Concept, design and implementation of a cardiovascular gene-centric 50 k SNP array for large-scale genomic association studies

Abstract

A wealth of genetic associations for cardiovascular and metabolic phenotypes in humans has been accumulating over the last decade, in particular a large number of loci derived from recent genome wide association studies (GWAS). True complex disease-associated loci often exert modest effects, so their delineation currently requires integration of diverse phenotypic data from large studies to ensure robust meta-analyses. We have designed a gene-centric 50 K single nucleotide polymorphism (SNP) array to assess potentially relevant loci across a range of cardiovascular, metabolic and inflammatory syndromes. The array utilizes a "cosmopolitan" tagging approach to capture the genetic diversity across approximately 2,000 loci in populations represented in the HapMap and SeattleSNPs projects. The array content is informed by GWAS of vascular and inflammatory disease, expression quantitative trait loci implicated in atherosclerosis, pathway based approaches and comprehensive literature searching. The custom flexibility of the array platform facilitated interrogation of loci at differing stringencies, according to a gene prioritization strategy that allows saturation of high priority loci with a greater density of markers than the existing GWAS tools, particularly in African HapMap samples. We also demonstrate that the IBC array can be used to complement GWAS, increasing coverage in high priority CVD-related loci across all major HapMap populations. DNA from over 200,000 extensively phenotyped individuals will be genotyped with this array with a significant portion of the generated data being released into the academic domain facilitating in silico replication attempts, analyses of rare variants and cross-cohort meta-analyses in diverse populations. These datasets will also facilitate more robust secondary analyses, such as explorations with alternative genetic models, epistasis and gene-environment interactions.

Figure 1. Power to detect main effect with 550 K SNPs using various case control sizes & MAFs.

Genome wide association power calculated based on n unrelated cases and n unrelated controls. The disease model is assumed to be multiplicative with disease minor allele frequency (MAF) = 0.05, 0.1, and 0.2, and the odds ratio = 1.2, 1.4, and 1.6. Significance is assessed at the 5% level using Bonferroni correction assuming 550 K tests.

Figure 2. MAF distribution for IBCv1 assays across studies of Caucasians, African Americans and South Asians.

Proportion of polymorphic variants (as a % of total) are described on the y-axis with the x-axis illustrating bins of the minor allele frequencies (MAF) for six different studies; Caucasian Study 1 (n = 2094 European); Caucasian Study 2 (n = 2150 European); Caucasian Study 3 (n = 1054 European American); African American Study 1 (n = 254); African American Study 2 (n = 130) and South Asians (n = 385).

Figure 3. Average number of SNPs per Group 1 and 2 loci, respectively, on IBCv1 compared with the major GWAS products.

IBCv1 Group 1 and Group 2 loci (n = 435 and 1,349) are captured at MAF>0.02, r² 0.8 and MAF>0.05, r² 0.5 in HapMap populations and SeattleSNPs, and illustrated in blue and red respectively. IBCv1 refers to the ITMAT-Broad- CARE version1 array, Illum650, Illum550 & Illum300 refer to Illumina HumanHap650Y, HumanHap550 and HumanHap300 Infinium products containing ∼660 K, ∼550 K & ∼317 K SNPs respectively and Affy 6.0 and Affy 5.0 refers to Affymetrix SNP array products containing ∼907 K & 500 K respectively.

Figure 4. Coverage of IBCv1 versus GWAS products for Group1 loci in HapMap populations.

Cumulative coverage (y axis) of the HapMap 22 release was assessed using Max r² (× axis), at an MAF cutoff of 0.02, in CEPH (CEU) (A), Chinese (CHB) plus Japanese (JPT), (B) and Yoruba (YRI) HapMap individuals (C). Coverage was also assessed at an MAF cutoff of 0.05 in CEU (D), CHB+JPT (E), and YRI individuals (F). IBCv1 refers here to version1 ITMAT-Broad-CARe array using 45,237 SNPs passed by the manufacture. ILMN_1M, ILMNHap550 and ILMNHap300 refer to Illumina's Human1M, HumanHap550 (555,352 SNPs) and HumanHap300 (317,503 SNPs) products, respectively. Affy_6.0 and Affy_5.0 refers to Affymetrix 6.0 & Affymetrix 5.0 array products containing ∼906,600 SNPs & 500,568 SNPs respectively.

Figure 5. Combined coverage of IBCv1 with the 500 K and one million SNP products for Group1 loci in HapMap populations.

For the combined coverage of IBCv1 with the 500 K SNP products (A–F): coverage (y axis) was assessed using Max r² (× axis), at an MAF cutoff of 0.02, for the HapMap 22 release in CEPH (CEU) (A), Chinese (CHB) plus Japanese (JPT) (B), and Yoruba (YRI) HapMap individuals (C). Coverage was also assessed at an MAF cutoff of 0.05 in CEU (D), CHB+JPT (E), and YRI individuals (F). For combined coverage with the one million SNP products (G–L): Coverage (y axis) was also assessed using Max r² (× axis), at an MAF cutoff of 0.02, for the HapMap 22 release in CEPH (CEU) (G), Chinese (CHB) plus Japanese (JPT) (H), and Yoruba (YRI) HapMap individuals (I) and coverage with an MAF cutoff of 0.05 in CEU (J), CHB+JPT (K), and YRI individuals (L). IBCv1 refers here to the version1 ITMAT-Broad-CARe array using 45,237 SNPs that passed manufacturers criteria. Affy_5.0 refers to the Affymetrix 5.0 array and ILMN_HapMap550 refers to the Infinium 550 K HumanHap array which contains 500,568 and 555,352 SNPs respectively. Affy_6.0 refers to the Affymetrix 6.0 array and ILMN_1M refers to the Illumina Infinium one million SNP array containing ∼906,600 and ∼1,050,000 SNPs respectively.