Association between p 53 Gene Variants and Oral Cancer Susceptibility in Population from Gujarat , West India

Oral cancer has emerged as a major health problem worldwide. In India, 69,820 new cases and 47,653 deaths were registered in 2008 (Ferlay et al., 2010). This high death rate is mainly due to problems in clinical management of oral cancer i.e. lack of early detection and high incidence of local regional recurrence. Epidemiological studies revealed that tobacco chewing is an important risk factor for oral cancer development in India. Moreover, differences in the clinico-pathological and molecular pathological profile in the tobacco (smoking) and alcohol associated oral cancers in the western countries and tobacco chewing associated oral cancers, mainly in the Indian subcontinent have been reported (Paterson et al., 1996). Although, tobacco use plays a major role in the etiology of oral cancer, only a fraction of tobacco users develop this disease, suggesting that genetic susceptibility may contribute to carcinogenic mechanisms in the population. In addition, complex interactions between these genetic and environmental factors may play a major role in oral carcinogenesis (Imyanitov et al., 2004). This susceptibility is modulated by polymorphisms in genes encoding DNA repair proteins, cell cycle control proteins and metabolic enzymes (Wu et al., 2002).


Introduction
Oral cancer has emerged as a major health problem worldwide.In India, 69,820 new cases and 47,653 deaths were registered in 2008 (Ferlay et al., 2010).This high death rate is mainly due to problems in clinical management of oral cancer i.e. lack of early detection and high incidence of local regional recurrence.Epidemiological studies revealed that tobacco chewing is an important risk factor for oral cancer development in India.Moreover, differences in the clinico-pathological and molecular pathological profile in the tobacco (smoking) and alcohol associated oral cancers in the western countries and tobacco -chewing associated oral cancers, mainly in the Indian subcontinent have been reported (Paterson et al., 1996).Although, tobacco use plays a major role in the etiology of oral cancer, only a fraction of tobacco users develop this disease, suggesting that genetic susceptibility may contribute to carcinogenic mechanisms in the population.In addition, complex interactions between these genetic and environmental factors may play a major role in oral carcinogenesis (Imyanitov et al., 2004).This susceptibility is modulated by polymorphisms in genes encoding DNA repair proteins, cell cycle control proteins and metabolic enzymes (Wu et al., 2002).
1 Biochemistry Research Division, 3 The Gujarat Cancer and Research Institute, Ahmedabad, 2 Department of Biochemistry, The M. S. University of Baroda, Vadodara, Gujarat, India *For correspondence: prabhudas_p@hotmail.com

RESEARCH ARTICLE
Association between p53 Gene Variants and Oral Cancer Susceptibility in Population from Gujarat, West India Kinjal R Patel 1,3 , Bhairavi N Vajaria 1,3 , Rasheedunnisa Begum 2 , Franky D Shah 1,3 , Jayendra B Patel 1,3 , Shilin N Shukla 3 , Prabhudas S Patel 1,3 * p53 gene encode important tumor suppressor protein, plays a critical role in regulating cell cycle arrest, apoptosis and DNA repair.Inactivation of p53 is frequently observed in human cancers.In addition, numerous sequence variations are present in the p53 gene.Most of these variations are intronic and have no cancer related biological consequences (Whibley et al., 2009).However, two intronic polymorphisms in p53 gene; 16 bp duplication in intron 3 and G>A transition in intron 6 have been suggested to affect the levels of p53 gene expression as well as its function (Chumakov and Jenkins, 1991;Lazar et al., 1993).Introns were originally believed to be non functional because they do not code for proteins, still they have been implicated in regulation of gene expression and DNA -protein interactions (Beenken et al., 1991;Lozano and Levine, 1991;Mattick, 1994;Shamsher and Montano, 1996).Sequence variations in introns may affect function of proteins and hence cancer risk.Studies of the association between certain types of cancer and intron 3 and intron 6 polymorphisms have showed conflicting results (Wang-Gohrke et al., 1999;Gallì et al., 2009;Hu et al., 2010).
Of the 19 exonic polymorphisms, 11 polymorphisms in p53 are non-synonymous, resulting in an amino-acid change and only four of these have been validated.Out of these, there are sufficient molecular evidences for two polymorphisms (Pro47Ser and Arg72Pro) suggesting their role in functional change of the p53 protein.The rest two polymorphisms (V217M and G360A) have not been associated with an altered cancer risk till date (Whibley et al., 2009).The codon 47 polymorphism results in proline to serine substitution is rare whereas arginine to proline substitution in codon 72 is common (Felley-Bosco et al., 1993;Beckman et al., 1994).The relationship between the polymorphism in codon 72 and oral cancer has been studied but the results are inconsistent (Zhuo et al., 2009).
It has been speculated that both ethnic back-ground and lifestyle differences contribute to the differences in predisposing genetic factors to cancer development (Chakrabarti et al., 2001).This fact is very crucial for country like, India where wide variations in ethnicity and life style exist.There is a single study from east region of India, analyzed association between these three polymorphisms and oral cancer till date (Mitra et al., 2005).It has also been reported that the frequencies of the polymorphisms tend to differ among Indians and this is mainly due to differences in ethnicity (Mitra et al., 2003).Therefore, the present study aimed to analyze whether the polymorphisms in exon 4, intron 3 and intron 6 of the p53 gene are associated with differential oral cancer risk in Gujarati population from West India where incidence of oral cancer is dramatically high.

