Reduced methylation correlates with diabetic nephropathy risk in type 1 diabetes
Ishant Khurana, … , Per-Henrik Groop, Assam El-Osta
Ishant Khurana, … , Per-Henrik Groop, Assam El-Osta
Published January 12, 2023
Citation Information: J Clin Invest. 2023;133(4):e160959. https://doi.org/10.1172/JCI160959.
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Research Article Metabolism Nephrology

Reduced methylation correlates with diabetic nephropathy risk in type 1 diabetes

Abstract

Diabetic nephropathy (DN) is a polygenic disorder with few risk variants showing robust replication in large-scale genome-wide association studies. To understand the role of DNA methylation, it is important to have the prevailing genomic view to distinguish key sequence elements that influence gene expression. This is particularly challenging for DN because genome-wide methylation patterns are poorly defined. While methylation is known to alter gene expression, the importance of this causal relationship is obscured by array-based technologies since coverage outside promoter regions is low. To overcome these challenges, we performed methylation sequencing using leukocytes derived from participants of the Finnish Diabetic Nephropathy (FinnDiane) type 1 diabetes (T1D) study (n = 39) that was subsequently replicated in a larger validation cohort (n = 296). Gene body–related regions made up more than 60% of the methylation differences and emphasized the importance of methylation sequencing. We observed differentially methylated genes associated with DN in 3 independent T1D registries originating from Denmark (n = 445), Hong Kong (n = 107), and Thailand (n = 130). Reduced DNA methylation at CTCF and Pol2B sites was tightly connected with DN pathways that include insulin signaling, lipid metabolism, and fibrosis. To define the pathophysiological significance of these population findings, methylation indices were assessed in human renal cells such as podocytes and proximal convoluted tubule cells. The expression of core genes was associated with reduced methylation, elevated CTCF and Pol2B binding, and the activation of insulin-signaling phosphoproteins in hyperglycemic cells. These experimental observations also closely parallel methylation-mediated regulation in human macrophages and vascular endothelial cells.

Authors

Ishant Khurana, Harikrishnan Kaipananickal, Scott Maxwell, Sørine Birkelund, Anna Syreeni, Carol Forsblom, Jun Okabe, Mark Ziemann, Antony Kaspi, Haloom Rafehi, Anne Jørgensen, Keith Al-Hasani, Merlin C. Thomas, Guozhi Jiang, Andrea O.Y. Luk, Heung Man Lee, Yu Huang, Yotsapon Thewjitcharoen, Soontaree Nakasatien, Thep Himathongkam, Christopher Fogarty, Rachel Njeim, Assaad Eid, Tine Willum Hansen, Nete Tofte, Evy C. Ottesen, Ronald C.W. Ma, Juliana C.N. Chan, Mark E. Cooper, Peter Rossing, Per-Henrik Groop, Assam El-Osta

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Figure 2

FinnDiane T1D methylome at sites of regulation.

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FinnDiane T1D methylome at sites of regulation. (A) Hierarchical profile...
(A) Hierarchical profile-based clustering of DMRs. DNA methylation differences detected between controls and cases with normoalbuminuria (Normo), macroalbuminuria (Macro), and end-stage renal disease (ESRD). Heatmap shows loss (black) and gain (green) of methylation. We observed clustering of DMRs by the prevalence of DN (P value < 0.01). (B) Binned orientation and distance of DMRs relative to transcription start site (TSS). (C) Graphical representation of genomic features and CG islands, shelves, and shores. (D) Distribution of DMR clusters shown at gene promoters, exons, introns, CG islands (CGI), and CG island shores (±1 kb from CGI) and shelves (±1–5 kb from CGI). The majority of CG differences occur at gene intronic regions. (E) Scatterplot of DMR overlap with TFBSs (ENCODE data). The x axis shows increased methylation at sites compared with reduced methylation at sites in T1D cases (y axis). TFBSs that associate with DMRs are shown as increased in methylation (pink) circles and reduced in methylation (purple) in T1D cases when compared with healthy controls. Criteria for TFBS overlap with DMRs was set to 50%. (F) CTCF and Pol2B binding sites are overrepresented at DMRs with reduced methylation in T1D cases when compared with healthy controls. Within each box, horizontal black lines denote median values; boxes extend from the 25th to the 75th percentile of each group’s distribution and the whisker denote the 5th and 95th percentiles. CTCF and Pol2B binding sites were overrepresented at sites of reduced methylation in diabetics that developed renal complications. All P values < 0.001. (G) Atlas of DNA methylation from FinnDiane discovery cohort. Human chromosome ideogram of DMRs clustered by DN (P < 0.01). The outermost track is organized by autosomes, showing several methylation-dependent genes. The centromere of each chromosome (chr) is represented by a double red line. The second track represents a genomic view of 3362 regions with increased methylation in all cases (hypomethylated in healthy group) (green, P < 0.01). The second track represents 1792 regions with decreased methylation in the Normo group (purple, P < 0.01). The fourth track represents the Macro group with 3227 regions with decreased methylation (blue, P < 0.01). The fifth track represents the genomic view of 1697 regions with decreased methylation detected in the ESRD group (orange, P < 0.01).