Genetically proxied HTRA1 protease activity and circulating levels independently predict risk of ischemic stroke and coronary artery disease

Abstract

HTRA1 has emerged as a major risk gene for stroke and cerebral small vessel disease with both rare and common variants contributing to disease risk. However, the precise mechanisms mediating this risk remain largely unknown as does the full spectrum of phenotypes associated with genetic variation in HTRA1 in the general population. Using a family-history informed approach, we first show that rare variants in HTRA1 are linked to ischemic stroke in 425,338 European individuals from the UK Biobank with replication in 143,149 individuals from the Biobank Japan. Integrating data from biochemical experiments on 76 mutations occurring in the UK Biobank, we next show that rare variants causing loss of protease function in vitro associate with ischemic stroke, coronary artery disease, and skeletal traits. In addition, a common causal variant (rs2672592) modulating circulating HTRA1 mRNA and protein levels enhances the risk of ischemic stroke, small vessel stroke, and coronary artery disease while lowering the risk of migraine and age-related macular dystrophy in GWAS and UK Biobank data from > 2,000,000 individuals. There was no evidence of an interaction between genetically proxied HTRA1 activity and levels. Our findings demonstrate a central role of HTRA1 for human disease including stroke and coronary artery disease and identify two independent mechanisms that might qualify as targets for future therapeutic interventions.

Figure 1. Analytic workflow of human phenotypes linked to rare and common HTRA1 variants.

(1) 78 rare missense variants in the HTRA1 protease domain were selected for biochemical analysis. Variants targeting residues conserved in human HTRA1–4 were prioritized. Amino acids strictly conserved in 3 or 4 human HTRAs are highlighted in bright or dark red, respectively; asterisks label the 119 missense protease domain variants identified in the UKB; red asterisks mark the 78 selected variants. Protease activity was measured in secretomes from HEK-293E cells transfected to overexpress wt or mutant HTRA1. (2) Common variant assessment included GWAS, eQTL, pQTL and colocalization analyzes. (3) Rare and common HTRA1 variants were investigated for their associations with human disease through a PheWAS framework. (4) The interaction of rare and common HTRA1 variants on stroke and coronary artery disease (CAD) risk was determined. HTRA1: High-Temperature Requirement A serine peptidase 1; IGFBP7: Insulin-like Growth Factor-Binding Protein 7; PDZ: Post synaptic density protein (PSD95), Drosophila disc large tumor suppressor (DlgA), and Zonula occludens-1 protein (zo-1, PheWAS: Phenome-Wide Association Study; eQTL: expression Quantitative Trait Loci; pQTL: protein Quantitative Trait Loci.

Figure 2. Consequences of rare missense protease domain variants on enzymatic activity.

Upper panel: HTRA1 protease activity was measured using LTBP1 as a substrate and corrected for HTRA1 levels. Histogram depicts the average activity +SD measured in 5–10 experiments; circles: data points. The ratio cleaved / intact LTBP1, normalized to HTRA1 levels of wt HTRA1 (set to 1) and of the active site mutant S328A (SA) are marked by dashed lines. Variants were stratified into the following categories: residual activity < 0.25 (dark red; n=28); ≥ 0.25 and < 0.5 (light red; n=18); ≥ 0.5 and < 1.0 (dark pink; n=23), and ≥ 1.0 (light pink; n=7). Lower panels: i) minor allele counts in the UKB), ii) percentage of cardiovascular events (CVD), defined as the combination of ischemic stroke and CAD events) in the UKB, iii) mutations reported in familial and sporadic cSVD cases, and iv) mutations targeting the HTRA1 protomer-protomer interface.

Figure 3. The HTRA1 protomer-protomer interface is a hotspot for severe loss of function mutations.

Structure of the HTRA1 trimer (PDB ID 3TJO, catalytic domain of the inactive S328A mutant). a, The predicted protomer-protomer interface (PyMOL plugin ‘InterfaceResidues’) is highlighted in purple. b, Rare missense variants with experimental protease activity data are highlighted as follows: residual activity < 0.25 (dark red); ≥0.25 and < 0.5 (light red); >0.5 and < 1 (dark pink), and ≥ 1.0 (light pink). c, For all identified interface variants, we counted the percentage of mutations belonging to each protease activity category.

Figure 4. PheWAS of imputed HTRA1 protease activity reveals associations with neurovascular, skeletal and CAD-related traits.

PheWAS results using predicted HTRA1 protease activity for each individual as the dependent variable. The x-axis shows the Odds Ratio for a 10% increase in HTRA1 activity, the y-axis displays a log-transformed p-value. The FDR cutoff of 0.05 is presented as a dashed line.

Figure 5. A common variant in HTRA1 shows colocalization with five neurovascular phenotypes.

For each of the phenotypes (any ischemic stroke; coronary artery disease; small vessel stroke; lacunar stroke; migraine) the HTRA1 gene ±150kb is depicted on the x-axis. The y-axis depicts the −log10 p-value of the respective GWAS. The dashed lines depict genome-wide significance (p=5E⁻⁸). We used Hyprcoloc to compute the posterior probability of colocalization (PIP) for each SNP in the genomic interval. The highest PIP was estimated for rs2672592. For this SNP, we depict the p-value for each of the five phenotypes.

Figure 6. Genetically proxied HTRA1 activity and levels independently predict risk of stroke and coronary artery disease.

Forest plot for each of the strata defined by rs2672592 genotype (GG, TG, or TT) and presence of a rare damaging variant. Cardiovascular phenotype (CVD): pooled ischemic stroke and CAD events.