Relationship between GSTs Gene Polymorphism and Susceptibility to End Stage Renal Disease among North Indians

Abstract

Background and Objective. Glutathione-S-transferase (GST) is the superfamily of genes that provides protection to the cells against reactive oxygen species and plays a vital role in phase II of biotransformation of many substances. Overexpression of GST (EC 2.5.1.18) has been documented in the erythrocytes of patients with chronic renal failure, which may be of clinical relevance. Keeping this background in mind, we have investigated the relationship between human GST gene polymorphism in end stage renal disease (ESRD) patients. Design and Methods. We have assessed 184 patients with ESRD and 569 age-and sex-matched controls from North India. The GSTT1 and GSTM1 null genotypes were identified by polymerase chain reaction (PCR). GSTP1–313 A/G mutation was determined by PCR followed by restriction enzyme digestion. Results. The gene frequency of GSTM1, GSTT1, and GSTP1 polymorphism were evaluated. We observed that GSTM1 null genotype was present in 46.74% of the ESRD patients while GSTT1 null genotype was present in 58.7% of the ESRD subjects. The genotypic distribution of GSTP1 was Ile₁₀₅/Ile₁₀₅ in 47.3%, Ile₁₀₅/Val₁₀₅ in 30.97% and Val₁₀₅/Val₁₀₅ in 21.74% of ESRD patients. There was a significant association of null alleles of the GSTM1 (p = 0.0386; OR = 1.445, 95% CI = 1.033–2.021) and GSTT1 (p ≤ 0.0001; OR = 4.568, 95% CI = 3.215–6.492) and in the -313 G alleles (Val) of the GSTP1 gene (p = 0.0032; OR = 1.956, 95% CI = 1.265–3.024) with end stage renal disease. The combined analysis of the three genotypes showed a further increased risk to ESRD (p ≤ 0.0001; OR = 9.01, 95% CI = 5.55–14.626). Interpretations and Conclusions. The null / low polymorphism of the detoxifying enzymes GSTT1, GSTM1, and GSTP1 are associated with the risk of developing ESRD in North Indian patients.

Keywords:

• end stage renal disease (ESRD)
• hypertension
• glutathione S-transferase
• polymorphism
• chronic renal failure

INTRODUCTION

Soluble glutathione S-transferase (GST) represents a superfamily of inducible enzymes, comprising at least seven classes of cytoplasmic proteins (α,μ,π,σ,θ,κ,ζ)[1] that catalyze the conjugation of glutathione (GSH) with different species of electrophilic compound. GST consists of a superfamily of dimeric phase II metabolic enzymes that catalyze the conjugation of reduced glutathione with various electrophilic enzymes.[2] The human GST genes are divided into four major subfamilies designated as GST α or A, GST μ or M, GST θ or T, and GST π or P.[3] The π GST gene exists as a single functional gene in humans, whereas α, μ, and θ families contain multiple distinct genes, sharing ∼55, 65, and 50% homology, respectively.[4–8] For both GSTM1 and GSTT1, the variant allele is a deletion of the gene; individuals who are homozygous for the deleted allele are said to possess the “null” genotype and do not express the enzyme.[9] The GSTP1 gene shows polymorphism within its coding region, of which the most well-known are an A to G transition at nucleotide position 1,578, causing an isoleucine to valine substitution at codon 105 (Ile¹⁰⁵ Val) in exon 5; and C to T base change at position 2,293, giving rise to the replacement of alanine to valine at the amino acid position 114 (Ala¹¹⁴ Val) in the exon 6.[8],[10] This results in the decreased enzyme activity.[8]

