Abstract 1923: Massively Parallel Resequencing of Phenotypically Defined DNA Pools Identifies Rare Pathological Gene Variants in Human Non-ischemic Cardiomyopathy
Common heart failure (HF) tends to occur in families, suggesting a genetic component. Genome-wide scans are beginning to identify loci associated with HF and its clinical antecedents, but in most instances these loci do not alter encoded protein. Some may affect regulatory domains or non-coding RNAs, but most disease-associated polymorphisms are probably linked to causal variants that must be identified through resequencing. Here, we describe targeted resequencing that applies next-generation Illumina GAII sequencing technology to a large case-control study of HF, and discovered rare pathogenic alleles. Based on existing gene-association data, we resequenced coding exons of five HF genes: ADRA1A (α1-adrenergic receptor), ADRB1 (β1-adrenergic receptor), ADRB2 (β2-adrenergic receptor), HSPB7 (cardiovascular heat shock protein), and PLN (phospholamban) in 2,741 individuals, consisting of 1865 HF cases and 876 controls. For resequencing, DNA from the primary study cohort was pooled according to phenotype: a set of twelve pools (8 cases, 4 controls) averaging ~250 individual DNAs grouped by race and HF etiology (ischemic vs non-ischemic), and a set of 29 pools (21 cases, 8 controls) averaging ~100 individual DNAs grouped by race, HF etiology, and diabetes status. Sequence coverage depth averaged 25x. Resequencing identified 83 SNPs, 55 that are in dbSNP and 28 novel. Allele frequencies were reproducible in different sequencing runs (r2=0.96 for identical pools; r2=0.92 for similar pools), and were highly correlated with HapMap data (Caucasian r2=0.90; African-Americans r2=0.96). Fourteen polymorphisms (2 novel) had significantly different (P<0.0001) allele frequencies between HF cases and controls. Of 19 non-synonymous SNPs, 7 were predicted to be deleterious using functional analysis prediction algorithms. Results were confirmed by pooled resequencing in an independent HF population, and in some instances, by Sanger sequencing of individual DNAs from the entire study cohort. These findings demonstrate the utility of pooled genotyping with next generation platforms for rapid, accurate, and cost-effective profiling of genetic variants in large populations affected with common polygenic diseases, such as sporadic heart failure.