Association of a p 53 Codon 72 Gene Polymorphism with Environmental Factors and Risk of Lung Cancer : a Case Control Study in Mizoram and Manipur , a High Incidence Region in North East India

The p53 tumor suppressor gene plays a central role in modulating cellular process that governs the major defenses against tumor growth, maintenance of genetic integrity, cell cycle arrest and apoptosis (Whibley et al., 2009; Cheng et al., 2012; Azlin et al., 2014; Ren et al., 2013). p53 gene is one of the most studied genes in cancer research. The gene harbors high-frequency, functional single-nucleotide polymorphisms (SNPs) which may alter p53 protein function (Grochola et al., 2010). Several functional p53 SNPs have been reported to be associated with risk of developing different human cancers, including lung cancer (Yan et al., 2009; Francisco et al., 2011; Bellini et al., 2012; Karim et al., 2014; Zeichner et al., 2014). Among these the most common is codon 72 polymorphisms is a single-base substitution of cytosine


Introduction
The p53 tumor suppressor gene plays a central role in modulating cellular process that governs the major defenses against tumor growth, maintenance of genetic integrity, cell cycle arrest and apoptosis (Whibley et al., 2009;Cheng et al., 2012;Azlin et al., 2014;Ren et al., 2013).p53 gene is one of the most studied genes in cancer research.The gene harbors high-frequency, functional single-nucleotide polymorphisms (SNPs) which may alter p53 protein function (Grochola et al., 2010).Several functional p53 SNPs have been reported to be associated with risk of developing different human cancers, including lung cancer (Yan et al., 2009;Francisco et al., 2011;Bellini et al., 2012;Karim et al., 2014;Zeichner et al., 2014).Among these the most common is codon 72 polymorphisms is a single-base substitution of cytosine for guanine, leading to arginine (A72) being replaced by proline (p72) (Pietsch et al., 2006) and has been associated by with several types of cancer (Ishan et al., 2011;Li et al., 2013;Malakar et al., 2014).However few studies examined if there are interaction between p53 codon 72 polymorphisms and smoking or other environmental factors on lung cancer risk.
Studies on p53 codon 72 polymorphisms reveals stricking difference on ethnicity (Sjalander et al., 1995).In a study conducted by Beckam et al (1994) have observed that the frequency of p53 variant allele varies with latitude as its increases in a linear trend as population near equator suggesting that ethnicity might be related to allelic distribution of the gene and its determinacy in disease involvement; however, some studies do refute the ethnicity-risk confounding relationship (Fan et al., 2000;Lin et al., 2014).

Bhaskar Jyoti Saikia et al
Lung cancer is the most common cause of death from cancer worldwide, estimated to be responsible for nearly one in five (1.59 million deaths, 19.4% of the total) (Ferlay et al., 2012).India contributes 6.2% cases of lung cancer with approximately 58,000 incidence cases reported in 2008 (Ferlay et al., 2010;D'Souza et al., 2013).North Eastern parts of India represent a unique, strategic geographic location with demographically diverse population.These areas also shows similarities in occurrences of cancer incidence patterns for various cancer sites of North East India with South and East Asian region which reflect that genetic linkage between these regions (Sharma et al., 2014).Mizoram and Manipur are two states from North East parts of India where lung cancer is mostly predominant, with highest age-adjusted rate (AAR) in Mizoram (28.3 per 10 5 in males and 28.7 per 10 5 in females).Similarly, Manipur also contributes a very high incidence of lung cancer (with AAR of 14.1 per 10 5 in males and 11.9 per 10 5 in females) (NCRP, 2013).
The area also reports for a unique consumption of tobacco, betel quids and dietary habits that are different from other places (Phukan et al., 2001(Phukan et al., , 2005(Phukan et al., , 2006(Phukan et al., , 2014;;Saikia et al., 2014).A high risk of lung cancer can be an outcome of either environmental and genetic risk factors or a complex interaction of both.Therefore the present study was an age, sex and ethnicity matched population based case control study carried out to find out any relevance of p53 codon 72 polymorphisms as well as their interaction with environmental factors for the increase risk of lung cancer in Mizoram and Manipur North East India.

