Genetic variants affecting mitochondrial function provide further insights for kidney disease.

BACKGROUND: Chronic kidney disease (CKD) is a complex disorder that has become a high prevalence global health problem, with diabetes being its predominant pathophysiologic driver. Autosomal genetic variation only explains some of the predisposition to kidney disease. Variations in the mitochondrial genome (mtDNA) and nuclear-encoded mitochondrial genes (NEMG) are implicated in susceptibility to kidney disease and CKD progression, but they have not been thoroughly explored. Our aim was to investigate the association of variation in both mtDNA and NEMG with CKD (and related traits), with a particular focus on diabetes. METHODS: We used the UK Biobank (UKB) and UK-ROI, an independent collection of individuals with type 1 diabetes mellitus (T1DM) patients. RESULTS: Fourteen mitochondrial variants were associated with estimated glomerular filtration rate (eGFR) in UKB. Mitochondrial variants and haplogroups U, H and J were associated with eGFR and serum variables. Mitochondrial haplogroup H was associated with all the serum variables regardless of the presence of diabetes. Mitochondrial haplogroup X was associated with end-stage kidney disease (ESKD) in UKB. We confirmed the influence of several known NEMG on kidney disease and function and found novel associations for SLC39A13, CFL1, ACP2 or ATP5G1 with serum variables and kidney damage, and for SLC4A1, NUP210 and MYH14 with ESKD. The G allele of TBC1D32-rs113987180 was associated with higher risk of ESKD in patients with diabetes (OR:9.879; CI95%:4.440-21.980; P = 2.0E-08). In UK-ROI, AGXT2-rs71615838 and SURF1-rs183853102 were associated with diabetic nephropathies, and TFB1M-rs869120 with eGFR. CONCLUSIONS: We identified novel variants both in mtDNA and NEMG which may explain some of the missing heritability for CKD and kidney phenotypes. We confirmed the role of MT-ND5 and mitochondrial haplogroup H on renal disease (serum variables), and identified the MT-ND5-rs41535848G variant, along with mitochondrial haplogroup X, associated with higher risk of ESKD. Despite most of the associations were independent of diabetes, we also showed potential roles for NEMG in T1DM.

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.

Differential methylation in CD44 and SEC23A is associated with time preference in older individuals.

Time preference is a measure used to ascertain the level of which individuals prefer smaller, immediate rewards over larger, delayed rewards. We explored how an individual's time preference associates with their epigenetic profile. Time preferences were ascertained by asking participants of the Northern Ireland COhort for the Longitudinal study of Ageing to make a series of choices between two hypothetical income scenarios. From these, eight 'time preference' categories were derived, ranging from "patient" to "impatient" on an ordinal scale. The Infinium High Density Methylation Assay, MethylationEPIC (Illumina) was used to evaluate the status of 862,927 CpGs. Time preference and DNA methylation data were obtained for 1648 individuals. Four analyses were conducted, assessing the methylation patterns at single site resolution between patient and impatient individuals using two adjustment models. In this discovery cohort analysis, two CpG sites were identified with significantly different levels of methylation (p < 9 × 10-8) between the individuals allocated to the patient group and the remaining population following adjustment for covariates; cg08845621 within CD44 and cg18127619 within SEC23A. Neither of these genes have previously been linked to time preference. Epigenetic modifications have not previously been linked to time preference using a population cohort but they may represent important biomarkers of accumulated, complex determinants of this trait. Further analysis is warranted of both the top-ranked results and of DNA methylation as an important link between measurable biomarkers and health behaviours.

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.

Genome-wide meta-analysis and omics integration identifies novel genes associated with diabetic kidney disease.