The phase II metabolizing enzymes (e.g., GST, N-acetyltransferase epoxide hydroxylase, and sulphotransferase) are involved in detoxifying chemical carcinogens; subsequently, their role is expected to be protective.[11] GSTs are generally involved in detoxification, but they are also important in activating and inactivating oxidative metabolites of carcinogenic compounds associated with cancers.[12] Other functions of GST include protection against oxidative damage to lipids and nucleic acid and participation in the metabolism of some steroid and leukotrienes.[13] In human erythrocytes, GST is present in large amounts[14] and in two forms: a highly cationic enzyme, which accounts for <5% of the total GST activity, and the main anionic enzyme, corresponding to the P form (π).[15] Because of its abundance and overlapping substrate specificity with the ρ form, the GSTP1 dimer is often considered to be sole GST enzyme in the erythrocyte.[13],[16] In the present study, we have determined the genotypic frequency of the GSTM1 null, GSTT1 null, and GSTP1 A/G polymorphism to understand whether the GST polymorphism is associated with ESRD in North Indians. The data were analyzed to evaluate the frequencies of the major polymorphisms of GSTM1, GSTT1, and GSTP1 in North Indian population. The present study is the first of its kind from this part of the country on ESRD patients. Our aim was to provide a basic database for future clinical and genetic studies in India.

METHODS

Subjects

Patients included in the present study were randomly selected from the Department of Nephrology, which is one of the super specialty centers in Sanjay Gandhi Post Graduate Institute of Medical Sciences (SGPGIMS) in Lucknow, India. The inclusion criteria for patients included a constantly elevated serum creatinine level above normal range (ranging from 3.4 to 15.8), diastolic blood pressure <90 mmHg and/or systolic blood pressure <140 mm Hg, and no previous anti-hypertensive medication; further, the patients were recommended for renal transplantation. The creatinine clearance rate was <15mL/min, and all of these patients were on regular dialysis. For each of the patient, the information was collected for various other criteria such as age, gender, urinary protein level, blood urea nitrogen, systolic and diastolic blood pressure, and complete lipid profile. Depending on the type and the severity of renal disorders, patients were categorized on the basis of histologically proven chronic glomerular nephropathy (CGN, n = 127), chronic intestinal nephropathy (CIN, n = 49), and hypertensive nephrosclerosis (HN, n = 4). Polycystic kidney (PK, n = 4) disease was diagnosed on the basis of ultrasound and CT scan. A total of 184 patients were included in the study. All patients with diabetic nephropathy were excluded from the study. A total of 569 randomly selected normal healthy controls were also genotyped for GSTs gene polymorphism. Because ∼85% of the patients were male, care was taken to include more male controls in order to rule out gender bias. Furthermore, the collection sites of control samples included the districts of Lucknow, Kanpur, Raebareilly, Barabanki, Faizabad, Agra, Jhansi, Gonda, and Basti, as more than 80% of the patients also belonged to these places. The study was approved by the ethical committee of SGPGIMS.

Blood Collection

Blood samples for measuring serum biochemical and lipid profiles were obtained in the morning after fasting for 8 hours. Three ml of venous blood sample were collected in EDTA vials for the extraction of genomic DNA.

DNA Extraction

DNA was extracted from blood by a high-salting out method using phenol-chloroform, as described by Comey et al.,[17] and was purified by ethanol precipitation. Various markers used for the present study were GSTM1, GSTT1, and GSTP1. The forward and reverse primers and different PCR conditions are listed in Table 1.

Table 1 List of primers and PCR conditions for all the GSTs markers

Marker	Primer sequence	PCR condition	References
GSTM1	F 5′-GAACTCCCTGAAAAGCTAAAGC -3′ R 5′-GTTGGGCTCAAATATACGGTG -3′	Initial denaturation 94°C for 5 min followed by 28 cycles at 94°C for 1 min, annealing at 59°C for 30 s, extension at 72°C for 30 s with a final extension at 72°C for 5 min.	35
GSTT1	F 5′-TTCCTTACTGGTCCTCACATCTC -3′ R 5′-TCACGGGATCATGGCCAGCA -3′	Initial denaturation 95°C for 4 min followed by 30 cycles at 94°C for 30 s, annealing at 59°C for 30 s, extension at 72°C for 30 s with a final extension at 72°C for 15 min.	35
GSTP1	F 5′-ACCCCAGGGCTCTATGGGAA -3′ R 5′-TGAGGGCACAAGAAGCCCCT -3′	Initial denaturation 95°C for 5 min followed by 30 cycles at 94°C for 30 s, annealing at 55.5°C for 30 s, extension at 72°C for 1 min with a final extension at 72°C for 5 min.	35
CYP1A1	F 5′-GAACTGCCACTTCAGCTGTCT -3′ R 5′-CAGCTGCATTTGGAAGTGCTC -3′	Initial denaturation 94°C for 5 min followed by 28 cycles at 94°C for 1 min, annealing at 59°C for 30 s, extension at 72°C for 30 s with a final extension at 72°C for 5 min.	21

