LIFE09200604007 Tabish - Homi Bhabha National Institute

More documents

Recommendations

Info

Introduction most of the genetic variants contribute to relatively small risk. Therefore studies investigating genotypic effect of a panel of polymorphisms in multiple pathways regulating critical cellular functions are of immense importance. The unique feature of our study is that potential susceptibility of 22 SNPs, together or in combinations, has been identified. Genotyping involved documentation of 22 SNPs in candidate genes and calculating a Genotype Score (G Score) from the number of variant alleles. Subsequently, the G Score was correlated with phenotypic effects using Epstein Barr Virus (EBV) transformed lymphoblastoid cell lines (LCLs) generated from UADT MPN patients (n=20) and healthy individuals (n=10) as controls. Using these LCLs, the effect to exposure to γ-radiation or Benzo[a]pyrenediol-epoxide (BPDE) was studied. Intermediate phenotypes including transient arrest in cell cycle, proper function of DNA damage repair machinery and activation of cell death program upon exposure to genotoxic agents are known to be disrupted in variety of cancers and are associated with neoplastic evolution. We assumed that individuals with inherited defects in cell cycle control, apoptosis, and/or DNA repair, owing to inter-individual difference at the genetic level, might be susceptible to UADT MPN development. We have correlated these genotypic and phenotypic measurements to investigate their potential relationships to have a better understanding of pathogenesis of UADT MPN. Thus the study has the potential to identify a signature of polymorphisms and their probable phenotypic effects associated with UADT MPN etiology. The potential susceptibility markers could assist in early detection of patients who are genetically predisposed to develop second primary neoplasia. These susceptibility markers could increase the possibility of identifying cohort of clinically relevant patients that can be accrued for chemo prevention studies. Hypothesis Our hypothesis is that UADT MPN is a manifestation of inter individual difference in the susceptibility to carcinogen exposure. Individuals carrying multiple, less efficient variant forms of gene products involved in DNA damage/repair, apoptosis, carcinogen metabolism and cell cycle regulation, are possibly at a greater risk of having compromised/dysfunctional intermediate phenotypes like DNA damage/repair, apoptosis or cell cycle regulation after genotoxic exposure, and hence an increased UADT MPN risk. Therefore, if genetic polymorphisms in genes falling in 27
Introduction important carcinogenesis pathways have functional significance, then a correlation between genotype and intermediate phenotypes needs to be unraveled. 1.3 Aim and objectives The aim of present study was to develop a correlation between genotype - performed by evaluating SNPs in candidate genes involved in DNA repair, apoptosis, cell cycle regulation and carcinogen metabolism, with the phenotype - measured as defect in DNA repair, apoptosis or cell cycle regulation, using lymphoblastoid cell lines established from MPN patients and healthy controls following genotoxic exposure in vitro. The objectives of the study were as follows: 1. To generate EBV LCLs from peripheral blood lymphocytes (PBLs) isolated from MPN patients and healthy controls - an established in vitro model for genetic studies. 2. To compare the response of MPN patients with tobacco habits and appropriate controls in vitro, to the exposure to DNA damaging agents such as γ-radiations and Benzo[a]pyrene-diol-epoxide (BPDE, a tobacco specific carcinogen), by assessing DNA damage and repair, cell cycle profiling, apoptosis and global gene expression profiling. 3. To genotype selected candidate genes involved in DNA repair, carcinogen metabolism, apoptosis and cell cycle regulation. 4. To establish a correlation between the phenotype (e.g., poor DNA repair capacity, apoptosis) with the genotype (e.g., polymorphisms in the genes). 28
