Functional and Molecular Characterization of Novel <i>GDF2 (BMP9</i> ) and <i>BMP10</i> Variants From the French PAH and HHT Cohorts

BACKGROUND: GDF2 (encoding BMP9) variants have been described in pulmonary arterial hypertension (PAH) and hereditary hemorrhagic telangiectasia, as well as a few BMP10 variants in PAH. The purpose of the present study was to develop a functional assay capable of discriminating benign from pathogenic variants and to characterize the underlying molecular mechanisms responsible for their loss of function. METHODS: We developed a single-step functional assay in which C2C12 cells stably expressing a BMP (bone morphogenetic protein)-responsive element upstream of a firefly luciferase reporter would be stimulated by the autocrine secretion of BMP9 or BMP10 variants produced by transfected expression plasmids. RESULTS: Using this functional assay, we reclassified all GDF2 variants and 2 out of 5 BMP10 variants identified in patients with PAH as likely pathogenic. In contrast, only 2 of the 4 GDF2 variants identified in suspected patients with hereditary hemorrhagic telangiectasia were found to be likely pathogenic; nevertheless, none of the patients met the diagnostic criteria for hereditary hemorrhagic telangiectasia. We also showed, using ELISAs and Western blots, that the loss of function of GDF2 and BMP10 variants was mostly due to altered processing (folding/stability defects). Moreover, we found that loss-of-function GDF2 variants impaired the secretion of BMP10, suggesting a potential dominant-negative mechanism. CONCLUSIONS: We developed a functional assay for GDF2 and BMP10 variants, enabling the reclassification of variants of unknown significance. Together, this study further supports the involvement of GDF2 and BMP10 as predisposing genes in PAH. This single-step assay will be transferable to clinical genetic laboratories and will improve diagnosis of patients with PAH and hereditary hemorrhagic telangiectasia.