... Introduction 1. INTRODUCTION

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2 ... Introduction 1. INTRODUCTION The field of life sciences has ever been a valuable ensemble of extremely gifted biologists deeply devoted to the cause of unravelling the challenging mysteries of human life, its origins, its progress and its various states- the normal and the diseased ones. Right from the beginning of mankind's quest to know the unknown, enthusiasts in the garb of scientists have been craving and toiling to zero in on the driving force of life which is key to the transfer of information (both good and bad) from one generation to its successors. After centuries of fruitful inquisition and relentless experimentation had culminated in the discovery of genes as the perfect hereditary material, focus then shifted towards unearthing the key factor which could serve as causative agent for some diseases and as markers for others - as a genetic marker which could differentiate human beings right from population to the individual levels. Single Nucleotide Polymorphisms (SNPs, popularly called 'snips') are considered best markers inhabiting the genome, due to their wide prevalence, easier automation for scoring, highly polymorphic & biallelic nature, co-dominance, easy reproducibility and their presence in both exonic as well as intronic regions of the genes, furnishing large sets of markers near or within the locus of interest. SNPs are the most widespread and abundant types of variations in the human genome occurring within vital genes like regulatory regions, drug responding elements, drug metabolizing enzymes, drug transporters, drug receptors, proton pumps and noncoding regions linked to disease susceptibility (Brookes, 1999;Zhang et al., 2005a). Because of their high abundance, low mutation rate and accessibility to highthroughput genotyping, SNPs are highly preferred for association studies on complex diseases worldwide including susceptibility towards cancers (Syvanen et al., 1999). Investigations have projected that the known high risk cancer alleles are rare and account for less than 5% of all cancer cases, (King et al., 1983;Kolodner, 1995;Marcus et al., 1996;Szabo and King, 1997;Renan, 1997) while on the other hand, in the remaining 95% of the cases, interplay between low and medium penetrance genes and exposure to carcinogens and lifestyle factors carry a high cumulative risk towards cancer (King et al., 1983;Kelsey, 1993;Pierce et al., 1996;Talamini et al., 1997;Whiteman et al., 1997). The ability to metabolize carcinogens or procarcinogens, repair DNA damage and control cell signalling and the cell cycle are major examples of low and medium-penetrance genes fundamental to homoeostasis, defects in which contribute to cancers (Borish et al., 1985;Parkin et al., 1993;Blot et 1

3 ... Introauction al., 1996). Therefore, the degree of susceptibility to cancers is hypothesised to be the final product of a mishmash of high-risk genetic polymorphic variants or SNPs in a subset of medium and low penetrance genes like DNA repair genes. Consequently, the existence of SNPs as exemplified by cancer susceptibility alleles have emerged as key factor in determining an individual's risk of cancer which, even in the absence of the highly penetrant variant cancer-associated alleles, may increase the degree of susceptibility towards cancers a few fold thus having a major impact on the population incidence of cancer (Shen et al., 1998). Most of the population cancer risk is thus the result of the interaction of susceptibility alleles at a series of loci and moderate or even low level exposure to carcinogenic agents. Modem epidemiological studies have therefore, incorporated such genetic variations, having alleles with small individual effect but existing at high frequency in the population, into models concerning assessment of genetic association with complex diseases (Mohrenweiser and Jones, 1998). DNA repair genes are gammg widespread attention throughout the scientific initiatives around the world as low penetrance candidate genes for genetic association studies on human cancers owing to their critical role in the maintenance of genome integrity in both somatic and germinal cells through the minimisation of replication errors, removal of DNA damage and reduction of deleterious rearrangements arising through aberrant recombination. DNA repair, therefore, has a critical role in protecting the genome against mutations that lead to cancer and/or inherited genetic diseases which therefore implies that DNA repair plays a pivotal role in cancer biology whereby individuals are at a very high risk of cancer owing to the inheritance of polymorphic variants in genes of DNA repair and metabolism (Ma et al., 1995;Tlsty et al., 1995;Radman et al., 1995;Bohr, 1995). Therefore, mutations in these candidate genes (DNA repair genes) in the form of single nucleotide polymorphisms are suggested to be involved in the modulation of DNA repair capacity and thus their relatedness to cancer risk is being increasingly explored. (Spitz et al., 2003;Hung et al., 2005). Sequence variants in DNA repair genes are thought to modulate DNA repair capacity and consequently are suggested to be associated with altered cancer risk by modifying an individual's susceptibility to cancer (Spitz et al., 2003). Polymorphisms in DNA repair genes have been identified and reported to be associated with various cancers including Squamous Cell Carcinomas of the Head and Neck (SCCHN) and Breast cancer (Sturgis et al., 1999a;Sturgis et al., 1999b;Olshan 2

