1Thoraxklinik - Heidelberg gGmbH Pneumologie und Beatmungsmedizin / Zentrum für Pulmonale Hypertonie Heidelberg, Deutschland; 2Institute of Cardiology Pulmonary Hypertension Center Kiew, Ukraine; 3Universitätsklinikum Giessen und Marburg GmbH Klinik für Innere Medizin, Pneumologie und Intensivmedizin Marburg, Deutschland; 4Universitätsklinikum Heidelberg Institut für Humangenetik Heidelberg, Deutschland; 5Universitätsklinikum Heidelberg Klinik für Innere Med. III, Kardiologie, Angiologie u. Pneumologie Heidelberg, Deutschland
Background: Pulmonary arterial hypertension (PAH) is characterized by a downregulation of the TGF-β-signaling (including SMAD genes) leading to plexiform lesions in the small pulmonary arteries with an aberrant cell viability of pulmonary artery smooth muscle cells (PASMCs). In an analysis of 325 patients, including 47 heritable PAH (HPAH) families, we identified in 23 % mutations in 18 previously described PAH genes. However, in some patients with HPAH no mutations could be identified. In these patients we analyzed further genes which do not belong to the 18 PAH genes. Using this approach, we were able to identify new mutations in the SMAD5 gene in two HPAH families/patients. In this abstract we will present methods and results of the clinical and functional assessment of the new not yet described SMAD5 mutations.
Methods: To assess functional implications, the variants were introduced into PASMCs using transient transfection. Variant expression was confirmed by Western blotting, quantitative polymerase chain reaction, and Sanger sequencing. Cell viability was evaluated by cck-8, cell proliferation by BrdU and apoptosis by annexin V assay. Familial cosegregation and clinical parameters were evaluated.
Results: Both SMAD5 variants were absent in >60.000 healthy controls (GnomAD v.2.1 database), each located in different protein domains and predicted to be pathogenic by in silico prediction programs. The first variant c.1175T>C p.(Leu392Pro) was identified in a heritable PAH family. The p.(Leu392Pro) variant led to significantly higher PASMC viability (cell number) due to higher proliferation in comparison to the wild type, which is typical for the development of PAH. The index patient and her healthy 9-year old son were carriers of the variant. Her mother had died of suspected PAH aged 42 years. The index patients’ sister was healthy and had the SMAD5 wild type allele. The presence of the likely pathogenic variant in the healthy son could be explained by a reduced penetrance as known for other PAH genes (Figure 1).
The second SMAD5 variant c.277T>A p.(Trp93Arg) was identified in a patient with corrected congenital heart disease associated PAH who had a surgically repaired ventricle septal defect in childhood and developed PAH aged 30 years. This p.(Trp93Arg) variant led to significant lower PASMC viability due to increased apoptosis.
Conclusion: Due to the familial aggregation, clinical findings and the functional evidence the variants could be classified as likely pathogenic. This study is the first description of SMAD5 as a novel PAH gene.
Fig. 1 Pedigree of heritable PAH family with the SMAD5 variant c.1175T>C p.(Leu392Pro).
The index patient’s mother died of PAH aged 42 years. The index patient carried the SMAD5 variant c.1175C>T p.(Leu392Pro) as did her healthy son. The index’s sister had the wild type (wt) of SMAD5. Circles denote females, squares males, strike through deceased individuals.