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. © 2022 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).