Exon-7 of the CYP1A1 gene was co-amplified and used as an internal control. All products were separated on 2% agarose gel and subsequently stained with ethidium bromide to visualize the bands. DNA from samples positive for GSTM1 and GSTT1 genotypes yielded bands of 215 bp and 480 bp, while the internal positive controls (CYP1A1) PCR product corresponded to 312 bp. The GSTP1 genotype showed a band of 176 bp of amplified product, which was digested with Alw261 and electrophoresed on 3% agarose gel. The presence of restriction site resulted in two fragments of 91 and 85 bp size indicating the G allele; if there were A/G polymorphism, then there were three fragments of 176, 91 and 85 bp.

Statistical Analysis

All of the statistical calculations for the continuous data of biochemical and physiological factors were performed using SPSS version 13 statistical software packages. Normality test was completed, and depending upon the distribution, parametric/non-parametric tests were applied. For each variable, the values are expressed as mean ± SD. Allele and genotypes were calculated by the gene-counting method. To investigate the inter relationship between GSTM1, GSTT1, and GSTP1 genotypes and their association with age, sex, and different biochemical parameters, the Spearman correlation coefficient was computed. The association between GST genotypes and different biochemical parameters were examined with conditional logistic regression analysis to calculate odds ratios (OR) and their 95% confidence intervals (CI). Comparison of the categorical data (i.e., different GSTP1 genotypes) among controls and patients was done by Fischer's exact test and χ² test. Odds ratios were calculated with a 95% confidence interval. A p value <0.05 was considered significant.

RESULTS

We completed a logistic regression that included all significant variables. The genotypes were available for 184 ESRD patients (161 male, 23 female) with a mean age of 34.6 (±10.2). Similarly, the mean value of TC, TG, HDL, LDL, and VLDL was found to be 162.4±39.4, 151.8±68.2, 37.9±8.1, 94.6±34.9, and 31.1±14.3, respectively (see Table 2). The mean value of the serum creatinine and proteinuria among ESRD patients was found to be 8.81±3.27 and 3.1±0.99, respectively (see Table 2). There were significant differences between the ESRD patients and controls in biochemical parameters.

