LIFE09200604007 Tabish - Homi Bhabha National Institute

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Synopsis A synopsis of the thesis submitted to Homi Bhabha National Institute For the degree of Doctor of Philosophy In life Sciences Name of the candidate : TABISH Title of the thesis : Genotype-Molecular Phenotype Correlation using Lymphoblastoid Cell Lines from Patients with Multiple Primary Neoplasms Subject : Life Science Registration number : LIFE09200604007 Date of registration : 01/09/2006 Date of Synopsis submission : 21.2.2012 Institute : ACTREC, Tata Memorial Centre, Kharghar Navi Mumbai-410210. Name and designation of the guide : Dr. R. Mulherkar, S.O.„H‟, ACTREC 1
Synopsis Genotype-Molecular Phenotype Correlation using Lymphoblastoid Cell Lines from Patients with Multiple Primary Neoplasms Introduction: Squamous cell carcinomas of upper aero-digestive tract (UADT) are most prevalent cancers amongst Indian men and account for at least one third of total cancer deaths 1, 2 . Advancement in early detection and treatment has improved overall disease management, however, a 3-7% annual risk of development of a second/ multiple primary neoplasm(s) (MPN) among the survivors of early stage UADT cancer poses an additional threat in terms of morbidity and mortality 3-5 . Convincing evidence from case-control studies analyzing cumulative polymorphisms suggests aberrant geneenvironment interactions to be an important etiological factor in the genesis of MPN 6, 7 . Collective effect of genetic polymorphisms and tobacco habit has been studied earlier in the lab and an association of polymorphisms in genes falling in major carcinogenesis pathways (SULT1A1-Arg 213 His, hOGG1-Ser 326 cys, BRCA2-Asn 372 His, mEH-Try 113 His, XRCC1-Arg 280 His) was observed in MPN patients with tobacco habit 8 . Therefore it was considered important to validate the findings of genotyping study in at least a subset of the patients by phenotypic assays and develop a genotype-phenotype correlation. We hypothesized that MPN may be a manifestation of differences in the polygenic susceptibility to carcinogens between different individuals. Hence if genetic polymorphisms in important carcinogenesis pathways like DNA damage/repair, apoptosis, carcinogen metabolism and cell cycle regulation have functional significance then there may be a correlation between genotype and intermediate phenotypes like defect in DNA repair, apoptosis or cell cycle regulation. One of the prime requisites for undertaking such studies is continuous supply of starting parent material. Being a spontaneous replicating source, Epstein Barr Virus (EBV) transformed Lymphoblastoid cell lines (LCLs) very well fulfil the requirement of constant supply of starting material for a variety of assays, sparing the need of resampling 9 . Using these LCLs as a model system we have compared the difference in the phenotypic response between MPN patient and control group in terms of percent cell death, DNA damage/repair, transcriptional response and cell cycle regulation after γ-radiation and Benzo[a]pyrene-diol-epoxide (BPDE, a tobacco specific carcinogen) exposure in vitro. In addition we have genotyped 22 candidate single nucleotide polymorphisms (SNPs) in candidate genes involved in DNA repair, apoptosis, cell 2
Page 1: Genotype-Molecular Phenotype Correl
Page 7 and 8: CONTENTS Page No Synopsis 1 List of
Page 9: Synopsis
Page 13 and 14: Synopsis Materials and Methods: 1.