AIMS/HYPOTHESIS: Diabetic kidney disease (DKD) is the leading cause of kidney failure and has a substantial genetic component. Our aim was to identify novel genetic factors and genes contributing to DKD by performing meta-analysis of previous genome-wide association studies (GWAS) on DKD and by integrating the results with renal transcriptomics datasets. METHODS: We performed GWAS meta-analyses using ten phenotypic definitions of DKD, including nearly 27,000 individuals with diabetes. Meta-analysis results were integrated with estimated quantitative trait locus data from human glomerular (N=119) and tubular (N=121) samples to perform transcriptome-wide association study. We also performed gene aggregate tests to jointly test all available common genetic markers within a gene, and combined the results with various kidney omics datasets. RESULTS: The meta-analysis identified a novel intronic variant (rs72831309) in the TENM2 gene associated with a lower risk of the combined chronic kidney disease (eGFR<60 ml/min per 1.73 m2) and DKD (microalbuminuria or worse) phenotype (p=9.8×10-9; although not withstanding correction for multiple testing, p>9.3×10-9). Gene-level analysis identified ten genes associated with DKD (COL20A1, DCLK1, EIF4E, PTPRN-RESP18, GPR158, INIP-SNX30, LSM14A and MFF; p<2.7×10-6). Integration of GWAS with human glomerular and tubular expression data demonstrated higher tubular AKIRIN2 gene expression in individuals with vs without DKD (p=1.1×10-6). The lead SNPs within six loci significantly altered DNA methylation of a nearby CpG site in kidneys (p<1.5×10-11). Expression of lead genes in kidney tubules or glomeruli correlated with relevant pathological phenotypes (e.g. TENM2 expression correlated positively with eGFR [p=1.6×10-8] and negatively with tubulointerstitial fibrosis [p=2.0×10-9], tubular DCLK1 expression correlated positively with fibrosis [p=7.4×10-16], and SNX30 expression correlated positively with eGFR [p=5.8×10-14] and negatively with fibrosis [p<2.0×10-16]). CONCLUSIONS/INTERPRETATION: Altogether, the results point to novel genes contributing to the pathogenesis of DKD. DATA AVAILABILITY: The GWAS meta-analysis results can be accessed via the type 1 and type 2 diabetes (T1D and T2D, respectively) and Common Metabolic Diseases (CMD) Knowledge Portals, and downloaded on their respective download pages ( https://t1d.hugeamp.org/downloads.html ; https://t2d.hugeamp.org/downloads.html ; https://hugeamp.org/downloads.html ).

An investigation into DNA methylation patterns associated with risk preference in older individuals.

Risk preference is a complex trait governed by psycho-social, environmental and genetic determinants. We aimed to examine how an individual's risk preference associates with their epigenetic profile.Risk preferences were ascertained by asking participants of the Northern Ireland COhort for the Longitudinal study of Ageing to make a series of choices between hypothetical income scenarios. From these, four risk preference categories were derived, ranging from risk-averse to risk-seeking. Illumina's Infinium High-Density Methylation Assay was used to evaluate the status of 862,927 CpGs.Risk preference and DNA methylation data were obtained for 1,656 individuals. The distribution of single-site DNA methylation levels between risk-averse and risk-seeking individuals was assessed whilst adjusting for age, sex and peripheral white cell counts. In this discovery cohort, 55 CpGs were identified with significantly different levels of methylation (p≤x10-5) between risk-averse and risk-seeking individuals when adjusting for the maximum number of covariates. No CpGs were significantly differentially methylated in any of the risk preference groups at an epigenome-wide association level (p<9x10-8) following covariate adjustment.Protein-coding genes NWD1 and LRP1 were among the genes in which the top-ranked dmCpGs were located for all analyses conducted. Mutations in these genes have previously been linked to neurological conditions.Epigenetic modifications have not previously been linked to risk-aversion using a population cohort, but may represent important biomarkers of accumulated, complex determinants of this trait. Several striking results from this study support further analysis of DNA methylation as an important link between measurable biomarkers and health behaviours.

DNA Methylation Associated With Diabetic Kidney Disease in Blood-Derived DNA.

