Clinical and molecular genetic features of pulmonary hypertension in patients with hereditary hemorrhagic telangiectasia

BACKGROUND: Most patients with familial primary pulmonary hypertension have defects in the gene for bone morphogenetic protein receptor II (BMPR2), a member of the transforming growth factor beta (TGF-beta) superfamily of receptors. Because patients with hereditary hemorrhagic telangiectasia may have lung disease that is indistinguishable from primary pulmonary hypertension, we investigated the genetic basis of lung disease in these patients. METHODS: We evaluated members of five kindreds plus one individual patient with hereditary hemorrhagic telangiectasia and identified 10 cases of pulmonary hypertension. In the two largest families, we used microsatellite markers to test for linkage to genes encoding TGF-beta-receptor proteins, including endoglin and activin-receptor-like kinase 1 (ALK1), and BMPR2. In subjects with hereditary hemorrhagic telangiectasia and pulmonary hypertension, we also scanned ALK1 and BMPR2 for mutations. RESULTS: We identified suggestive linkage of pulmonary hypertension with hereditary hemorrhagic telangiectasia on chromosome 12q13, a region that includes ALK1. We identified amino acid changes in activin-receptor-like kinase 1 that were inherited in subjects who had a disorder with clinical and histologic features indistinguishable from those of primary pulmonary hypertension. Immunohistochemical analysis in four subjects and one control showed pulmonary vascular endothelial expression of activin-receptor-like kinase 1 in normal and diseased pulmonary arteries. CONCLUSIONS: Pulmonary hypertension in association with hereditary hemorrhagic telangiectasia can involve mutations in ALK1. These mutations are associated with diverse effects, including the vascular dilatation characteristic of hereditary hemorrhagic telangiectasia and the occlusion of small pulmonary arteries that is typical of primary pulmonary hypertension

Chronic Allergic Inflammation Causes Vascular Remodeling and Pulmonary Hypertension in Bmpr2 Hypomorph and Wild-Type Mice

Loss-of-function mutations in the bone morphogenetic protein receptor type 2 (BMPR2) gene have been identified in patients with heritable pulmonary arterial hypertension (PAH); however, disease penetrance is low, suggesting additional factors play a role. Inflammation is associated with PAH and vascular remodeling, but whether allergic inflammation triggers vascular remodeling in individuals with BMPR2 mutations is unknown. Our goal was to determine if chronic allergic inflammation would induce more severe vascular remodeling and PAH in mice with reduced BMPR-II signaling. Groups of Bmpr2 hypomorph and wild-type (WT) Balb/c/Byj mice were exposed to house dust mite (HDM) allergen, intranasally for 7 or 20 weeks to generate a model of chronic inflammation. HDM exposure induced similar inflammatory cell counts in all groups compared to controls. Muscularization of pulmonary arterioles and arterial wall thickness were increased after 7 weeks HDM, more severe at 20 weeks, but similar in both groups. Right ventricular systolic pressure (RVSP) was measured by direct cardiac catheterization to assess PAH. RVSP was similarly increased in both HDM exposed groups after 20 weeks compared to controls, but not after 7 weeks. Airway hyperreactivity (AHR) to methacholine was also assessed and interestingly, at 20 weeks, was more severe in HDM exposed Bmpr2 hypomorph mice versus WT. We conclude that chronic allergic inflammation caused PAH and while the severity was mild and similar between WT and Bmpr2 hypomorph mice, AHR was enhanced with reduced BMPR-II signaling. These data suggest that vascular remodeling and PAH resulting from chronic allergic inflammation occurs independently of BMPR-II pathway alterations

Integrative Multiomics to Dissect the Lung Transcriptional Landscape of Pulmonary Arterial Hypertension