4 I ntroauction et al., 2002;Shen et al., 2002a;Shen et al., 2002b;Spitz et al., 2003;Cho et al., 2003;Tae et al., 2004;Benhamou et al., 2004;Shen et al., 2005b). There are over 130 human DNA repair genes, grouped into six major DNA repair pathways which include Direct damage reversal pathways such as Base-excision repair (BER) pathway involving the genes apurinic/apyrimidinic endonucleases (APEXl), DNA glycosylases, Flap endonuclease (FENl), Proliferating cell nuclear antigen (PCNA), X-ray repair crosscomplementing group 1 (XRCCl), etc; Nucleotide excision repair (NER) pathway involving excision repair cross-complementing rodent repair deficiency, complementation group 2 (ERCC2/XPD), etc; Double-strand break (DSB) repair pathway and Mismatched repair (MMR) pathway (Yu et al., 1999;Wood et al., 2001;Mohrenweiser et al., 2003). Base excision DNA repair (BER) is the main proactive DNA repair pathway and the most important cellular defense mechanism that has evolved to avert the deleterious effects of the most frequent damaged or inappropriate bases in DNA (Lindahl, 1974;Krokan et al., 2000;Fortini et al., 2003;Fan and Wilson, Ill, 2005). Polymorphisms in base excision repair genes have been investigated for association with various cancers across a wide range of world populations (Li et al., 2007;De et al., 2007;Bemdt et al., 2007;Huang et al., 2007). However, similar literatures on the Indian population subgroups are relatively rare (Ramachandran et al., 2006;Pachouri et al., 2007;Shekari et al., 2008;Sreeja et al., 2008) although incidences of cancers have been found to differ between populations of different geographic and ethnic origin. Furthermore, quite intriguingly, uptil now Squamous Cell Carcinomas of the Head and Neck (SCCHN) had been almost absolutely overlooked as a possible venture for cancer association studies both among literatures worldwide and India in particular overlooking its high incidence across the world (Parkin et al., 1993) and within the Indian subcontinent (Liu et al., 1998). The need of the present hour is to demonstrate the involvement of genetic differences in such vital candidate genes like DNA repair genes, apart from the known environmental factors, in the variable incidence of disease (including SCCHN and breast cancer) prevalence among various diverse subpopulations of the world, especially India. Under neutrality and in the absence of mutation, genetic variation across populations is expected to be cjetermined by genetic drift only, which in tum is determined by the demographic history of populations. A priori, all loci in the genome have the same expected degree of differentiation, which may be used to detect the action of natural 3

5 Introduction selection (Cavalli-Sforza, 1966a;Cavalli-Sforza, 1966b;Lewontin and Krakauer, 1973;Fullerton et al., 2002a;Fullerton et al., 2002b). The diversification of allelic frequencies and incidences of different cancers between populations of different geographic and ethnic origin could have been caused by neutral demographic mechanisms, such as drift or migrations, population bottlenecks, and founder effects accompanying the out-of-africa expansion (Mountain and Cavalli-Sforza, 1994;Lahr and Foley, 1998). The Human genome project (HGP), the Hapmap project and other SNP projects including the National Center for Biotechnology Information (NCBI)'s dbsnp and The SNP Consortium (TSC) and smaller SNP mapping projects like the Seattle SNPs and Asian SNP project of Vita Genomics have been involved in the identification and cataloguing of SNPs in a major way through creation and maintenance of SNP databases (Holden, 2002;The International HapMap Consortium, 2003;Greenhalgh, 2005;Tanaka, 2005;Shih-Hsin et al., 2007). However, in all these enterprises, the huge diversity of the Indian population comprising of more than a billion people (116th of the world population), covering 4693 communities with several thousands of endogamous groups, 325 functioning languages and 25 scripts (Indian Genome Variation Consortium, 2005), encompassing the largest human genetic diversity among comparable global regions, second only to Africa (Majumder, 1998;Majumder, 2001) have been noticeably ignored. Only a fraction of SNPs reported in public databases available at present would be relevant for Indian populations. Evidently, to address the questions related to ethnic diversity, migrations, founder populations, predisposition to complex disorders or pharmacogenomics, there is a need to understand the diversity and relatedness at the genetic level in such a diverse population. like India. Therefore, the present research initiative was undertaken to first, identify and validate SNPs among representative subpopulations of India within some selected genes involved primarily in the DNA repair pathway, with focus on the base excision repair genes, using a two-stage design so as to get the complete list of functional SNPs relevant to the Indian population. Reliable knowledge on which base excision repair sequence variants are associated with cancer risk would help to elucidate the disease mechanism. Therefore, subsequent to SNP detection and validation, selected nonsynonymous SNPs located in the exonic region of some DNA repair genes were further analyzed using Polymerase Chain Reaction followed by Restriction Fragment 4

6 ... Introduction Length Polymorphism (PCR-RFLP) and DNA sequencing analysis for association with the risk of Squamous Cell Carcinomas of the Head and Neck (SCCHN) and Breast cancer in a subpopulation cluster-matched (Indo-Europeans + Caucasoids) case-control based genetic association study among north Indian subpopulations. Gene expression analysis was performed to assess the overall change in expression of the selected genes among SCCHN patients with respect to normal healthy controls. Additionally, DNA ploidy status and Y chromosome microdeletion status were investigated among SCCHN samples for possible alterations at the chromosomal level. Eight genes, mostly involved in the human DNA repair mechanism, viz. Apurinic/apyrimidinic Exonuclease/endonuclease (APEX!), Flap Endonuclease 1 (FENl), Proliferating Cell Nuclear Antigen (PCNA), DNA ligase I (LIGl), X-ray repair Cross Complementing in Chinese Hamster 1 (XRCC 1), human Oxo-Guanine Glycosylase 1 (hogg 1 ), Excision Repair Cross-Complementing Rodent Repair Deficiency, Complementation Group 2 (ERCC2) and the gene 2',3'-Cyclic Nucleotide 3' Phosphodiesterase (CNP), were selected for the study mainly on the basis of their relevance as functional and positional candidates in many cancers and complex diseases. This is the first large-scale comprehensive study of DNA repair genes on the Indian population, in terms of the samples studied, both for population genetic studies and genetic epidemiological studies, and the genes under investigation. Therefore, the results and inferences drawn from this study may have wide-reaching implications. The findings of this endeavour will provide an Indian subpopulation-specific resource on the polymorphic variants of the genes under study and shall be instrumental in offering further insights into the complex analysis of individual and population risk estimation thus having positive implications towards cancer screening, prevention, treatment and management. Biological markers of structural and functional damage to DNA can prove useful for identifying persons at risk of developing cancer, assist in prognostic predictions and provide baseline to assess the efficacy of potential therapeutic interventions for prevention, treatment and management of diseases. 5