Genetic analysis of coronary artery disease single nucleotide polymorphisms in diabetic nephropathy

Background. Diabetic nephropathy is a leading cause of end-stage renal disease. Premature mortality is common in patients with nephropathy, largely due to cardiovascular disease. Genetic variants implicated in macrovascular disease are therefore excellent candidates to assess for association with diabetic nephropathy. Recent genome-wide association studies have identified a total of 15 single-nucleotide polymorphisms (SNPs) that are reproducibly associated with cardiovascular disease.

Methods. We initially assessed these SNPs for association in UK type 1 diabetic patients with (cases; n = 597) and without (controls; n = 502) nephropathy using iPLEXTM and TaqMan® assays. Replication studies were performed with DNA genotyped in a total of 2668 individuals from the British Isles.

Results. One SNP (rs4420638) on chromosome 19q13 was found to be significantly associated with diabetic nephropathy before (P = 0.0002) and after correction for multiple testing (Pcorrected = 0.002). We replicated this finding in a phenotypically similar case–control collection comprising 709 individuals with type 1 diabetes (P = 0.002; combined P < 0.00001; OR = 1.54, 95% CI: 1.29–1.84).

Conclusions. Our case–control data suggest that rs4420638, or a functional SNP in linkage disequilibrium with this SNP, may be associated with diabetic nephropathy.

Is hyperfiltration associated with the future risk of developing diabetic nephropathy? A meta-analysis.

Aims/hypothesis Glomerular hyperfiltration is a well established phenomenon occurring early in some patients with type 1 diabetes. However, there is no consistent answer regarding whether hyperfiltration predicts later development of nephropathy. We performed a systematic review and meta-analysis of observational studies that compared the risk of developing diabetic nephropathy in patients with and without glomerular hyperfiltration and also explored the impact of baseline GFR.

Methods A systematic review and meta-analysis was carried out. Cohort studies in type 1 diabetic participants were included if they contained data on the development of incipient or overt nephropathy with baseline measurement
of GFR and presence or absence of hyperfiltration.

Results We included ten cohort studies following 780 patients. After a study median follow-up of 11.2 years, 130 patients had developed nephropathy. Using a random effects model, the pooled odds of progression to a minimum
of microalbuminuria in patients with hyperfiltration was 2.71 (95% CI 1.20–6.11) times that of patients with normofiltration. There was moderate heterogeneity (heterogeneity test p=0.05, measure of degree of inconsistency=48%) and some evidence of funnel plot asymmetry, possibly due to publication bias. The pooled weighted mean difference in baseline GFR was 13.8 ml min-1 1.73 m-2 (95% CI 5.0–22.7) greater in the group progressing to nephropathy than in those not progressing (heterogeneity test p<0.01).

Conclusions/interpretation In published studies, individuals with glomerular hyperfiltration were at increased risk of progression to diabetic nephropathy using study level data. Further larger studies are required to explore this relationship and the role of potential confounding variables.

Investigation of DNA polymorphisms in SMAD genes for genetic predisposition to diabetic nephropathy in patients with type 1 diabetes mellitus

Aims/hypothesis: SMAD proteins are involved in multiple signalling pathways and are key modulators of gene expression. We hypothesised that genetic variation in selected SMAD genes contributes to susceptibility to diabetic nephropathy. Methods: We selected 13 haplotype tag (ht) single nucleotide polymorphisms (SNPs) from 67 variants identified by resequencing the SMAD2 and SMAD3 genes. For SMAD1, SMAD4 and SMAD5 genes, genotype data were downloaded for 217 SNPs from Phase II of the International HapMap project. Of these, 85 SNPs met our inclusion criteria, resulting in the selection of 13 tag SNPs for further investigation. A case-control approach was employed, using 267 nephropathic patients and 442 controls with type 1 diabetes from Ireland. Two further populations (totalling 1,407 patients, 2,238 controls) were genotyped to validate initial findings. Genotyping was conducted using iPLEX, TaqMan and gel electrophoresis.
Results: The distribution of genotypes was in Hardy-Weinberg equilibrium. Analysis by the ? 2 test of genotype and allele frequencies in patients versus controls in the Irish population (n?=?709) revealed evidence for the association of one allele at 5% level of significance (rs10515478, p uncorrected?=?0.006; p corrected?=?0.04). This finding represents a relatively small difference in allele frequency of 6.4% in the patient group compared with 10.7% in the control group; this difference was not supported in subsequent investigations using DNA from European individuals with similar phenotypic characteristics.
Conclusions/interpretation: We selected an appropriate subset of variants for the investigation of common genetic risk factors and assessed SMAD1 to SMAD5 genes for association with diabetic nephropathy. We conclude that common polymorphisms in these genes do not strongly influence genetic susceptibility to diabetic nephropathy in white individuals with type 1 diabetes mellitus.

