HLA class II is a factor in cardiovascular morbidity and mortality rates in patients with type 1 diabetes.

AIMS/HYPOTHESIS: Patients with type 1 diabetes and kidney disease have a higher risk of cardiovascular events. HLA class II genes are expressed on infiltrated inflammatory cells and smooth-muscle cells in atherosclerotic plaques. We hypothesised that HLA class II haplotypes or genotypes might influence the risk of cardiovascular complications and death in Finnish type 1 diabetic patients. METHODS: We included 3,082 patients with type 1 diabetes from the Finnish Diabetic Nephropathy Study. We analysed the 12 and ten most common HLA II haplo- and genotypes, respectively, using χ (2) tests. The positive findings were analysed with three differently adjusted regression models with cardiovascular morbidity and death as endpoints. Different kidney status groups were analysed separately. RESULTS: At baseline, the common (DR1/10)-DQB1*05:01 haplotype (20.4%) and the (DR1/10)-DQB1*05:01/DRB1*04:01-DQB1*03:02 genotype (8.7%) were independently associated with cardiovascular disease in all kidney status groups, except in patients with normal AER. At follow-up (9.45 years; range 0.1-16.1 years), the (DR1/10)-DQB1*05:01/DRB1*04:01-DQB1*03:02 genotype was associated with cardiovascular mortality rates in patients with normal AER and microalbuminuria. CONCLUSIONS/INTERPRETATION: The (DR1/10)-DQB1*05:01 haplotype and the (DR1/10)-DQB1*05:01/DRB1*04:01-DQB1*03:02 genotype are independently associated with cardiovascular events and death in Finnish type 1 diabetic patients.

SNP in the genome-wide association study hotspot on chromosome 9p21 confers susceptibility to diabetic nephropathy in type 1 diabetes.

AIMS/HYPOTHESIS: Parental type 2 diabetes mellitus increases the risk of diabetic nephropathy in offspring with type 1 diabetes mellitus. Several single nucleotide polymorphisms (SNPs) that predispose to type 2 diabetes mellitus have recently been identified. It is, however, not known whether such SNPs also confer susceptibility to diabetic nephropathy in patients with type 1 diabetes mellitus. METHODS: We genotyped nine SNPs associated with type 2 diabetes mellitus in genome-wide association studies in the Finnish population, and tested for their association with diabetic nephropathy as well as with severe retinopathy and cardiovascular disease in 2,963 patients with type 1 diabetes mellitus. Replication of significant SNPs was sought in 2,980 patients from three other cohorts. RESULTS: In the discovery cohort, rs10811661 near gene CDKN2A/B was associated with diabetic nephropathy. The association remained after robust Bonferroni correction for the total number of tests performed in this study (OR 1.33 [95% CI 1.14, 1.56], p = 0.00045, p (36tests) = 0.016). In the meta-analysis, the combined result for diabetic nephropathy was significant, with a fixed effects p value of 0.011 (OR 1.15 [95% CI 1.02, 1.29]). The association was particularly strong when patients with end-stage renal disease were compared with controls (OR 1.35 [95% CI 1.13, 1.60], p = 0.00038). The same SNP was also associated with severe retinopathy (OR 1.37 [95% CI 1.10, 1.69] p = 0.0040), but the association did not remain after Bonferroni correction (p (36tests) = 0.14). None of the other selected SNPs was associated with nephropathy, severe retinopathy or cardiovascular disease. CONCLUSIONS/INTERPRETATION: A SNP predisposing to type 2 diabetes mellitus, rs10811661 near CDKN2A/B, is associated with diabetic nephropathy in patients with type 1 diabetes mellitus.

Genetic polymorphisms in nitric oxide synthase 3 gene and implications for kidney disease: a meta-analysis.