Page 1: Genotype-Molecular Phenotype Correl
Page 7 and 8: CONTENTS Page No Synopsis 1 List of
Page 9 and 10: Synopsis
Page 11 and 12: Synopsis Genotype-Molecular Phenoty
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: Introduction In previous studies fr
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
Page 61 and 62: Review of Literature MPO gene polym
Page 63 and 64: Review of Literature important role
Page 65 and 66: Materials and Methods Source of che
Page 67 and 68: Materials and Methods Method: EBV-t
Page 69 and 70: Materials and Methods Immunophenoty
Page 71 and 72: Materials and Methods with 500 μl
Page 73 and 74: Materials and Methods cDNA it can b
Page 75 and 76: Materials and Methods Cobalt-60 iso
Page 77 and 78: Materials and Methods specific bind
Page 79 and 80: Materials and Methods h half of the
Page 81 and 82: Materials and Methods phenol and th
Page 83 and 84: Materials and Methods 7. Filter ste
Page 85 and 86: Materials and Methods viz. EXO-SAP
Page 88 and 89:
Materials and Methods Table 2: List
Page 90 and 91:
Materials and Methods SMAD6 AREG HM
Page 92 and 93:
Materials and Methods 3.9 Effect of
Page 94 and 95:
Materials and Methods fresh eppendo
Page 96 and 97:
Materials and Methods Power SYBR Gr
Page 98 and 99:
Results Objective 1 To generate EBV
Page 100 and 101:
Results Fig. 8: Frequency of second
Page 102 and 103:
Results Table 6: Demographics of he
Page 104 and 105:
Results Fig. 10: Cell population do
Page 106 and 107:
Results Fig. 12: Cell surface marke
Page 108 and 109:
Results 4.1.5 Expression of ATM gen
Page 110 and 111:
Results Objective 2 To compare the
Page 112 and 113:
Results Fig. 17: Standardization of
Page 114 and 115:
Results Fig. 19: Standardisation of
Page 116 and 117:
Results The cells were incubated fu
Page 118 and 119:
Results 4.2.3.1 Percent G2 delay af
Page 120 and 121:
Results 4.2.3.2 Effect of BPDE expo
Page 122 and 123:
Results Fig. 29: Comparison of perc
Page 124 and 125:
Results Fig. 31: Percent cell death
Page 126 and 127:
Results normalization with baseline
Page 128 and 129:
Results 4.2.5.2 Real time PCR valid
Page 130 and 131:
Results homozygous wild-type, heter
Page 132 and 133:
Results Fig. 36b: Electrophoresis o
Page 134 and 135:
Results Fig. 37a: Representative el
Page 136 and 137:
Results Table 13: Consolidated geno
Page 138 and 139:
Results LCL Genes NAT2(1) NAT2(2) N
Page 140 and 141:
Results Fig. 38: Distribution of Ge
Page 142 and 143:
Results Fig. 39: Various combinatio
Page 144 and 145:
Results Table 14: Genotype-phenotyp
Page 146 and 147:
Discussion Introduction Squamous ce
Page 148 and 149:
Discussion stimulated lymphocytes w
Page 150 and 151:
Discussion H2AX is currently the mo
Page 152 and 153:
Discussion or G2/M phase arrest. As
Page 154 and 155:
Discussion differential expression
Page 156 and 157:
Discussion DNA repair genes and MPN
Page 158 and 159:
Chapter 6 Summary and Conclusions
Page 160 and 161:
Summary and Conclusions Statistical
Page 162 and 163:
Bibliography 1. Dikshit, R.; Gupta,
Page 164 and 165:
Bibliography 43. Lei, D.; Sturgis,
Page 166 and 167:
Bibliography 76. Fry, R. C.; Svenss
Page 168 and 169:
Bibliography 115. Kote-Jarai, Z.; M
Page 170 and 171:
Bibliography 149. Hashibe, M.; Bren
Page 172 and 173:
Bibliography 183. Bergamaschi, D.;
Page 174 and 175:
Bibliography 216. Bondy, M. L.; Wan
Page 176 and 177:
Indian J Med Res 135, June 2012, pp
Page 178 and 179:
822 INDIAN J MED RES, JUNE 2012 mar
Page 180 and 181:
824 INDIAN J MED RES, JUNE 2012 (h)
Page 182 and 183:
826 INDIAN J MED RES, JUNE 2012 Tab
Page 184 and 185:
828 INDIAN J MED RES, JUNE 2012 lik
Page 186 and 187:
CASE REPORT Eben L. Rosenthal, MD,
Page 188 and 189:
FIGURE 2. Chromosomal breakage anal
Page 190 and 191:
FA along with ataxia telangiectasia
show all

LIFE09200604007 Tabish - Homi Bhabha National Institute

You also want an ePaper? Increase the reach of your titles

Delete template?

Save as template?