Genome-Wide Association Study of Diabetic Kidney Disease Highlights Biology Involved in Glomerular Basement Membrane Collagen.

BACKGROUND: Although diabetic kidney disease demonstrates both familial clustering and single nucleotide polymorphism heritability, the specific genetic factors influencing risk remain largely unknown. METHODS: To identify genetic variants predisposing to diabetic kidney disease, we performed genome-wide association study (GWAS) analyses. Through collaboration with the Diabetes Nephropathy Collaborative Research Initiative, we assembled a large collection of type 1 diabetes cohorts with harmonized diabetic kidney disease phenotypes. We used a spectrum of ten diabetic kidney disease definitions based on albuminuria and renal function. RESULTS: Our GWAS meta-analysis included association results for up to 19,406 individuals of European descent with type 1 diabetes. We identified 16 genome-wide significant risk loci. The variant with the strongest association (rs55703767) is a common missense mutation in the collagen type IV alpha 3 chain (COL4A3) gene, which encodes a major structural component of the glomerular basement membrane (GBM). Mutations in COL4A3 are implicated in heritable nephropathies, including the progressive inherited nephropathy Alport syndrome. The rs55703767 minor allele (Asp326Tyr) is protective against several definitions of diabetic kidney disease, including albuminuria and ESKD, and demonstrated a significant association with GBM width; protective allele carriers had thinner GBM before any signs of kidney disease, and its effect was dependent on glycemia. Three other loci are in or near genes with known or suggestive involvement in this condition (BMP7) or renal biology (COLEC11 and DDR1). CONCLUSIONS: The 16 diabetic kidney disease-associated loci may provide novel insights into the pathogenesis of this condition and help identify potential biologic targets for prevention and treatment.

The association of previously reported polymorphisms for microvascular complications in a meta-analysis of diabetic retinopathy.

We investigated the association of signals from previous GWAS and candidate gene meta-analyses for diabetic retinopathy (DR) or nephropathy (DN), as well as an EPO variant in meta-analyses of severe (SDR) and mild diabetic retinopathy (MDR). Meta-analyses of SDR (≥severe non-proliferative diabetic retinopathy (NPDR) or history of panretinal photocoagulation) and MDR (≥mild NPDR), defined based on seven-field stereoscopic fundus photographs, were performed in two well-characterized type 1 diabetes (T1D) cohorts: the Diabetes Control and Complications Trial/Epidemiology of Diabetes Interventions and Complications (DCCT/EDIC, n = 1,304) and Wisconsin Epidemiologic Study of Diabetic Retinopathy (WESDR, n = 603). Among 34 previous signals for DR, after controlling for multiple testing, no association was replicated in our meta-analyses. rs1571942 and rs12219125 at PLXDC2 locus showed nominally significant (<0.05) association with SDR in the same direction as previous report, as did rs1801282 in PPARG gene with MDR. Among 55 loci previously associated with DN, three showed suggestive associations with SDR in our study without maintaining significance after correction for multiple testing. Of particular interest, rs1617640 (EPO) was not significantly associated with DR status, combined SDR-DN phenotype, time to SDR or time to DN (all P > 0.05). Lack of replication of previous DR hits and EPO despite reasonable statistical power implies that many of these may be false positives. Consistent with pleiotropy, we provide suggestive collective evidence for association between DR and variants previously associated with DN without reaching statistical significance at any single locus.

New susceptibility loci associated with kidney disease in type 1 diabetes.

Diabetic kidney disease, or diabetic nephropathy (DN), is a major complication of diabetes and the leading cause of end-stage renal disease (ESRD) that requires dialysis treatment or kidney transplantation. In addition to the decrease in the quality of life, DN accounts for a large proportion of the excess mortality associated with type 1 diabetes (T1D). Whereas the degree of glycemia plays a pivotal role in DN, a subset of individuals with poorly controlled T1D do not develop DN. Furthermore, strong familial aggregation supports genetic susceptibility to DN. However, the genes and the molecular mechanisms behind the disease remain poorly understood, and current therapeutic strategies rarely result in reversal of DN. In the GEnetics of Nephropathy: an International Effort (GENIE) consortium, we have undertaken a meta-analysis of genome-wide association studies (GWAS) of T1D DN comprising ~2.4 million single nucleotide polymorphisms (SNPs) imputed in 6,691 individuals. After additional genotyping of 41 top ranked SNPs representing 24 independent signals in 5,873 individuals, combined meta-analysis revealed association of two SNPs with ESRD: rs7583877 in the AFF3 gene (P = 1.2 × 10(-8)) and an intergenic SNP on chromosome 15q26 between the genes RGMA and MCTP2, rs12437854 (P = 2.0 × 10(-9)). Functional data suggest that AFF3 influences renal tubule fibrosis via the transforming growth factor-beta (TGF-ß1) pathway. The strongest association with DN as a primary phenotype was seen for an intronic SNP in the ERBB4 gene (rs7588550, P = 2.1 × 10(-7)), a gene with type 2 diabetes DN differential expression and in the same intron as a variant with cis-eQTL expression of ERBB4. All these detected associations represent new signals in the pathogenesis of DN.

