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