Genome-wide DNA methylation analysis for diabetic nephropathy in type 1 diabetes mellitus

BACKGROUND: Diabetic nephropathy is a serious complication of diabetes mellitus and is associated with considerable morbidity and high mortality. There is increasing evidence to suggest that dysregulation of the epigenome is involved in diabetic nephropathy. We assessed whether epigenetic modification of DNA methylation is associated with diabetic nephropathy in a case-control study of 192 Irish patients with type 1 diabetes mellitus (T1D). Cases had T1D and nephropathy whereas controls had T1D but no evidence of renal disease. METHODS: We performed DNA methylation profiling in bisulphite converted DNA from cases and controls using the recently developed Illumina Infinium(R) HumanMethylation27 BeadChip, that enables the direct investigation of 27,578 individual cytosines at CpG loci throughout the genome, which are focused on the promoter regions of 14,495 genes. RESULTS: Singular Value Decomposition (SVD) analysis indicated that significant components of DNA methylation variation correlated with patient age, time to onset of diabetic nephropathy, and sex. Adjusting for confounding factors using multivariate Cox-regression analyses, and with a false discovery rate (FDR) of 0.05, we observed 19 CpG sites that demonstrated correlations with time to development of diabetic nephropathy. Of note, this included one CpG site located 18 bp upstream of the transcription start site of UNC13B, a gene in which the first intronic SNP rs13293564 has recently been reported to be associated with diabetic nephropathy. CONCLUSION: This high throughput platform was able to successfully interrogate the methylation state of individual cytosines and identified 19 prospective CpG sites associated with risk of diabetic nephropathy. These differences in DNA methylation are worthy of further follow-up in replication studies using larger cohorts of diabetic patients with and without nephropathy

Association Analysis of Canonical Wnt Signalling Genes in Diabetic Nephropathy

Several studies have provided compelling evidence implicating the Wnt signalling pathway in the pathogenesis of diabetic nephropathy. Gene expression profiles associated with renal fibrosis have been attenuated through Wnt pathway modulation in model systems implicating Wnt pathway members as potential therapeutic targets for the treatment of diabetic nephropathy. We assessed tag and potentially functional single nucleotide polymorphisms (SNPs; n = 31) in four key Wnt pathway genes (CTNNB1, AXIN2, LRP5 and LRP6) for association with diabetic nephropathy using a case-control design.SNPs were genotyped using Sequenom or Taqman technologies in 1351 individuals with type 1 diabetes (651 cases with nephropathy and 700 controls without nephropathy). Cases and controls were white and recruited from the UK and Ireland. Association analyses were performed using PLINK, to compare allele and haplotype frequencies in cases and controls. Adjustment for multiple testing was performed by permutation testing.Following logistic regression analysis adjusted by collection centre, duration of T1D, and average HbA1c as covariates, a single SNP in LRP6 (rs1337791) was significantly associated with DN (OR = 0.74; CI: 0.57-0.97; P = 0.028), although this was not maintained following correction for multiple testing. Three additional SNPs (rs2075241 in LRP6; rs3736228 and rs491347 both in LRP5) were marginally associated with diabetic nephropathy, but none of the associations were replicated in an independent dataset. Haplotype and subgroup analysis (according to duration of diabetes, and end-stage renal disease) also failed to reveal an association with diabetic nephropathy.Our results suggest that analysed common variants in CTNNB1, AXIN2, LRP5 and LRP6 are not strongly associated with diabetic nephropathy in type 1 diabetes among white individuals. Our findings, however, cannot entirely exclude these genes or other members of the Wnt pathway, from involvement in the pathogenesis of diabetic nephropathy as our study had limited power to detect variants with small effect size

Association of eGFR-Related Loci Identified by GWAS with Incident CKD and ESRD

Family studies suggest a genetic component to the etiology of chronic kidney disease (CKD) and end stage renal disease (ESRD). Previously, we identified 16 loci for eGFR in genome-wide association studies, but the associations of these single nucleotide polymorphisms (SNPs) for incident CKD or ESRD are unknown. We thus investigated the association of these loci with incident CKD in 26,308 individuals of European ancestry free of CKD at baseline drawn from eight population-based cohorts followed for a median of 7.2 years (including 2,122 incident CKD cases defined as eGFR <60ml/min/1.73m2 at follow-up) and with ESRD in four case-control studies in subjects of European ancestry (3,775 cases, 4,577 controls). SNPs at 11 of the 16 loci (UMOD, PRKAG2, ANXA9, DAB2, SHROOM3, DACH1, STC1, SLC34A1, ALMS1/NAT8, UBE2Q2, and GCKR) were associated with incident CKD; p-values ranged from p = 4.1e-9 in UMOD to p = 0.03 in GCKR. After adjusting for baseline eGFR, six of these loci remained significantly associated with incident CKD (UMOD, PRKAG2, ANXA9, DAB2, DACH1, and STC1). SNPs in UMOD (OR = 0.92, p = 0.04) and GCKR (OR = 0.93, p = 0.03) were nominally associated with ESRD. In summary, the majority of eGFR-related loci are either associated or show a strong trend towards association with incident CKD, but have modest associations with ESRD in individuals of European descent. Additional work is required to characterize the association of genetic determinants of CKD and ESRD at different stages of disease progression

