GSTP1 I105V Polymorphism and Susceptibility to Colorectal Cancer in Kashmiri Population

Abstract

The glutathione S-transferase (GST) enzyme encoded by the GSTP1 gene is one of the critical enzymes involved in detoxification of carcinogens. The substitution of isoleucine to valine residue at position 105 of the GSTP1 protein results in decreased enzyme activity and hence less capability of effective detoxification. Hence, we investigated the role of GSTP1 I105V polymorphism in modulating the risk of colorectal cancer (CRC) associated in a Kashmiri population. We designed a case–control study in which 86 CRC cases were studied for GSTP1 I105V polymorphism against 160 controls taken from the general population employing the polymerase chain reaction–restriction length fragment polymorphism technique. There was no significant association between GSTP1 I105V genotypes and the disease, but the Val/Val genotype was associated with an increased risk with some clinicopathological parameters (odds ratio=1.5; 95% confidence interval=0.55–4.57). This study suggests that the GSTP1 I105V polymorphism may modulate CRC risk in the Kashmiri population.

Introduction

C olorectal cancer (CRC) is a leading malignancy and major cause of mortality and morbidity. CRC is the fourth most common cancer in men and the third most common cancer in women worldwide (Center et al., 2009). Kashmir is a high-incidence area of gastrointestinal (GIT) cancers (Shah and Jan, 1990; Dhar et al., 1993; Mir et al., 2005; Murtaza et al., 2006). In Kashmir valley, CRC represents the third most common GIT cancer after esophageal and gastric cancers (Sameer et al., 2010a, 2010b; Javid et al., 2011).

Kashmir, also called Pir-e-Waer, is located in the northern part of India in between the Himalayas. It is home of one of the oldest ethnic populations that has been exposed to a special set of environmental and dietary risks, which include consumption of sun-dried and smoked fish and meat, dried and pickled vegetables, red chili, Hakh (a leafy vegetable of Brassica family), hot noon chai (salted tea), and Hukka (water pipe) smoke (Mir et al., 2005; Murtaza et al., 2006; Sameer et al., 2010a, 2010b). As previously reported, the etiology and incidence of various GIT cancers in this population have been attributed to a probable exposure to nitroso compounds, amines, and nitrates—reported to be present in these local food stuffs and most of which have been shown to contain important irritant and carcinogens (Siddiqi et al., 1992; Murtaza et al., 2006).

Glutathione S-transferases (GSTs) are a superfamily of dimeric phase II metabolic enzymes, which play an important role in the cellular defense system. GST enzymes catalyze the conjugation of toxic and carcinogenic electrophilic molecules with glutathione and thereby protect cellular macromolecules from damage (Stoehlmacher et al., 2002). Most GST substrates are thought as xenobiotics or products of oxidative stress, including polycyclic aromatic hydrocarbons present in the diet or from tobacco smoke. The GST enzymes also conjugate isothiocyanates, potent enzyme inducers, detoxify mutagens to glutathione, and divert them from the enzyme induction pathway to excretion (Yeh et al., 2005). GST superfamily enzymes are categorised into five subclasses—Alpha, Pi, Mu, Theta, and Zeta (Board et al., 1997). The subclass GSTP1 is widely expressed in normal human epithelial tissues and has been shown to be highly overexpressed in colon cancer (Moscow et al., 1989).

The GSTP1 gene, located on chromosome 11q13.2, is involved in the detoxification of electrophillic and heterocyclic amine carcinogens by conjugation (Zimniak et al., 1994; Henderson et al., 1998) and protection of DNA from oxidative damage (Ryberg et al., 1997). A single-nucleotide substitution (A→G) at polymorphic site 313 (codon 105) of the GSTP1 gene results in replacing isoleucine with valine (I105V) (Watson et al., 1998). The substitution of the less bulkier and more hydrophobic valine results in substrate-dependent alterations of GSTP1 catalytic activity. The presence of this valine residue in close proximity to the hydrophobic binding site for electrophilic substrates has been associated with decreased enzyme activity and a propensity to develop different neoplasms (García-Sáez et al., 1994).

Owing to the role of GSTP1 enzyme in detoxification of carcinogens and its decreased activity in variant allele form, we carried out a case–control study in our population to determine whether this GSTP1 I105V gene polymorphism may alter the risk of developing CRC.

