@article{187437a146004abca2c1c243b7b4adc8,
title = "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 ACVR1R206H 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 BMPR1AR443C and ACVR2AV173I as candidate modifiers. Molecular modeling predicted significant structural perturbations. Neither variant decreased BMP signaling in ACVR1R206H HEK 293T cells at baseline or after stimulation with BMP4 or activin A (AA), ligands that activate ACVR1R206H signaling. Overexpression of BMPR1AR443C in a Tg(ACVR1-R206Ha) embryonic zebrafish model, in which overactive BMP signaling yields ventralized embryos, did not alter ventralization severity, while ACVR2AV173I exacerbated ventralization. Co-expression of both variants did not affect dorsoventral patterning. In contrast, BMPR1A knockdown in ACVR1R206H 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 ACVR1R206H HEK cells and had no effect in Tg(ACVR1-R206Ha) zebrafish. Co-knockdown in ACVR1R206H 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 ACVR1R206H signaling, particularly in response to AA, and that ACVR2AV173I unexpectedly increased ACVR1R206H-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.",
keywords = "BMP/TGFβ, DEVELOPMENTAL MODELING, FIBRODYSPLASIA OSSIFICANS PROGRESSIVA, GENETIC ANIMAL MODELS, PRECLINICAL STUDIES",
author = "Wentworth, {Kelly L.} and Lalonde, {Robert L.} and Groppe, {Jay C.} and Niambi Brewer and Tania Moody and Steven Hansberry and Taylor, {Kimberly E.} and Shore, {Eileen M.} and Kaplan, {Frederick S.} and Pignolo, {Robert J.} and Yelick, {Pamela C.} and Hsiao, {Edward C.}",
note = "Funding Information: This manuscript was supported by an NIH-LRP (1L30 DE029063-01) to KLW; NIH/NIAMS R01AR066735 to ECH and NIH/NIAMS (R21 AR072085) to PCY, the Radiant Hope Foundation to ECH, a UCSF Cohort Development Grant to ECH, a Robert L Koch Chair in Rheumatic and Connective Tissue Diseases III to ECH, and an International FOP Association ACT Grant to ECH and PCY. We thank Melissa LaBonty and Alex Cintolo for their work generating the zebrafish model used in this manuscript; and Tea Chan, Angela Liang, Christine Chang, Carlos de Sousa, Lauren Dickey, and Samuel Kou for providing clinical research coordinator support. We also thank the patients and their families for their generous participation in this study. Authors{\textquoteright} roles: EMS, FSK, ECH, and NB generated the initial hypothesis on which this study was based. ECH, KLW, and PCY developed the analytical strategy. FSK and RJP provided clinical data on the index patient and advised study design. ECH and KLW recruited the other patients with FOP for this study and provided clinical data. KLW and KLW, ECH and TM performed the whole exome sequencing (WES) and conducted the human cellular experiments. TM, NB, and KET assisted with the WES analysis. RL and PY generated the zebrafish data. JCG performed the detailed three-dimensional protein structural analyses. EMS provided DNA and advice on the in vitro cellular studies and wrote the original draft for these sections. SH developed the ACVR1R206H HEK 293T cell lines. PCY and ECH were responsible for project design, oversight, coordination, and obtaining funding to support this project. KLW and RLL wrote the manuscript. KLW, RLL, ECH, and PCY revised the manuscript. All authors reviewed this manuscript and agree with the data presented. Publisher Copyright: {\textcopyright} 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).",
year = "2022",
month = nov,
doi = "10.1002/jbmr.4711",
language = "English (US)",
volume = "37",
pages = "2058--2076",
journal = "Journal of Bone and Mineral Research",
issn = "0884-0431",
publisher = "Wiley-Blackwell",
number = "11",
}