A genome-wide association study in American Indians implicates DNER as a susceptibility locus for type 2 diabetes

Abstract

Most genetic variants associated with type 2 diabetes mellitus (T2DM) have been identified through genome-wide association studies (GWASs) in Europeans. The current study reports a GWAS for young-onset T2DM in American Indians. Participants were selected from a longitudinal study conducted in Pima Indians and included 278 cases with diabetes with onset before 25 years of age, 295 nondiabetic controls ≥45 years of age, and 267 siblings of cases or controls. Individuals were genotyped on a ∼1M single nucleotide polymorphism (SNP) array, resulting in 453,654 SNPs with minor allele frequency >0.05. SNPs were analyzed for association in cases and controls, and a family-based association test was conducted. Tag SNPs (n = 311) were selected for 499 SNPs associated with diabetes (P < 0.0005 in case-control analyses or P < 0.0003 in family-based analyses), and these SNPs were genotyped in up to 6,834 additional Pima Indians to assess replication. Rs1861612 in DNER was associated with T2DM (odds ratio = 1.29 per copy of the T allele; P = 6.6 × 10(-8), which represents genome-wide significance accounting for the number of effectively independent SNPs analyzed). Transfection studies in murine pancreatic β-cells suggested that DNER regulates expression of notch signaling pathway genes. These studies implicate DNER as a susceptibility gene for T2DM in American Indians.

Figure 1

A: “Manhattan” plot of genome-wide association results for young-onset T2DM in American Indians in case-control study. The negative base 10 logarithm of the P value for an association with diabetes is plotted against chromosome and position (determined in Build 37). Results are shown after genomic control. B: “Manhattan” plot of genome-wide association results for young-onset T2DM in American Indians in family-based analysis. The negative base 10 logarithm of the P value for an association with diabetes is plotted against chromosome and position (determined in Build 37). C: Quantile–quantile plot of observed vs. expected (given the null distribution) P value for the case-control study. The observed distribution of P values without genomic control is shown in the dotted line, and the distribution with genomic control is shown in the solid line. D: Quantile–quantile plot of observed vs. expected (given the null distribution) P value for the family-based study. The expected distribution was estimated from simulation of 10⁸ test statistics with the observed correlation (r = 0.29) between those for the conditional logistic regression and case-control analyses. GC, genomic control.

Figure 2

A: Association results for 63 variants across DNER with T2DM in Pima Indians. The negative base 10 logarithm of the P value for association is shown at the Build 37 position. Variants include 48 “tags” (r² >0.8) for 153 SNPs from the GWAS and 14 variants identified from sequencing ∼5,300 base pairs in DNER; one variant identified by sequencing (a short insertion/deletion at position 230.580216 Mb) is not in public databases. Results obtained in all 7,674 individuals are shown as boxes, while those obtained in the GWAS and first replication samples are shown as triangles. Symbols are shaded according to r² with rs1861612. B: The prevalence of T2DM by genotype at rs1861612 and age-group in 7,674 Pima Indians. P values for association with T2DM in each age-group are as follows: P = 0.0005 (5–24), P = 0.0517 (25–34), P = 0.0265 (35–44), P = 0.0004 (45–54), and P = 0.0028 (55 and up). C: Relative mRNA level in murine pancreatic β-cells after transfection experiments to overexpress DNER (gray bars) or to knockdown DNER expression (open bars) compared with negative control (black bars, which = 1 by definition). mRNA levels are shown for DNER itself, for notch pathway genes (Notch1, Hes1, Neurog3) and for Rtn2 (which is not involved in the notch pathway). *P < 0.001; **P < 0.0001.