Materials and Methods

Study design

This study was conducted at Sher-I-Kashmir Institute of Medical Sciences (SKIMS) Srinagar in Kashmir, India. The study was previously approved by the appropriate Institutional Ethics Committee.

Colorectal cases and controls

This study included 86 CRC cases recruited from Department of Surgery, SKIMS, Srinagar. Tumor and adjacent normal tissue samples from the cases were resected in the General Surgery Department (SKIMS). Data on all CRC cases were obtained from personal interviews with patients and/or guardians and medical records. All patients and/or guardians were informed about the study and their willingness to participate in this study was noted on a predesigned questionnaire (available on request). Blood samples of 160 age- and sex-matched controls with no signs of any malignancy were collected from Outpatients Department of the same institute to serve as external controls. The individuals of control group were previously interviewed and only those who fulfilled the predetermined age/sex group were selected after obtaining the consent to participate in the study. The controls belonged to the same racial Kashmiri population to which the cases belonged (see Table 1 for details).

Table 1.

Frequency Distribution Analysis of Selected Demographic and Risk Factors in Colorectal Cancer Cases and Controls

Variable	Cases (n=86)	Controls (n=160)	p-value
Age group			1.00
≤50	30 (34.9%)	56 (35.0%)
>50	56 (65.1%)	104 (65.0%)
Gender			0.76
Female	37 (43.0%)	72 (45.0%)
Male	49 (67.0%)	88 (55.0%)
Dwelling			0.56
Rural	59 (68.6%)	104 (65.0%)
Urban	27 (31.4%)	56 (35.0%)

DNA extraction

Samples were immediately snap-frozen after collection and stored at −70°C until further analysis. DNA was then isolated from both tissue and blood samples by using an ammonium acetate method and proteinase-K digestion.

Polymerase chain reaction

Genotyping for the GSTP1 polymorphism was determined by the method described by Yeh et al. (2005). Oligonucleotide primers used for the amplification of the target regions are listed in Table 2. Polymerase chain reactions (PCRs) were carried out in a 25 μL volume containing about 50 ng of genomic DNA template, 200 μm each dNTP, 200 ng each primer, 1.5 mm MgCl₂, 1X PCR buffer (50 mm KCl and 10 mm Tris-HCl [pH 8.3]), and 1 U Taq DNA polymerase (Fermentas). After an initial denaturation step of 10 min at 95°C, the samples were processed through 30 temperature cycles of 30 s at 94°C, 30 s at 55°C, and 30 s at 72°C. A final extension step of 72°C for 10 min was performed.

Table 2.

Primers for GSTP1 Gene Polymorphism

Target codon	Sequence	Amplicon (bp)	T_m (°C)
GSTP1	F5′-ACC CCA GGG CTC TAT GGG AA-3′	176	55
Ile105Val	R5′-TGA GGG CAC AAG AAG CCC CT-3′

Restriction length fragment polymorphism

The 176-bp PCR products (20 μL) obtained were digested for 2 h at 37°C with 2 U of Alw26I (Fermentas). The Val allele was cut into 91- and 85-bp fragments (Ile allele is not digested). The detection of different alleles was carried out by horizontal ethidium bromide 3% agarose gel electrophoresis, along with a 100-bp DNA ladder.

DNA amplicons as well as the digestion products were electrophoresed through a 2%–3% agarose gel (Genie) for resolution. The genotypes of >20% of the samples were double-blindly reassessed to confirm the results by two independent researchers. To validate the results, 15% of the amplified samples were randomly selected for genomic sequencing, and the results were 100% concordant. A positive control was used for each reaction to assure that the PCR was working.

Statistical analysis

Observed frequencies of genotypes in colorectal patients were compared with controls using chi-square or Fisher exact tests when expected frequencies were small. The chi-square test was used to verify whether genotype distributions were in Hardy–Weinberg equilibrium. Statistical significance was set at p≤0.05. The correlation between a polymorphism and various clinicopathological parameters was assessed using the relative risk ratio (odds ratio [OR]; hazard ratio) and a 95% confidence interval (CI). Statistical analyses were performed with PASW version 18 Software.