BACKGROUND/AIMS: The NOS3 gene is a biological and positional candidate for diabetic nephropathy. However, the relationship between NOS3 polymorphisms and renal disease is inconclusive. This study aimed to clarify the association of NOS3 variants with nephropathy in individuals with type 1 diabetes. METHODS: We conducted a case-control study examining all common SNPs in the NOS3 gene by a tag SNP approach. Individuals with type 1 diabetes and persistent proteinuria (cases, n = 718) were compared with individuals with type 1 diabetes but no evidence of renal disease (controls, n = 749). Our replication collection comprised 1,105 individuals with type 1 diabetes recruited to a nephropathy case group and 862 control individuals with normal urinary albumin excretion rates. Meta-analysis was conducted for SNPs where more than three genotype datasets were available. RESULTS: A novel association was identified in the discovery collection (rs1800783, p(genotype) = 0.006, p(allele) = 0.002, OR = 1.26, 95% CI: 1.08-1.47) and supported by independent replication using a tag SNP (rs4496877, pairwise r² = 0.96 with rs1800783) in the replication collection (p(genotype) = 0.002, p(allele) = 0.0006, OR = 1.27, 95% CI: 1.10-1.45). CONCLUSION: The A allele of rs1800783 is a significant risk factor for nephropathy in individuals with type 1 diabetes, and further comprehensive studies are warranted to confirm the definitive functional variant in the NOS3 gene.

DDOST, PRKCSH and LGALS3, which encode AGE-receptors 1, 2 and 3, respectively, are not associated with diabetic nephropathy in type 1 diabetes.

AIMS/HYPOTHESIS: The AGE receptors 1, 2 and 3, which are encoded by DDOST, PRKCSH and LGALS3, respectively, may be involved in the pathogenesis of diabetic complications. We sought to find out whether these genes are associated with diabetic nephropathy, cardiovascular disease and type 1 diabetes or related quantitative traits. METHODS: Using the Tagger program, we selected 28 single nucleotide polymorphisms (SNPs) based on the HapMap Centre d'Etude du Polymorphisme (Utah residents with northern and western European ancestry) data. The SNPs were genotyped in 2,719 Finnish patients with type 1 diabetes and tested for association with diabetic nephropathy (821 cases, 1,060 controls), cardiovascular disease and related quantitative traits. For association analysis with type 1 diabetes, 703 non-diabetic control participants were genotyped. RESULTS: We found evidence of genotype association between diabetic nephropathy and the SNPs rs2170336 in DDOST (p = 0.03), rs311788 in PRKCSH (p = 0.04) and rs311778 in PRKCSH (p = 0.02). However, these associations did not reach the significance limit of 0.0008 adjusted for multiple testing. None of the DDOST, PRKCSH or LGALS3 SNPs were associated with quantitative traits related to diabetic nephropathy, including AER and estimated GFR. No associations were found between the SNPs and cardiovascular disease, blood pressure, serum lipid levels or type 1 diabetes. CONCLUSIONS/INTERPRETATION: The common SNPs tested in DDOST, PRKCSH and LGALS3 do not seem to be associated with diabetic micro- or macrovascular complications or with type 1 diabetes in Finnish patients.

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 chi(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.

Association analysis of podocyte slit diaphragm genes as candidates for diabetic nephropathy.

AIMS/HYPOTHESIS: The slit diaphragm is an adhesion and signalling protein complex linking the interdigitating podocyte foot processes in the kidney glomerulus, and mutations in slit diaphragm-associated genes result in severe proteinuria. Here we report a genetic association analysis of four slit diaphragm genes, LRRC7, KIRREL, NPHS2 and ACTN4, in a Finnish diabetic nephropathy cohort. MATERIALS AND METHODS: A total of 40 single nucleotide polymorphisms (SNPs) were genotyped in 1103 patients with type 1 diabetes. The patients were classified according to their renal status, and the genotype data were analysed in a cross-sectional case-control setting. To confirm positive associations, four SNPs were genotyped in 1,025 additional patients with type 1 diabetes. RESULTS: No associations with diabetic nephropathy were observed for any of the analysed SNPs. The SNPs were not associated with the time from the onset of diabetes to the diagnosis of nephropathy or with glomerular filtration rate or AER as quantitative variables. In a sex-specific sub-analysis, the variants rs979972 and rs749701 in the first intron of ACTN4 were nominally associated with diabetic nephropathy in females, with odds ratios of 1.81 (95% CI 1.18-2.79, p = 0.007) and 1.93 (95% CI 1.26-2.96, p = 0.003) respectively. CONCLUSIONS/INTERPRETATION: Our study has not found any evidence that common variants in LRRC7, KIRREL, NPHS2 and ACTN4 contribute to susceptibility to diabetic nephropathy in Finnish patients with type 1 diabetes.

Novel sequence variants in the human xylosyltransferase I gene and their role in diabetic nephropathy.

