Whole exome sequencing to identify a novel gene (caveolin-1) associated with human pulmonary arterial hypertension

Abstract

Background: Heritable and idiopathic pulmonary arterial hypertension (PAH) are phenotypically identical and associated with mutations in several genes related to transforming growth factor (TGF) beta signaling, including bone morphogenetic protein receptor type 2, activin receptor-like kinase 1, endoglin, and mothers against decapentaplegic 9. Approximately 25% of heritable cases lack identifiable mutations in any of these genes.

Methods and results: We used whole exome sequencing to study a 3-generation family with multiple affected family members with PAH, but no identifiable TGF beta mutation. We identified a frameshift mutation in caveolin-1 (CAV1), which encodes a membrane protein of caveolae abundant in the endothelium and other cells of the lung. An independent de novo frameshift mutation was identified in a child with idiopathic PAH. Western blot analysis demonstrated a reduction in caveolin-1 protein, while lung tissue immunostaining studies demonstrated a reduction in normal caveolin-1 density within the endothelial cell layer of small arteries.

Conclusions: Our study represents successful elucidation of a dominant Mendelian disorder using whole exome sequencing. Mutations in CAV1 are associated in rare cases with PAH. This may have important implications for pulmonary vascular biology, as well as PAH-directed therapeutic development.

Figure 1

(A) Pedigree of family with pulmonary arterial hypertension. c.474delA, P158PfsX22 was identified in nine family members. In the four-generation family, 6 members were clinically affected (filled squares or circles). Subjects’ current ages and ages at diagnosis are labeled under each family member tested for the CAV1 mutation. CAV1 genotyping results are represented for PAH patients in this family; however, CAV1 genotyping results for healthy subjects are not displayed out of respect for the family represented. : male; : female; : affected male; : affected female; : affected female deceased. + indicates heterozygous for the c.474delA CAV1 mutation and − indicates normal CAV1 sequence. (B) Sequencing results of the CAV1 mutations. c.474delA was identified in a family with pulmonary arterial hypertension (label: II-4). c.463G>A V155I and c473delC, P158HfsX22 were identified in one patient previously diagnosed as IPAH (label: IPAH929).

Figure 1

(A) Pedigree of family with pulmonary arterial hypertension. c.474delA, P158PfsX22 was identified in nine family members. In the four-generation family, 6 members were clinically affected (filled squares or circles). Subjects’ current ages and ages at diagnosis are labeled under each family member tested for the CAV1 mutation. CAV1 genotyping results are represented for PAH patients in this family; however, CAV1 genotyping results for healthy subjects are not displayed out of respect for the family represented. : male; : female; : affected male; : affected female; : affected female deceased. + indicates heterozygous for the c.474delA CAV1 mutation and − indicates normal CAV1 sequence. (B) Sequencing results of the CAV1 mutations. c.474delA was identified in a family with pulmonary arterial hypertension (label: II-4). c.463G>A V155I and c473delC, P158HfsX22 were identified in one patient previously diagnosed as IPAH (label: IPAH929).

Figure 2

Reduced caveolin-1 expression by cells derived from patients with CAV1-PAH. Caveolin-1 expression by CAV1-PAH fibroblasts in culture compared to healthy wild-type controls. While present, CAV1-PAH cells exhibited reduced expression of Caveolin-1 compared to healthy wild type control fibroblasts cultured under identical conditions. β-actin was used as loading control. The highest density protein band (Lane 5) was considered 1.0 U, and the other lanes were normalized to this value.

Figure 3

Lung biopsy from the patient previously diagnosed with IPAH. Medial thickening was in pulmonary arteries, most recognizable in small peripheral pulmonary artery branches (Panel A). Medial thickening was confirmed by immunohistochemistry for alpha smooth muscle actin (Panel B). Alveoli did not show hypercellularity or fibrosis (Panel C), and increase in endothelial cell number was not observed on CD31 immunohistochemistry (Panel D) (Hematoxylin and eosin, Original magnification, (A) × 100 and (C) × 150. DAB chromogen, Original magnification (B) × 100 and (D) × 150).

Figure 4

Reduced caveolin-1 expression by lung small artery endothelial cells in CAV1-PAH. Caveolin-1 expression by CAV1-PAH lung endothelial cells was reduced along the plasma membrane of the vessel lumen compared to wild-type healthy control lung small arteries. Representative micrographs of immunostaining of lung sections with anti-caveolin-1 (green). Nuclei were counterstained with DAPI (blue). 100× magnification. Arrows represent the arterial lumen membrane of the endothelial cells.

Figure 5

Conservation of amino acids of caveolin-1 and mutations identified in PAH patients. (A) Conservation of amino acids of CAV1 among human, mouse, chicken, xenopus and zebrafish are indicated. Variant identified is highlighted at amino acid 155 and mutated region is highlighted starting at amino acid 158. (B) Comparison of normal and mutated amino acids sequences as predicted by open reading frame finder are shown in Panel B. Mutation is highlighted.