Background A subset of individuals with type 1 diabetes mellitus (T1DM) are predisposed to developing diabetic kidney disease (DKD), the most common cause globally of end-stage kidney disease (ESKD). Emerging evidence suggests epigenetic changes in DNA methylation may have a causal role in both T1DM and DKD. The aim of this exploratory investigation was to assess differences in blood-derived DNA methylation patterns between individuals with T1DM-ESKD and individuals with long-duration T1DM but no evidence of kidney disease upon repeated testing to identify potential blood-based biomarkers. Blood-derived DNA from individuals (107 cases, 253 controls and 14 experimental controls) were bisulphite treated before DNA methylation patterns from both groups were generated and analysed using Illumina’s Infinium MethylationEPIC BeadChip arrays (n = 862,927 sites). Differentially methylated CpG sites (dmCpGs) were identified (false discovery rate adjusted p ≤ × 10–8 and fold change ± 2) by comparing methylation levels between ESKD cases and T1DM controls at single site resolution. Gene annotation and functionality was investigated to enrich and rank methylated regions associated with ESKD in T1DM. Results Top-ranked genes within which several dmCpGs were located and supported by functional data with methylation look-ups in other cohorts include: AFF3, ARID5B, CUX1, ELMO1, FKBP5, HDAC4, ITGAL, LY9, PIM1, RUNX3, SEPTIN9 and UPF3A. Top-ranked enrichment pathways included pathways in cancer, TGF-β signalling and Th17 cell differentiation. Conclusions Epigenetic alterations provide a dynamic link between an individual’s genetic background and their environmental exposures. This robust evaluation of DNA methylation in carefully phenotyped individuals has identified biomarkers associated with ESKD, revealing several genes and implicated key pathways associated with ESKD in individuals with T1DM.

Gremlin, a cell growth and differentiation factor, promotes the development of diabetic nephropathy in animal models, but whether GREM1 gene variants associate with diabetic nephropathy is unknown. We comprehensively screened the 5Ј upstream region (including the predicted promoter), all exons, intronexon boundaries, complete untranslated regions, and the 3Ј region downstream of the GREM1 gene. We identified 31 unique variants, including 24 with a minor allele frequency exceeding 5%, and 9 haplotypetagging single nucleotide polymorphisms (htSNPs). We selected one additional variant that we predicted to alter transcription factor binding. We genotyped 709 individuals with type 1 diabetes of whom 267 had nephropathy (cases) and 442 had no evidence of kidney disease (controls). Three individual SNPs significantly associated with nephropathy at the 5% level, and two remained significant after adjustment for multiple testing. Subsequently, we genotyped a replicate population comprising 597 cases and 502 controls: this population supported an association with one of the SNPs (rs1129456; P ϭ 0.0003). Combined analysis, adjusted for recruitment center (n ϭ 8), suggested that the T allele conferred greater odds of nephropathy (OR 1.69; 95% CI 1.36 to 2.11). In summary, the GREM1 variant rs1129456 associates with diabetic nephropathy, perhaps explaining some of the genetic susceptibility to this condition.

Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant multisystemic vascular dysplasia, characterized by arteriovenous malformations (AVMs), mucocutaneous telangiectasia and nosebleeds. HHT is caused by a heterozygous null allele in ACVRL1, ENG, or SMAD4, which encode proteins mediating bone morphogenetic protein (BMP) signaling. Several missense and stop-gain variants identified in GDF2 (encoding BMP9) have been reported to cause a vascular anomaly syndrome similar to HHT, however none of these patients met diagnostic criteria for HHT. HHT families from UK NHS Genomic Medicine Centres were recruited to the Genomics England 100,000 Genomes Project. Whole genome sequencing and tiering protocols identified a novel, heterozygous GDF2 sequence variant in all three affected members of one HHT family who had previously screened negative for ACVRL1, ENG, and SMAD4. All three had nosebleeds and typical HHT telangiectasia, and the proband also had severe pulmonary AVMs from childhood. In vitro studies showed the mutant construct expressed the proprotein but lacked active mature BMP9 dimer, suggesting the mutation disrupts correct cleavage of the protein. Plasma BMP9 levels in the patients were significantly lower than controls. In conclusion, we propose that this

