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Background: Up to 25% of idiopathic and > 70% of cases with familial pulmonary arterial hypertension (PAH) can be explained by rare deleterious variants in established PAH risk genes. We hypothesised that integrating deep phenotyping with whole-genome sequencing (WGS) data will reveal additional disease variants that are ultra-rare and/or have a unique phenotypic signature. Methods: We analysed WGS data from 1,148 PAH patients and 11,889 controls enrolled in the NIHR BioResource-Rare Diseases study. We used a Bayesian model comparison method (BeviMed) to test for genotype-phenotype associations. Case-control labels were defined by diagnostic groupings and KCO and age strata. Competing models were fitted to identify associations between labels and rare variants under the dominant and recessive mode of inheritance and different variant consequence types. Results: We discovered a strong association between the protein-truncating variants (PTV) in KDR, which encodes VEGFR2, and PAH with low KCO (posterior probability (PP)=0.99) and older age at diagnosis (PP=0.91). Lung CT scans of patients harbouring PTV in KDR revealed a range of mild parenchymal abnormalities. One KDR subject had a family history of PAH. KCO stratification also highlighted an association between IDH3G and moderately reduced KCO in patients with PAH (PP=0.92). The US PAH Biobank was used to validate these findings and identified 4 additional PAH cases with PTVs in KDR and 3 in IDH3G. Conclusions: The smart study design allowed the discovery of new PAH risk, which further implicate central roles for the endothelium and alterations in mitochondrial respiration in PAH

Endostatin (ES) is an angiostatic peptide encoded by Col18a1. Our group has shown that a single nucleotide polymorphism (SNP) resulting in a missense mutation in Col18a1 is associated with altered disease severity and survival in PAH. We hypothesized that associations would exist between circulating serum ES levels and 1) clinical metrics of PAH severity, including survival and 2) SNPs in Col18a1. Serum samples and clinical data were obtained from 2,017 adult subjects in the PAH Biobank. Serum ES was measured with an electrochemiluminescent immunosorbent assay. Statistical associations between ES and clinical variables were examined with regression models. Genotyping was performed for protein quantitative trait loci (pQTL) analysis. Each log-unit increase in ES was associated with higher right atrial pressure (1.8 mmHg, 95% CI 1.3-2.3), higher mean pulmonary arterial pressure (2.0 mmHg, 95% CI 0.7 to 3.2), higher pulmonary vascular resistance (1.0 Wood unit, 95% CI 0.4 to 1.5), and lower 6 minute walk distance (-53.5m, 95 % CI -70.7 to -36.2). Mortality doubled for each log-unit increase in ES (2.3, 95% CI 1.6 to 3.4). In pQTL analysis, 4 SNPs in Col18a1 were associated with differences in circulating ES levels (Figure). Increased ES levels are associated with disease severity and survival in PAH, and several SNPs in Col18a1 are associated with differences in circulating ES levels. ES is a genetically-influenced determinant of disease severity in PAH.