Pulmonary arterial hypertension (PAH) remains an incurable and often fatal disease despite currently available therapies. Multiomics systems biology analysis can shed new light on PAH pathobiology and inform translational research efforts. Using RNA sequencing on the largest PAH lung biobank to date (96 disease and 52 control), we aim to identify gene co-expression network modules associated with PAH and potential therapeutic targets. Co-expression network analysis was performed to identify modules of co-expressed genes which were then assessed for and prioritized by importance in PAH, regulatory role, and therapeutic potential via integration with clinicopathologic data, human genome-wide association studies (GWAS) of PAH, lung Bayesian regulatory networks, single-cell RNA-sequencing data, and pharmacotranscriptomic profiles. We identified a co-expression module of 266 genes, called the pink module, which may be a response to the underlying disease process to counteract disease progression in PAH. This module was associated not only with PAH severity such as increased PVR and intimal thickness, but also with compensated PAH such as lower number of hospitalizations, WHO functional class and NT-proBNP. GWAS integration demonstrated the pink module is enriched for PAH-associated genetic variation in multiple cohorts. Regulatory network analysis revealed that BMPR2 regulates the main target of FDA-approved riociguat, GUCY1A2, in the pink module. Analysis of pathway enrichment and pink hub genes (i.e. ANTXR1 and SFRP4) suggests the pink module inhibits Wnt signaling and epithelial-mesenchymal transition. Cell type deconvolution showed the pink module correlates with higher vascular cell fractions (i.e. myofibroblasts). A pharmacotranscriptomic screen discovered ubiquitin-specific peptidases (USPs) as potential therapeutic targets to mimic the pink module signature. Our multiomics integrative study uncovered a novel gene subnetwork associated with clinicopathologic severity, genetic risk, specific vascular cell types, and new therapeutic targets in PAH. Future studies are warranted to investigate the role and therapeutic potential of the pink module and targeting USPs in PAH

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

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

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

Muscularization of small pulmonary arteries.

<p><b>A</b>: Representative images of α-SMA immunostaining of non-muscular (NM), partially muscularized (PM) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032468#pone.0032468-McCormack1" target="_blank">[53]</a>, and fully muscularized (FM) small arterioles used to determine percent muscularization of vessels after HDM exposure (40×). <b>B</b>: WT and Bmpr2 ΔE2 (ΔE2) mice demonstrated increases in the number of arterioles that were fully muscularized (FM) and fewer arterioles that were non-muscular (NM) compared to saline controls after 7 weeks (wk) HDM (left). The percentage of FM arterioles was further enhanced after 20 weeks of HDM and the proportion of NM arterioles was further reduced. No differences were detected between groups of WT and Bmpr2 ΔE2 mice exposed to saline or HDM (n = 5–13 mice/group). *P<0.05 vs saline control, ∧P<0.05 vs ΔE2 saline PM.</p

Inflammatory cell response in HDM exposed mice.

<p><b>A</b>: Total inflammatory cell counts were increased in BALF in both WT (white bars) and Bmpr2 ΔE2 (ΔE2; black bars) mice after 7 weeks (wk) and 20 weeks of HDM (n = 4–13 mice/group in two independent experiments). *P<0.05 vs saline control, ∧P<0.05 vs saline WT 20 weeks. <b>B</b>: Representative images of cytospins from BALF demonstrating specific inflammatory cell types in the lung after 7 and 20 weeks of saline or HDM exposure. The inset in the top panel shows a high power image of an eosinophil and the bottom panel inset is a representative image of a neutrophil. M = Macrophage, L = Lymphocyte, N = Neutrophil, and E = Eosinophil. Scale bar larger panels = 20 µm. Insets scale bar = 10 µm. <b>C</b>: Changes in macrophage cell numbers were only observed after 20 weeks of HDM and only in BMPR2 ΔE2 mice. Neutrophils and eosinophil numbers were increased in HDM exposed groups at both time points, however, the number of eosinophils was lower after 20 weeks. *P<0.05 vs saline control, ∧P<0.05 vs saline ΔE2 lymphocyte 20 weeks, #P<0.05 vs saline WT macrophage 20 weeks, +P<0.05 vs HDM WT macrophages 20 weeks, @P<0.05 vs HDM WT neutrophil 20 weeks.</p

Allergic sensitization in HDM exposed mice.

<p>Levels of HDM specific IgG1 and IgE were increased in both WT and Bmpr2 ΔE2 mice after 7 weeks (top panels) and 20 weeks (bottom panels) of HDM exposure. At 7 weeks, HDM specific IgG1 levels in WT mice were slightly higher compared to Bmpr2 ΔE2 mice (n = 4–12 mice/group). *P<0.05 vs saline control, ∧P<0.05 vs HDM WT 7 weeks.</p

Genotype analysis of Bmpr2 hypomorph allele and levels of P-Smad1/5 in HDM exposed mice.

<p><b>A</b>: Tail DNA from mice carrying the wild-type (WT) and/or the Bmpr2 hypomorph (ΔE2) alleles were genotyped by PCR using the primers Bmpr2 MT Neo R1 to detect the hypomorph allele (ΔE2 450 bp) and Bmpr2 WT R2 to detect the WT allele (WT 230 bp). PCR products were run on a 4% agarose gel containing ethidium bromide and photographed. <b>B</b>: Western blot analysis of P-Smad1/5, a downstream mediator of BMPR-II signaling, was decreased by approximately 50% after HDM exposure compared to saline controls (<i>n</i> = 5 mice/group). *P<0.05 vs saline.</p