AIMS: Decreased content of heparan sulphate proteoglycans (HSPGs) is a characteristic of the glomerular basement membrane (GBM) in diabetes and contributes to the development of diabetic nephropathy (DN). Xylosyltransferase I (XT-I) is the chain-initiating enzyme involved in the biosynthesis of HSPGs. This study investigated a possible association between XYLT-I sequence variants and susceptibility to DN. METHODS: Screening of all XYLT-I exons was performed in 74 caucasians with Type 1 diabetes (48 with and 26 without DN) and in 13 non-diabetic control subjects using denaturing high-performance liquid chromatography. RESULTS: Fifteen XYLT-I sequence variants were identified. Of these, six were previously unknown. There were significant differences in the allele frequencies of the three polymorphisms (c.343G-->T (p.A115S), IVS3+10C-->T, IVS3+30G-->C) in Type 1 diabetic patients and healthy controls. CONCLUSIONS: The occurrence of DN is independent of the XYLT-I variants detected in our study. However, three XYLT-I polymorphisms may be linked to Type 1 diabetes. Since we have previously proposed that one of these polymorphisms was not associated with Type 1 diabetes (Schön S et al. Kidney Int 2005; 68: 1483-1490), larger-scale analysis is clearly necessary to pinpoint the significance of this mutation.

Polymorphisms in the gene encoding angiotensin I converting enzyme 2 and diabetic nephropathy.

AIMS/HYPOTHESIS: Substantial evidence exists for the involvement of the renin-angiotensin system (RAS) in diabetic nephropathy. Angiotensin I converting enzyme 2 (ACE2), a new component of the RAS, has been implicated in kidney disease, hypertension and cardiac function. Based on this, the aim of the present study was to evaluate whether variations in ACE2 are associated with diabetic nephropathy. MATERIALS AND METHODS: We used a cross-sectional, case-control study design to investigate 823 Finnish type 1 diabetic patients (365 with and 458 without nephropathy). Five single-nucleotide polymorphisms (SNPs) were genotyped using TaqMan technology. Haplotypes were estimated using PHASE software, and haplotype frequency differences were analysed using a chi(2)-test-based tool. RESULTS: None of the ACE2 polymorphisms was associated with diabetic nephropathy, and this finding was supported by the haplotype analysis. The ACE2 polymorphisms were not associated with blood pressure, BMI or HbA(1)c. CONCLUSIONS/INTERPRETATION: In Finnish type 1 diabetic patients, ACE2 polymorphisms are not associated with diabetic nephropathy or any studied risk factor for this complication. Further studies are necessary to assess a minor effect of ACE2.

Genomewide scan for familial combined hyperlipidemia genes in finnish families, suggesting multiple susceptibility loci influencing triglyceride, cholesterol, and apolipoprotein B levels.

Familial combined hyperlipidemia (FCHL) is a common dyslipidemia predisposing to premature coronary heart disease (CHD). The disease is characterized by increased levels of serum total cholesterol (TC), triglycerides (TGs), or both. We recently localized the first locus for FCHL, on chromosome 1q21-q23. In the present study, a genomewide screen for additional FCHL loci was performed. In stage 1, we genotyped 368 polymorphic markers in 35 carefully characterized Finnish FCHL families. We identified six chromosomal regions with markers showing LOD score (Z) values >1.0, by using a dominant mode of inheritance for the FCHL trait. In addition, two more regions emerged showing Z>2.0 with a TG trait. In stage 2, we genotyped 26 more markers and seven additional FCHL families for these interesting regions. Two chromosomal regions revealed Z>2.0 in the linkage analysis: 10p11.2, Z=3.20 (theta=.00), with the TG trait; and 21q21, Z=2.24 (theta=.10), with the apoB trait. Furthermore, two more chromosomal regions produced Z>2.0 in the affected-sib-pair analysis: 10q11.2-10qter produced Z=2.59 with the TC trait and Z=2.29 with FCHL, and 2q31 produced Z=2.25 with the TG trait. Our results suggest additional putative loci influencing FCHL in Finnish families, some potentially affecting TG levels and some potentially affecting TC or apoB levels.

Mapping of a gene for type 2 diabetes associated with an insulin secretion defect by a genome scan in Finnish families.