Subjects
The study was approved by the Institutional Review Board of The Gujarat Cancer and Research Institute, Ahmedabad, India and informed consent was obtained from all the subjects.Histopathologically confirmed and previously untreated oral cancer patients from the out patient's department of The Gujarat Cancer and Research Institute were enrolled.The controls were volunteer blood donors, genetically unrelated to patients and had no previous history of cancer.Both subjects should not have any major illness in past.A detailed questionnaire was administered to all participants for gathering information on age, sex, occupation and tobacco habits.Both patients and controls were found to have tobacco habits such as smoking and chewing in different forms.Habit of tobacco chewing was more prevalent in this population.

Methods
Five milliliters of blood was drawn by venipuncture from all the subjects.WBCs were separated and stored at -80 0 C until analysis.DNA isolation was done using DNA blood mini isolation kit (Qiagen, USA) following manufacturer's instructions and stored at -20 0 C until analysis.
Genotyping was performed by Polymerase Chain Reaction -Restriction Fragment Length Polymorphism (PCR-RFLP) method.Intron 3 and exon 4 were amplified together within which the polymorphisms fall, using primers as mentioned previously (Mitra et al., 2003).The PCR products were resolved by 6% polyacrylamide gel electrophoresis (PAGE) and visualized after staining with ethidium bromide.16 bp duplication in intron 3 was directly interpreted from 6% PAGE analysis of the PCR products.Resulting PCR products were either 432 or 448 bp DNA fragments depending on the absence or presence of 16 bp duplication in intron 3 in template genomic DNA.Homozygotes for the absence of duplication (A1/A1) produced band of 432 bp DNA fragment; heterozygotes produced both the bands (A1/A2); homozygotes for the presence of duplication (A2/A2) produced band of 448 bp DNA fragment (Figure 1).
Further, aliquots of the same PCR products were subjected to restriction digestion with BstUI (Fermetas, USA).The products were separated by 6% PAGE and visualized after staining with ethidium bromide.There were four DNA fragments of different sizes.Pro/Pro homozygotes produced band of 448 bp DNA fragment with the intron 3 duplication or produced band of 432 bp DNA fragment without the intron 3 duplication; Pro/ Arg heterozygotes produced bands of 448 bp/432 bp, 246 bp/230 bp (depending upon the absence or presence of intron 3 duplication) and 202 bp DNA fragments.Arg/Arg homozygotes produced two bands of 246 bp and 202 bp DNA fragments with the intron 3 duplication or produced two bands of 230 bp and 202 bp DNA fragments without intron 3 duplication (Figure 2).
Intron 6 was amplified separately using primers as described previously (Mitra et al., 2003).The 913 bp PCR products were digested with NciI (Fermentas, USA), separated by 6% PAGE and visualized after staining with ethidium bromide.Homozygotes for the absence of NciI restriction site produced band of 563 bp DNA fragment (A/A); heterozygotes for the NciI restriction site produced bands of 563 bp, 286 bp and 277 bp DNA fragments (G/A); homozygotes for the presense of NciI restriction site produced two bands of 286 bp and 277 bp  doi.org/10.7314/APJCP.2013.14.2.1093 p53 Polymorphisms andOral Cancer Risk in Gujarat, India DNA fragments (G/G).All the three types of individuals contained band of 350 bp DNA fragment due to presence of a nonpolymorphic NciI site in the amplicon (Figure 3).Reproducibility of the assay was confirmed by repeating the samples.

