity. Results and conclusion: Exposure to different doses of ionizing radiation showed a marked difference in the survival of CD34+ stem cells in both dose and time dependant manner. Differentiation of stem cells into their adult progenitor cells were also altered with varying doses of radiation treatment. Our findings show variations on response of the human CD34+ stem cells when exposed to ionizing radiation which is significantly comparable to normal blood lymphocytes and cancerous K562 cells. THERAPEUTI CS P129 Impact of Vedic Chants Intervention Programme on Autistic Spectrum Disorder Kandasamy Dinesh Kumar 1* , Sushruth Badhe 1,2 , Sathiyavedu Santhiya 1 1 Dept of Genetics, Dr ALM PG IBMS, University of Madras, Taramani Campus, Chennai, India; 2 Midham charitable trust, Puduchery, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):P129 Background: Autism Spectrum Disorder (ASD) is a developmental disorder that affects the behavior and social communication of the child. In India, awareness about Autism coupled with shortage of skilled professionals poses severe constraints in management of children with ASD. There arises a need to explore the avenues of alternate therapy in the form of Group Therapy. A 44-bp insertion/deletion polymorphism in the promoter region of the serotonin receptor gene 5-HTT gene (5-HTTLPR) has been identified to modify the transcription rate of the gene (Lesch et al., 1996; Greenberg et al., 1999). The short allelic variant has been shown to reduce 5-HTT transcription resulting in diminished 5-HTT levels and reduced serotonin (5-HTT) reuptake into human platelets. We speculate the possibility of altered hormone levels (Melatonin and Serotonin) in individuals with ASD. As per earlier reports an association between 5-HTT gene polymorphism and autism has been indicated. Recent evidences from clinical and neuroscience research suggests an approach based on yogic principles and meditative tools is worth experimenting in children with autism. The same could be addressed in ASD individuals to ameliorate the drastic effect of these hormones on ASD individuals. Methods: A single group pre-/ post- test study design was employed. With the aid of a social and behavioral assessment scale for ASD, fifteen children with ASD between the age group of 7 - 14 years were evaluated for assessing the effect of the Vedic Chants Intervention Program recruited from special schools in and around Chennai. Chanting of select Sanskrit Mantras by an experienced meditator were given as an auditory stimulus in a closed room to the children for a period of 20 min daily at a fixed time. Alterations in the endocrine profile (Melatonin and Serotonin) were measured using ELISA Kits. Polymorphism in serotonin transporter (5-HTT) is to be analyzed to correlate the endocrine findings. Results: Study shows that there is a difference in the pre & post-test mean achievement scores due to the Vedic Chants Intervention Program. Some of the qualitative changes observed by the special educators were a reduction in the hyper activity, self-biting and other common traits of ASD like head nodding, hand flapping etc. This reflected in their class room sitting as well. Some of the children were remembering certain lines of the mantras and imitating the sound ‘Om’ and responding to the ‘Namaste’ gesture. Most of the children including those with ADHD sat throughout the entire session. The endocrine profile of the subjects was altered. Conclusions: The auditory stimulus has the potential to provoke the cognitive abilities of the children. Vedic Chants therapy might be one of the efficient Group therapies for the management of children with ASD. P130 Molecular basis of lysosomal storage disorders in India Shweta P Kondurkar 1* , Parag Tamhankar 1 , Pratima Kondurkar 1 , Ashwin Dalal 2 , Jayesh Sheth 3 1 ICMR Genetic Research Centre (NIRRH), Mumbai, India; 2 Center for DNA Fingerprinting and Diagnostics, Hyderabad, India; 3 Foundation for Research in Genetics and Endocrinology, Ahmedabad, Gujarat, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):P130 Background: Lysosomal Storage Diseases(LSD) are a group of rare recessive inherited metabolic disorders that result from the deficiency of a single enzyme required for the metabolism of lipids, glycoproteins or mucopolysaccharides. There is a lack of data on molecular basis of LSDs in India . The current study involves molecular analysis of patients with Tay Sachs disease(TSD) (HEXA gene), Sandhoff disease(SD) (HEXB gene), Gaucher disease (GD) (GBA gene), GM1 gangliosidosis (GG) (GLB1 gene), metachromatic leukodystrophy (MLD) (ARSA gene), and Pompe disease (PD) (GAA gene). Materials and methods: Patients presenting during the year 2011-2013 with characteristic clinical features of the above LSDs underwent specific biochemical testing (leucocyte enzyme assay) followed by sequencing of the respective gene. Enzyme assays performed include total hexosaminidase and hexosaminidase B (TSD and SD), glucocerebrosidase (GD), beta galactosidase (GG), arylsulphatase A (MLD) and alpha glucosidase (PD). The molecular basis of disease in patients with enzyme deficiency was confirmed by bidirectional Sanger sequencing covering all the exons and exon-intron boundaries of the respective genes. Molecular Cytogenetics 2014, Volume 7 Suppl 1 http://www.molecularcytogenetics.org/supplement/7/S1 Page 58 of 59 Results: During the study period, 131 unrelated families across India were studied. These included 47 families of TSD, 36 families with SD, 15 families with GD, 8 families with GG, 15 families with MLD, and 10 families with PD. Ninety two families (70. 