Genome-wide association identifies the ABO blood group as a major locus associated with serum levels of soluble E-selectin.

BACKGROUND: Elevated serum soluble E-selectin levels have been associated with a number of diseases. Although E-selectin levels are heritable, little is known about the specific genetic factors involved. E-selectin levels have been associated with the ABO blood group phenotype. METHODS AND RESULTS: We performed a high-resolution genome-wide association study of serum soluble E-selectin levels in 685 white individuals with type 1 diabetes from the Diabetes Control and Complications Trial (DCCT)/Epidemiology of Diabetes Intervention and Complications (EDIC) study to identify major loci influencing levels. Highly significant evidence for association (P=10(-29)) was observed for rs579459 near the ABO blood group gene, accounting for 19% of the variance in E-selectin levels. Levels of E-selectin were higher in O/O than O/A heterozygotes, which were likewise higher than A/A genotypes. Analysis of subgroups of A alleles reveals heterogeneity in the association, and even after this was accounted for, an intron 1 SNP remained significantly associated. We replicate the ABO association in nondiabetic individuals. CONCLUSIONS: ABO is a major locus for serum soluble E-selectin levels. We excluded population stratification, fine-mapped the association to sub-A alleles, and also document association with additional variation in the ABO region.

LDL composition in E2/2 subjects and LDL distribution by Apo E genotype in type 1 diabetes.

Apo E plays an important role in chylomicron and VLDL remnant processing, uptake or conversion to LDL. The type of lipoprotein that isolates in the LDL density of E2/2 subjects was investigated and the effect of the apo E isoforms on LDL mass was determined in all genotypes in a large group of Type 1 diabetics. Analysis of the LDL composition of E2/2 homozygotes (n=6) compared to subjects with the common E3/3 isoform (n=6) demonstrated an enrichment in apo E, unesterified cholesterol, phospholipid and triglyceride relative to apo B in E2/2 subjects, more typical of a dense IDL remnant than of LDL. Although diabetics were studied, these findings are considered to reflect those of the general population. Comparison of the lipoprotein distribution of homozygous and heterozygous subjects revealed that, as genotype changed from E4/4 (n=22) to E3/4 (n=262), E3/3 (n=710)=E2/4 (n=30), E2/3 (n=151), E2/2 (n=6), LDL cholesterol decreased significantly in a stepwise manner. The decrease was not in a specific subgroup of LDL. In conclusion, for E2/2 subjects, lipoproteins isolated in the LDL density range appear to be composed mainly of dense IDL remnants and some Lp(a). The apo E isoform also has a significant effect on LDL concentration in both homozygotes and heterozygotes.

Genetic variation at the ACE gene is associated with persistent microalbuminuria and severe nephropathy in type 1 diabetes: the DCCT/EDIC Genetics Study.

