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 ACVR1R206H-Specific Human Cellular and Zebrafish Models

Abstract

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 ACV-R2A 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 andThis is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.

Functional comparison of human ACVR1 and zebrafish Acvr1l FOP‐associated variants in embryonic zebrafish

“…Here, we used the zebrafish embryo as an in vivo model to define the functional domains and type II receptor signaling partners of FOP-associated human Hs-ACVR1 R206H and orthologous zebrafish Dr-Acvr1l R203H , taking advantage of the well-established embryonic dorsalization (C1-C5) and ventralization (V1-V5) phenotypes as a read-out for global BMP signaling. 30,37,[52][53][54] Using HS inducible transgenic lines, mRNA injections, anti-sense morpholino oligomer (MO) injections, and CRISPR/Cas9 Homology Directed Repair (HDR) approaches, we showed that Hs-ACVR1 R206H and Dr-acvr1l R203H exhibit functional differences in embryonic zebrafish. While Dr-acvr1l R203H exhibits WT Dr-acvr1l functions in early embryonic zebrafish; in contrast, Hs-ACVR1 R206H induced ventralized embryonic phenotypes consistent with upregulated BMP signaling.…”

Shedding Light on Bone Morphogenetic Protein (BMP) Signaling Modifiers to Modulate Fibrodysplasia Ossificans Progressiva Severity

“…In this issue, Wentworth and colleagues 17 and Yamamoto and colleagues 18 provide new insight on FOP pathophysiology pointing to possible future new therapeutic targets.…”