Whole-exome and whole-genome sequencing of 1064 individuals with type 1 diabetes reveals novel genes for diabetic kidney disease

Abstract

Aims/hypothesis: Diabetic kidney disease (DKD) is a severe diabetic complication that affects one third of individuals with type 1 diabetes. Although several genes and common variants have been shown to be associated with DKD, much of the predicted inheritance remains unexplained. Here, we performed next-generation sequencing to assess whether low-frequency variants, extending to a minor allele frequency (MAF) ≤10% (single or aggregated) contribute to the missing heritability in DKD.

Methods: We performed whole-exome sequencing (WES) of 498 individuals and whole-genome sequencing (WGS) of 599 individuals with type 1 diabetes. After quality control, next-generation sequencing data were available for a total of 1064 individuals, of whom 541 had developed either severe albuminuria or end-stage kidney disease, and 523 had retained normal albumin excretion despite a long duration of type 1 diabetes. Single-variant and gene-aggregate tests for protein-altering variants (PAV) and protein-truncating variants (PTV) were performed separately for WES and WGS data and combined in a meta-analysis. We also performed genome-wide aggregate analyses on genomic windows (sliding window), promoters and enhancers using the WGS dataset.

Results: In the single-variant meta-analysis, no variant reached genome-wide significance, but a suggestively associated common THAP7 rs369250 variant (p=1.50 × 10^-5, MAF=49%) was replicated in the FinnGen general population genome-wide association study (GWAS) data for chronic kidney disease and DKD phenotypes. The gene-aggregate meta-analysis provided suggestive evidence (p<4.0 × 10^-4) at four genes for DKD, of which NAT16 (MAF_PAV≤10%) and LTA (also known as TNFβ, MAF_PAV≤5%) are replicated in the FinnGen general population GWAS data. The LTA rs2229092 C allele was associated with significantly lower TNFR1, TNFR2 and TNFR3 serum levels in a subset of FinnDiane participants. Of the intergenic regions suggestively associated with DKD, the enhancer on chromosome 18q12.3 (p=3.94 × 10^-5, MAF_variants≤5%) showed interaction with the METTL4 gene; the lead variant was replicated, and predicted to alter binding of the MafB transcription factor.

Conclusions/interpretation: Our sequencing-based meta-analysis revealed multiple genes, variants and regulatory regions that were suggestively associated with DKD. However, as no variant or gene reached genome-wide significance, further studies are needed to validate the findings.

Keywords: Diabetic kidney disease; METTL4; TNF receptors; TNFβ; Type 1 diabetes; Whole-exome sequencing; Whole-genome sequencing.

Fig. 1

Study setup for single-variant and gene and intergenic region aggregate analyses. We performed single-variant and gene-aggregate meta-analyses on the WES and WGS cohorts, covering the exome regions and surrounding regions. The gene-aggregate analysis was run only for low-frequency PAVs and PTVs. We also performed a single-variant analysis and genome- and regulome-wide window scans using only WGS data. We replicated the results against FinnDiane and THL Biobank GWAS data, publicly available FinnGen data and TOPMed WGS data

Fig. 2

Manhattan plot for the WES/WGS single-variant meta-analysis. The meta-analysis resulted in identification of six variants: five common ones (chr2 FAM161A rs3736598 [p=1.30 × 10⁻⁵], rs6748320 [p=1.58 × 10⁻⁵], chr22 THAP7 rs369250 [p=1.50 × 10⁻⁵], chr7 AP1S1 rs1048365 [p=2.07 × 10⁻⁵] and KMT2E rs117986340 [p=2.71 × 10⁻⁵]), and one with a low frequency (chr9 EPB41L4B rs10979729 [p=6.76 × 10⁻⁶])

Fig. 3

Association of the LTA rs2229092 C allele and lower levels of TNF receptors. Serum levels of three TNF receptors that bind LT-α (TNFR1, TNFR2 and TNFR3) were measured using the Olink platform, and serum TNF receptor levels were measured for 128 individuals with WES and WGS data. Linear regression was adjusted for age and sex. The alternative C allele of LTA rs2229092 had significantly lower levels of (a) TNFR1, (b) TNFR2 and (c) TNFR3 compared with carriers of the reference allele. Differences between the groups are indicated by asterisks: *p<0.05, **p<0.01