Functional analysis of the BMP9 response of ALK1 mutants from HHT2 patients: a diagnostic tool for novel ACVRL1 mutations

Hereditary Hemorrhagic Telangiectasia (HHT) is an autosomal dominant genetically inheritable vascular dysplasia caused by mutations in genes encoding receptors of the TGFβ family: ENG, encoding endoglin (HHT1), and ACVRL1, encoding activin receptor-like kinase-1 (ALK1) (HHT2). Our recent discovery of BMP9 as the specific ligand for ALK1 allowed us to re-evaluate the functional significance of ACVRL1 mutations. We generated nineteen ALK1 mutants reproducing HHT2 mutations (four were novel mutations) found throughout the protein. We show that all ALK1 mutant proteins were expressed by transfected cells, most of them were present at the cell surface and retained their ability to bind BMP9 (except for the extracellular mutants). However, most were defective in BMP9 signaling. None of the ALK1 mutants had a dominant negative effect on wild-type ALK1 activity. These data demonstrate that mutations of ACVRL1 fit with a functional haploinsufficiency model affecting BMP9 signaling. Our study also identified four ACVRL1 mutations (D179A, R386C, R454W and A482V) that did not alter the BMP9 responses that are polymorphisms and two novel mutations that are pathogenic (L381P and I485F). This demonstrates that the analysis of BMP9 responses can be used as a diagnostic tool by geneticists confronted with novel or conflicting ACVRL1 mutations.