Table 2 Clinical characteristics of ESRD patients and controls

Parameters (SI)	Patients = 184 (Mean ± SD)	Controls = 569 (Mean ± SD)	p value
S. glucose (F) (mg/dL)	104.19 ± 34.5	77.9 ± 6.7	<0.0001
S. uric (mg/dL)	6.4 ± 1.9	5.03 ± 1.04	<0.0001
S. BUN (mg/dL)	68.9 ± 22.8	9.8 ± 2.7	<0.0001
S. ALKP (U/L)	185.9 ± 9.2	87.8 ± 16.7	<0.0001
S. SGOT (U/L)	35.5 ± 2.1	25.3 ± 0.7	<0.0001
S. SGPT (U/L)	36.29 ± 2.34	26.6 ± 0.52	0.0001
S. Na (mmol/L)	135.9 ± 8.4	138.4 ± 2.6	0.0005
S. K (mmol/L)	4.6 ± 0.7	3.7 ± 0.24	<0.0001
S. PHOS (mg/dL)	5.60 ± 0.36	3.7 ± 0.31	<0.0001
S. creatinine (mg/dL)	8.81 ± 3.27	0.81 ± 0.2	<0.0001
S. TG (mg/dL)	151.8 ± 68.2	146.5 ± 62.2	NS
S. TC (mg/dL)	162.4 ± 39.4	133.6 ± 33.7	<0.0001
S. HDL (mg/dL)	37.9 ± 8.1	23.8 ± 7.7	<0.0001
S. LDL (mg/dL)	94.6 ± 34.9	81.6 ± 29.6	<0.0001
S. VLDL (mg/dL)	31.1 ± 14.3	30.3 ± 12.4	NS
Systolic blood pressure	149.8 ± 20.5	116.6 ± 11.6	<0.0001
Diastolic blood pressure	91.14 ± 9.8	76.9 ± 5.6	<0.0001
Proteinuria	3.1 ± 0.99	0.28 ± 0.52	<0.0001
Age	34.6 ± 10.2	36.1 ± 11.4	NS
Gender, % (male/female)	87.5/12.5	85.2/ 14.8

*NS = not significant.

The allele and genotype frequencies of GSTM1, GSTT1, and GSTP1 were calculated in controls and patients (see Table 3). The frequency of GSTM1 and GSTT1 null alleles in patients group (n = 184) was 46.74% and 58.7%, respectively, whereas GSTP1 Ile allele was present in 62.8% and Val-105 allele was present 37.2% in patients. In the control group (n = 569), it was 37.7% and 23.8% for GSTM1 and GSTT1 null type, respectively, and the frequency of GSTP1 for Val-105 allele was 23.25%. When GSTM1 and GSTT1 were compared between patients and controls, we found 46.7% cases and 37.7% of controls for GSTM1, and 58.7% of cases and 23.8% of controls were of null type. There was a significant association of the null genotype with ESRD patients as compared to controls (p = 0.0386, OR = 1.445, 95% CI = 1.033–2.021; p ≤ 0.0001, OR = 4.568, 95% CI = 3.215–6.492; see Table 3). At GSTP1 locus, the frequency for Val-105 allele was 37.23% for cases and 23.25 for controls; 21.74% of individuals were homozygous and 30.97% were heterozygous for Val-105, and 41.28% were homozygous for Ile-105. In controls, the frequency was 10.19, 26.11, and 63.69%, respectively. There was a highly significant difference between cases and individually matched controls in the frequency of homozygote who possessed at least one Val-105 allele (p ≤ 0.0001, OR = 1.958, 95% CI = 1.398–2.741; see Table 3).

Table 3 Distribution of GST genotypes among ESRD patients and controls

Marker: GSTM1	Patient (n = 184)	Control (n = 569)
Genotypes
Null (0)	86 (46.7%)	215 (37.7%)
Present (1)	98 (53.3%)	354 (62.3%)
Comparison of different allelic and genotypic states
Null vs. present	p = 0.0386	OR (95% CI limit):1.445 (1.033–2.021)
Marker: GSTT1	Patient (n = 184)	Control (n = 569)
Genotypes
Null (0)	108 (58.70%)	135 (23.8%)
Present (1)	76 (41.30%)	434 (76.2%)
Comparison of different allelic and genotypic states
Null vs. present	p ≤ 0.0001	OR (95% CI limit): 4.568 (3.215–6.492)
Marker: GSTP1	Patient (n = 184)	Control (n = 569)
Genotypes
AA	87 (47.28%)	362 (63.69%)
AG	57 (30.97%)	148 (26.11%)
GG	40 (21.74%)	59 (10.19%)
Alleles
A	231 (62.77%)	872 (76.75%)
G	137 (37.23%)	266 (23.25%)
Comparison of different allelic and genotypic states
G vs. A	p < 0.0001	OR (95% CI limit): 1.958 (1.398–2.741)
GG vs. GA + AA	p = 0.0051	OR (95% CI limit): 2.448 (1.311–4.572)
AA vs. GG + AG	p = 0.0032	OR (95% CI limit): 1.956 (1.265–3.024)