Materials and Methods
Present study was an age (±5 years), sex and ethnicity matched population based case-control study.The study duration was from February 2010 -January 2014.The work was carried out at Regional Medical Research Centre (RMRC) North East Region, Indian Council of Medical Research (ICMR); India in collaboration with civil hospital Aizawl, Mizoram and Population Based Cancer Registry (PBCR), Imphal, Manipur.Incident cases and controls subjects willing to participate in the study were indigenous people of Manipur and Mizoram.Information of smoking, betel quid chewing, consumption of alcohol, exposure to household combustion were recorded in a structured pre-designed questionnaire.All subject provide written inform consent for participation in the study was done under a protocol approved by the institutional ethics committee of Regional Medical Research Centre (RMRC) North East Region, Indian Council of Medical Research (ICMR).
The inclusion criteria for the cases includes histopathologically or cytologically confirmed lung cancer cases with no evidence of pulmonary inflammation or benign lung tumours and were resident of Mizoram and Manipur.The exclusion criteria for the cases were patient's diagnosis reported by death certificate or at autopsy, diagnosed with stomach, colon, liver, pancreatic, breast or rectal cancers, cases too old or too debilitated to be interviewed elaborately and those who refuse to participate in the study.The inclusion criteria for controls were those who had no blood relation with cases, non malignant individual, residence of Mizoram and Manipur, age, sex and ethnicity matched with controls.A total of 272 lung cancer cases and 544 controls matched for age (±5 years), sex and ethnicity were enrolled in the study.

DNA extraction
Four ml. of blood sample was collected from all subjects in EDTA vials.DNA was isolated by using standard phenol chloroform method and stored at -80º C till further analysis (Landi et al., 2006).

Genotyping
Genotyping of p53 gene polymorphisms were ascertained by PCR amplification using gene specific primers followed by Restriction Fragment Length Polymorphisms (RFLP).PCR reaction for p53  MgCl 2 (Roche, Germany), 200 ul dNTPs (Roche, Germany) and 1.5 unit of Taq DNA Polymerase (Roche).Primer sequences for PCR amplification were 5"TTGCCGTCCCAAGCAATGGATGA3"and 5"TCTGGGAAGGGACAGAAGATGAC3" which produced a 199 base-pair band (Ihsan et al., 2011).Amplifications for p53 genotype were performed under the following conditions: 96°C for 5 minutes; 30 cycles of 94°C for 1 minute (denaturation), 55°C for 1 minute (annealing) and 72°C for 1 minute (extension); followed by 72°C for 4 minutes.After amplification of the PCR product, it was digested at 55˚C for 3 hours with BstU1 restriction enzyme.The RFLP product were then electrophoresed in 2.5% agarose gel and then visualized in gel documentation system (Cell Biosciences).Arg/Arg genotype yields two small fragments of 113 bp and 86 bp.Pro/Pro genotype yield single 199bp.The heterozygote Arg/Pro genotype has three fragments of 199, 113 and 86 bp size (Figure 1).Genotyping of 10% of the randomly selected cases and controls were confirmed by sequencing and the results were observed 100% concordance was observed (Figure 2).

Statistical analysis
Multiple logistic regression analysis was used to analyze the data.The conditional maximum likelihood method was used to estimate the parameters of the regression model because of the matched design and significance was taken at p≤0.05 (two tailed).The crude measure of association between single putative risk factors and lung cancer was expressed as the odds ratio (OR) and its 95% confidence interval (CI) was calculated from the standard error of the regression coefficient.To control for confounding variables and other covariates, the data was analyzed by conditional multiple logistic regression to evaluate the extent of risk association.The statistical package used for the analysis was SPSS version 19.
Tests for Hardy-Weinberg equilibrium in both the subject were conducted using observed genotype frequencies and a chi-square test featuring one degree of freedom.

Results
Socio demographic characteristics of lung cancer cases and controls were presented in Table 1.No statistically significant difference in age and sex were observed between cases and controls.66.5% of the cases are of non small squamous cell carcinoma, 26.5% are of non-small adenocarcinoma and 7.0% are of small cell carcinoma.Illiteracy (OR=2.10,CI=1.15-3.86,p=0.016) is significantly associated with increased risk of lung cancer of the study population.Among occupational status cultivators (OR=1.76,CI=1.13-2.75,p=0.013) and those of unskilled workers (OR=1.85,CI=1.09-3.16,p=0.024) were also significantly associated with increased risk of lung cancer in the study population.
Frequency distribution of Arg/Arg, Arg/Pro and Pro/ Pro genotype among lung cancer cases were 34.9%, 46% and 19.1% while that of control were 41.4%, 47.8% and 10.8% respectively.The p53 Pro/Pro genotype were found to be significantly associated with increased risk of lung cancer of the study population (adjusted OR=2.14,p=0.001).In gender wise stratified analysis, the p53 Pro/Pro genotype demonstrated slightly higher risk of lung cancer risk association among female (adjusted OR=2.02,CI=1.05-3.90, p=0.035) than that of male (adjusted OR=1.96,CI=1.01-3.82,p=0.048).The distribution of p53 genotype in both cases and controls were in Hardy-Weinberg Equilibrium (HWE) (Table 2).Interaction analysis of p53 codon 72 polymorphisms with environmental factors was analysed.Significantly higher risk association for lung cancer was observed when p53 Pro/Pro genotype interact with exposure of wood combustion (adjusted OR=3.60,p<0.001) and exposure of cooking oil fumes (adjusted OR=3.27,p=0.002).Interaction combination of p53 Pro/Pro genotype with that of betel quid chewing (adjusted OR=3.85,p<0.001),tobacco smoking (adjusted OR=4.42,p<0.001) and that of alcohol consumption (adjusted OR=3.31,p=0.034)were also reveals highly significant association for increased risk of lung cancer in the study population (Table 3).