A subset of individuals with type 1 diabetes will develop diabetic kidney disease (DKD). DKD is heritable and large-scale genome-wide association studies have begun to identify genetic factors that influence DKD. Complementary to genetic factors, we know that a person's epigenetic profile is also altered with DKD. This study reports analysis of DNA methylation, a major epigenetic feature, evaluating methylome-wide loci for association with DKD. Unique features (n = 485,577; 482,421 CpG probes) were evaluated in blood-derived DNA from carefully phenotyped White European individuals diagnosed with type 1 diabetes with (cases) or without (controls) DKD (n = 677 samples). Explicitly, 150 cases were compared to 100 controls using the 450K array, with subsequent analysis using data previously generated for a further 96 cases and 96 controls on the 27K array, and de novo methylation data generated for replication in 139 cases and 96 controls. Following stringent quality control, raw data were quantile normalized and beta values calculated to reflect the methylation status at each site. The difference in methylation status was evaluated between cases and controls; resultant P-values for array-based data were adjusted for multiple testing. Genes with significantly increased (hypermethylated) and/or decreased (hypomethylated) levels of DNA methylation were considered for biological relevance by functional enrichment analysis using KEGG pathways. Twenty-two loci demonstrated statistically significant fold changes associated with DKD and additional support for these associated loci was sought using independent samples derived from patients recruited with similar inclusion criteria. Markers associated with CCNL1 and ZNF187 genes are supported as differentially regulated loci (P < 10-8), with evidence also presented for AFF3, which has been identified from a meta-analysis and subsequent replication of genome-wide association studies. Further supporting evidence for differential gene expression in CCNL1 and ZNF187 is presented from kidney biopsy and blood-derived RNA in people with and without kidney disease from NephroSeq. Evidence confirming that methylation sites influence the development of DKD may aid risk prediction tools and stimulate research to identify epigenomic therapies which might be clinically useful for this disease.

A scoping review and proposed workflow for multi-omic rare disease research.

BACKGROUND: Patients with rare diseases face unique challenges in obtaining a diagnosis, appropriate medical care and access to support services. Whole genome and exome sequencing have increased identification of causal variants compared to single gene testing alone, with diagnostic rates of approximately 50% for inherited diseases, however integrated multi-omic analysis may further increase diagnostic yield. Additionally, multi-omic analysis can aid the explanation of genotypic and phenotypic heterogeneity, which may not be evident from single omic analyses. MAIN BODY: This scoping review took a systematic approach to comprehensively search the electronic databases MEDLINE, EMBASE, PubMed, Web of Science, Scopus, Google Scholar, and the grey literature databases OpenGrey / GreyLit for journal articles pertaining to multi-omics and rare disease, written in English and published prior to the 30th December 2018. Additionally, The Cancer Genome Atlas publications were searched for relevant studies and forward citation searching / screening of reference lists was performed to identify further eligible articles. Following title, abstract and full text screening, 66 articles were found to be eligible for inclusion in this review. Of these 42 (64%) were studies of multi-omics and rare cancer, two (3%) were studies of multi-omics and a pre-cancerous condition, and 22 (33.3%) were studies of non-cancerous rare diseases. The average age of participants (where known) across studies was 39.4 years. There has been a significant increase in the number of multi-omic studies in recent years, with 66.7% of included studies conducted since 2016 and 33% since 2018. Fourteen combinations of multi-omic analyses for rare disease research were returned spanning genomics, epigenomics, transcriptomics, proteomics, phenomics and metabolomics. CONCLUSIONS: This scoping review emphasises the value of multi-omic analysis for rare disease research in several ways compared to single omic analysis, ranging from the provision of a diagnosis, identification of prognostic biomarkers, distinct molecular subtypes (particularly for rare cancers), and identification of novel therapeutic targets. Moving forward there is a critical need for collaboration of multi-omic rare disease studies to increase the potential to generate robust outcomes and development of standardised biorepository collection and reporting structures for multi-omic studies.

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.

Genetic Strategies to Understand Human Diabetic Nephropathy: In Silico Strategies for Molecular Data-Association Studies.

Multiple genetic strategies are available to help improve understanding of diabetic nephropathy. This chapter provides detailed methodology for a single-nucleotide polymorphism association study and meta-analysis, using a protocol suitable for targeted SNP or genome-wide association studies, to identify genetic risk factors for diabetic nephropathy.

The impact of donor and recipient common clinical and genetic variation on estimated glomerular filtration rate in a European renal transplant population.

Genetic variation across the human leukocyte antigen loci is known to influence renal-transplant outcome. However, the impact of genetic variation beyond the human leukocyte antigen loci is less clear. We tested the association of common genetic variation and clinical characteristics, from both the donor and recipient, with posttransplant eGFR at different time-points, out to 5 years posttransplantation. We conducted GWAS meta-analyses across 10 844 donors and recipients from five European ancestry cohorts. We also analyzed the impact of polygenic risk scores (PRS), calculated using genetic variants associated with nontransplant eGFR, on posttransplant eGFR. PRS calculated using the recipient genotype alone, as well as combined donor and recipient genotypes were significantly associated with eGFR at 1-year posttransplant. Thirty-two percent of the variability in eGFR at 1-year posttransplant was explained by our model containing clinical covariates (including weights for death/graft-failure), principal components and combined donor-recipient PRS, with 0.3% contributed by the PRS. No individual genetic variant was significantly associated with eGFR posttransplant in the GWAS. This is the first study to examine PRS, composed of variants that impact kidney function in the general population, in a posttransplant context. Despite PRS being a significant predictor of eGFR posttransplant, the effect size of common genetic factors is limited compared to clinical variables.

