Background: Pulmonary arterial hypertension (PAH) is a rare, progressive disease of pulmonary arterioles, ultimately leading to right heart failure. While most disease-causing variants are found in the bone morphogenetic protein receptor type 2 (BMPR2) gene, there are still heritable PAH families without a known genetic cause.

Objective: Identifying novel PAH genes and determining their role in disease development.

Method: Using a PAH-specific gene panel, we sequenced 325 PAH patients and classified variants by ACMG guidelines. Variants and wildtype were transiently overexpressed in pulmonary artery smooth muscle cells (PASMCs) and proliferation was assessed by BrdU assay. A transient gene knockdown by silencing RNA and for BMPR2 pathway inhibition by ALK1/2/3/6 inhibitor LDN-193189 was performed.

Results: We identified 2 missense variants in the SMAD Family Member 5 (SMAD5) gene: c.277T>A p.(Trp93Arg) in a hereditary PAH, and c.1175T>C p.(Leu392Pro) in a congenital heart disease associated PAH patient. The variants were classified as variants of uncertain significance but functional evidence could suggest pathogenicity. They had opposite effects on cell proliferation compared to wildtype: c.1175T>C increased and c.277T>A decreased it. In addition, SMAD5 silencing and BMPR2 pathway inhibition resulted in a lower proliferation rate than control.

Conclusions: Our results show that the c.277T>A variant in SMAD5 may be a loss-of-function variant, as both the variant and the knockdown showed decreased proliferation. In contrast, the c.1175T>C variant increases proliferation and could be classified as a gain-of-function variant. However, further experiments are required to confirm this hypothesis.