Co-regulation of Gremlin and Notch signalling in diabetic nephropathy

Diabetic nephropathy is currently the leading cause of end-stage renal disease worldwide, and occurs in approximately one third of all diabetic patients. The molecular pathogenesis of diabetic nephropathy has not been fully characterized and novel mediators and drivers of the disease are still being described. Previous data from our laboratory has identified the developmentally regulated gene Gremlin as a novel target implicated in diabetic nephropathy in vitro and in vivo. We used bioinformatic analysis to examine whether Gremlin gene sequence and structure could be used to identify other genes implicated in diabetic nephropathy. The Notch ligand Jagged1 and its downstream effector, hairy enhancer of split-1 (Hes1), were identified as genes with significant similarity to Gremlin in terms of promoter structure and predicted microRNA binding elements. This led us to discover that transforming growth factor-beta (TGFß1), a primary driver of cellular changes in the kidney during nephropathy, increased Gremlin, Jagged1 and Hes1 expression in human kidney epithelial cells. Elevated levels of Gremlin, Jagged1 and Hes1 were also detected in extracts from renal biopsies from diabetic nephropathy patients, but not in control living donors. In situ hybridization identified specific upregulation and co-expression of Gremlin, Jagged1 and Hes1 in the same tubuli of kidneys from diabetic nephropathy patients, but not controls. Finally, Notch pathway gene clustering showed that samples from diabetic nephropathy patients grouped together, distinct from both control living donors and patients with minimal change disease. Together, these data suggest that Notch pathway gene expression is elevated in diabetic nephropathy, co-incident with Gremlin, and may contribute to the pathogenesis of this disease.

Targeted genome-wide investigation identifies novel SNP's associated with diabetic nephropathy

Loci contributing to complex disease have been identified by focusing on genome-wide scans utilising non-synonymous single nucleotide polymorphisms (nsSNPs). We employed Illumina’s HNS12 BeadChip (13,917 high-value SNPs) which was specifically designed to capture nsSNPs and ideally complements more dense genome-wide association studies that fail to consider many of these putatively functional variants. The HNS12 panel also includes 870 tag SNPs covering the major histocompatibility region. All individuals genotyped in this study were Caucasians with (cases) and without (controls) diabetic nephropathy. About 449 individuals with type 2 diabetes (203 cases, 246 controls) were genotyped in the initial study. 1,467 individuals with type 1 diabetes (718 cases, 749 controls) were genotyped in the follow up study. 11,152 SNPs were successfully analysed and ranked for association with diabetic nephropathy based on significance (P) values. The top ranked 32 SNPs were subsequently genotyped using MassARRAY iPLEX™ and TaqMan technologies to investigate association of these polymorphisms with nephropathy in individuals with type 1 diabetes. The top ranked nsSNP, rs1543547 (P = 10-5), is located in RAET1L, a major histocompatibility class I-related gene at 6q25.1. Of particular interest, multiple nsSNPs within the top ranked (0.2%) SNPs are within several plausible candidate genes for nephropathy on 3q21.3 and 6p21.3.

DNA methylation profiling in cell models of diabetic nephropathy

We have previously identified differentially expressed genes in cell models of diabetic nephropathy and renal biopsies. Here we have performed quantitative DNA methylation profiling in cell models of diabetic nephropathy. Over 3,000 CpG units in the promoter regions of 192 candidate genes were assessed in unstimulated human mesangial cells (HMCs) and proximal tubular epithelial cells (PTCs) compared to HMCs or PTCs exposed to appropriate stimuli. A total of 301 CpG units across 38 genes (similar to 20%) were identified as differentially methylated in unstimulated HMCs versus PTCs. Analysis of amplicon methylation values in unstimulated versus stimulated cell models failed to demonstrate a >20% difference between amplicons. In conclusion, our results demonstrate that specific DNA methylation signatures are present in HMCs and PTCs, and standard protocols for exposure of renal cells to stimuli that alter gene expression may be insufficient to replicate possible alterations in DNA methylation profiles in diabetic nephropathy.

