An ARHGAP25 variant links aberrant Rac1 function to early-onset skeletal fragility

Ras homologous guanosine triphosphatases (RhoGTPases) control several cellular functions, including cytoskeletal actin remodeling and cell migration. Their activities are downregulated by GTPase-activating proteins (GAPs). Although RhoGTPases are implicated in bone remodeling and osteoclast and osteoblast function, their significance in human bone health and disease remains elusive. Here, we report defective RhoGTPase regulation as a cause of severe, early-onset, autosomal-dominant skeletal fragility in a three-generation Finnish family. Affected individuals (n = 13) presented with multiple low-energy peripheral and vertebral fractures despite normal bone mineral density (BMD). Bone histomorphometry suggested reduced bone volume, low surface area covered by osteoblasts and osteoclasts, and low bone turnover. Exome sequencing identified a novel heterozygous missense variant c.652G>A (p.G218R) in ARHGAP25, encoding a GAP for Rho-family GTPase Rac1. Variants in the ARHGAP25 5 ' untranslated region (UTR) also associated with BMD and fracture risk in the general population, across multiple genomewide association study (GWAS) meta-analyses (lead variant rs10048745). ARHGAP25 messenger RNA (mRNA) was expressed in macrophage colony-stimulating factor (M-CSF)-stimulated human monocytes and mouse osteoblasts, indicating a possible role for ARHGAP25 in osteoclast and osteoblast differentiation and activity. Studies on subject-derived osteoclasts from peripheral blood mononuclear cells did not reveal robust defects in mature osteoclast formation or resorptive activity. However, analysis of osteosarcoma cells overexpressing the ARHGAP25 G218R-mutant, combined with structural modeling, confirmed that the mutant protein had decreased GAP-activity against Rac1, resulting in elevated Rac1 activity, increased cell spreading, and membrane ruffling. Our findings indicate that mutated ARHGAP25 causes aberrant Rac1 function and consequently abnormal bone metabolism, highlighting the importance of RhoGAP signaling in bone metabolism in familial forms of skeletal fragility and in the general population, and expanding our understanding of the molecular pathways underlying skeletal fragility. (c) 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.Peer reviewe

Functional characterization of a GGPPS variant identified in atypical femoral fracture patients and delineation of the role of GGPPS in bone-relevant cell types

Atypical femoral fractures (AFFs) are a rare but potentially devastating event, often but not always linked to bisphosphonate (BP) therapy. The pathogenic mechanisms underlying AFFs remain obscure, and there are no tests available that might assist in identifying those at high risk of AFF. We previously used exome sequencing to explore the genetic background of three sisters with AFFs and three additional unrelated AFF cases, all previously treated with BPs. We detected 37 rare mutations (in 34 genes) shared by the three sisters. Notably, we found a p.Asp188Tyr mutation in the enzyme geranylgeranyl pyrophosphate synthase, a component of the mevalonate pathway, which is critical to osteoclast function and is inhibited by N-BPs. In addition, the CYP1A1 gene, responsible for the hydroxylation of 17β-estradiol, estrone, and vitamin D, was also mutated in all three sisters and one unrelated patient. Here we present a detailed list of the variants found and report functional analyses of the GGPS1 p.Asp188Tyr mutation, which showed a severe reduction in enzyme activity together with oligomerization defects. Unlike BP treatment, this genetic mutation will affect all cells in the carriers. RNAi knockdown of GGPS1 in osteoblasts produced a strong mineralization reduction and a reduced expression of osteocalcin, osterix, and RANKL, whereas in osteoclasts, it led to a lower resorption activity. Taken together, the impact of the mutated GGPPS and the relevance of the downstream effects in bone cells make it a strong candidate for AFF susceptibility. We speculate that other genes such as CYP1A1 might be involved in AFF pathogenesis, which remains to be functionally proved. The identification of the genetic background for AFFs provides new insights for future development of novel risk assessment tools.We thank the patients for their kind participation. Funds for the study include grants SAF2014‐56562R, SAF2016‐75948‐R (Spanish MINECO), PI12/02315 (FIS, ISCII), 2014SGR932 (Catalan Government), and CIBERER (U720). This work was also supported by the Centro de Investigación Biomédica en Red en Fragilidad y Envejecimiento Saludable (CIBERFES; CB16/10/00245) and FEDER funds. JED was supported by the NIHR Biomedical Research Centre, Oxford, UK. NR is recipient of an FPU predoctoral fellowship from the Spanish Ministerio de Educación Cultura y Deporte. The work was also supported by a grant from the US government, NIH, NIAMS (R01 AR062054) to RB