Differential Methylation of Telomere-Related Genes Is Associated with Kidney Disease in Individuals with Type 1 Diabetes.

Diabetic kidney disease (DKD) represents a major global health problem. Accelerated ageing is a key feature of DKD and, therefore, characteristics of accelerated ageing may provide useful biomarkers or therapeutic targets. Harnessing multi-omics, features affecting telomere biology and any associated methylome dysregulation in DKD were explored. Genotype data for nuclear genome polymorphisms in telomere-related genes were extracted from genome-wide case-control association data (n = 823 DKD/903 controls; n = 247 end-stage kidney disease (ESKD)/1479 controls). Telomere length was established using quantitative polymerase chain reaction. Quantitative methylation values for 1091 CpG sites in telomere-related genes were extracted from epigenome-wide case-control association data (n = 150 DKD/100 controls). Telomere length was significantly shorter in older age groups (p = 7.6 × 10-6). Telomere length was also significantly reduced (p = 6.6 × 10-5) in DKD versus control individuals, with significance remaining after covariate adjustment (p = 0.028). DKD and ESKD were nominally associated with telomere-related genetic variation, with Mendelian randomisation highlighting no significant association between genetically predicted telomere length and kidney disease. A total of 496 CpG sites in 212 genes reached epigenome-wide significance (p ≤ 10-8) for DKD association, and 412 CpG sites in 193 genes for ESKD. Functional prediction revealed differentially methylated genes were enriched for Wnt signalling involvement. Harnessing previously published RNA-sequencing datasets, potential targets where epigenetic dysregulation may result in altered gene expression were revealed, useful as potential diagnostic and therapeutic targets for intervention.

Longitudinal Epigenome-Wide Analysis of Kidney Transplant Recipients Pretransplant and Posttransplant.

Introduction: Kidney transplantation remains the gold standard of treatment for end-stage renal disease (ESRD), with improved patient outcomes compared with dialysis. Epigenome-Wide Association Analysis (EWAS) of DNA methylation may identify markers that contribute to an individual's risk of adverse transplant outcomes, yet only a limited number of EWAS have been conducted in kidney transplant recipients. This EWAS aimed to interrogate the methylation profile of a kidney transplant recipient cohort with minimal posttransplant complications, exploring differences in samples pretransplant and posttransplant. Methods: We compared differentially methylated cytosine-phosphate-guanine sites (dmCpGs) in samples derived from peripheral blood mononuclear cells of the same kidney transplant recipients, collected both pretransplant and posttransplant (N = 154), using the Infinium MethylationEPIC microarray (Illumina, San Diego, CA). Recipients received kidneys from deceased donors and had a mean of 17 years of follow-up. Results: Five top-ranked dmCpGs were significantly different at false discovery rate (FDR) adjusted P ≤ 9 × 10-8; cg23597162 within JAZF1, cg25187293 within BTNL8, cg17944885, located between ZNF788P and ZNF625-ZNF20, cg14655917 located between ASB4 and PDK4 and cg09839120 located between GIMAP6 and EIF2AP3. Conclusion: Five dmCpGs were identified at the generally accepted EWAS critical significance level of FDR adjusted P (P FDRadj) ≤ 9 × 10-8, including cg23597162 (within JAZF1) and cg17944885, which have prior associations with chronic kidney disease (CKD). Comparing individuals with no evidence of posttransplant complications (N = 105) demonstrated that 693,555 CpGs (89.57%) did not display any significant difference in methylation (P FDRadj ≥ 0.05), thereby this study establishes an important reference for future epigenetic studies that seek to identify markers of posttransplant complications.

Epigenome-wide meta-analysis identifies DNA methylation biomarkers associated with diabetic kidney disease.

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.

A Formative Study of the Implementation of Whole Genome Sequencing in Northern Ireland.

Background: The UK 100,000 Genomes Project was a transformational research project which facilitated whole genome sequencing (WGS) diagnostics for rare diseases. We evaluated experiences of introducing WGS in Northern Ireland, providing recommendations for future projects. Methods: This formative evaluation included (1) an appraisal of the logistics of implementing and delivering WGS, (2) a survey of participant self-reported views and experiences, (3) semi-structured interviews with healthcare staff as key informants who were involved in the delivery of WGS and (4) a workshop discussion about interprofessional collaboration with respect to molecular diagnostics. Results: We engaged with >400 participants, with detailed reflections obtained from 74 participants including patients, caregivers, key National Health Service (NHS) informants, and researchers (patient survey n = 42; semi-structured interviews n = 19; attendees of the discussion workshop n = 13). Overarching themes included the need to improve rare disease awareness, education, and support services, as well as interprofessional collaboration being central to an effective, mainstreamed molecular diagnostic service. Conclusions: Recommendations for streamlining precision medicine for patients with rare diseases include administrative improvements (e.g., streamlining of the consent process), educational improvements (e.g., rare disease training provided from undergraduate to postgraduate education alongside genomics training for non-genetic specialists) and analytical improvements (e.g., multidisciplinary collaboration and improved computational infrastructure).

Identification and validation of a novel pathogenic variant in GDF2 (BMP9) responsible for hereditary hemorrhagic telangiectasia and pulmonary arteriovenous malformations.

