Rare, Damaging DNA Variants in CORIN and Risk of Coronary Artery Disease: Insights From Functional Genomics and Large-Scale Sequencing Analyses.

BACKGROUND: Corin is a protease expressed in cardiomyocytes that plays a key role in salt handling and intravascular volume homeostasis via activation of natriuretic peptides. It is unknown if Corin loss-of-function (LOF) is causally associated with risk of coronary artery disease (CAD). METHODS: We analyzed all coding CORIN variants in an Italian case-control study of CAD. We functionally tested all 64 rare missense mutations in Western Blot and Mass Spectroscopy assays for proatrial natriuretic peptide cleavage. An expanded rare variant association analysis for Corin LOF mutations was conducted in whole exome sequencing data from 37 799 CAD cases and 212 184 controls. RESULTS: We observed LOF variants in CORIN in 8 of 1803 (0.4%) CAD cases versus 0 of 1725 controls (P, 0.007). Of 64 rare missense variants profiled, 21 (33%) demonstrated <30% of wild-type activity and were deemed damaging in the 2 functional assays for Corin activity. In a rare variant association study that aggregated rare LOF and functionally validated damaging missense variants from the Italian study, we observed no association with CAD-21 of 1803 CAD cases versus 12 of 1725 controls with adjusted odds ratio of 1.61 ([95% CI, 0.79-3.29]; P=0.17). In the expanded sequencing dataset, there was no relationship between rare LOF variants with CAD was also observed (odds ratio, 1.15 [95% CI, 0.89-1.49]; P=0.30). Consistent with the genetic analysis, we observed no relationship between circulating Corin concentrations with incident CAD events among 4744 participants of a prospective cohort study-sex-stratified hazard ratio per SD increment of 0.96 ([95% CI, 0.87-1.07], P=0.48). CONCLUSIONS: Functional testing of missense mutations improved the accuracy of rare variant association analysis. Despite compelling pathophysiology and a preliminary observation suggesting association, we observed no relationship between rare damaging variants in CORIN or circulating Corin concentrations with risk of CAD.

Role of angiopoietin-like 3 (ANGPTL3) in regulating plasma level of low-density lipoprotein cholesterol.

BACKGROUND AND AIMS: Angiopoietin-like 3 (ANGPTL3) has emerged as a key regulator of lipoprotein metabolism in humans. Homozygous loss of ANGPTL3 function causes familial combined hypolipidemia characterized by low plasma levels of triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C). While known effects of ANGPTL3 in inhibiting lipoprotein lipase and endothelial lipase contribute to the low TG and HDL-C, respectively, the basis of low LDL-C remains unclear. Our aim was to explore the role of ANGPTL3 in modulating plasma LDL-C. METHODS: We performed RNAi-mediated gene silencing of ANGPTL3 in five mouse models and in human hepatoma cells. We validated results by deleting ANGPTL3 gene using the CRISPR/Cas9 genome editing system. RESULTS: RNAi-mediated Angptl3 silencing in mouse livers resulted in very low TG, HDL-C and LDL-C, a pattern similar to the human phenotype. The effect was observed in wild-type and obese mice, while in hCETP/apolipoprotein (Apo) B-100 double transgenic mice, the silencing decreased LDL-C and TG, but not HDL-C. In a humanized mouse model (Apobec1-/- carrying human ApoB-100 transgene) deficient in the LDL receptor (LDLR), Angptl3 silencing had minimum effect on LDL-C, suggesting the effect being linked to LDLR. This observation is supported by an additive effect on LDL-C between ANGPTL3 and PCSK9 siRNAs. ANGPTL3 gene deletion induced cellular long-chain TG and ApoB-100 accumulation with elevated LDLR and LDLR-related protein (LRP) 1 expression. Consistent with this, ANGPTL3 deficiency by gene deletion or silencing reduced nascent ApoB-100 secretion and increased LDL/VLDL uptake. CONCLUSIONS: Reduced secretion and increased uptake of ApoB-containing lipoproteins may contribute to the low LDL-C observed in mice and humans with genetic ANGPTL3 deficiency.

Identification of new susceptibility loci for type 2 diabetes and shared etiological pathways with coronary heart disease.

