Abstract 2817: Allelic Expression Imbalance Analysis in Heart Failure Samples Finds Novel cis-acting Alleles in ACE and SOD2
INTRODUCTION: Despite identification of probable candidate genes, elucidation of the genetic elements that influence human susceptibility to cardiovascular disease is elusive. Heterozygous SNPs in DNA can be used to measure allele expression imbalances (AEI) in RNA from individual chromosomes, yielding a direct marker for genetic contributions to variability of RNA expression.
HYPOTHESIS: Functional cis-acting differences are present in genes affecting susceptibility to cardiovascular disease. Quantitation of mRNA AEI can provide direct identification of cis-acting contributions to genetic variability.
METHODS: DNA and total RNA were extracted from 65 human heart failure explants (10 African-Americans, 55 Caucasians). Frequent SNPs in the coding regions of candidate genes ACE, FLT1, HIF1a, LPL, MCP1, NOS3, PTGDS, and SOD2 were selected and genotyped. Heterozygotes were used for AEI analysis. Replicate cDNAs and negative controls were synthesized from 1 ug RNA. RNA levels were analyzed for all genes by qRTPCR. AEI was measured using a primer extension assay. Expression of cDNA alleles was normalized to DNA alleles. Genetic analysis was performed with HelixTree software.
RESULTS: We found significant and frequent AEI for ACE (p<0.001) and SOD2 (p<0.001) in human heart failure explants. Other genes showed rarer and smaller AEI (FLT1, MCP1, NOS3, PTGDS) or no AEI (HIF1a, LPL). ACE results were validated by a second marker SNP (r2=0.98) and dilution-mixing experiments (r2=0.99). Samples with high ACE AEI (>2-fold) were exclusively African-American, and displayed significantly lower total RNA expression of ACE (p<0.02). SOD2 AEI was common (88% of samples) and imbalance was in a single direction.
CONCLUSIONS: We report a haplotype (~17% frequency) in an African-American heart failure population that associates with lower ACE expression and is unexplained by the known I/D polymorphism. We find a SNP in SOD2 is tightly linked to AEI of that mRNA. Our results show that measuring AEI is effective in locating cis-acting alleles in candidate genes. The ACE alleles we identified may account for disparities in clinical findings on the I/D polymorphism and differences in African-American heart disease progression and response to ACE inhibitors.