Resumen de The Clinical description, molecular etiology, and pathophysiological studies in cutaneous skeletal hypophosphatemia syndrome: a mosaic disorder of activating RAS mutations /
Introduction: Cutaneous Skeletal Hypophosphatemia Syndrome (CSHS) is an ultra-rare mosaic disorder caused by postzygotic RAS mutations that is defined by the association of epidermal and/or melanocytic nevi, fibroblast growth factor-23 (FGF23) excess, and a skeletal dysplasia. Because of its rarity, CSHS has been inadequately characterized. Further, unknown are the roles of RAS mutations in dysplastic bone formation and the source and physiopathology of FGF23 overproduction in CSHS. Thesis hypotheses: 1) A thorough phenotypic assessment of patients with CSHS, and the analysis of all potential reported CSHS cases will provide important clinical information, 2) Dysplastic bone is the source of FGF23 overproduction in CSHS and, 3) The generation of appropriate experimental models will inform of CSHS’s physiopathology. Objectives: 1) To clinically characterize CSHS through the study of a cohort of subjects and a review of all reported cases, 2) To identify the physiopathology of FGF23 overproduction in CSHS, 3) To investigate the effects of hyperactive RAS on dysplastic bone formation. Subjects and methods: Five CSHS subjects were phenotyped and underwent prospective data collection. A review of the literature identified 51 subjects in whom the findings were compatible with CSHS. Data on nevi, bone histology, mineral and skeletal alterations, abnormalities in other tissues, and response to treatments of hypophosphatemia were analyzed. To assess the effects of RAS mutations on FGF23 production in “bone-like” cells, FGF23 production/gene expression was measured in 1) human bone marrow stromal cells (hBMSCs) and 2) IDG-SW3 cells transduced with mutated adenoviral RAS constructs, in 3) mutant hBMSCs from a CSHS patient, and in 4) IDG-SW3 cells treated with media in which mutant CSHS hBMSCs had been cultured. To further characterize CSHS bone histology, an ossicle formation assay was performed by transplanting mutant CSHS hBMSCs subcutaneously into immunocompromised mice. Finally, 3 tissue-specific mutant Ras knock-in mouse models were designed through Cre-Lox recombination technology to investigate the effects of activating Ras mutations in bone. The models consisted in the activation of a LoxP-STOP-LoxP-KrasG12D (LSL-KrasG12D) transgene through 3 different Cre-recombinases driven by the following promoters: 1) tamoxifen-inducible 3.6 kb collagen type I α-1, 2) tamoxifen-inducible paired-related homeobox gene-1(Prx1), and 3) non-inducible Prx1. Results: From a clinical standpoint, fractures, limb deformities, and scoliosis affected most CSHS subjects. Hypophosphatemia was not present at birth. Bone histology only revealed severe osteomalacia without other obvious abnormalities. Skeletal dysplasia was reported in all anatomical compartments. Phosphate and calcitriol supplementation was effective in treating rickets in most patients. Convincing data that nevi removal improved blood phosphate levels was lacking. An age-dependent improvement in mineral abnormalities was observed. A spectrum of extra-osseous/extra-cutaneous manifestations that included both benign and malignant neoplasms was present in many subjects, although osteosarcoma remains unreported. From an experimental standpoint, increased FGF23 production/expression was not detected with any of the experimental conditions in the in vitro experiments. Poor bone formation with the ossicle assay hampered histological characterization of the ossicles. No abnormal findings were detected in the models featuring the tamoxifen-inducible promoters. No double transgenic offspring harboring both LSL-KrasG12D and the non-inducible Prx1-Cre recombinase were identified. Conclusions: The study of patients and reports of subjects with CSHS provided relevant and novel clinical information. Also, the analysis of affected patient tissues allowed further appreciation of the effects of hyperactive RAS in different organs. Still, the source of excess FGF23 in CSHS remains unknown, although data indicate it is probably not produced by the nevi. Other conclusions include the lack of suitability of BMSCs to study FGF23 production/expression, the poor reproducibility of the subcutaneous ossicle formation assay, and the lethality of widespread Kras mutations in early embyrionic skeletal cells in mice.