Non-insulin dependent diabetes mellitus (NIDDM) affects more than 100 million people worldwide and is associated with severe metabolic defects, including peripheral insulin resistance, elevated hepatic glucose production, and inappropriate insulin secretion. Family studies point to a major genetic component, but specific susceptibility genes have not yet been identified-except for rare early-onset forms with monogenic or mitochondrial inheritance. We have screened over 4,000 individuals from a population isolate in western Finland, identified 26 families (comprising 217 individuals) enriched for NIDDM and performed a genome-wide scan using non-parametric linkage analysis. We found no significant evidence for linkage when the families were analysed together, but strong evidence for linkage when families were classified according to mean insulin levels in affecteds (in oral glucose tolerance tests). Specifically, families with the lowest insulin levels showed linkage (P = 2 x 10(-6)) to chromosome 12 near D12S1349. Interestingly, this region contains the gene causing the rare, dominant, early-onset form of diabetes MODY3. Unlike MODY3 families, the Finnish families with low insulin have an age-of-onset typical for NIDDM (mean = 58 years). We infer the existence of a gene NIDDM2 causing NIDDM associated with low insulin secretion, and suggest that NIDDM2 and MODY3 may represent different alleles of the same gene.

The effect of (steroid) immunosuppression on skeletal muscle glycogen metabolism in patients after kidney transplantation.

To examine the mechanisms by which immunosuppression by steroids impairs glycogen synthesis in human skeletal muscle, we measured glycogen synthase protein content and activity in muscle samples from 14 patients receiving corticosteroid therapy after kidney transplantation and in 20 healthy control subjects. A percutaneous muscle sample was taken before and at the end of a euglycemic hyperinsulinemic insulin clamp. Insulin-stimulated glucose disposal was reduced by 33% in kidney transplant patients compared with healthy controls (33.8 +/- 4.2 vs. 50.5 +/- 2.7 mumol (kg LBM)-1 min-1; P<0.01), primarily due to a decrease in nonoxidative glucose metabolism (14.2 +/- 3.3 vs. 32.3 +/- 2.7 mumol (kg LBM)-1 min-1; P<0.001). Glycogen synthase activity measured at both 0.1 mmol/L (17.6 +/- 2.6 vs. 24.0 +/- 2.2 nmol min-1 mg protein-1; P<0.05), and at 10 mmol/L glucose 6-phosphate (24.1 +/- 3.5 vs. 33.7 +- 2.4 nmol min-1 mg protein-1; P<0.05) and glycogen synthase protein concentrations (8.8 +/- 1.8 vs. 18.9 +/- 1.9 relative units per ng DNA; P<0.01) were lower in kidney transplant patients compared with controls. Glycogen synthase protein correlated with nonoxidative glucose metabolism (r=0.42; P=0.04). Alpha-actinin (used as a control of general protein degradation) was lower in kidney transplant patients compared with controls (4.4 +/- 0.8 vs. 9.6 +/- 1.1 cpm/ng DNA; P<0.01). In conclusion, corticosteroids cause insulin resistance, which correlates with impaired activation of glycogen synthase and decreased enzyme protein content. The decrease in glycogen synthase protein may reflect increased degradation rather than a defect in translation.

Increased concentrations of glycogen synthase protein in skeletal muscle of patients with NIDDM.

To examine whether changes in the glycogen synthase protein concentration contribute to impaired insulin-stimulated glycogen metabolism in patients with noninsulin-dependent diabetes mellitus (NIDDM), muscle biopsies were taken before and after a 4-h euglycemic hyperinsulinemic clamp to measure glycogen synthase activity and glycogen synthase protein concentrations in 14 patients with NIDDM and in 17 control subjects. Nonoxidative glucose metabolism was reduced by 64% in patients with NIDDM compared with control subjects and correlated with insulin-stimulated glycogen synthase activity (r = 0.55, P < 0.05). The concentration of glycogen synthase protein in skeletal muscle was higher in patients with NIDDM than in control subjects (6.75 +/- 0.88 vs. 4.41 +/- 0.50 counts.min-1.micrograms protein-1, P < 0.05), whereas there was no significant difference in glycogen synthase mRNA concentration between the two groups. The glycogen synthase protein concentration correlated inversely with the rate of nonoxidative glucose metabolism (r = -0.63, P < 0.05). These findings indicate that the amount of glycogen synthase protein is increased in skeletal muscle of patients with NIDDM. The increase in the glycogen synthase protein may serve to compensate for a functional defect in the activation of the enzyme by insulin.