Functional Characterization of a GGPPS Variant Identified in Atypical Femoral Fracture Patients and Delineation of the Role of GGPPS in Bone-Relevant Cell Types

Abstract

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. © 2018 American Society for Bone and Mineral Research.

Bone development and remodeling in metabolic disorders

“…One of the most interesting genes is CYP1A1 , an enzyme involved in the metabolism of drugs and xenobiotics and responsible for the hydroxylation of steroid hormones, such as estrogens (Figure B). Mutations in CYP1A1 were found in the three sisters and one unrelated patient in our study and also reported elsewhere in two patients with AFF and glucocorticoid‐induced osteoporosis . Another study identified a missense variant in the PPEF2 gene significantly associated with AFF by exon array analysis of a small cohort .…”

“…We also performed cellular functional assays, such as RNAi knockdown of the GGPS1 gene in osteoblasts and in osteoclasts. In osteoblasts, RNAi produced a strong mineralization reduction and a reduced expression of the typical osteoblastic markers osteocalcin, osterix, and RANKL, whereas in osteoclasts, it led to an increase of the osteoclast number with a lower resorption activity …”

Atypical Femoral Fracture: A Fascinating Story in Evolution

“…In this issue of the journal, Roca‐Ayats et al present functional studies of the GGPS1 variant and further MPS detail . Expression of wild‐type and mutant GGPPS constructs in E. coli BL‐21 cells demonstrated markedly reduced enzymatic activity of mutant GGPSS (<6%) compared with wild type, with disruption of the usual GGPPS hexameric conformation.…”

“…The three sisters share 36 other rare (minor allele frequency <0.5%) variants in 34 genes, including a variant in CYP1A1 predicted damaging and “likely pathogenic.” Rare “likely pathogenic” CYP1A1 variants have been identified in two other AFF cases . CYP1A1 belongs to the cytochrome P450 enzyme family, which affects multiple drugs.…”

“…CYP1A1 belongs to the cytochrome P450 enzyme family, which affects multiple drugs. Functionality of the CYP1A1 variant is yet to be assessed, which the authors acknowledge …”