We completed a combined analysis for all the GSTs by using logistic regression analysis. There was a significant association of null genotype of the GSTM1 and GSTT1 and of heterozygote (A/G) of the GSTP1 gene with ESRD risk (see Table 4). The combination of two high-risk genotype GSTM1 null and GSTT1 null or GSTP1–313 (A/G and G/G) genotype showed that the risk increased 4.3-fold for GSTP1 and GSTT1 and 4.8-fold for the GSTM1 and GSTT1 genotypes. However, there was no association of null genotypes of the GSTM1 and GSTP1 (A/G and G/G) genotype.

Table 4 Combined analysis of GST genotypes among ESRD patients and controls

Genotype	Patients (n = 184)	Controls (n = 569)	p	OR (95% CI)
Double: GSTM1 and GSTT1
M1 & T1	76	229	0.8667	1.045 (0.7451–1.465)
M0 & T0	86	88	<0.0001	4.797 (3.319–6.932)
M1 & T0	0	85	<0.0001	2091 (128.98–33389)
M0 & T1	22	167	<0.0001	3.059 (1.892–4.947)
Double: GSTM1 and GSTP1
M1 & P1	48	184	0.1325	1.354 (0.9324–1.966)
M0 & P0	47	124	0.3398	1.231 (0.8364–1.812)
M1 & P0	50	119	0.0954	1.411 (0.9624–2.069)
M0 & P1	39	142	0.3480	0.8088 (0.5414–1.208)
Double: GSTT1 and GSTP1
T1 & P1	39	214	<0.0001	2.241 (1.514–3.318)
T0 & P0	60	58	<0.0001	4.263 (2.827–6.429)
T1 & P0	37	184	0.0021	1.899 (1.271–2.836)
T0 & P1	48	113	0.0915	1.424 (0.966–2.1)
Triple: GSTT1, GSTM1, and GSTP1
P1, M1, & T1	39	131	0.6789	1.112 (0.7425–1.665)
P0, M0, & T0	60	29	<0.0001	9.01 (5.55–14.626)
P1, M0, & T0	26	59	0.205	1.422 (0.8673–2.333)
P0, M0, & T1	0	95	<0.0001	1833.4 (113.18–29699)
P0, M1, & T0	0	30	<0.0001	6527.1 (396.89–107348)
P0, M1, & T1	37	89	0.1729	1.357 (0.8869–2.078)
P1, M0, & T1	0	83	<0.0001	63.333 (3.906–1027)
P1, M1, & T0	22	53	0.3217	1.322 (0.7801–2.241)

We further investigated the risk associated with all three genotypes. GST genotypes were compared to no-risk genotype (positive genotypes of GSTM1), and GSTT1 and 313 A/A genotype of GSTP1 were designated as the reference group (see Table 4). The OR was 9.0 for the three high-risk genotypes vs. no-risk genotypes.

DISCUSSION

The present study is the first report from North India regarding the role of different genetic variants such as GSTM1, GSTT1, and GSTP1 gene in the causation of ESRD. There are some studies on other diseases on Indian population, but no study is available to evaluate the role of GST polymorphism in chronic renal failure from the Indian subcontinent. In the present study, we have observed that there were no significant differences of the biochemical renal function parameters (serum creatinine and protein urea) among the risk and non-risk genotypes for all three studied polymorphisms. Our results show that the GSTs variant may not be one of the causative factors involved in the renal damage but may be responsible for the aggravation or progression of the disease. Our hospital is a tertiary care center; hence, most of the patients reported to us had incomplete records of various parameters related to ESRD, as they visit our center quite late for renal transplantation. Data required for the regression analysis of serum creatinine profile were missing in most of the cases; therefore, we were not able to evaluate the role of different risk genotypes in progression of end stage renal disease as suggested by McLaughlin et al.[see [23]

Our results revealed that the null genotype of GSTM1 and GSTT1 and the G allele of GSTP1 were associated with the higher risk of chronic renal failure. The combination of the two high risk genotypes, GSTM1 null and GSTT1 null, or GSTP1–313 (A/G and G/G) genotypes increased the risk 4.2 times for GSTP1 and GSTT1 and 4.7 times for the GSTM1 and GSTT1 genotypes. When the three risk genotypes were combined, the risk increased to 9.0 times.

