SNP genotyping We searched the HapMap database (http://hapmap.ncbi.nlm.nih.gov/) for SNPs within the genes encoding sirtuin families, and selected 55 SNPs (39 tagging SNPs) for genotyping; 11 in SIRT1 (rs12778366, rs3740051, rs2236318, rs2236319, Crenolanib in vivo rs10823108, rs10997868, rs2273773, rs3818292, rs3818291, rs4746720, rs10823116), 7 in
SIRT2 (rs1001413, rs892034, rs2015, rs2241703, rs2082435, rs11575003, rs2053071), 15 in SIRT3 (rs11246002, rs2293168, rs3216, rs10081, rs511744, rs6598074, rs4758633, rs11246007, rs3782117, rs3782116, rs3782115, rs1023430, rs12576565, rs536715, rs3829998), 7 in SIRT4 (rs6490288, rs7298516, rs3847968, rs12424555, rs7137625, rs2261612, rs2070873), 11 in SIRT5 (rs2804923, rs9382227, rs2804916, rs2804918, rs9370232, rs4712047, rs3734674, rs11751539, rs3757261, ATM Kinase Inhibitor cell line rs2253217, rs2841514), and 4 in SIRT6 (rs350852, rs7246235, rs107251, rs350844). We could not identify any confirmed SNPs within SIRT7 in the Japanese population. The genotyping of these SNPs was performed by using multiplex polymerase chain reaction (PCR)-invader assays, as described previously [7–10]. Statistical analyses We tested the genotype distributions for Hardy–Weinberg equilibrium (HWE) proportions by using the chi-squared test. We analyzed
the differences between the case−control groups in terms of the distribution of genotypes with the Cochran–Armitage trend test. The analyses TNF-alpha inhibitor for haplotype
structures within each gene were performed using Haploview software version 4.1 [20]. CB-839 clinical trial A combined meta-analysis was performed using the Mantel–Haenszel procedure with a fixed effects model after testing for heterogeneity. Results Among the 55 SNPs examined, genotype distributions of 3 SNPs, rs12576565 in SIRT3, and rs2804923 and rs2841514 in SIRT 5, showed significant deviation from HWE proportion in control groups (P < 0.01, Supplementary Table 2), and these 3 SNPs were excluded from the association study. As shown in Table 1, 8 out of 11 SNPs in SIRT1 showed a directionally consistent association with diabetic nephropathy in all 3 studies, although individual associations were not significant (P > 0.05, Supplementary Table 2). In a combined meta-analysis, we could identify a nominally significant association between rs4746720 and proteinuria, and between 4 SNPs, rs2236319, rs10823108, rs3818292, rs4746720, and combined phenotypes (proteinuria + ESRD, P < 0.05). Subsequent haplotype analysis revealed that the 11 SNPs formed one haplotype block (Fig. 1), and 7 common haplotypes covered >99% of the present Japanese population. Among them one haplotype had a stronger association with diabetic nephropathy than single SNPs alone (P = 0.016, odds ratio (OR) 1.31 95% confidence interval (CI) 1.05–1.62].