End-stage renal disease (ESRD) represents the terminal stage of chronic kidney disease and carries substantial morbidity and mortality worldwide. Progression to ESRD is driven by common comorbidities such as diabetes, hypertension, obesity and aging, and is aggravated in low- and middle-income settings by delayed diagnosis and limited access to renal replacement therapies. Oxidative stress has been implicated in progressive renal injury, motivating investigation of genetic variants in antioxidant pathways as potential determinants of ESRD susceptibility.
Glutathione S-transferases (GSTs) are detoxifying enzymes involved in cellular defense against reactive oxygen species and electrophiles. Polymorphisms that alter expression or function of GST isoforms, including GSTM1, GSTT1, and GSTP1, have been associated with a range of diseases. The tumor suppressor TP53 (p53) also modulates responses to DNA damage and oxidative stress; the rs1042522 polymorphism in TP53 has been evaluated in relation to kidney disease susceptibility. This study evaluated the association of presence/absence of GSTM1 and GSTT1 and SNPs in GSTP1 (rs1695) and TP53 (rs1042522) with ESRD in a Pakistani population.
A case–control design enrolled 610 participants (305 ESRD cases and 305 matched healthy controls) from District Multan in Punjab, Pakistan. Recruitment occurred between 30 October 2023 and 28 February 2024. Cases were clinically confirmed ESRD patients undergoing dialysis at the Nephrology Department of Nishtar Medical University and Hospital, Multan. Controls were age-, sex- and ethnicity-matched individuals without a history of ESRD recruited from the same district.
ESRD diagnosis was established using clinical evaluation, laboratory testing (including Albumin-to-creatinine ratio and blood urea nitrogen) and imaging; a GFR below 15 mL/min was used to confirm ESRD. Epidemiological and clinical data captured by standardized questionnaire included age, gender, family history, hypertension, diabetes, hepatitis C status, smoking, alcohol and drug use. Informed verbal consent was obtained from all participants under institutional ethical approval.
Genomic DNA was extracted from whole blood using an inorganic DNA extraction protocol. Presence or absence of GSTM1 and GSTT1 was determined by multiplex PCR with CYP1A1 exon 7 serving as an internal amplification control. Genotypes at GSTP1 rs1695 and TP53 rs1042522 were determined using tetra-primer amplification refractory mutation system PCR (T-ARMS–PCR). Representative amplicon sizes and banding patterns were used to assign genotypes.
PCR products were separated on 1.8% agarose gels, visualized under UV light, and imaged. Quality control measures included negative controls in each PCR run and re-genotyping of approximately 10% of samples, which showed 100% concordance.
Analyses were performed using Minitab (version 19). Genotype and allele frequencies in cases and controls were compared using Chi-square tests. Hardy–Weinberg equilibrium was assessed for the SNPs. Combined genotype combinations across loci were considered to evaluate joint effects. Associations were further examined using logistic regression to estimate odds ratios (ORs) with 95% confidence intervals; statistical significance was set at P ≤ 0.05.
Multiplex PCR produced expected fragment sizes for GSTM1 (215 bp) and GSTT1 (480 bp) when present; absence of these bands indicated a null genotype. The CYP1A1 internal control (312 bp) amplified across all samples.
The GSTM1 null genotype was significantly more frequent among ESRD cases than controls (P < 0.001). The reported odds ratio indicated that cases had an approximately 2.6-fold higher risk of being GSTM1 null compared with controls.
For GSTT1, genotype frequencies also differed between groups (P = 0.05) but in the opposite direction: a higher number of controls were GSTT1 null than ESRD patients. The OR reported (≈0.69) indicated that absence of GSTT1 was not associated with increased ESRD risk in this cohort.
At GSTP1 rs1695, the study detected three genotypes by expected band sizes. Genotype distributions differed significantly between ESRD cases and controls (P < 0.001). In controls, the AA (wild-type) genotype was most frequent (70%), AG was 23%, and GG (polymorphic) was 7%. Among ESRD cases, AA was 59%, AG 24%, and GG 17%.
Allelic frequencies also differed: controls had a higher frequency of the A allele (82%) versus the G allele (18%); cases had A = 67% and G = 33%. The GG genotype was associated with an increased risk of ESRD relative to AA, with an OR reported in the source indicating about a 4.4-fold higher risk.
For TP53 rs1042522, three genotype patterns were observed by expected amplicons. Genotype frequencies differed significantly between cases and controls (P < 0.001). Controls most commonly carried the GG wild-type genotype (69%), with CG 21% and CC 10%. ESRD cases showed GG 55%, CG 24%, and CC 21%.
Allelic distributions followed the same trend: controls had G = 74% and C = 26%; cases had G = 65% and C = 35%. The higher proportion of the polymorphic C allele and CC/CG genotypes in cases suggests a potential association with ESRD susceptibility in this cohort. The source reports that OR values indicated comparability of susceptibility in some comparisons; detailed effect sizes for each genotype comparison are presented in the study tables.
The investigators constructed genotype combinations (haplotypes) across GSTM1, GSTT1, GSTP1 rs1695 and TP53 rs1042522 to evaluate joint effects on ESRD susceptibility. The source reports that combined analyses were performed using Chi-square and logistic regression; however, specific combined-haplotype frequencies and effect estimates are not fully detailed in the summarized text.
Demographic associations noted in the enrolled cohort included higher ESRD burden among participants aged 26–40 years, male sex, and smokers.
In this hospital-based case–control study from Multan, Pakistan, genetic variation in detoxification and DNA-damage response genes showed significant associations with ESRD. The GSTM1 null genotype and the polymorphic GSTP1 rs1695 G allele (particularly GG genotype) were more frequent among ESRD patients and associated with increased risk. The TP53 rs1042522 polymorphic C allele and CC/CG genotypes were also enriched in cases. In contrast, absence of GSTT1 was more common in controls and was not associated with increased ESRD risk in this sample.
These results support a contribution of antioxidant and apoptosis-regulating gene variants to ESRD susceptibility in this population. The reported quality controls and matching of controls strengthen internal validity. The source indicates that the authors conclude these genetic variations may influence progression to ESRD.
Limitations explicitly reported in the summarized source text are limited; the detailed manuscript contains supplementary tables and full data. The source states that all relevant data are within the manuscript. Funding was not provided for the work and the authors declared no competing interests.
Clinicians and researchers should interpret these associations in the context of population-specific genetics, environmental exposures, and the need for replication in independent cohorts before clinical application.