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 heterozygous GDF2 variant is a rare cause of HHT associated with pulmonary AVMs.

Genetic Strategies to Understand Human Diabetic Nephropathy: Wet-Lab Approaches.

Multiple genetic strategies are available to help improve understanding of diabetic nephropathy. This methods chapter provides an overview of phenotype considerations specific to diabetic nephropathy and biobank essentials, and provides detailed methodology for a common benchtop wet-lab approach (Ion Torrent semiconductor sequencing) including in silico genetic variant identification from next-generation sequencing data to identify genetic risk factors for diabetic nephropathy.

Validation of differentially methylated microRNAs identified from an epigenome-wide association study; Sanger and next generation sequencing approaches.

OBJECTIVES: Altered DNA methylation and microRNA profiles are associated with diabetic kidney disease. This study compared different sequencing approaches to define the genetic and epigenetic architecture of sequences surrounding microRNAs associated with diabetic kidney disease. RESULTS: We compared Sanger and next generation sequencing to validate microRNAs associated with diabetic kidney disease identified from an epigenome-wide association study (EWAS). These microRNAs demonstrated differential methylation levels in cases with diabetic kidney disease compared to controls with long duration of type 1 diabetes and no evidence of kidney disease (Padjusted < 10-5). Targeted next generation sequencing analysis of genomic DNA and matched cell-line transformed DNA samples identified four genomic variants within the microRNAs, two within miR-329-2 and two within miR-429. Sanger sequencing of genomic DNA replicated these findings and confirmed the altered methylation status of the CpG sites identified by the EWAS in bisulphite-treated DNA. This investigation successfully fine-mapped the genetic sequence around key microRNAs. Variants have been detected which may affect their methylation status and methylated CpG sites have been confirmed. Additionally, we explored both the fidelity of next generation sequencing analysis and the potential efficacy of cell-line transformed DNA samples in place of finite patient samples in discovery genetic and epigenetic research.

Design and implementation of a custom next generation sequencing panel for selected vitamin D associated genes.

BACKGROUND: Biologically active vitamin D has an important regulatory role within the genome. It binds the vitamin D receptor (VDR) in order to control the expression of a wide range of genes as well as interacting with the epigenome to modify chromatin and methylation status. Vitamin D deficiency is associated with several human diseases including end-stage renal disease. METHODS: This article describes the design and testing of a custom, targeted next generation sequencing (NGS) panel for selected vitamin D associated genes. Sequencing runs were used to determine the effectiveness of the panel for variant calling, to compare efficiency and data across different sequencers, and to perform representative, proof of principle association analyses. These analyses were underpowered for significance testing. Amplicons were designed in two pools (163 and 166 fragments respectively) and used to sequence two cohorts of renal transplant recipients on the Ion Personal Genome Machine (PGM)™ and Ion S5™ XL desktop sequencers. RESULTS: Coverage was provided for 43.8 kilobases across seven vitamin D associated genes (CYP24A1, CUBN, VDR, GC, NADSYN1, CYP27B1, CYP2R1) as well as 38 prioritised SNPs. Sequencing runs provided sufficient sequencing quality, data output and validated the effective library preparation and panel design. CONCLUSIONS: This novel, custom-designed, validated panel provides a fast, cost effective, and specific approach for the analysis of vitamin D associated genes in a wide range of patient cohorts. This article does not report results from a controlled health-care intervention.

Development of next generation sequencing panel for UMOD and association with kidney disease.

Chronic kidney disease (CKD) has a prevalence of approximately 10% in adult populations. CKD can progress to end-stage renal disease (ESRD) and this is usually fatal unless some form of renal replacement therapy (chronic dialysis or renal transplantation) is provided. There is an inherited predisposition to CKD with several genetic risk markers now identified. The UMOD gene has been associated with CKD of varying aetiologies. An AmpliSeq next generation sequencing panel was developed to facilitate comprehensive sequencing of the UMOD gene, covering exonic and regulatory regions. SNPs and CpG sites in the genomic region encompassing UMOD were evaluated for association with CKD in two studies; the UK Wellcome Trust Case-Control 3 Renal Transplant Dysfunction Study (n = 1088) and UK-ROI GENIE GWAS (n = 1726). A technological comparison of two Ion Torrent machines revealed 100% allele call concordance between S5 XL™ and PGM™ machines. One SNP (rs183962941), located in a non-coding region of UMOD, was nominally associated with ESRD (p = 0.008). No association was identified between UMOD variants and estimated glomerular filtration rate. Analysis of methylation data for over 480,000 CpG sites revealed differential methylation patterns within UMOD, the most significant of these was cg03140788 p = 3.7 x 10-10.

Resequencing of the CCL5 and CCR5 genes and investigation of variants for association with diabetic nephropathy.

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 (n=58), 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.

A GREM1 gene variant associates with diabetic nephropathy.

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 5' upstream region (including the predicted promoter), all exons, intron-exon boundaries, complete untranslated regions, and the 3' region downstream of the GREM1 gene. We identified 31 unique variants, including 24 with a minor allele frequency exceeding 5%, and 9 haplotype-tagging 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 = 0.0003). Combined analysis, adjusted for recruitment center (n = 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.