In an attempt to refine the possible TRPC1 association with diabetic nephropathy in type 1 diabetes, seven tag SNPs extending across the TRPC1 gene were genotyped in type 1 diabetes patients with and without diabetic nephropathy from the GoKinD samples. In addition to the 1,177 European American (Caucasian) type 1 diabetes patients, this cohort also included 109 non-Caucasian samples (8.5% of the total). To reduce type 1 or type 2 error resulting from population-specific variants, we analyzed the data in European Americans. All examined SNPs were satisfied in Hardy-Weinberg equilibrium assumption. Figure demonstrates the location and LD matrix of examined SNPs in the TRPC1 gene. Pairwise marker correlations suggested strong LD in GoKinD European Americans, in agreement with the haplotype block structure in European Caucasians in HapMap.
Fig. 1. Tag SNP markers examined in the TRPC1 gene. Seven examined Tag SNP markers and their linkage disequilibrium (LD) matrix along the TRPC1 gene in the GoKinD population. The coding exons in the gene are marked with the blocks. The LD values (D′) (more ...)
Single marker association analysis was conducted on the genotype distribution and allele frequency in the GoKinD population. Detailed information of examined SNPs and their minor allele frequencies (MAFs) in GoKinD European Americans are summarized in table . Genotype distribution and the association of the TRPC1 polymorphisms with diabetic nephropathy in GoKinD European American type 1 diabetes patients are shown in table . The MAF of SNPs rs7621642 and rs2033912 were of borderline significant difference (p = 0.045 and 0.047, respectively) in female European American type 1 diabetes patients with versus without diabetic nephropathy, but not in males (p = 0.661 and 0.694, respectively). The MAF of SNP rs3821647, residing in a haplotype block with rs7610200, trended toward a higher frequency in female type 1 diabetes patients with diabetic nephropathy, compared to those without diabetic nephropathy (0.187 vs. 0.151, p = 0.096). To examine independent variables associated with diabetic nephropathy among female type 1 diabetes patients in the GoKinD population, further multivariate logistic regression analysis was performed. No significant association of SNPs rs7621642 (p = 0.233, OR = 1.457, CI 95% 0.785–2.705), rs2033912 (p = 0.232, OR = 1.458, CI 95% 0.785–2.707) and rs3821647 (p = 0.389, OR = 1.328, CI 95% 0.696–2.532) with diabetic nephropathy among female type 1 diabetic patients with and without diabetic nephropathy was observed.
TRPC1 SNPs and MAFs in GoKinD Caucasian type 1 diabetic subjects with and without diabetic nephropathy
Genotype distributions and minor allele frequencies of TRPC1 polymorphisms: American Caucasians (a) and African Americans (b)
Tests in the entire GoKinD population were then performed. When African Americans from the GoKinD population were combined with European Americans, the difference in MAFs of these three polymorphisms were statistically significant in type 1 diabetes patients with versus without diabetic nephropathy (p = 0.003, 0.003 and 0.004, respectively; Pc = 0.042, 0.042 and 0.056, respectively, after Bonferroni correction). In order to understand whether TRPC1 polymorphisms are the population-specific variants or confer the susceptibility to diabetic nephropathy in African Americans, we further genotyped the three SNPs, rs7621642, rs2033912 and rs3821647, in a second cohort of African Americans, including 284 with type 2 diabetes-associated ESRD, 284 with hypertension-associated ESRD and 282 nondiabetic subjects [19
]. The genotype distribution and MAF of these polymorphisms in the population of Wake Forest African Americans are listed in table . The genotype distributions of these SNPs were reversed in African Americans, compared to what had been detected in European Americans (table ); major alleles of the SNPs in European Americans were minor alleles in African Americans. No significant associations for these three polymorphisms were detected with type 2 diabetes-ESRD or hypertensive-ESRD in African Americans.
We further investigated TRPC1 gene expression at mRNA levels with a mouse model of diabetic nephropathy to determine whether reduced TRPC1 gene expression was a primary or subsequent defect in the development of diabetic nephropathy. TRPC1 gene expression in kidney tissue from db/db mice at 12 and 26 weeks of age, respectively, was examined using a TaqMan real time RT-PCR protocol. Data indicated that TRPC1 mRNA expression levels kidney tissue from db/db mice at the age of 12 weeks compared to the controls were tendency to reduce (p = 0.295, fig. ), while the expression levels of this gene in 26-week-old db/db mice were significantly decreased (p = 0.013, fig. ).
Fig. 2. TRPC1 gene mRNA expression in kidney tissues of db/db mice. TRPC1 gene mRNA expression was examined using real-time RT-PCR in kidney tissue from db/db mice at 12 and 26 weeks of age, respectively. TRPC1 mRNA expression levels between db/db and db/m (control) (more ...)