Statistical analysis
The cases as well as controls were compared for genotype frequencies with the expected frequencies by the Hardy-Weinberg Equilibrium (HWE) by goodnessof-fit χ 2 test.Pearson's χ 2 test was performed to compare genotypic and allelic distributions between cases and controls.Odds ratios (ORs) and 95% Confidence Intervals (CIs) were calculated for the risk estimation of oral cancer development.The highest proportion of a homozygous genotype at any particular p53 locus in the control group was considered as referent genotype to estimate risk associated with these polymorphisms (Mitra et al., 2005).Linkage disequilibrium analysis was performed for the study groups.The disequilibrium parameter (D) and the Pearson's χ 2 values for statistical significance were calculated.Statistical analysis was performed using SPSS (version 15).Frequencies of the pair-wise and extended haplotypes resulting from 3 polymorphisms in the p53 gene were estimated using PHASE software (Stephens et al., 2001).p value<0.05 was considered to be statistically significant.

Distribution of genotypes and allele frequencies among subjects
The genotype and allele frequency distribution of 16 bp duplication in intron 3, Arg72Pro in codon 72 of exon 4 and G>A transition in intron 6 among cases and controls is provided in Table 2. Controls and cases showed good fit to HWE for all three loci.There was marginal difference in the distribution of Arg72Pro genotypes at exon 4 locus between cases and controls (χ 2 =5.19, p=0.075).However, no significant difference in the distribution of intron 3 and intron 6 genotypes between cases and controls was observed.Controls represent more Arg/Pro genotype compared to cases.The frequency of Arg and G allele was higher in cancer patients as compared to the controls.

Risk estimation of oral cancer associated with p53 genotypes
Table 3 depicts risk of oral cancer development associated with these polymorphisms.16 bp duplication in intron 3 and G>A transition in intron 6 did not show any association with oral cancer risk.The Arg/Pro genotype at

Determination of genotype combinations and risk of oral cancer
Present study also determined the genotype distribution of all the three polymorphisms in combination and risk associated with these combined genotypes (Table 4).Combination of genotypes for two loci of p53 gene revealed that A1/A1 and A1/A2 genotypes of intron 3 polymorphism in combination with the Arg/Pro genotype at codon 72 significantly protect from oral cancer development (OR=0.5;95%CI:0.2-1.0;p=0.044,OR=0.4; 95%CI:0.2-1.0;p=0.049, respectively).Individuals who were heterozygous at codon 72 and intron 6 locus showed marginal protection towards oral cancer development (OR=0.4;95%CI:0.2-1.0;p=0.057).Also, combination of Arg/Pro and G/G genotypes at codon 72 and intron 6 locus, respectively showed marginal protection from oral cancer development (OR=0.5;95%CI:0.2-1.1;p=0.070).Other combinations of intron 3 and intron 6 genotypes did not confer association with the risk of oral cancer development.When genotype combination was made for all the three polymorphisms of p53 gene, heterozygous genotypes at all the three loci in combination was found to have protective effect from oral cancer development (OR=0.4;95%CI:0.2-1.1;p=0.069).Further, Arg/Pro at codon 72, A1/A1 at intron 3 and G/G at intron 6 genotypes in combination also showed marginal protection from oral cancer development (OR=0.5;95%CI:0.2-1.1;p=0.077).Individuals who were carriers of proline allele at codon 72 of exon 4 in combination with A1 and G allele at intron 3 and intron 6, respectively were significantly protected from oral cancer development (OR=0.5;95%CI:0.3-1.0;p=0.047).We also explored the role of genetic and environmental factors in combination which could further modify the risk of oral cancer.The results suggested that tobacco habituates with Pro/Pro and Pro/Arg genotypes at exon 4 locus were protected from oral cancer development (OR=0.6;95%CI: 0.3-1.2;p=0.16) (Table 5).