Human cytosolic GSTs have been well characterized and are known to be polymorphic, with variable frequency in different ethnic groups.[18] The percentage of individuals who do not express the GSTM1 and GSTT1 enzymes due to homozygous gene deletion is higher in Caucasians and Asians than in Africans.[19] About 60% of Asians, 40% of Africans, and 20% of Caucasians do not express the GSTM1 and GSTT1 enzyme. Polymorphism of GSTM1, GSTT1, and GSTP1 is shown to be associated with susceptibility to various forms of cancer, particular those caused by cigarette smoking,[20] resistance to chemotherapy treatment, and disease outcomes.[21] GST overexpression has been documented in the RBC of patients with CRF.[22] In the present study, the frequency of G allele of GSTP1 in control population was 10.19% and the GSTT1 null genotype was 23.8%, whereas in cases, it was 21.74% and 58.70%, respectively.

There was a significant difference at the allele frequency level in the two groups. However, recent reports and meta-analysis show that single GST gene polymorphism does not significantly increase risk to various diseases.[24–26] There was higher frequency of homozygotes Val₁₀₅/Val₁₀₅ genotype. It has been shown that the Val₁₀₅/Val₁₀₅ genotypes were more frequent in Caucasians than in Asians.[27] However, to date, few studies have been reported to see the relationship between combination of the GST genotype and risk of various diseases such as chronic renal failure, chronic lymphocytic leukemia, thyroid cancer and breast cancer,[28–30] and some of them have suggested a possible synergistic effect between GST genotypes. Our results revealed that there may be a high risk genotype GSTM1 null and GSTT1 null or GSTP1–313 (A/G and G/G) genotypes that have conferred an increased risk up to 4.3-fold for GSTP1 and GSTT1 and 4.8-fold for the GSTM1 and GSTT1 genotypes. However, more populations from India need to be evaluated to make an assumption that these polymorphisms may be helpful in evaluating the progression and effect of various drugs, as GST includes protection against oxidative damage to lipids and nucleic acids and participation in the metabolism of some steroid and leutriens.[13] If there are inactive forms of the enzyme (null genotypes of GSTM1 and GSTT1 and G allele of GSTP1), then detoxification of oxidative metabolism is reduced, which may lead to the progression of ESRD.

Our results of GSTP1–313 A/G polymorphism are similar with the findings from Italian population,[22] where there was a greater risk of renal failure with the GSTP1 313 A/G or G/G polymorphism. Yet there was no such association in the German and Austrian population.[31–33] Therefore, it appears that the association of ESRD risk with null alleles of GSTM1, GSTT1, and G allele of GSTP1 vary greatly in different populations.[24–26] Combined analyses of GSTM1/GSTT1 and GSTP1 loci and their significant association were also reported in Indian, German, and Japanese studies, among others.[21],[33–36] The present study revealed a greater risk with several risk alleles of GST, and suggests that gene interaction may contribute to a causal propensity for developing ESRD in the North Indian population.

We are reporting for the first time from India that the GSTP1–313 G allele (Val) and null allele of GSTM1 and GSTT1 are strong predisposing risk factors for ESRD. Moreover, the combination of three GSTs genotypes further increases the risk of ESRD in the North Indian population. Further studies are required to precisely define the biochemical mechanism of action of GSTs genes that can help in the development of methods for the prediction, prevention, and treatment of hypertension and further the end stage renal disease.

ACKNOWLEDGMENT

We are indebted to department of biotechnology New Delhi for the financial assistance and Mr. Sanjay Kumar Johari for technical help.