Identifying additional patients with diabetic nephropathy using the UK primary care initiative

AIMS:
The aim of this study was to use general practice data to estimate the prevalence of diabetic nephropathy within the registered diabetes patients and examine variation in practice prevalence and management performance since introduction of this initiative.
METHODS:
Reported quality indicators from the Northern Ireland General Practice Quality and Outcomes Framework were analysed for diabetes and diabetic nephropathy prevalence and management in the period 2004-2008. Variation in prevalence at practice level was assessed using multiple linear regression adjusting for age, practice size, deprivation and glycaemic control.
RESULTS:
In 2006-2007, 57,454 (4.1%) adult diabetic patients were registered in the denominator population of 1.4 million compared with 51,923 (3.8%) in 2004-2005 (mean practice range 0.5-7.7%). Diabetic nephropathy prevalence was 15.1 and 11.5%, respectively (8688 and 5955 patients). Documented diabetic nephropathy prevalence showed marked variation across practices (range 0-100%) and was significantly negatively correlated with diabetes list size, albumin creatinine ratio testing rates and renin-angiotensin-aldosterone system blockade use and positively correlated with exception reporting rates. Specifically, for every increase in 100 diabetic patients to a register, documented diabetic nephropathy prevalence reduced by 40% (P=0.003). On the positive side, median albumin-creatinine ratio testing rates doubled to 82% compared with figures in the pre-Framework era.
CONCLUSIONS:
Implementation of the Northern Ireland General Practice Quality and Outcomes Framework has positively benefitted testing for diabetic nephropathy and increased numbers of detected patients in a short space of time. Large variation in diabetic nephropathy prevalence remains and is associated with diabetes registry size, screening and treatment practices, suggesting that understanding this variation may help detect and better manage diabetic nephropathy.

Association analysis of Notch pathway signalling genes in diabetic nephropathy

Several studies have provided compelling evidence implicating the Notch signalling pathway in diabetic nephropathy. Co-regulation of Notch signalling pathway genes with GREM1 has recently been demonstrated and several genes involved in the Notch pathway are differentially expressed in kidney biopsies from individuals with diabetic nephropathy. We assessed single-nucleotide polymorphisms (SNPs; n = 42) in four of these key genes (JAG1, HES1, NOTCH3 and ADAM10) for association with diabetic nephropathy using a case-control design.
Tag SNPs and potentially functional SNPs were genotyped using Sequenom or Taqman technologies in a total of 1371 individuals with type 1 diabetes (668 patients with nephropathy and 703 controls without nephropathy). Patients and controls were white and recruited from the UK and Ireland. Association analyses were performed using PLINK (http://pngu.mgh.harvard.edu/similar to purcell/plink/) and haplotype frequencies in patients and controls were compared. Adjustment for multiple testing was performed by permutation testing.
In analyses stratified by centre, we identified six SNPs, rs8708 and rs11699674 (JAG1), rs10423702 and rs1548555 (NOTCH3), rs2054096 and rs8027998 (ADAM10) as being associated with diabetic nephropathy before, but not after, adjustment for multiple testing. Haplotype and subgroup analysis according to duration of diabetes also failed to find an association with diabetic nephropathy.
Our results suggest that common variants in JAG1, HES1, NOTCH3 and ADAM10 are not strongly associated with diabetic nephropathy in type 1 diabetes among white individuals. Our findings, however, cannot entirely exclude these genes from involvement in the pathogenesis of diabetic nephropathy.

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.

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. Journal of Human Genetics (2010) 55, 248-251; doi:10.1038/jhg.2010.15; published online 5 March 2010

A rare haplotype of the vitamin D receptor gene is protective against diabetic nephropathy

Background. Vitamin D and its analogues are reported to have renoprotective effects in chronic kidney disease including diabetic nephropathy (DN). Vitamin D3 is converted to 1,25(OH) D3 by CYP2R1 and CYP27B1. The biological action of 1,25(OH) D3 is mediated via its receptor. VDR, CYP27B1 or CYP2R1 gene variants could modify the biological activity of vitamin D3. We have conducted the first case- control association study to determine the relationship between polymorphisms in VDR, CYP27B1 and CYP2R1 genes, and the risk of DN in individuals with type 1 diabetes.