Rare variant analysis of 4241 pulmonary arterial hypertension cases from an international consortium implicates FBLN2, PDGFD, and rare de novo variants in PAH

Abstract

Background: Pulmonary arterial hypertension (PAH) is a lethal vasculopathy characterized by pathogenic remodeling of pulmonary arterioles leading to increased pulmonary pressures, right ventricular hypertrophy, and heart failure. PAH can be associated with other diseases (APAH: connective tissue diseases, congenital heart disease, and others) but often the etiology is idiopathic (IPAH). Mutations in bone morphogenetic protein receptor 2 (BMPR2) are the cause of most heritable cases but the vast majority of other cases are genetically undefined.

Methods: To identify new risk genes, we utilized an international consortium of 4241 PAH cases with exome or genome sequencing data from the National Biological Sample and Data Repository for PAH, Columbia University Irving Medical Center, and the UK NIHR BioResource - Rare Diseases Study. The strength of this combined cohort is a doubling of the number of IPAH cases compared to either national cohort alone. We identified protein-coding variants and performed rare variant association analyses in unrelated participants of European ancestry, including 1647 IPAH cases and 18,819 controls. We also analyzed de novo variants in 124 pediatric trios enriched for IPAH and APAH-CHD.

Results: Seven genes with rare deleterious variants were associated with IPAH with false discovery rate smaller than 0.1: three known genes (BMPR2, GDF2, and TBX4), two recently identified candidate genes (SOX17, KDR), and two new candidate genes (fibulin 2, FBLN2; platelet-derived growth factor D, PDGFD). The new genes were identified based solely on rare deleterious missense variants, a variant type that could not be adequately assessed in either cohort alone. The candidate genes exhibit expression patterns in lung and heart similar to that of known PAH risk genes, and most variants occur in conserved protein domains. For pediatric PAH, predicted deleterious de novo variants exhibited a significant burden compared to the background mutation rate (2.45×, p = 2.5e-5). At least eight novel pediatric candidate genes carrying de novo variants have plausible roles in lung/heart development.

Conclusions: Rare variant analysis of a large international consortium identified two new candidate genes-FBLN2 and PDGFD. The new genes have known functions in vasculogenesis and remodeling. Trio analysis predicted that ~ 15% of pediatric IPAH may be explained by de novo variants.

Keywords: Case-control association testing; De novo variant analysis; Exome sequencing; Genetics; Genome sequencing; Pulmonary arterial hypertension.

Fig. 1

Gene-based association analysis using 1647 European IPAH cases and 18,819 European controls. a Results of a binomial test confined to rare, likely gene damaging (LGD) and predicted deleterious missense (D-Mis) variants or D-Mis only variants in 20,000 protein-coding genes. The control group included 11,101 unaffected SPARK parents and 7718 NFE gnomAD v2.1.1 individuals. Horizontal gray line indicates the Bonferroni-corrected threshold for significance. b Complete list of top association genes (FDR < 0.1)

Fig. 2

Locations of PAH-associated rare variants within FBLN2 and PDGFD protein structures. a Variants and conserved domains within two-dimensional protein structures. The numbers of variants at each amino acid position is indicated along the y-axes. D-MIS, predicted deleterious missense; LGD, likely gene-disrupting (stopgain, frameshift, splicing). FBLN2: ANATO, anaphylatoxin-like 2; EGF-ca, calcium-binding endothelial growth factor-like 1; EGF, non-calcium-binding EGF domain. PDGFD: CUB, complement subcomponent; PDGF/VEGF, platelet-derived growth factor/vascular endothelial-derived growth factor domain. b FBLN2 residues 858-900: p.(Gly880Val) and p.(Gly889Asp) change the conserved i+2 glycine residues of type II reverse turns within an EGF domain. Residues 981-1011: recurrent p.(Asp982Tyr) changes a residue within the highly conserved DXXE motif/calcium-binding site within an EGF domain. c PDGFD residues 43-180: p.(Asp148Asn) predicted to destroy the Ca++ binding site of the CUB domain. Residues 264-364: p.(Arg295Cys) disrupts a hydrogen bond and p.(Ser309Cys) may create a new disulfide bond in the PDGF/VEGF domain

Fig. 3

Gene expression patterns of PAH risk genes using murine single-cell RNA-seq data. a Heat map showing fraction of cells with > 0 reads in specific cell types of lung, heart, and aorta for 11 known PAH risk genes and 3 new candidate risk genes (KDR, FBLN2, and PDGFD). L, lung; H, heart; A, aorta. b PCA analysis of gene expression for PAH risk genes and a set of 100 randomly selected genes, overlaid on a plot of all other 16,744 sequenced genes expressed in both human and mouse cells. c Histogram of PC2 values for PAH risk genes and a set of 100 randomly selected genes indicates a right shift for PC2 among PAH risk genes. d Relative rank of PC2 values for PAH risk genes among 16,744 sequenced genes expressed in both human and mouse cells.