Discussion
p53 genes act as a central regulatory node of multiple cellular response pathways to endogenous or exogenous stresses (Robles et al., 2002).p53 protein has the capacity to regulate activity of key effectors of cellular processes, such as senescence, DNA repair, cell cycle arrest and apoptosis (Levine et al., 1997;Riley et al., 2008, Ren et al., 2013).Functional inactivation of p53 pathways affects p53 signaling and further alters cancer risk (Robles et al., 2002;Roy et al., 2012).
In this case control study we had examined whether association of p53 codon 72 polymorphisms and their interaction with environmental factors are associated with increased risk of lung cancer in the study population.Present study reveals significant association of p53 codon 72 polymorphisms and risk of lung cancer from high incidence region of North East India.Moreover we found synergistic effects of p53 gene with exposure of wood combustion, exposure of cooking oil fumes, tobacco smoking, betel quid chewing, tobacco smoking and alcohol consumption.
Tobacco smoking and environmental air pollution have been linked with high frequency of p53 polymorphisms (Pfeifer et al., 2002;Dagher et al., 2006;Patel et al., 2013).Tobacco smoking, betel quid and wood smoke constituents contains large number of polycyclic aromatic hydrocarbons, alkaloids and other carcinogens (Lissowska et al., 2005;Sellappa et al., 2009;Hosgood et al., 2010;Ishan et al., 2011).The positive interactions observed between p53 codon 72 polymorphisms with tobacco smoking, betel-quid chewing and other environmental factors or exposure in the present might suggest that individuals carrying the variant p53 Pro/Pro genotype may have compromised p53 function and may have been respond poorly to the adverse effects of smoking and environmental factors or exposure and thus have an elevated risk of developing lung cancer.
Association of p53 codon 72 polymorphisms and risk of lung cancer also varies across ethnicity (Wang et al., 2013).Significantly increased risks for lung cancer were found among Asians, but not in Africans for both p53 Pro/Pro and the Pro allele carriers in a previous meta-analysis by Li et al (2009).These inconsistencies ethnicities different may be attributed to different genetic backgrounds and environments.Furthermore, the effect of p53 codon 72 Arg/Pro polymorphisms on lung cancer risk differs in Asians.In a study conducted by Jung et al (2008), demonstrated that the p53 codon 72 Arg/Pro polymorphism was not significantly associated with lung cancer susceptibility in a Korean population (Jung et al., 2008).However in a study conducted by Piao et al (2011), the p53 codon 72 polymorphism was associated with an increase risk of lung cancer in another Korean population (Piao et al., 2011).Different study design, sample size, genotyping method, and source of controls may be responsible for the conflicting findings among individual studies.
In conclusion, the present study provides preliminary evidence that p53 codon 72 polymorphisms is novel single nucleotide polymorphisms that may effect lung cancer risk, especially among smokers and indoor environmental exposure.Taking into account, other confounding variables such as dietary habits, and infectious agents such as H. pylori status, HPV, etc will give us more probable factors for increased risk of lung cancer in the study population.

Table 1 . Distribution of Demographic Characteristics and Risk of Lung Cancer
*Significant; †For independent samples T-test; # OR were estimated through conditional multiple logistic regression model Bhaskar JyotiSaikia et al

Table 3 . Distributions of p53 Genotype and Risk of Lung Cancer
*Significant; # Adjusted OR were estimated through conditional multiple logistic regression model; † Exposure of cooking oil fumes, betel-quid chewing, tobacco smoking and alcohol consumption were adjusted to estimate adjusted OR in the model; ‡ Exposure of wood combustion, betel-quid chewing, tobacco smoking and alcohol consumption were adjusted to estimate adjusted OR in the model; § Exposure of wood combustion, exposure of cooking oil fumes, tobacco smoking and alcohol consumption were adjusted to estimate adjusted OR in the model; ¶ Exposure of wood combustion, exposure of cooking oil fumes, betel-quid chewing and alcohol consumption were adjusted to estimate adjusted OR in the model; ¥ Exposure of wood combustion, exposure of cooking oil fumes, betel-quid chewing and tobacco smoking were adjusted to estimate adjusted OR in each model.