The development and progression of microvascular complications have been extensively documented in a cohort of type 1 diabetic subjects enrolled in the Diabetes Control and Complications Trial (DCCT) and followed in the Epidemiology of Diabetes Interventions and Complications (EDIC) study. We describe the association of genetic variation in the ACE gene in 1,365 DCCT/EDIC subjects with incident persistent microalbuminuria (n = 312) and severe nephropathy (n = 115). We studied three markers (rs1800764, insertion/deletion, and rs9896208) in the ACE gene that allowed us to capture genetic variation in the common haplotypes occurring at frequencies of >5% in Caucasians. Compared with the more frequent genotype (D/I) for the insertion/deletion polymorphism, in multivariate models, the I/I genotype conferred a lower risk for persistent microalbuminuria (hazard ratio [HR] 0.62 [95% CI 0.43-0.89], P = 0.009) and severe nephropathy (0.56 [0.32-0.96], P = 0.033). Variation at the two other markers, rs1800764 and rs9896208, were also associated with these renal outcomes. In addition, homozygosity for the common haplotype TIC (which corresponded to the T, insertion, and C alleles at the three markers, rs1800764, insertion/deletion, and rs9896208, respectively) versus the CDT/TIC haplotype pair was associated with lower risk for development of persistent microalbuminuria (HR 0.49 [0.32-0.75], P = 0.0009) and severe nephropathy (0.41 [0.22-0.78], P = 0.006). Our findings in the DCCT/EDIC cohort provide strong evidence that genetic variation at the ACE gene is associated with the development of nephropathy in patients with type 1 diabetes.

A genome-wide association study identifies a novel major locus for glycemic control in type 1 diabetes, as measured by both A1C and glucose.

OBJECTIVE: Glycemia is a major risk factor for the development of long-term complications in type 1 diabetes; however, no specific genetic loci have been identified for glycemic control in individuals with type 1 diabetes. To identify such loci in type 1 diabetes, we analyzed longitudinal repeated measures of A1C from the Diabetes Control and Complications Trial. RESEARCH DESIGN AND METHODS: We performed a genome-wide association study using the mean of quarterly A1C values measured over 6.5 years, separately in the conventional (n = 667) and intensive (n = 637) treatment groups of the DCCT. At loci of interest, linear mixed models were used to take advantage of all the repeated measures. We then assessed the association of these loci with capillary glucose and repeated measures of multiple complications of diabetes. RESULTS: We identified a major locus for A1C levels in the conventional treatment group near SORCS1 (10q25.1, P = 7 x 10(-10)), which was also associated with mean glucose (P = 2 x 10(-5)). This was confirmed using A1C in the intensive treatment group (P = 0.01). Other loci achieved evidence close to genome-wide significance: 14q32.13 (GSC) and 9p22 (BNC2) in the combined treatment groups and 15q21.3 (WDR72) in the intensive group. Further, these loci gave evidence for association with diabetic complications, specifically SORCS1 with hypoglycemia and BNC2 with renal and retinal complications. We replicated the SORCS1 association in Genetics of Diabetes in Kidneys (GoKinD) study control subjects (P = 0.01) and the BNC2 association with A1C in nondiabetic individuals. CONCLUSIONS: A major locus for A1C and glucose in individuals with diabetes is near SORCS1. This may influence the design and analysis of genetic studies attempting to identify risk factors for long-term diabetic complications.

Genome-wide association scan for diabetic nephropathy susceptibility genes in type 1 diabetes.

OBJECTIVE: Despite extensive evidence for genetic susceptibility to diabetic nephropathy, the identification of susceptibility genes and their variants has had limited success. To search for genes that contribute to diabetic nephropathy, a genome-wide association scan was implemented on the Genetics of Kidneys in Diabetes collection. RESEARCH DESIGN AND METHODS: We genotyped approximately 360,000 single nucleotide polymorphisms (SNPs) in 820 case subjects (284 with proteinuria and 536 with end-stage renal disease) and 885 control subjects with type 1 diabetes. Confirmation of implicated SNPs was sought in 1,304 participants of the Diabetes Control and Complications Trial (DCCT)/Epidemiology of Diabetes Interventions and Complications (EDIC) study, a long-term, prospective investigation of the development of diabetes-associated complications. RESULTS: A total of 13 SNPs located in four genomic loci were associated with diabetic nephropathy with P < 1 x 10(-5). The strongest association was at the FRMD3 (4.1 protein ezrin, radixin, moesin [FERM] domain containing 3) locus (odds ratio [OR] = 1.45, P = 5.0 x 10(-7)). A strong association was also identified at the CARS (cysteinyl-tRNA synthetase) locus (OR = 1.36, P = 3.1 x 10(-6)). Associations between both loci and time to onset of diabetic nephropathy were supported in the DCCT/EDIC study (hazard ratio [HR] = 1.33, P = 0.02, and HR = 1.32, P = 0.01, respectively). We demonstratedexpression of both FRMD3 and CARS in human kidney. CONCLUSIONS: We identified genetic associations for susceptibility to diabetic nephropathy at two novel candidate loci near the FRMD3 and CARS genes. Their identification implicates previously unsuspected pathways in the pathogenesis of this important late complication of type 1 diabetes.