Estimation of haplotype frequency and risk of oral cancer
Pair-wise haplotype frequencies for all possible combinations of these three p53 polymorphisms were also estimated.The frequencies of pair-wise haplotypes did not differ significantly between controls and cases (Table 6).Linkage disequilibrium analysis was also performed to examine the linkage among the three loci (intron 3-exon 4-intron 6).There was significant linkage disequilibrium between these three loci in both the studied groups (Table 6).
We also determined the extended p53 haplotypes (3 loci haplotypes) in controls and cases which are reported in the order of intron 3-exon 4-intron 6 (Table 7).For haplotype analysis, absence of 16 bp duplication at intron 3, Pro at exon 4 and A at intron 6 is defined as allele 1 at each of the three loci, respectively.The predominant haplotype consisted of the highly prevalent alleles (1-2-2).It was followed by 1-1-2 and 2-1-1, respectively.1-1-1 haplotype was completely absent in cases and present in controls whereas 2-1-2 haplotype was more frequent in cases compared to the controls.There was no significant difference in the distribution of haplotypes between cases and controls (Table 8).Risk associated with various extended p53 haplotypes are presented in table VIII.2-1-2 haplotype showed 3 times risk of oral cancer development.

Discussion
Present study evaluated the relationship of three polymorphisms i.e. 16 bp duplication in intron 3, Arg72Pro in exon 4 and G>A transition in intron 6 of p53 gene in order to predict the oral cancer risk in West Indian population.The controls as well as the cases belonged to same ethnicity and were from the same geographic location.An allele frequency of Arg72Pro polymorphism has been reported to vary with respect to ethnicity and latitude (Nagpal et al., 2002).The allele frequency of proline at codon 72 varies from 0.12-0.69worldwide (Francisco et al., 2011) whereas for the Indian population; it ranges from 0.42-0.72 (Nagpal et al., 2002;Tandle et al., 2001;Mitra et al., 2003;2005;Mittal et al., 2011;Suresh et al., 2011).In our population, the frequency of proline was 0.46.A2 and A allele frequencies at intron 3 and intron 6 in our population were 0.18 and 0.19, respectively.The frequencies of these alleles range from 0.10-0.23 for A2 allele of intron 3 and 0.19-0.32 for A allele of intron 6 among different populations (Wang-Gohrke et al., 1999;Wu et al., 2002;Sprague et al., 2007;Hrstka et al., 2009;Hu et al., 2010).
Risk of oral cancer was also estimated in association with p53 genotypes of all three polymorphic loci.The Arg/Pro genotype of p53 codon 72 singly as well as in combination with Pro/Pro genotypes conferred protection towards oral cancer development.There are evidences that the codon 72 polymorphism had a profound effect on the primary structure of p53 protein and its biochemical and biological activities (Matlashewski et al., 1987;Ozeki et al., 2011).It has been shown that the Pro-72 form of p53 has increased transcriptional trans-activation capacity, induces a higher level of G1 arrest and senescence compared to the Arg-72 form (Thomas et al., 1999;Pim and Banks 2004;Frank et al., 2011).It was found that the Arg-72 form of the p53 protein was more susceptible to human papilloma virus (HPV) E6-mediated degradation compared to the Pro-72 form (Storey et al., 1998).Hsieh et al. (2005) found that oral squamous cell carcinoma (OSCC) patients with the Arg allele had a significantly higher frequency of p53 mutation than those with Pro/ Pro genotype among patients with common alleles of intron 3 and intron 6.In addition, p53 mutant acts as a more potent inhibitor of p73, which is responsible for apoptosis when p53 has Arg-72 rather than Pro-72 (Bergamaschi et al., 2003).In contrast, both Pro-72 and Arg-72 form of p53 are capable of inducing equal levels of apoptosis but with different kinetics (Thomas et al., 1999).Dumont et al. (2003) observed that the Arg-72 form has a much stronger capacity to induce apoptosis than the Pro-72 form of p53 in tumor cells but not in normal cells.Cell-line based studies suggest that the Arg-72 has superior proapoptotic function in human tumor cell-lines.This effect of Arg72Pro polymorphism may inhibit p53 function in vitro but whether this effect also occur in vivo remains to be determined.Recently, studies on mouse model indicate that the Arg-72 variant induces increased apoptosis in mouse embryo fibroblast (MEF) and in the small intestines of mice along with decreased apoptosis in the thymus compared to Pro-72 (Zhu et al., 2010;Azzam et al., 2011).Thus, there is tissue specific influence of Arg72Pro polymorphism on apoptosis.However, in primary head and neck tumors, retention of the Arg allele in codon 72 correlates with poor apoptosis (Schneider-Stock et al., 2004).Recently Hu et al. (2008) also observed that Pro allele at codon 72 of p53 gene has significantly higher level