Rare Variants in HTRA1, SGTB, and RBM12 Confer Risk of Atherosclerotic Cardiovascular Disease Independent of Traditional Cardiovascular Risk Factors

Abstract

Background: Atherosclerosis is a pathophysiological process common to a range of cardiovascular diseases. We reasoned that considering clinical presentations of atherosclerosis across the coronary, peripheral, and cerebrovasculature as a single entity would enhance statistical power to identify rare genetic variation driving pathological processes across multiple vascular beds.

Methods: We performed an exome-wide association study of atherosclerotic cardiovascular disease in 434 438 UK Biobank participants of European ancestry.

Results: We identified rare, predicted damaging variants in HTRA1, SGTB, and RBM12 to be associated with risk of atherosclerotic cardiovascular disease, independent of known risk factors. Both SGTB and HTRA1 were downregulated in the aorta of patients with coronary artery disease compared with controls. Loss-of-function variants in the RNA-binding protein RBM12 increased the risk of coronary, cerebrovascular, and peripheral vascular diseases to a similar extent. SGTB increased the risk of atherosclerotic cardiovascular disease in the coronary and peripheral circulations but not the cerebrovasculature. While loss-of-function variants in HTRA1 are known to cause monogenic stroke syndromes, we found that damaging missense variants in HTRA1 are associated with increased risk of disease in both the cerebrovascular and coronary circulation. Surprisingly, the increased risk of coronary artery disease was driven predominantly by a single missense variant (p.R227W; minor allele frequency, 0.009). In vitro, the R227W mutant HTRA1 efficiently proteolyzed the disordered substrate casein but not aggregated α-synuclein. In contrast, a stroke risk-raising variant (D320N) could not efficiently process any of the tested substrates.

Conclusions: We identified novel genetic variants predisposing to atherosclerotic cardiovascular diseases that act independently of established cardiovascular risk factors. The observed phenotypic and functional heterogeneities between HTRA1 variants suggest that distinct biochemical mechanisms drive HTRA1-related vascular disease in the brain and heart.

Keywords: atherosclerosis; cardiovascular diseases; myocardial ischemia; stroke.

Figure 1.

An exome-wide association study of atherosclerotic cardiovascular disease in UK Biobank (UKBB) participants of European ancestry. Manhattan plot summarizing an exome-wide association study of atherosclerotic cardiovascular disease in UKBB. Each dot represents an individual gene×mask combination. The dotted line represents a Bonferroni-corrected multiple testing threshold of 1.33×10-6. The labels represent gene names where any single gene×mask combination is significantly associated with atherosclerotic cardiovascular disease. Test statistics were derived from a linear mixed model of European participants in UKBB (implemented in BOLT-LMM, Bayesian, Orthogonal, at Large-scale, Linear Mixed Model) with adjustment for age, age-squared, sex, the first 10 genetic principal components, and whole exome–sequencing batch.

Figure 2.

Atherosclerotic cardiovascular disease risk genes increase disease risk via actions in multiple vascular beds. To provide insight into the tissue of action of the candidate genes, we partitioned our atherosclerotic cardiovascular disease phenotype into vascular beds and assessed the effect of the most strongly associated mask for each of the 3 exome-wide significant atherosclerotic cardiovascular risk genes. The test statistics plotted are from a generalized linear model after adjustment for the first 10 genetic principal components, age, age-squared, and sex. P values from collapsing burden tests implemented in BOLT-LMM (Bayesian, Orthogonal, at Large-scale, Linear Mixed Model) are available in Table S5. LDLR indicates low-density lipoprotein receptor; OR, odds ratio; and SGTB, small glutamine-rich tetratricopeptide repeat co-chaperone beta.

Figure 3.

Effects of pLOF variants in atherosclerotic cardiovascular disease risk genes on selected cardiovascular risk factors. The effects of the atherosclerotic risk genes on continuous (top) and dichotomized cardiovascular risk factors (bottom) are shown. For each gene, the variant mask most significantly associated with atherosclerotic cardiovascular disease is plotted. The points coloured red are significantly associated with the indicated risk factor after adjustment for multiple testing. ApoB indicates apolipoprotein-B; BMI, body mass index; OR, odds ratio; SBP, systolic blood pressure; CRP, c-reactive protein; DBP, diastolic blood pressure; HbA1c, Haemoglobin A1c; eGFR, estimated glomerular filtration rate; WHR, waist-hip ratio; HDL, High density lipoprotein; Lp(a), Lipoprotein(a); LDL, low density lipoprotein; pLOF, predicted loss of function.

Figure 4.

p.R227W increases coronary artery disease risk but not risk of cerebrovascular disease. Lolliplots of variants in HTRA1 and their effect on coronary artery disease (A) and cerebrovascular disease risk (B). The size of each dot is proportional to the number of carriers. The red dot represents p.R227W, which has differential effects on cerebrovascular and coronary artery disease. Test statistics plotted were derived from a linear mixed model implemented in BOLT. C through F, Effect of mask composition on coronary artery disease, cerebrovascular disease, acute myocardial infarction, and ischemic stroke, specifically focused on the differential effects of R227W inclusion/exclusion. G, The effect of the R227W variant on vascular disease, where controls are restricted to predicted damaging missense variants in HTRA1 (minor allele frequency [MAF] <0.0001; Rare Exome Variant Ensemble Learner [REVEL] >0.5). Test statistics plotted and P values in C through G were derived from a generalized linear model. MI indicates myocardial infarction; and OR, odds ratio.

Figure 5.

Substrate-specific impairment of proteolysis by R227W HTRA1. α-Synuclein sedimentation (A) and inhibition assays (B). α-Synuclein monomer (25 μM) was incubated in buffer, with and without the indicated HTRA variants (5 μM) for 72 hours at 37 °C with agitation of 1500 rpm in a thermomixer. For sedimentation assays, samples were collected at 72 hours and centrifuged to separate soluble (S) and pellet (P) fractions and analyzed by SDS-PAGE. For inhibition assays, samples were mixed with thioflavin T (ThT) dye to quantify α-synuclein aggregation. Fluorescence at 482 nm was measured after excitation at 450 nm using a Tecan Spark plate reader. C, Illustration of CryoEM structure of HTRA1 bound to inhibitory fragment antibody (7SJO, only 1 monomer is shown) R227 binds to Fab and cannot interact with E198 and E239. D, Ability of HTRA1 mutants to cleave fluorescein isothiocyanate (FITC)–labeled casein, with fluorescence indicative of casein proteolysis. Proteolysis assays of casein 40 μM (E) or α-synuclein (25 μM; F) were incubated with HTRA1 mutants for 24 hours at 37 °C. Samples were collected at 0 and 24 hours and then processed by SDS-PAGE. In B, the average and SEM of 3 independent experiments are plotted. In A and D through F, representative results of 3 independent experiments are displayed.