Page 15 and 16: Synopsis loading control. PCR produ
Page 17 and 18: Synopsis 7.1 Percent cell death aft
Page 19 and 20: Synopsis slides were then dried and
Page 21 and 22: Synopsis range 41.77% - 90.95% at 5
Page 23 and 24: Synopsis 5. Genotyping of genes: Ge
Page 25 and 26: Synopsis G2/M arrest and delaying e
Page 27 and 28: Synopsis may have an important bear
Page 29 and 30: Synopsis References: 1. Bobba, R.;
Page 31 and 32: Abbreviations MOPS : 3-(N-morpholin
Page 33 and 34: List of Figures Fig. 28: Percent re
Page 35 and 36: Chapter 1 Introduction
Page 37 and 38: Introduction In previous studies fr
Page 39 and 40: Introduction important carcinogenes
Page 41 and 42: Review of Literature The yet undeci
Page 43 and 44: Review of Literature Fig. 2: Incide
Page 45 and 46: Review of Literature sustained angi
Page 47 and 48: Review of Literature 2.6 Genotype p
Page 49 and 50: Review of Literature 2.7.2 Biologic
Page 51 and 52: Review of Literature Table 1: Steps
Page 53 and 54: Review of Literature future analysi
Page 55 and 56: Review of Literature Inefficient ce
Page 57 and 58: Review of Literature XRCC1 (X-ray r
Page 59 and 60: Review of Literature 2.9.2 Gene inv
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Review of Literature MPO gene polym
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Review of Literature important role
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Materials and Methods Source of che
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Materials and Methods Method: EBV-t
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Materials and Methods Immunophenoty
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Materials and Methods with 500 μl
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Materials and Methods cDNA it can b
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Materials and Methods Cobalt-60 iso
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Materials and Methods specific bind
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Materials and Methods h half of the
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Materials and Methods phenol and th
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Materials and Methods 7. Filter ste
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Materials and Methods viz. EXO-SAP
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Materials and Methods Table 2: List
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Materials and Methods SMAD6 AREG HM
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Materials and Methods 3.9 Effect of
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Materials and Methods fresh eppendo
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Materials and Methods Power SYBR Gr
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Results Objective 1 To generate EBV
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Results Fig. 8: Frequency of second
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Results Table 6: Demographics of he
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Results Fig. 10: Cell population do
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Results Fig. 12: Cell surface marke
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Results 4.1.5 Expression of ATM gen
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Results Objective 2 To compare the
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Results Fig. 17: Standardization of
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Results Fig. 19: Standardisation of
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Results The cells were incubated fu
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Results 4.2.3.1 Percent G2 delay af
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Results 4.2.3.2 Effect of BPDE expo
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Results Fig. 29: Comparison of perc
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Results Fig. 31: Percent cell death
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Results normalization with baseline
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Results 4.2.5.2 Real time PCR valid
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Results homozygous wild-type, heter
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Results Fig. 36b: Electrophoresis o
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Results Fig. 37a: Representative el
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Results Table 13: Consolidated geno
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Results LCL Genes NAT2(1) NAT2(2) N
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Results Fig. 38: Distribution of Ge
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Results Fig. 39: Various combinatio
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Results Table 14: Genotype-phenotyp
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Discussion Introduction Squamous ce
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Discussion stimulated lymphocytes w
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Discussion H2AX is currently the mo
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Discussion or G2/M phase arrest. As
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Discussion differential expression
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Discussion DNA repair genes and MPN
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Chapter 6 Summary and Conclusions
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Summary and Conclusions Statistical
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Bibliography 1. Dikshit, R.; Gupta,
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Bibliography 43. Lei, D.; Sturgis,
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Bibliography 76. Fry, R. C.; Svenss
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Bibliography 115. Kote-Jarai, Z.; M
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Bibliography 149. Hashibe, M.; Bren
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Bibliography 183. Bergamaschi, D.;
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Bibliography 216. Bondy, M. L.; Wan
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Indian J Med Res 135, June 2012, pp
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822 INDIAN J MED RES, JUNE 2012 mar
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824 INDIAN J MED RES, JUNE 2012 (h)
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826 INDIAN J MED RES, JUNE 2012 Tab
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828 INDIAN J MED RES, JUNE 2012 lik
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CASE REPORT Eben L. Rosenthal, MD,
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FIGURE 2. Chromosomal breakage anal
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FA along with ataxia telangiectasia
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LIFE09200604007 Tabish - Homi Bhabha National Institute

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