Abstract
Recent genome-wide association studies (GWAS) have identified multiple new loci which appear to alter coronary artery disease (CAD) risk via arterial wall-specific mechanisms. One of the annotated genes encodes LMOD1 (Leiomodin 1), a member of the actin filament nucleator family that is highly enriched in smooth muscle-containing tissues such as the artery wall. However, it is still unknown whether LMOD1 is the causal gene at this locus and also how the associated variants alter LMOD1 expression/function and CAD risk. Using epigenomic profiling we recently identified a non-coding regulatory variant, rs34091558, which is in tight linkage disequilibrium (LD) with the lead CAD GWAS variant, rs2820315. Herein we demonstrate through expression quantitative trait loci (eQTL) and statistical fine-mapping in GTEx, STARNET, and human coronary artery smooth muscle cell (HCASMC) datasets, rs34091558 is the top regulatory variant for LMOD1 in vascular tissues. Position weight matrix (PWM) analyses identify the protective allele rs34091558-TA to form a conserved Forkhead box O3 (FOXO3) binding motif, which is disrupted by the risk allele rs34091558-A. FOXO3 chromatin immunoprecipitation and reporter assays show reduced FOXO3 binding and LMOD1 transcriptional activity by the risk allele, consistent with effects of FOXO3 downregulation on LMOD1. LMOD1 knockdown results in increased proliferation and migration and decreased cell contraction in HCASMC, and immunostaining in atherosclerotic lesions in the SMC lineage tracing reporter mouse support a key role for LMOD1 in maintaining the differentiated SMC phenotype. These results provide compelling functional evidence that genetic variation is associated with dysregulated LMOD1 expression/function in SMCs, together contributing to the heritable risk for CAD.
Highlights
Coronary artery disease (CAD) is the leading cause of mortality worldwide, and poses considerable public health burden despite current treatment and lifestyle interventions
By implementing several complementary approaches, we extend previous findings demonstrating that this genetic variant is associated with attenuated expression of Leiomodin 1
We show that reduced Leiomodin 1 results in phenotypic modulation of smooth muscle cells, a process related to diseases such as atherosclerosis
Summary
Coronary artery disease (CAD) is the leading cause of mortality worldwide, and poses considerable public health burden despite current treatment and lifestyle interventions. Around 60% of these loci have been associated independently of classical risk factors[7], and given previous functional evidence of candidate causal genes at these loci[8,9,10,11], suggests an involvement of pathways intrinsic to the vessel wall This is supported by the recent CAD GWAS meta-analysis, which combined targeted genotyped, multi-ethnic individuals with individuals from the CARDIoGRAMplusC4D consortium, to identify 15 new loci, within or near genes having arterial wall-specific functions[4]. One of these new loci (lead SNP rs2820315) is found to reside in a smooth muscle cell (SMC)-restricted gene annotated as Leiomodin 1 (LMOD1)[4]. We determined this variant resides in a region of accessible chromatin defined by the Assay for Transpose Accessible Chromatin (ATAC-seq) and is marked by the H3K27ac active enhancer histone modification, the underlying mechanism for this association remains unknown
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