of benzo[a]pyrene-7,8-9,10-diol Controls 0.527 0.264 0.023 0.013 0.005 0.167 6.905 Cases 0.581 0.254 0 0.007 0.018 0.139 p=0.228 *1=A1 at intron 3, Proline at exon 4, A at intron 6; 2=A2 at intron 3, Arginine at exon 4, G at intron 6 epoxide (BPDE) induced apoptotic index compared to Arg allele in primary lymphocytes.Such tissue specific function of this polymorphism may explain why most of the epidemiological studies remain inconclusive.We observed no association between intron 3 and intron 6 polymorphisms and oral cancer risk.Recent researches suggest that intronic polymorphisms may affect the function of wild type p53 protein and hence cancer risk (Avigad et al., 1997;Lehman et al., 2000;Gemignani et al., 2004).However, in cell culture analysis, these two intronic polymorphisms (16 bp duplication in intron 3 and G>A in intron 6) did not seem to be sufficient to impair p53 function during the neoplastic transformation but required an additional coding region mutation.They have also reported that there were no splicing errors linked to these polymorphisms (Wang-Gohrke et al., 1999).In contrast, Hu et al. (2008) observed that variant alleles of intron 3 and intron 6 (A2 and A allele respectively) have significantly higher level of BPDE induced apoptotic index compared to its homozygous wild-type genotypes.Wu et al. (2002) have reported low apoptotic index and less repair capacity of lymphoblastoid cell lines that harbor A2 and A alleles of intron 3 and intron 6 polymorphisms, respectively.Gemignani et al. (2004) have also found that 16 bp duplication in intron 3 reduced the mRNA level of p53 but because of strong linkage disequilibrium between 3 and exon 4 polymorphism, it remains to be determined that whether intron 3 alone influence mRNA stability or need the presence of exon 4 polymorphism.Overall, the functional role of these 2 intronic polymorphisms of p53 in cancer risk remains uncertain.
When genotypes of these three polymorphisms were assessed in combination for the association with oral cancer risk, it was found that Arg/Pro genotypes in combination with A1/A1 and A1/A2 genotypes were significantly protected from oral cancer Moreover combination of heterozygous genotypes of exon 4 and intron 6 loci were also marginally protected from oral cancer development and individuals who were heterozygous at all three loci were protected from oral cancer development.It may be noted from the genotype combination analysis that individual who were carriers of proline allele at codon 72 of exon 4 in combination with A1 and G allele at intron 3 and intron 6 respectively were significantly protected from oral cancer development.More interestingly, Wu et al. (2002) have observed that proline at exon 4 in conjugation with intron 3 and 6 variant alleles exert a protective effect rather than a detrimental effect for lung and colorectal cancers though they found significant risk of lung cancer associated with these variants.The present study found that proline at exon 4 in conjugation with intron 3 and 6 variant alleles was associated with lower risk but not have the statistical power.To the best of our knowledge, there are no reports on the association of these three genotypes combinations and oral cancer risk from India till date.As there was significant linkage disequilibrium between these 3 loci in our population, these intronic p53 polymorphisms may confer protection from oral cancer risk through linkage disequilibrium with a functional variant of the p53 gene.
There are only few reports which have assessed the role of these p53 polymorphisms in oral cancer from India.Among them, most of reports are on Arg72Pro polymorphism of p53 gene.There is only one study from eastern region of India, explored the role of these three polymorphisms in oral cancer (Mitra et al., 2005).They have observed that the Proline allele was more frequent in control population and no association was found between oral cancer risk and Arg72Pro polymorphism.Another study from east region of India noted that Arginine allele was more frequent in population (Nagpal et al., 2002).
In the present study, Arginine allele was more frequent in this population.Other studies from India did not find any association between Arg72Pro polymorphism and oral cancer risk (Tandle et al., 2001;Nagpal et al., 2002;Katiyar et al., 2003).Meta-analysis by Zhuo et al. (2009) on this polymorphism revealed that there was no association between Arg72Pro polymorphism and oral cancer risk.When comparison was made for all other cancers from India, proline allele was at risk for colorectal cancer and bladder cancer but it was found to be protective in breast cancer and prostate cancer and no association was observed for head and neck cancer (Pandith et al., 2010;Sameer et al., 2010;Suresh et al., 2010;2011;Mittal et al., 2011).Studies from other populations showed conflicting results for the association of Arg72Pro