Buffy coat specimens remain viable as a DNA source for highly multiplexed genome-wide genetic tests after long term storage

<p>Abstract</p> <p>Background</p> <p>Blood specimen collection at an early study visit is often included in observational studies or clinical trials for analysis of secondary outcome biomarkers. A common protocol is to store buffy coat specimens for future DNA isolation and these may remain in frozen storage for many years. It is uncertain if the DNA remains suitable for modern genome wide association (GWA) genotyping.</p> <p>Methods</p> <p>We isolated DNA from 120 Action to Control Cardiovascular Risk in Diabetes (ACCORD) clinical trial buffy coats sampling a range of storage times up to 9 years and other factors that could influence DNA yield. We performed TaqMan SNP and GWA genotyping to test whether the DNA retained integrity for high quality genetic analysis.</p> <p>Results</p> <p>We tested two QIAGEN automated protocols for DNA isolation, preferring the Compromised Blood Protocol despite similar yields. We isolated DNA from all 120 specimens (yield range 1.1-312 ug per 8.5 ml ACD tube of whole blood) with only 3/120 samples yielding < 10 ug DNA. Age of participant at blood draw was negatively associated with yield (mean change -2.1 ug/year). DNA quality was very good based on gel electrophoresis QC, TaqMan genotyping of 6 SNPs (genotyping no-call rate 1.1% in 702 genotypes), and excellent quality GWA genotyping data (maximum per sample genotype missing rate 0.64%).</p> <p>Conclusions</p> <p>When collected as a long term clinical trial or biobank specimen for DNA, buffy coats can be stored for up to 9 years in a -80degC frozen state and still produce high yields of DNA suitable for GWA analysis and other genetic testing.</p> <p>Trial Registration</p> <p>The Action to Control Cardiovascular Risk in Diabetes (ACCORD) trial is registered with ClinicalTrials.gov, number <a href="http://www.clinicaltrials.gov/ct2/show/NCT00000620">NCT00000620</a>.</p

Rs1888747 polymorphism in the FRMD3 gene, gene and protein expression: Role in diabetic kidney disease

© 2016 Buffon et al. Background: We carried out a case-control study in patients with type 2 diabetes mellitus (T2DM) to evaluate the association between seven single nucleotide polymorphisms (SNPs) previously described to be linked to diabetic kidney disease (DKD) in type 1 diabetes mellitus (T1DM). Additionally, we evaluated gene and protein expression related to the polymorphism associated with DKD. Methods: The association study included 1098 T2DM patients (718 with DKD and 380 without DKD). Out of the 13 polymorphisms associated with DKD in a previous study with T1DM, seven were chosen for evaluation in this sample: rs1888747, rs9521445, rs39075, rs451041, rs1041466, rs1411766 and rs6492208. The expression study included 91 patients who underwent nephrectomy. Gene expression was assessed by RT-qPCR and protein expression in kidney samples was quantified by western blot and it localization by immunohistochemistry. Results: The C/C genotype of rs1888747 SNP was associated with protection for DKD (OR = 0.6, 95 % CI 0.3-0.9; P = 0.022). None of the other SNPs were associated with DKD. rs1888747 is located near FRMD3 gene. Therefore, FRMD3 gene and protein expression were evaluated in human kidney tissue according to rs1888747 genotypes. Gene and protein expression were similar in subjects homozygous for the C allele and in those carrying the G allele. Conclusions: Replication of the association between rs1888747 SNP and DKD in a different population suggests that this link is not the result of chance. rs1888747 SNP is located at the FRMD3 gene, which is expressed in human kidney. Therefore, this gene is a candidate gene for DKD. However, in this study, no rs1888747 genotype or specific allele effect on gene and/or protein expression of the FRMD3 gene was demonstrated

Genetic associations in diabetic nephropathy: a meta-analysis

Diabetes mellitus: pathophysiological changes and therap

A Single Nucleotide Polymorphism within the Acetyl-Coenzyme A Carboxylase Beta Gene Is Associated with Proteinuria in Patients with Type 2 Diabetes

