Spontaneous Fractures in the Mouse Mutant sfxAre Caused by Deletion of the Gulonolactone OxidaseGene, Causing Vitamin C Deficiency*

Using a mouse mutant that fractures spontaneously and dies at a very young age, we identified that a deletion of the GULOgene, which is involved in the synthesis of vitamin C, is the cause of impaired osteoblast differentiation, reduced bone formation, and development of spontaneous fractures.Introduction: A major public health problem worldwide, osteoporosis is a disease characterized by inadequate bone mass necessary for mechanical support, resulting in bone fracture. To identify the genetic basis for osteoporotic fractures, we used a mouse model that develops spontaneous fractures (sfx) at a very early age.Materials and Methods: Skeletal phenotype of the sfxphenotype was evaluated by DXA using PIXImus instrumentation and by dynamic histomorphometry. The sfxgene was identified using various molecular genetic approaches, including fine mapping and sequencing of candidate genes, whole genome microarray, and PCR amplification of candidate genes using cDNA and genomic DNA as templates. Gene expression of selected candidate genes was performed using real‐time PCR analysis. Osteoblast differentiation was measured by bone marrow stromal cell nodule assay.Results: Femur and tibial BMD were reduced by 27% and 36%, respectively, in sfxmice at 5 weeks of age. Histomorphometric analyses of bones from sfxmice revealed that bone formation rate is reduced by >90% and is caused by impairment of differentiated functions of osteoblasts. The sfxgene was fine mapped to a 2 MB region containing ∼30 genes in chromosome 14. By using various molecular genetic approaches, we identified that deletion of the gulonolactone oxidase(GULO) gene, which is involved in the synthesis of ascorbic acid, is responsible for the sfxphenotype. We established that ascorbic acid deficiency caused by deletion of the GULOgene (38,146‐bp region) contributes to fractures and premature death because the sfxphenotype can be corrected in vivo by treating sfxmice with ascorbic acid and because osteoblasts derived from sfxmice are only able to form mineralized nodules when treated with ascorbic acid. Treatment of bone marrow stromal cells derived from sfx/sfxmice in vitro with ascorbic acid increased expression levels of type I collagen, alkaline phosphatase, and osteocalcin several‐fold.Conclusion: The sfxis a mutation of the GULOgene, which leads to ascorbic acid deficiency, impaired osteoblast cell function, and fractures in affected mice. Based on these and other findings, we propose that ascorbic acid is essential for the maintenance of differentiated functions of osteoblasts and other cell types.