Critical Genomic Networks and Vasoreactive Variants in Idiopathic Pulmonary Arterial Hypertension

Abstract

Rationale: Idiopathic pulmonary arterial hypertension (IPAH) is usually without an identified genetic cause, despite clinical and molecular similarity to bone morphogenetic protein receptor type 2 mutation-associated heritable pulmonary arterial hypertension (PAH). There is phenotypic heterogeneity in IPAH, with a minority of patients showing long-term improvement with calcium channel-blocker therapy.

Objectives: We sought to identify gene variants (GVs) underlying IPAH and determine whether GVs differ in vasodilator-responsive IPAH (VR-PAH) versus vasodilator-nonresponsive IPAH (VN-PAH).

Methods: We performed whole-exome sequencing (WES) on 36 patients with IPAH: 17 with VR-PAH and 19 with VN-PAH. Wnt pathway differences were explored in human lung fibroblasts.

Measurements and main results: We identified 1,369 genes with 1,580 variants unique to IPAH. We used a gene ontology approach to analyze variants and identified overrepresentation of several pathways, including cytoskeletal function and ion binding. By mapping WES data to prior genome-wide association study data, Wnt pathway genes were highlighted. Using the connectivity map to define genetic differences between VR-PAH and VN-PAH, we found enrichment in vascular smooth muscle cell contraction pathways and greater genetic variation in VR-PAH versus VN-PAH. Using human lung fibroblasts, we found increased stimulated Wnt activity in IPAH versus controls.

Conclusions: A pathway-based analysis of WES data in IPAH demonstrated multiple rare GVs that converge on key biological pathways, such as cytoskeletal function and Wnt signaling pathway. Vascular smooth muscle contraction-related genes were enriched in VR-PAH, suggesting a potentially different genetic predisposition for VR-PAH. This pathway-based approach may be applied to next-generation sequencing data in other diseases to uncover the contribution of unexpected or multiple GVs to a phenotype.

Keywords: pulmonary arterial hypertension; vasodilator responsive; whole-exome sequencing.

Figure 1.

Filtering strategy to identify rare genetic variants in whole-exome sequencing data. A total of 11,981 variants were filtered to 1,580 gene variants. CIDR = Center for Integrated Disease Research; IPF = idiopathic pulmonary fibrosis; PolyPhen2 = Polymorphism Phenotyping version 2; SIFT = Sorting Intolerant from Tolerant.

Figure 2.

Circos plot to facilitate the identification and analysis of similarities and differences arising from comparisons of the 36 idiopathic pulmonary arterial hypertension exomes. Variation in genome structure between chromosomes (1–22, X, and Y) is demonstrated in this plot, which allows for demonstration of the frequency of variations according to phenotype. From outside to inside, the outer rim demonstrates the frequency of variants in the vasodilator-nonresponsive pulmonary arterial hypertension (VN-PAH) group according to chromosomal location, and the second-most outer rim demonstrates the frequency of variants in the vasodilator-responsive pulmonary arterial hypertension (VR-PAH) group according to chromosomal location. Next the frequency of genome-wide association study (GWAS) common variations (single-nucleotide polymorphisms) are sandwiched between the abundance of novel VN-PAH variants (outer faint orange circle) and the abundance of novel VR-PAH variants (inner faint orange circle). Relevant identified genes are shown in the innermost circle (see Figure 3).

Figure 3.

Region of interest on chromosome 5. Magnification of a region of chromosome 5 on the Circos plot in Figure 2 for closer evaluation of a portion with a high degree of intersection between our whole-exome sequencing data and the published genome-wide association study (GWAS) dataset. This region included a gene cluster with nine protocadherin genes related to the Wnt signaling pathway. PAH = pulmonary arterial hypertension; VN-PAH = vasodilator-nonresponsive pulmonary arterial hypertension; VR-PAH = vasodilator-responsive pulmonary arterial hypertension.

Figure 4.

Connectivity map (cMAP) analysis was used to define expression arrays associated with vasodilator drugs and prioritize genetic variants associated with vasodilator-nonresponsive pulmonary arterial hypertension (VN-PAH) and vasodilator-responsive pulmonary arterial hypertension (VR-PAH). (A) The areas of intersection are shown on this Venn diagram. There was overrepresentation of vascular smooth muscle genes in the VR-PAH cohort using this analysis (P < 0.05). (B) Heat map showing frequency of variants in specific pathway and number of affected pathways per patient. Rows represent individual patients and columns are affected pathways. Color key represents number of affected genes. down exp = decreased expression; IPAH = idiopathic pulmonary arterial hypertension; up exp = increased expression.

Figure 5.

Central mutation map for idiopathic pulmonary arterial hypertension (IPAH). We performed a network analysis focusing on key pathways or gene ontology groups and high-variant gene clusters. The central mutation map for IPAH shows 75 nodes and 107 total links. Implicated linker genes were not identified in the variant analysis but are required to connect other identified gene variants in this analysis. At a minimum, four steps linked the candidate pathways of Wnt and transforming growth factor (TGF)-β; starting from WIF1 to CDC25C to SRC to PLA2G4A to TGF-β₁.

Figure 6.

Analysis of Wnt pathway in human lung fibroblasts. Lung fibroblasts were isolated from human patients with idiopathic pulmonary arterial hypertension and healthy, failed donor control subjects and used for these studies (n = 3–4 each). (A) Polymerase chain reaction of cell homogenates for SFRP1, SFRP2 (Wnt antagonists), and AXIN2 (canonical Wnt signaling) show increased expression of Wnt antagonists and the Wnt target gene, WISP1. (B) Western analysis for Wisp1 demonstrated a trend to increased Wisp1. (C) Wnt pathway activity was assessed using a dual luciferase assay at baseline and after 72-hour stimulation with Wnt3a and LiCl. Idiopathic pulmonary arterial hypertension cells demonstrated significantly greater stimulation of Wnt activity after LiCl stimulation. *P < 0.05. HPRT = hypoxanthine phosphoribosyltransferase; LiCl = lithium chloride; PAH = pulmonary arterial hypertension.