It has been suggested that genetic susceptibility plays an important role in the pathogenesis of diabetic nephropathy. A large-scale genotyping analysis of gene-based single nucleotide polymorphisms (SNPs) in Japanese patients with type 2 diabetes identified the gene encoding acetyl-coenzyme A carboxylase beta (ACACB) as a candidate for a susceptibility to diabetic nephropathy; the landmark SNP was found in the intron 18 of ACACB (rs2268388: intron 18 +4139 C > T, p = 1.4×10−6, odds ratio = 1.61, 95% confidence interval [CI]: 1.33–1.96). The association of this SNP with diabetic nephropathy was examined in 9 independent studies (4 from Japan including the original study, one Singaporean, one Korean, and two European) with type 2 diabetes. One case-control study involving European patients with type 1 diabetes was included. The frequency of the T allele for SNP rs2268388 was consistently higher among patients with type 2 diabetes and proteinuria. A meta-analysis revealed that rs2268388 was significantly associated with proteinuria in Japanese patients with type 2 diabetes (p = 5.35×10−8, odds ratio = 1.61, 95% Cl: 1.35–1.91). Rs2268388 was also associated with type 2 diabetes–associated end-stage renal disease (ESRD) in European Americans (p = 6×10−4, odds ratio = 1.61, 95% Cl: 1.22–2.13). Significant association was not detected between this SNP and nephropathy in those with type 1 diabetes. A subsequent in vitro functional analysis revealed that a 29-bp DNA fragment, including rs2268388, had significant enhancer activity in cultured human renal proximal tubular epithelial cells. Fragments corresponding to the disease susceptibility allele (T) had higher enhancer activity than those of the major allele. These results suggest that ACACB is a strong candidate for conferring susceptibility for proteinuria in patients with type 2 diabetes

Different molecular patterns in glioblastoma multiforme subtypes upon recurrence

One of the hallmarks of glioblastoma is its inherent tendency to recur. At this point patients with relapsed GBM show a survival time of only few months. The molecular basis of the recurrence process in GBM is still poorly understood. The aim of the present study was to investigate the genetic profile of relapsed GBM compared to their respective primary tumors. We have included 20 paired GBMs. In all tumor samples, we have analyzed p53 and PTEN status by sequencing analysis, EGFR amplification by semiquantitative PCR and a wide-genome fingerprinting was performed by microsatellite analysis. Among primary GBM, we observed twelve type 2 GBM, four type 1 GBM and four further GBM showing neither p53 mutations nor EGFR amplification (non-type 1–non-type 2 GBM). Upon recurrence, we have detected two molecular patterns of tumor progression: GBM initially showing either type 1 or type 2 profiles conserved them at the time of relapse. In contrast, non-type 1–non-type 2 GBM acquired the typical pattern of type 2 GBM and harbor EGFR amplification without p53 mutation. New PTEN mutations upon relapse were only detected in type 2 GBM. Additional LOH were more frequently identified in relapses of type 2 GBM than in those showing the type 1 signature. Taken together, our results strongly suggest that recurrences of GBM may display two distinct pattern of accumulation of molecular alterations depending on the profile of the original tumor

Differential Effects of MYH9 and APOL1 Risk Variants on FRMD3 Association with Diabetic ESRD in African Americans

Single nucleotide polymorphisms (SNPs) in MYH9 and APOL1 on chromosome 22 (c22) are powerfully associated with non-diabetic end-stage renal disease (ESRD) in African Americans (AAs). Many AAs diagnosed with type 2 diabetic nephropathy (T2DN) have non-diabetic kidney disease, potentially masking detection of DN genes. Therefore, genome-wide association analyses were performed using the Affymetrix SNP Array 6.0 in 966 AA with T2DN and 1,032 non-diabetic, non-nephropathy (NDNN) controls, with and without adjustment for c22 nephropathy risk variants. No associations were seen between FRMD3 SNPs and T2DN before adjusting for c22 variants. However, logistic regression analysis revealed seven FRMD3 SNPs significantly interacting with MYH9—a finding replicated in 640 additional AA T2DN cases and 683 NDNN controls. Contrasting all 1,592 T2DN cases with all 1,671 NDNN controls, FRMD3 SNPs appeared to interact with the MYH9 E1 haplotype (e.g., rs942280 interaction p-value = 9.3E−7 additive; odds ratio [OR] 0.67). FRMD3 alleles were associated with increased risk of T2DN only in subjects lacking two MYH9 E1 risk haplotypes (rs942280 OR = 1.28), not in MYH9 E1 risk allele homozygotes (rs942280 OR = 0.80; homogeneity p-value = 4.3E−4). Effects were weaker stratifying on APOL1. FRMD3 SNPS were associated with T2DN, not type 2 diabetes per se, comparing AAs with T2DN to those with diabetes lacking nephropathy. T2DN-associated FRMD3 SNPs were detectable in AAs only after accounting for MYH9, with differential effects for APOL1. These analyses reveal a role for FRMD3 in AA T2DN susceptibility and accounting for c22 nephropathy risk variants can assist in detecting DN susceptibility genes