Comparing strategies to fine-map the association of common SNPs at chromosome 9p21 with type 2 diabetes and myocardial infarction

Abstract

Noncoding variants at human chromosome 9p21 near CDKN2A and CDKN2B are associated with type 2 diabetes, myocardial infarction, aneurysm, vertical cup disc ratio and at least five cancers. Here we compare approaches to more comprehensively assess genetic variation in the region. We carried out targeted sequencing at high coverage in 47 individuals and compared the results to pilot data from the 1000 Genomes Project. We imputed variants into type 2 diabetes and myocardial infarction cohorts directly from targeted sequencing, from a genotyped reference panel derived from sequencing and from 1000 Genomes Project low-coverage data. Polymorphisms with frequency >5% were captured well by all strategies. Imputation of intermediate-frequency polymorphisms required a higher density of tag SNPs in disease samples than is available on first-generation genome-wide association study (GWAS) arrays. Our association analyses identified more comprehensive sets of variants showing equivalent statistical association with type 2 diabetes or myocardial infarction, but did not identify stronger associations than the original GWAS signals.

Figure 1. Comparison of targeted sequencing to 1000G Pilot 1 Data

Variant calls were made in all six regions of T2D association in the 32 individuals who were sequenced as part of both this targeted, high coverage sequencing effort (total 47 CEU HapMap individuals) as well as 1000G Pilot 1 (total 60 CEU HapMap individuals).

Figure 2. Fraction of variation on chromosome 9p21 captured in T2D disease cohort by different imputation scenarios

MACH imputation quality estimates (a, c, e) and overall fraction of variation captured in T2D samples (b, d, f) for different imputation scenarios. (a, c, e) The MACH-estimated r² for each SNP is plotted as a function of genomic position. SNPs not observed in the reference panel are assigned an r² of zero. Recombination rate (estimated from HapMap) is plotted to reflect local LD structure. Gene annotations were taken from the University of California-Santa Cruz Genome Browser. (b, d, f) The fraction of variants captured in T2D samples is shown as a function of MAF and MACH-estimated r². Imputation scenarios are: (a, b) Imputing from HapMap II (n=238 SNPs in 60 individuals) into the SNPs genotyped on the Affymetrix 500K array; (c, d) Imputing from 112 individuals genotyped at HapMap II sites and validated sequencing sites (total n=464 SNPs) into the SNPs genotyped on the Affymetrix 500K array; (e, f) Imputing from the same reference panel as c, d into SNPs genotyped on the Affymetrix 500K array plus additional tag SNPs genotyped in the T2D cohort (genotyped marker density in T2D samples ~1 SNP/1.5kb).

Figure 3. Comparison of imputation from a genotyped reference panel, directly from high coverage re-sequencing data, and directly from 1000G Pilot 1 data

(a) Variants present in the three reference panels and their overlap. The 67 variants present in the genotyped reference panel but not in the high coverage sequencing reference panel (denoted by asterisk) were called in high coverage sequencing as singletons and so were excluded from the sequencing reference panel. 40% of these variants are not singletons in the larger genotyped reference panel. (b) The fraction of sites within each reference panel captured with a MACH-estimated r² of at least 0.8. (c) The overall fraction of known variants captured with a MACH-estimated r² of at least 0.8 by imputation from each reference panel.

Figure 4. Association results for T2D and MI on chromosome 9p21

Regional plots showing association signal for (a) T2D and (b) MI. The signal for each SNP (represented as −log₁₀ p-value) is plotted as a function of genomic position. The size of the diamond for each SNP represents the LD (measured as r²) between that SNP and the original GWAS SNP (rs10811661 for T2D and rs4977574 for MI). Recombination rate (estimated from HapMap) is plotted to reflect the local LD structure in the region. Gene annotations were taken from the University of California-Santa Cruz Genome Browser.