To evaluate the shared genetic etiology of type 2 diabetes (T2D) and coronary heart disease (CHD), we conducted a genome-wide, multi-ancestry study of genetic variation for both diseases in up to 265,678 subjects for T2D and 260,365 subjects for CHD. We identify 16 previously unreported loci for T2D and 1 locus for CHD, including a new T2D association at a missense variant in HLA-DRB5 (odds ratio (OR) = 1.29). We show that genetically mediated increase in T2D risk also confers higher CHD risk. Joint T2D-CHD analysis identified eight variants-two of which are coding-where T2D and CHD associations appear to colocalize, including a new joint T2D-CHD association at the CCDC92 locus that also replicated for T2D. The variants associated with both outcomes implicate new pathways as well as targets of existing drugs, including icosapent ethyl and adipocyte fatty-acid-binding protein.

Human knockouts and phenotypic analysis in a cohort with a high rate of consanguinity.

A major goal of biomedicine is to understand the function of every gene in the human genome. Loss-of-function mutations can disrupt both copies of a given gene in humans and phenotypic analysis of such 'human knockouts' can provide insight into gene function. Consanguineous unions are more likely to result in offspring carrying homozygous loss-of-function mutations. In Pakistan, consanguinity rates are notably high. Here we sequence the protein-coding regions of 10,503 adult participants in the Pakistan Risk of Myocardial Infarction Study (PROMIS), designed to understand the determinants of cardiometabolic diseases in individuals from South Asia. We identified individuals carrying homozygous predicted loss-of-function (pLoF) mutations, and performed phenotypic analysis involving more than 200 biochemical and disease traits. We enumerated 49,138 rare (<1% minor allele frequency) pLoF mutations. These pLoF mutations are estimated to knock out 1,317 genes, each in at least one participant. Homozygosity for pLoF mutations at PLA2G7 was associated with absent enzymatic activity of soluble lipoprotein-associated phospholipase A2; at CYP2F1, with higher plasma interleukin-8 concentrations; at TREH, with lower concentrations of apoB-containing lipoprotein subfractions; at either A3GALT2 or NRG4, with markedly reduced plasma insulin C-peptide concentrations; and at SLC9A3R1, with mediators of calcium and phosphate signalling. Heterozygous deficiency of APOC3 has been shown to protect against coronary heart disease; we identified APOC3 homozygous pLoF carriers in our cohort. We recruited these human knockouts and challenged them with an oral fat load. Compared with family members lacking the mutation, individuals with APOC3 knocked out displayed marked blunting of the usual post-prandial rise in plasma triglycerides. Overall, these observations provide a roadmap for a 'human knockout project', a systematic effort to understand the phenotypic consequences of complete disruption of genes in humans.

High-Density Lipoprotein (HDL) Phospholipid Content and Cholesterol Efflux Capacity Are Reduced in Patients With Very High HDL Cholesterol and Coronary Disease.

OBJECTIVE: Plasma levels of high-density lipoprotein cholesterol (HDL-C) are strongly inversely associated with coronary artery disease (CAD), and high HDL-C is generally associated with reduced risk of CAD. Extremely high HDL-C with CAD is an unusual phenotype, and we hypothesized that the HDL in such individuals may have an altered composition and reduced function when compared with controls with similarly high HDL-C and no CAD. APPROACH AND RESULTS: Fifty-five subjects with very high HDL-C (mean, 86 mg/dL) and onset of CAD at the age of ≈ 60 years with no known risk factors for CAD (cases) were identified through systematic recruitment. A total of 120 control subjects without CAD, matched for race, sex, and HDL-C level (controls), were identified. In all subjects, HDL composition was analyzed and HDL cholesterol efflux capacity was assessed. HDL phospholipid composition was significantly lower in cases (92 ± 37 mg/dL) than in controls (109 ± 43 mg/dL; P=0.0095). HDL cholesterol efflux capacity was significantly lower in cases (1.96 ± 0.39) than in controls (2.11 ± 0.43; P=0.04). CONCLUSIONS: In people with very high HDL-C, reduced HDL phospholipid content and cholesterol efflux capacity are associated with the paradoxical development of CAD.