Objective: To determine the association of diabetic nephropathy with glyoxalase-1 enzyme levels and a genetic missense variation (SNP rs4746) in its gene (GLO-1). Methods: This cross-sectional comparative study was conducted at the Department of Biochemistry and Molecular Biology, Army Medical College, Rawalpindi from November 2020 to December 2022. One hundred patients and one hundred and thirteen healthy controls were enrolled using the nonprobability convenience sampling method. Medical history and 10ml blood were obtained from each individual after written informed consent. Blood samples were subjected to biochemical tests and DNA extraction which was later used for single nucleotide polymorphism (SNP) analysis (C332C variant of rs4741 GLO-1 gene) using Tetra primer ARMS PCR and gel electrophoresis. Glyoxalase-1 enzyme activity in serum was measured using ELISA. Results: There was a significant difference in serum glyoxalase-1 levels in the two groups (p-value< 0.001). The patient group had lower levels (16.24 ± 22.51mg/dl) of glyoxalase-1 as compared to the control group (48.70 ± 42.54mg/dl). In genotypic analysis, 98 out of 100 control individuals had AA genotype-while only one had CC and another AC genotype. In the patient group, 94 out of 100 patients showed AA genotype, three AC, and three CC genotypes. As the statistical significance (p-value) was 0.37, there was no significant association found between AC or CC genotype and diabetic nephropathy. Conclusion: Glyoxalase-1 levels are linked to the development of diabetic nephropathy in our patients while a known missense variant rs4746 in the GLO-1 gene is not associated with increased risk. doi: https://doi.org/10.12669/pjms.40.4.8258 How to cite this: Shah SZH, Rashid A, Majeed A. Determination of Glyoxalase-1 levels and Identification of Genetic Variants in GLO1 Gene in Patients of Diabetic Nephropathy. Pak J Med Sci. 2024;40(4):652-656. doi: https://doi.org/10.12669/pjms.40.4.8258 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Background and Objectives: Diabetes is a global health issue, with approximately 50% of patients developing diabetic nephropathy (DN) and 25% experiencing early and severe forms of the disease. The genetic factors contributing to rapid disease progression in a subset of these patients are unclear. This study investigates genetic variations in the GLO-1, CBR-1, and ACE genes associated with early and severe DN. Materials and Methods: Sanger DNA sequencing of the exons of CBR1, GLO1, and ACE genes was conducted in 113 patients with early and severe DN (defined as occurring within 10 years of the diagnosis of diabetes and with eGFR < 45 mL/min/1.73 m²) and 100 controls. The impact of identified genetic variations was analyzed using computational protein models created in silico with SWISS-Model and SWISS-Dock for ligand binding interactions. Results: In GLO1, two heterozygous missense mutations, c.102G>T and c.147C>G, and one heterozygous nonsense mutation, c.148G>T, were identified in patients. The SNP rs1049346 (G>A) at location 6:38703061 (GRCh38) was clinically significant. The c.147C>G mutation (C19S) was associated with ligand binding disruption in the GLO1 protein, while the nonsense mutation resulted in a truncated, non-functional protein. In CBR1, two heterozygous variations, one missense c.358G>A, and one silent mutation c.311G>C were observed, with the former (D120N) affecting the active site. No significant changes were noted in ACE gene variants concerning protein structure or function. Conclusions: The study identifies four novel and five recurrent mutations/polymorphisms in GLO1, ACE, and CBR1 genes associated with severe DN in Pakistani patients. Notably, a nonsense mutation in GLO1 led to a truncated, non-functional protein, while missense mutations in GLO1 and CBR1 potentially disrupt enzyme function, possibly accelerating DN progression.

Objective: To explore the association between SNP rs533864137 and diabetic nephropathy in a Pakistani cohort using tetra-primer amplification refractory mutation system-polymerase chain reaction (ARMS-PCR). Study Design: Case-Control study. Place and Duration of Study: Center for Research in Experimental and Applied Medicine (CREAM), within the Department of Biochemistry & Molecular Biology, Army Medical College, Rawalpindi, Pakistan from Nov 2020 to Aug 2024. Methodology: A total of 213 participants, comprising 113 DN Patients and 100 Controls with diabetes but without nephropathy, were included. Genomic DNA was extracted from blood samples, biochemical analysis was performed to ascertain cases and Controls, and SNP rs533864137 was genotyped using ARMS-PCR. Statistical analyses evaluated the distribution of genotypes and alleles between the Groups and assessed the SNP's association with DN. Results: Among Controls, 97(97%) individuals had the GG genotype, and 3(3%) had the GA genotype. In the DN Group, 100(99.1%) exhibited the GG genotype, while 1(0.9%) was homozygous for the AA genotype. No significant differences in genotype distribution were observed between Groups (p=0.310). Statistical analysis confirmed that rs533864137 was not significantly associated with DN (p>0.05). Conclusion: SNP rs533864137 does not appear to be associated with DN in this population. These findings suggest that rs533864137 may not serve as a reliable genetic marker for assessing DN risk in the studied cohort.