Results

A total of 86 CRC cases and 160 control subjects were included in this study. The patients comprised 49 men and 37 women (M/F ratio=1.32) and the control subjects consisted of 88 men and 72 women (M/F ratio=1.2). Mean age of the patients and controls was 52 years. No significant gender- or age-related differences were observed between the groups (p>0.05; Table 1). Further, of 86 confirmed cases of CRC, 81 cases were sporadic, 4 were FAP, and 1 was HNPCC. All but 1 case were adenocarcinoma and only 1 was squamous cell carcinoma of basal cell type, 59 rural and 27 urban, 36 cases had carcinoma in colon and 50 in rectum, and 55 were smokers and 31 nonmokers.

In our study, we found a difference in the genotype frequency of GSTP1 I105V between CRC cases and the matched controls. The incidence of the GSTP1 Val/Val genotype was slightly higher in CRC cases when compared with healthy controls (Table 3). The frequency of Ile/Val genotype in CRC cases was 16.3% and that of Val/Val was 8.1%, compared with healthy controls, where it was 16.9% and 5.0%, respectively. The overall hazard ratio of the GSTP1 Val allele in patients with CRC was 0.90 (95% CI=0.49–1.66). Overall, the homozygous variant Val/Val genotype was associated with a modestly elevated risk for CRC (OR=1.5; 95% CI=0.55–4.57).

Table 3.

Genotype Frequencies of GSTP1 Gene Polymorphism in Cases and Controls

GSTP1 genotype	Cases (n=86)	Controls (n=160)	OR (95% CI); χ² ^a; p-Value ^b	χ² ; p-Value (overall)
Ile/Ile	65 (75.6%)	118 (73.7%)	1.00 (Ref.)
Ile/Val	14 (16.3%)	34 (21.3%)	0.74 (0.37–1.49); 0.40; 0.49	1.64; 0.44
Val/Val	7 (8.1%)	8 (5.0%)	1.5 (0.55–4.57); 0.39; 0.41
Ile/Val + Val/Val	21 (24.4%)	42 (26.3%)	0.90 (0.49–1.66); 0.75; 0.76

Pearson's p-value.

Fisher's exact p-value.

CI, confidence interval; OR, odds ratio.

The correlation of GSTP1 I105V polymorphic status with clinicopathological characteristics was analyzed. We found a significant association (p-value <0.05) of the Val/Val variant genotype with the dwelling. We also found significant association of the Val allele and the site of the tumor but not with other variables (p-value <0.05; Table 4). We also found the hazard ratio for tumor location for all genotypes of GSTP1 to be greater than 2, showing more than twofold increase in the risk of CRC associated with each genotype (Table 4). The hazard ratio of the Ile/Val genotype was 2.53 (95% CI=0.75–7.88), and for the Val/Val genotype it was 4.56 (95% CI=0.82–25.41). We also found an increased hazard ratio for the age group for the Val/Val genotype (OR=1.47; 95% CI=0.30–7.13).

Table 4.

Association Between GSTP1 Gene Polymorphism and Clinicopathological Characteristics of Colorectal Cancer Cases

