Functional Testing of Bone Morphogenetic Protein (BMP) Pathway Variants Identified on Whole-Exome Sequencing in a Patient with Delayed-Onset Fibrodysplasia Ossificans Progressiva (FOP) Using ACVR1^R206H-Specific Human Cellular and Zebrafish Models

Bone morphogenetic protein (BMP) signaling is critical in skeletal development. Overactivation can trigger heterotopic ossification (HO) as in fibrodysplasia ossificans progressiva (FOP), a rare, progressive disease of massive HO formation. A small subset of FOP patients harboring the causative ACVR1^R206H mutation show strikingly mild or delayed-onset HO, suggesting that genetic variants in the BMP pathway could act as disease modifiers. Whole-exome sequencing of one such patient identified BMPR1A^R443C and ACVR2A^V173I as candidate modifiers. Molecular modeling predicted significant structural perturbations. Neither variant decreased BMP signaling in ACVR1^R206H HEK 293T cells at baseline or after stimulation with BMP4 or activin A (AA), ligands that activate ACVR1^R206H signaling. Overexpression of BMPR1A^R443C in a Tg(ACVR1-R206Ha) embryonic zebrafish model, in which overactive BMP signaling yields ventralized embryos, did not alter ventralization severity, while ACVR2A^V173I exacerbated ventralization. Co-expression of both variants did not affect dorsoventral patterning. In contrast, BMPR1A knockdown in ACVR1^R206H HEK cells decreased ligand-stimulated BMP signaling but did not affect dorsoventral patterning in Tg(ACVR1-R206Ha) zebrafish. ACVR2A knockdown decreased only AA-stimulated signaling in ACVR1^R206H HEK cells and had no effect in Tg(ACVR1-R206Ha) zebrafish. Co-knockdown in ACVR1^R206H HEK cells decreased basal and ligand-stimulated signaling, and co-knockdown/knockout (bmpr1aa/ab; acvr2aa/ab) decreased Tg(ACVR1-R206Ha) zebrafish ventralization phenotypes. Our functional studies showed that knockdown of wild-type BMPR1A and ACVR2A could attenuate ACVR1^R206H signaling, particularly in response to AA, and that ACVR2A^V173I unexpectedly increased ACVR1^R206H-mediated signaling in zebrafish. These studies describe a useful strategy and platform for functionally interrogating potential genes and genetic variants that may impact the BMP signaling pathway.