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Rationale: Coronary artery disease (CAD) is a pervasive and critical health care problem. Elevated high-density lipoprotein-associated cholesterol (HDL-C) is associated with improved atherosclerotic cardiovascular disease outcomes on a population level, but clinical trials aimed at HDL-C elevation have not succeeded in improving atherosclerotic cardiovascular disease event risk. Nevertheless, human variants in the HDL receptor, encoded by SCARB1 , are associated with dyslipidemia, suggesting that HDL metabolism, not HDL-C, is a suitable target for therapy. However, variants in SCARB1 have never been directly attributed to CAD by Mendelian inheritance. Objective: To determine if compound heterozygous variants in SCARB1 cause disease in 2 brothers with severe, early-onset CAD. Methods and Results: Using whole exome sequencing, we have identified rare, compound heterozygous variants in SCARB1 that segregate with severe, premature CAD, following patterns of Mendelian inheritance. Using induced pluripotent stem cell–derived hepatocyte-like cells from the proband, we discovered the maternal variant (c.754_755delinsC) to be the first identified SCARB1 null allele, characterized by the absence of RNA and protein expression. Further, we demonstrate that the variant on the paternal allele (c.956G>T [p.G319V]) results in decreased cholesterol uptake, decreased SR-BI:HDL binding, and increased affinity for SR-BI dimerization. Finally, we generated a p.G319V knock-in mouse model that displays nearly 100% homozygous lethality and elevated plasma cholesterol in heterozygous animals, confirming pathogenicity of this variant. Conclusions: In summary, our data provide the first molecular mechanism to show the Mendelian inheritance of CAD as a result of human SCARB1 variants. The rarity of these variants supports pathogenicity in this family. Furthermore, SR-BI p.G319V, which has previously been reported benign in the context of heterozygosity, was uniquely presented alongside a null allele, demonstrating the disease-contributing capability of loss-of-function SCARB1 variants within the population.

Dyslipidemia is a cardiovascular risk factor for coronary artery disease and atherosclerosis that is characterized by elevated serum cholesterol and lipid levels. Although high-density lipoprotein-associated cholesterol (HDL-C) is associated with reduced risk of cardiovascular events, targeted therapy to increase HDL-C levels have been unsuccessful in altering outcomes of associated atherosclerotic disease. Single nucleotide polymorphisms in SCARB1 , the gene that encodes HDL receptor Scavenger Receptor B1 (SR-BI), are associated with dyslipidemia and atherosclerotic cardiovascular disease. We were the first to identify inherited mutations in SCARB1 that segregate with disease in a family with severe coronary artery disease and dyslipidemia, including elevated HDL. Our findings suggest that HDL function (vs. HDL-C concentration) may be a promising target for cholesterol-based therapy. Here, we performed an unbiased high throughput drug screen with 788 FDA-approved compounds, using HepG2 cells to measure endogenous HDL binding. We identified five compounds that significantly increased endogenous HDL binding: imatinib, trimethoprim, eszopiclone, clemastine, and mepenzolate, of which, imatinib was the only compound to increase SR-BI expression. Imatinib is a tyrosine kinase inhibitor that is a chemotherapeutic agent designed to treat individuals with chronic myeloid leukemia. Limited clinical evidence suggests a reduction in total cholesterol with 400mg/day imatinib. Additionally, imatinib treatment (150mg/kg) in mouse models of atherosclerosis reduces total cholesterol. Yet, no data is available on the effects of imatinib on HDL and reverse cholesterol transport. We have found that imatinib promotes HDL binding and SR-BI expression in vitro . Furthermore, in wildtype C57Bl/6 mice on a high fat, high cholesterol diet, imatinib treatment (50mg/kg) was sufficient to decrease plasma total cholesterol, HDL-C and triglyceride levels and elevate hepatic SR-BI expression. In summary, our data supports the exploration of imatinib-mediated SR-BI regulation, HDL metabolism, and RCT pathway to identify new therapeutic targets for dyslipidemia.

Heritable thoracic aortic disease and familial thoracic aortic aneurysm/dissection are important causes of human morbidity/mortality, most without identifiable genetic cause. In a family with familial thoracic aortic aneurysm/dissection, we identified a missense p. (Ser178Arg) variant in PLOD1 segregating with disease, and evaluated PLOD1 enzymatic activity, collagen characteristics and in human aortic vascular smooth muscle cells, studied the effect on function. Comparison with homologous PLOD3 enzyme indicated that the pathogenic variant may affect the N-terminal glycosyltransferase domain, suggesting unprecedented PLOD1 activity. In vitro assays demonstrated that wild-type PLOD1 is capable of processing UDP-glycan donor substrates, and that the variant affects the folding stability of the glycosyltransferase domain and associated enzymatic functions. The PLOD1 substrate lysine was elevated in the proband, however the enzymatic product hydroxylysine and total collagen content was not different, albeit despite collagen fibril narrowing and preservation of collagen turnover. In VSMCs overexpressing wild-type PLOD1, there was upregulation in procollagen gene expression (secretory function) which was attenuated in the variant, consistent with loss-of-function. In comparison, si-PLOD1 cells demonstrated hypercontractility and upregulation of contractile markers, providing evidence for phenotypic switching. Together, the findings suggest that the PLOD1 product is preserved, however newly identified glucosyltransferase activity of PLOD1 appears to be affected by folding stability of the variant, and is associated with compensatory vascular smooth muscle cells phenotypic switching to support collagen production, albeit with less robust fibril girth. Future studies should focus on the impact of PLOD1 folding/variant stability on the tertiary structure of collagen and ECM interactions.