	Cases (n=86)
Variables	Group I	Group II	OR (95% CI); p-Value
Age group	≤50=30 (34.9%)	>50=56 (65.1%)
Ile/Ile; n=65 (75.6%)	22	43	1 (Ref.)
Ile/Val; n=14 (16.3%)	5	9	1.08 (0.32–3.63); 1.0
Val/Val; n=7 (8.1%)	3	4	1.47 (0.30–7.13); 0.68
Ile/Val + Val/Val; n=21 (24.4%)	8	13	1.20 (0.43–3.33); 0.79
Gender	Male=49 (67.0%)	Female=37 (43.0%)
Ile/Ile; n=65 (75.6%)	38	27	1 (Ref.)
Ile/Val; n=14 (16.3%)	8	6	0.95 (0.29–3.04); 1.0
Val/Val; n=7 (8.1%)	3	4	0.53 (0.11–2.57); 0.7
Ile/Val + Val/Val; n=21 (24.4%)	11	10	0.78 (0.29–2.09); 0.8
Dwelling	Rural=59 (68.6%)	Urban=27 (31.4%)
Ile/Ile; n=65 (75.6%)	45	20	1 (Ref.)
Ile/Val; n=14 (16.3%)	12	2	2.67 (0.54–13.03); 0.32
Val/Val; n=7 (8.1%)	2	5	0.17 (0.03–0.99); 0.04
Ile/Val + Val/Val; n=21 (24.4%)	14	7	0.89 (0.31–2.54); 1.0
Smoking status	Nonsmoker=31 (36.0%)	Smoker=55 (64.0%)
Ile/Ile; n=65 (75.6%)	24	41	1 (Ref.)
Ile/Val; n=14 (16.3%)	4	10	0.68 (0.19–2.42); 0.75
Val/Val; n=7 (8.1%)	3	4	1.28 (0.26–6.21); 1.0
Ile/Val + Val/Val; n=21 (24.4%)	7	14	0.85 (0.30–2.41); 0.8
Tumor location	Colon=36 (41.9%)	Rectum=50 (58.1%)
Ile/Ile; n=65 (75.6%)	23	42	1 (Ref.)
Ile/Val; n=14 (16.3%)	8	6	2.43 (0.75–7.88); 0.14
Val/Val; n=7 (8.1%)	5	2	4.56 (0.82–25.41); 0.1
Ile/Val + Val/Val; n=21 (24.4%)	13	8	2.96 (1.07–8.20); 0.04
Nodal status	Involved=48 (55.8%)	Not involved=38 (44.2%)
Ile/Ile; n=65 (75.6%)	36	29	1 (Ref.)
Ile/Val; n=14 (16.3%)	9	5	1.45 (0.44–4.80); 0.5
Val/Val; n=7 (8.1%)	3	4	0.60 (0.12–2.91); 0.7
Ile/Val plus; Val/Val; n=21 (24.4%)	12	9	1.07 (0.39–2.89); 1.0
Tumor grade	A+B=38 (44.2%)	C+D=48 (55.8%)
Ile/Ile; n=65 (75.6%)	29	36	1 (Ref.)
Ile/Val; n=14 (16.3%)	5	9	0.68 (0.21–2.28); 0.5
Val/Val; n=7 (8.1%)	4	3	1.65 (0.34–7.99); 0.7
Ile/Val + Val/Val; n=21 (24.4%)	9	12	0.93 (0.34–2.51); 1.0
Pesticide exposure	No=33 (38.4%)	Yes=53 (61.6%)
Ile/Ile; n=65 (75.6%)	25	40	1 (Ref.)
Ile/Val; n=14 (16.3%)	6	8	1.20 (0.37–3.86); 0.7
Val/Val; n=7 (8.1%)	2	5	0.64 (0.12–3.55); 0.7
Ile/Val + Val/Val; n=21 (24.4%)	8	13	0.98 (0.35–2.71); 1.0
Rectal bleeding/constipation	Yes=60 (69.8%)	No=26 (30.2%)
Ile/Ile; n=65 (75.6%)	48	17	1 (Ref.)
Ile/Val; n=14 (16.3%)	6	8	0.26 (0.08–0.87); 0.03
Val/Val; n=7 (8.1%)	6	1	2.12 (0.23–18.95); 0.7
Ile/Val + Val/Val; n=21 (24.4%)	12	9	0.47 (0.16–1.32); 0.2

Bold figures represent the statistical significance.

Discussion

As previously reported in various studies on Kashmiri population (Mir et al., 2005; Sameer et al., 2010a, 2010b), our population is exposed to a special set of environmental and dietary risks, one of which is the exposure to nitroso compounds, amines, and nitrates, present in local foodstuffs, many of which are irritants or carcinogens (Siddiqi et al., 1992; Murtaza et al., 2006).

In this study, we analyzed 86 CRC patients in relation to 160 healthy controls to examine the role of the I105V SNP of GSTP1 in modulating the CRC risk in Kashmiri population. Cancer patients and healthy control subjects were matched for age, gender, ethnic distribution, and tobacco use.

We found the frequency of the three different genotypes of GSTP1 in our ethnic Kashmir population, that is, Ile/Ile, Ile/Val, and Val/Val, to be 75.6%, 16.3%, and 8.1% among CRC cases and 73.7%, 21.3%, and 5.0% among general control population, respectively. The distribution frequency of the genotypes in CRC cases as well as controls were different from the previously reported ones (van der Logt et al., 2004; Sun et al., 2005; Kweekel et al., 2008; Pande et al., 2008), wherein the distribution of the Ile/Ile and Ile/Val genotypes were almost equal in the CRC cases (∼40%) but were similar to the study by Yeh et al. (2005) on a Taiwanese population. We also found that the CRC cases having the Val/Val genotype are at increased risk (OR=1.5; 95% CI=0.55–4.57), similar to the findings of Yeh et al. (2005) (OR=1.91; 95% CI=1.21–3.02).