2 %) families showed consanguinity. The age range for patients was between 3 months to 7 years; however, one patient with PD was adult (21 years). The youngest patient had PD. The study identified 218 mutant alleles in 119 patients. Only ten alleles were recurrent. Founder mutation was identified in TSD patients from Gujarat (p.E462V) which will help screening in patients from this state. Also in SD, the mutation hotspot R284X represented 23.3 % of alleles (7/30). No mutations could be identified in 12 patients and the second mutation could not be identified in 10 patients, despite being biochemically confirmed. Conclusion: The study shows allelic heterogeneity in Indian patients with LSDs. A molecular screening strategy for the common mutations could be adopted for TSD and SD patients. P131 Genome-wide analysis identifies common CNVs associated with primary open angle glaucoma Lalit Kaurani 1 , Mansi Vishal 2 , Dhirender Kumar 3 , Bharati Mehani 1 , Charu Sharma 4 , Anchal Sharma 1,5 , Debasis Dash 3 , Jharna Ray 6 , Abhijit Sen 7 , Kunal Ray 2,5 , Arijit Mukhopadhyay 1,5* 1 Genomics & Molecular Medicine, CSIR-Institute of Genomics & Integrative Biology, Delhi, India; 2 Molecular & Human Genetics Division, CSIR-Indian Institute of Chemical Biology, Kolkata, India; 3 G. N. Ramachandran Knowledge Centre for Genome Informatics, CSIR-Institute of Genomics & Integrative Biology, Delhi, India; 4 Mathematics Department, School of Natural Sciences, Shiv Nadar University, Uttar Pradesh, India; 5 Academy of Scientific and Innovative Research (AcSIR), Delhi, India; 6 S. N. Pradhan Centre for Neurosciences, University of Calcutta, Kolkata, India; 7 Drishti Pradip Eye Clinic, Kolkata, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):P131 Background: Copy number variation (CNV) is one of the major factors contributing to genomic diversity and diseases. Glaucoma is a major neurodegenerative disease causing irreversible vision loss across the globe. We wanted to analyze the impact of common CNVs in a genome- wide scale in patients of primary open angle glaucoma (POAG) collected from the West Bengal, India. Method: Genome-wide data was generated on 364 POAG cases and 365 controls on Illumina 660W-Quad arrays and CNVs were called using PennCNV. Copy number variant regions (CNVRs) were analyzed for association. A publicly available dataset of POAG cohort of 866 cases and 495 controls from Caucasian origin (GLAUGEN study) was used as a validation cohort. Representative CNVs were validated using real-time PCR. Results: We analyzed genome-wide CNV from 1928 samples. After association analysis we found 308 significantly associated (p<0.05) CNVRs in the Indian data. These POAG associated CNVRs were enriched in nervous system development. 113 CNVRs (37%) were significantly associated with the Caucasian data set. These contain 5 genes previously reported in eye diseases, namely, IDUA, FOXE3, NDUF7, PRPF6 and WNT3. We also found 6 associated CNVRs in previously known glaucoma loci. Conclusion: We have shown that common CNVRs are significantly associated in both datasets irrespective of the population background. We have also identified candidate genes/regions which are uniquely present in POAG cases and absent in controls. Our data might provide new insights into role of CNV in pathogenesis of POAG. P132 Microarray based global transcriptome profiling reveals involvement of non-Hsa21 genes and microRNAs in molecular mechanism of Down syndrome pathogenesis Ashutosh Pathak * , Divya Agarwal, Shubha R Phadke Department of Medical Genetics, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow, India E-mail: [email protected] Molecular Cytogenetics 2014, 7(Suppl 1):P132 Down syndrome (DS), the most frequent genetic disorder leading to mental retardation is caused by partial/complete triplication of human chromosome 21 (Hsa21). The differential expression of genes located on extra chromosome 21 is assumed responsible for phenotypic abnormalities but this gene dosage hypothesis has not been assessed on genome-wide basis. The gene expression patterns related to phenotypic abnormalities may provide insights into their roles in DS pathogenesis. To analyze the differential gene expression and understand the molecular mechanism underlying pathogenesis of DS, we performed global gene expression profiling in blood samples of 14 DS and 4 normal subjects using human whole transcriptome microarray. The microarray analysis revealed total of 624 genes (195 upregulated and 429 down regulated) were differentially expressed in DS patients as compared to control. Out of the genes present on chromosome-21, a total of 210 genes were differentially expressed ranging from 1.5 to 5 fold compared to normal individuals. Genes involved in physiological pathways such as apoptosis and cell cycle regulation, signal transduction, cell maturation, and immunity showed dysregulation. Several genes localized on Hsa21 such as APP, SOD1, DYRK1A, COL6A1 showed differential expression and the levels were conserved across all DS subjects. Interestingly, several non-Hsa21 genes such as RCAN3 (Hsa1), ANK3 (Hsa10), CDK17 (Hsa12) etc., having roles in cardiogenesis, signal transduction and differentiation of neurons showed conserved levels of expression across the DS subjects. Further to investigate the role of microRNAs (miRNAs) in regulation of gene expression, global miRNA profiling was performed in 4 DS patients and 1 control using Affymetrix miRNA 3.0 array. Several Hsa21 miRNAs like miR-99a, let-7c, miR-125b-2, miR155, miR-802 showed overexpression effecting the regulation of genes involved in DS pathogenesis. Our results substantiate that involvement of non-Hsa21 genes and miRNAs provide etiological basis for abnormal phenotypes during DS pathogenesis. Our data lead to more systematic understanding of molecular mechanism underlying DS pathogenesis and identification of miRNA based therapeutic targets for better management of the disease. Cite abstracts in this supplement using the relevant abstract number, e.g.: Pathak et al.: Microarray based global transcriptome profiling reveals involvement of non-Hsa21 genes and microRNAs in molecular mechanism of Down syndrome pathogenesis. Molecular Cytogenetics 2014, 7(Suppl 1):P132 Molecular Cytogenetics 2014, Volume 7 Suppl 1 http://www.molecularcytogenetics.org/supplement/7/S1 Page 59 of 59
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