Type 1 diabetes affects over nine million individuals globally, with approximately 40% developing diabetic kidney disease. Emerging evidence suggests that epigenetic alterations, such as DNA methylation, are involved in diabetic kidney disease. Here we assess differences in blood-derived genome-wide DNA methylation associated with diabetic kidney disease in 1304 carefully characterised individuals with type 1 diabetes and known renal status from two cohorts in the United Kingdom-Republic of Ireland and Finland. In the meta-analysis, we identify 32 differentially methylated CpGs in diabetic kidney disease in type 1 diabetes, 18 of which are located within genes differentially expressed in kidneys or correlated with pathological traits in diabetic kidney disease. We show that methylation at 21 of the 32 CpGs predict the development of kidney failure, extending the knowledge and potentially identifying individuals at greater risk for diabetic kidney disease in type 1 diabetes.

Chemokine (C-C motif) ligand 5 (CCL5) and chemokine (C-C motif) receptor 5 are implicated in the pathogenesis of diabetic nephropathy (DN). We hypothesize that variants in these genes may be associated with DN. The CCL5 and chemokine receptor type 5 (CCR5) genes were resequenced, variants identified (n¼58), allele frequencies determined in 46 individuals (92 chromosomes) and efficient haplotype tag single-nucleotide polymorphisms (htSNPs) selected to effectively evaluate the common variation in these genes. One reportedly functional gene variant and eight htSNPs were genotyped in a case-control association study involving Caucasian individuals with type 1 diabetes (267 cases with DN and 442 non-nephropathic diabetic controls). Genotyping was performed using MassARRAY iPLEX, TaqMan, gel electrophoresis and direct capillary sequencing. After correction for multiple testing, there were no statistically significant associations between variants in the CCL5 and CCR5 genes and DN. Keywords: association; chemokine; CCL5; CCR5; diabetic nephropathy; resequencing Diabetic nephropathy (DN) is the leading cause of end-stage renal disease in the Western world 1 with significant evidence that a subset of type 1 diabetic patients are genetically predisposed to developing DN. 2 Chemokine (C-C motif) ligand 5 (alternatively known as regulated upon activation, normal T cell expressed and secreted; CCL5) and its cognate receptor, chemokine receptor type 5 (CCR5), are biological candidate genes for DN.CCL5 is a potent chemoattractant produced by renal mesangial cells, which is active in the recruitment of monocytes and macrophages into the glomeruli and interstitium. CCL5 is nuclear factor -kB dependent and is upregulated by proteinuria 3 and other factors associated with the diabetic milieu. 4-6 Blockade of CCR5 has been shown to substantially reduce monocyte infiltration in experimental glomerulonephritis. 7 CCL5 and CCR5 have received considerable attention as candidate genes for DN, although these studies have focused on selected variants with inconsistent findings. [8][9][10][11][12][13][14][15] The CCL5 gene locus has been assigned to 17q11.2-q12, 16 and the CCR5 gene is located on chromosome 3p21. 17 Coding regions, intron-exon boundaries, untranslated and flanking regions of the CCL5 and CCR5 genes have been comprehensively screened to identify polymorphisms, and a robust case-control study was performed to assess the association between common haplotype tagging or functional variants and DN in a Caucasian population with type 1 diabetes.The Irish case-control collection investigated in this paper has been described previously. 18 Briefly, cases have type 1 diabetes with DN, while controls are type 1 diabetic patients without nephropathy. The average age at recruitment of cases and controls was 48.7 (s.d. 9.6) years and 42.2 (s.d. 11.7) years, respectively, with an average disease duration of 32.0 (s.d. 9.6) years for cases and 27.2 (s.d. 9.3) years for controls.