Abstract 18455: Genome Editing Identifies a Functional Role for the Lead Variant at the ZC3HC1 Coronary Artery Disease Risk Locus in Cell Cycle Regulation
Genome wide association studies have identified 46 chromosomal loci that are associated with coronary artery disease (CAD); however, for most of these loci the mechanism by which they affect CAD risk is unclear. The association signal at chromosome 7q32.2 is rare amongst CAD GWAS loci as the lead variant, rs11556924, introduces a coding change (His363Arg) in the ZC3HC1 gene. No other variants are in strong linkage disequilibrium, suggesting that this is the functional SNP. During interphase, ZC3HC1 degrades Cyclin B1, keeping its levels low. Inactivation of ZC3HC1 and subsequent accumulation of Cyclin B1 triggers entry into mitosis. We hypothesised that the effect of the SNP perturbs the function of ZC3HC1 in regulating the cell cycle. To investigate this, we used recombinant adeno-associated virus mediated genome editing to generate isogenic DLD-1 cell lines that are identical apart from at rs11556924. A proliferation assay comparing the rate of growth of cells of the homozygous non-risk and heterozygous isogenic lines showed a decrease in cellular growth in cells carrying one copy of the risk allele, with a mean of 25.4% fewer cells after 5 days growth (p=0.019). Counter-intuitively, cells carrying the risk allele also showed an increase in mitotic index, with the number of cells in mitosis after 2 hours of mitotic arrest induced with the microtubule-depolymerising drug colchicine, increasing from μ=7.25% (S.E.=0.71%) to μ=12.32% (S.E.=1.71%) (p=0.0001). This increase in mitotic index could be caused by chromosome segregation errors that may be causing cells to arrest in mitosis, which would be expected to result in a widening in the distribution of chromosome numbers in these cells. We tested this by counting the number of chromosomes in cells carrying the risk allele compared to those which are homozygous for the non-risk allele but found no difference in chromosome number (p=0.78). These data suggest that the CAD risk allele of rs11556924 perturbs Cyclin B1 dynamics causing a delay in the progression of mitosis. The mechanism by which this increases CAD risk requires further elucidation.
Author Disclosures: P.D. Jones: None. M. Kaiser: None. T.R. Webb: None. N.J. Samani: None.
- © 2014 by American Heart Association, Inc.