In two seminal population specific studies—one by Mishra et al. (2004) on 370 normal healthy unrelated individuals (age range: 30–85 years) from North India (Uttar Pradesh) and other by Vettriselvi et al. (2006) on 255 random healthy unrelated individuals from South India (Chennai)—the prevalence for wild (Ile/Ile), heterozygous (Ile/Val), and mutant (Val/Val) genotypes of GSTP1 was reported to be 164/370 (44.3%), 186/370 (50.3%), and 20/370 (5.4%), respectively, in North India and 149 (58.4%), 98 (38.4%), and 8 (3.2%), respectively, in South India. These frequencies are in striking contrast to our general healthy population, for which the prevalence for wild (Ile/Ile), heterozygous (Ile/Val), and mutant (Val/Val) genotypes of GSTP1 was found to be 118/160 (73.7%), 34/160 (21.3%), and 8/160 (5.0%), respectively. The most striking feature in our Kashmiri population is the relatively lower prevalence of the mutant Val allele (26.3%) when compared with both North and South Indian populations (Table 5).

Table 5.

Genotype Frequencies of GSTP1 in Kashmir and Other Indian States

	Kashmir (n=160)	Chennai (n=255)	Uttar Pradesh (n=370)	OR (95% CI); χ² ^a ; F ^b	OR (95% CI); χ² ^a ; F ^b
GSTP1 genotype	present study	Vettriselvi et al. (2006)	Mishra et al. (2004)	Kashmir vs. Chennai	Kashmir vs. Uttar Pradesh
Ile/Ile	118 (73.7%)	149 (58.4%)	164 (44.3%)	1.00 (Ref)	1.00 (Ref)
Ile/Val	34 (21.3%)	98 (38.4%)	186 (50.3%)	0.43 (0.27–0.69); <0.05; <0.05	0.25 (0.16–0.39); <0.05; <0.05
Val/Val	8 (5.0%)	8 (3.2%)	20 (5.4%)	1.26 (0.46–3.46); 0.64; 0.79	0.55 (0.23–1.3); 0.17; 0.22
Ile/Val + Val/Val	42 (26.3%)	106 (41.6%)	206 (55.7%)	0.50 (0.32–0.77); <0.05; <0.05	0.28 (0.19–0.42); <0.05; <0.05

Pearson's p-value.

Fisher's exact p-value.

Bold figures represent the statistical significance.

GSTP1 is more likely a susceptibility gene candidate, because it encodes the predominant GST isoenzyme (Nijhoff et al., 1995; de Bruin et al., 1999), which is directly involved in the inactivation of heterocyclic amine in colorectal tissue (Lin et al., 1994). The study by Harries et al. (1997) on the GSTP1 I105V polymorphism identified that CRC cases having GSTP1 with a Val allele are at an elevated risk when compared with those with the Ile/Ile genotype (OR=1.77; 95% CI=1.03–3.06).

This conflicting evidence regarding the role of GST polymorphisms in CRC susceptibility might be associated with the ethnic differences in allele frequency for these polymorphisms (Cotton et al., 2000). There may be differences in the carcinogen exposures among different populations. Inadequate study design parameters such as nonrandom sampling, limited sample size, and little attempt to adjust for potential confounders should also be considered (Yeh et al., 2005).

Conclusion

Although there is a nonsignificant relation between the GSTP1 I105V polymorphism and the CRC risk in our Kashmiri population, the presence of the Val allele contributes to increased risk in some clinicopathological variables of CRC in this population. These correlations need to be authenticated in a large sample study in future, so as to help in better discernment of racial differences and in determining aggressiveness of CRC.

Footnotes

Acknowledgment

The authors gratefully acknowledge the financial support provided by SKIMS, Kashmir, for this work. The authors' thanks are also due to the Head and Technical Staff of the operation theater of Department of General Surgery, who helped in tissue procurement. The authors also thank the anonymous pathologists of Department of Pathology for the histopathological assessment of the tumor tissues.

Author Contribution

A.S.S. designed the study, performed the lab work, analyzed the data statistically, and wrote the manuscript. Q.Q. helped in the lab work. M.A.S. supervised the study design and revised the manuscript. All authors read and approved the final manuscript.

Disclosure Statement

No competing financial interests exist.

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