DNA methylation profiling in diabetic nephropathy: A pilot study

Diabetes mellitus (DM) is a complex metabolic disorder with polygenic inheritance.The morbidity associated with DM and its complications, especially the microvascular complication of diabetic nephropathy (DN), is tremendous, with a high disease burden globally and in India. A literature search has identified various studies on genetic and epigenetic factors in the pathophysiological processes of DN. However, epigenetics knowledge is limited and requires ongoing research into DN pathogenesis, progression, and outcome mechanisms. Therefore, this study was proposed to conduct a genome-wide methylation study (GWAS) to identify the genes that have altered methylation, followed by validation of the shortlisted genes by real-time polymerase chain reaction (PCR). The results generated by this study would thus help to identify the genes affecting the etiopathogenesis of DN and thereby improve future patient outcomes.

In this pilot study, 200 cases were selected and divided into 04 groups of 50 patients each: healthy subjects, patients with nondiabetic chronic kidney disease (CKD), DN, and DM each. The GWAS by microarray was conducted on pooled samples of bisulfite-converted DNA from all groups. Genes showing the maximum amount of methylation with the maximum differential β, common to both DM and DN, were further selected for validation in all the sample groups. DNA extraction, bisulfite modification, and real-time PCR: methylation-specific PCR was undertaken. Computed tomography (CT) values for FAM156A, MOSPD1, OTUD5, FTSJ1, and SH3KBP1 in all 04 groups were noted. The 2^-ΔΔCT method was used as a relative quantification strategy for quantitative real-time polymerase chain reaction (qPCR) data analysis to calculate relative gene expression levels between different samples. Further data analysis was carried out using R 4.2.0 statistical software.

The methylation levels were higher in MOSPD1, OTUD, FTSJ1, FAM156A, and SH3KBP1 in the DN group as compared to the DM group and CKD group with a p value < <0.05, with a median of 0.855 in DN vis a vis -0.665 (DM) and -0.560 (CKD) for MOSPD1; 1.669 in DN vis a vis 0.280 (DM) and -2.051 (CKD) for OTUD5; 1.162 in DN vis a vis -2.0575 (DM) and -1.953 (CKD) for FTSJ1; 1.096 in DN vis a vis -1.395 (DM) and -1.449 (CKD) for FAM156A and 1.228 in DN vis a vis -1.155 (DM) and -1.425 (CKD) for SH3KBP1 respectively. The increased methylation levels in MOSPD1, OTUD5, FTSJ1, FAM156A, and SH3KBP1 genes in DN; decreased the expression of these genes in cases of DN.

The hypermethylation in the validated genes in DN patients shows the importance of these as likely novel biomarkers. However, being a pilot project, further studies with a comparison of DM and DN are required in the future for further validation in larger sample sizes. Baseline levels of methylation status are also required for newly diagnosed cases of type 2 DM and a longitudinal cohort study to track the change in methylation pattern as the disease progresses so that extrapolation to risk potential can be done in future.