Multiple superoxide dismutase 1/splicing factor serine alanine 15 variants are associated with the development and progression of diabetic nephropathy: the Diabetes Control and Complications Trial/Epidemiology of Diabetes Interventions and Complications Genetics study.

BACKGROUND: Despite familial clustering of nephropathy and retinopathy severity in type 1 diabetes, few gene variants have been consistently associated with these outcomes. RESEARCH DESIGN AND METHODS: We performed an individual-based genetic association study with time to renal and retinal outcomes in 1,362 white probands with type 1 diabetes from the Diabetes Control and Complications Trial/Epidemiology of Diabetes Interventions and Complications (DCCT/EDIC) study. Specifically, we genotyped 1,411 SNPs that capture common variations in 212 candidate genes for long-term complications and analyzed them for association with the time from DCCT baseline to event for renal and retinal outcomes using multivariate Cox proportion hazards models. To address multiple testing and assist interpretation of the results, false discovery rate q values were calculated separately for each outcome. RESULTS: We observed association between rs17880135 in the 3' region of superoxide dismutase 1 (SOD1) and the incidence of both severe nephropathy (hazard ratio [HR] 2.62 [95% CI 1.64-4.18], P = 5.6 x 10(-5), q = 0.06) and persistent microalbuminuria (1.82 [1.29-2.57], P = 6.4 x 10(-4), q = 0.46). Sequencing and fine-mapping identified additional SOD1 variants, including rs202446, rs9974610, and rs204732, which were also associated (P < 10(-3)) with persistent microalbuminuria, whereas rs17880135 and rs17881180 were similarly associated with the development of severe nephropathy. Attempts to replicate the findings in three cross-sectional case-control studies produced equivocal results. We observed no striking differences between risk genotypes in serum SOD activity, serum SOD1 mass, or SOD1 mRNA expression in lymphoblastoid cell lines. CONCLUSIONS: Multiple variations in SOD1 are significantly associated with persistent microalbuminuria and severe nephropathy in the DCCT/EDIC study.

Multiple variants in vascular endothelial growth factor (VEGFA) are risk factors for time to severe retinopathy in type 1 diabetes: the DCCT/EDIC genetics study.

OBJECTIVE: We sought to determine if any common variants in the gene for vascular endothelial growth factor (VEGFA) are associated with long-term renal and retinal complications in type 1 diabetes. RESEARCH DESIGN AND METHODS: A total of 1,369 Caucasian subjects with type 1 diabetes from the Diabetes Control and Complications Trial (DCCT)/Epidemiology of Diabetes Interventions and Complications (EDIC) Study had an average of 17 retinal photographs and 10 renal measures over 15 years. In the DCCT/EDIC, we studied 18 single nucleotide polymorphisms (SNPs) in VEGFA that represent all linkage disequilibrium bins (pairwise r(2) > or = 0.64) and tested them for association with time to development of severe retinopathy, three or more step progression of retinopathy, clinically significant macular edema, persistent microalbuminuria, and severe nephropathy. RESULTS: In a global multi-SNP test, there was a highly significant association of VEGFA SNPs with severe retinopathy (P = 6.8 x 10(-5))-the four other outcomes were all nonsignificant. In survival analyses controlling for covariate risk factors, eight SNPs showed significant association with severe retinopathy (P < 0.05). The most significant single SNP association was rs3025021 (hazard ratio 1.37 [95% CI 1.13-1.66], P = 0.0017). Family-based analyses of severe retinopathy provide evidence of excess transmission of C at rs699947 (P = 0.029), T at rs3025021 (P = 0.013), and the C-T haplotype from both SNPs (P = 0.035). Multi-SNP regression analysis including 15 SNPs, and allowing for pairwise interactions, independently selected 6 significant SNPs (P < 0.05). CONCLUSIONS: These data demonstrate that multiple VEGFA variants are associated with the development of severe retinopathy in type 1 diabetes.

Genescript: DNA sequence annotation pipeline.

UNLABELLED: Genescript uses a number of publicly available analysis programs to annotate a DNA sequence. It provides an integrated display of results from each program, and includes an evidence-based scoring system that gives informative summaries of predicted gene models. AVAILABILITY: Genescript is available for download from http:://tcag.bioinfo.sickkids.on.ca/genescript/