polymorphism with oral cancer risk (Kietthubthew et al., 2003;Kuroda et al., 2007;Saini et al., 2011).Francisco et al. (2011) suggested that ethnicity, allelic frequency, histological and anatomical sites may modulate the penetrance of Arg72Pro in cancer susceptibility.The results of intron 3 and intron 6 polymorphisms are also inconsistent and ranges from no association to increased risk in different population (Wang-Gohrke et al., 1999;Wu et al., 2002;Sprague et al., 2007;Gallì et al., 2009;Hrstka et al., 2009;Hu et al., 2010).From India, only one study from east region has analyzed the association between intron 3 and intron 6 polymorphisms in oral cancer risk, suggested no association between intron 3 polymorphism and oral cancer risk, however, A allele at intron 6 was protective for oral cancer development (Mitra et al., 2005).This is the first study from this region which has analyzed association of these intronic polymorphisms and oral cancer risk.The rising numbers of oral cancer cases each year in India is alarming.Buccal mucosa represents the primary site for cancer development that may be due to the habit of keeping the tobacco in contact with cheek for a long time.In this cohort of study, tobacco habituates were significantly higher in cases compared to controls and they were at significant risk to develop oral cancer.It was also found that tobacco users who were carriers of Pro allele at exon 4 locus may be protected from oral cancer development.However, the only study from India failed to observe any association between Arg72Pro polymorphism and oral cancer development in tobacco users (Mitra et al., 2005).They have suggested that combination of A/A and A/G genotypes at intron 6 locus showed protective effect towards oral cancer development but at a low smoking dose.However in our study, most of the subjects were tobacco chewers.
As the haplotype structure of a population is indicative of its evolutionary history and different haplotypes are  There are many studies which showed positive association with one or more haplotypes constructed from these three polymorphisms with cancer risk.Hu et al. (2008).observed that individuals who were carrier of 0 and 2 copies of 1-2-2 and 2-1-1 haplotypes, respectively have significantly higher level of BPDE induced apoptotic index.1-2-2 haplotype was more common in Caucasian population and 2-1-1 haplotype was associated with breast cancer risk in Caucasian population (Weston et al., 1998;Wu et al., 2002).Whereas haplotype 1-2-2 was found more frequent in breast cancer patients in Pakistani ethnic groups (Khaliq et al., 2000).They also observed that 1-1-2 haplotype was most common in the Makrani, Punjabis, and Sindhis.Significant differences in haplotype distribution among three Indian caste populations were also observed (Mitra et al., 2003).Thus, distributions of haplotype frequencies also tend to differ due to differences in the ethnicity among Indians.There is only one study from eastern India which has performed haplotype analysis in oral cancer (Mitra et al., 2005).In present study, all pair-wise haplotypes showed significant linkage disequilibrium.We did not find significant difference in the frequencies of haplotypes between cases and controls.On the other part, haplotype 1-2-2 and 2-1-2 were more prevalent in cases compared to controls while 1-1-1 was completely absent in cases.1-2-1 and 2-2-1 haplotypes were completely absent in our population whereas in the population from eastern India, 1-2-1 haplotype was present and 2-2-2 and 2-2-1 haplotypes were absent (Mitra et al., 2005).We did not find any significant association between haplotypes and oral cancer risk.Mitra et al. (2005) found that individuals who were carrier of haplotype 1-2-2 were at risk of developing oral cancer that was also observed to be more prevalent among our population especially in cases.Present study also observed that 2-1-2 haplotype was more frequent in cases compared to controls.Earlier study from India has also found that 2-1-2 haplotype was more prominent in head and neck cancer patients compared to controls (Mitra et al., 2003).
In conclusion, this is the first study on Gujarati population from west India.Our data suggest that the p53 polymorphisms especially Arg72Pro in exon 4 and its combination with 16 bp duplication in intron 3 and G>A transition in intron 6 could significantly modify the risk of oral cancer development in this population.Moreover, p53 codon 72 genotypes might be associated with tobacco associated oral cancer development.Both alleles at each locus and haplotype analysis give important information.Further larger sample size will give more valuable information which may be used for the detection of individuals who will be at risk for this malignancy.Identification of such candidates is very crucial for screening and prevention programs and also may help in early detection of this dreaded disease.