Information on Genetic Variants Does Not Increase Identification of Individuals at Risk of Esophageal Adenocarcinoma Compared to Clinical Risk Factors.

We previously developed a tool that identified individuals who later developed esophageal adenocarcinoma (based on age, sex, body mass index, smoking status, and prior esophageal conditions) with an area under the curve of 0.80. In this study, we collected data from 329,463 individuals in the UK Biobank cohort who were tested for genetic susceptibility to esophageal adenocarcinoma (a polygenic risk score based on 18 recognized genetic variants). We found that after inclusion of this genetic information, the area under the curve for identification of individuals who developed esophageal adenocarcinoma remained at 0.80. Testing for genetic variants associated with esophageal adenocarcinoma therefore seems unlikely to improve identification of individuals at risk of esophageal adenocarcinoma.

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.

DNA hypermethylation and DNA hypomethylation is present at different loci in chronic kidney disease.

Genetic risk factors for chronic kidney disease (CKD) are being identified through international collaborations. By comparison, epigenetic risk factors for CKD have only recently been considered using population-based approaches. DNA methylation is a major epigenetic modification that is associated with complex diseases, so we investigated methylome-wide loci for association with CKD. A total of 485,577 unique features were evaluated in 255 individuals with CKD (cases) and 152 individuals without evidence of renal disease (controls). Following stringent quality control, raw data were quantile normalized and ß values calculated to reflect the methylation status at each site. The difference in methylation status was evaluated between cases and controls with resultant P values adjusted for multiple testing. Genes with significantly increased and decreased levels of DNA methylation were considered for biological relevance by functional enrichment analysis using KEGG pathways in Partek Genomics Suite. Twenty-three genes, where more than one CpG per loci was identified with Padjusted<10(-8), demonstrated significant methylation changes associated with CKD and additional support for these associated loci was sought from published literature. Strong biological candidates for CKD that showed statistically significant differential methylation include CUX1, ELMO1, FKBP5, INHBA-AS1, PTPRN2, and PRKAG2 genes; several genes are differentially methylated in kidney tissue and RNA-seq supports a functional role for differential methylation in ELMO1 and PRKAG2 genes. This study reports the largest, most comprehensive, genome-wide quantitative evaluation of DNA methylation for association with CKD. Evidence confirming methylation sites influence development of CKD would stimulate research to identify epigenetic therapies that might be clinically useful for CKD.

c-Met inhibition in a HOXA9/Meis1 model of CN-AML.

BACKGROUND: Hematopoiesis is a paradigm for developmental processes, hierarchically organized, with stem cells at its origin. Hematopoietic stem cells (HSCs) replenish progenitor and precursor cells of multiple lineages, which normally differentiate into short-lived mature circulating cells. Hematopoiesis has provided insight into the molecular basis of tissue homeostasis and malignancy. Malignant hematopoiesis, in particular acute myeloid leukemia (AML), results from impaired development or differentiation of HSCs and progenitors. Co-overexpression of HOX and TALE genes, particularly the HOXA cluster and MEIS1, is associated with AML. Clinically relevant models of AML are required to advance drug development for an aging patient cohort. RESULTS: Molecular analysis identified altered gene, microRNA, and protein expression in HOXA9/Meis1 leukemic bone marrow compared to normal controls. A candidate drug screen identified the c-Met inhibitor SU11274 for further analysis. Altered cell cycle status, apoptosis, differentiation, and impaired colony formation were shown for SU11274 in AML cell lines and primary leukemic bone marrow. CONCLUSIONS: The clonal HOXA9/Meis1 AML model is amenable to drug screening analysis. The data presented indicate that human AML cells respond in a similar manner to the HOXA9/Meis1 cells, indicating pre-clinical relevance of the mouse model.