Site-, strain- and mutation-specific osteopetrosis phenotypes in mice with Clcn7 variants
Background: Autosomal dominant osteopetrosis type II (ADOII) is a rare bone disorder caused primarily by heterozygous mutations in CLCN7 and characterized by increased bone mass yet fragility and marked phenotypic heterogeneity among affected individuals. The biological factors underlying this variability remain poorly understood. We investigated the effects of age, sex, genetic background, and mutation type on bone phenotypes in murine models of ADOII carrying heterozygous Clcn7 F318L/+ or G213R/+ mutations. Methods: Bone microarchitecture was assessed by micro-computed tomography in vertebrae and femora from female and male mice across multiple ages (12, 24, and 52 weeks) and strain backgrounds (FVB/NJ, C57BL/6, and 129). Vertebral biomechanical properties were estimated by micro-finite element analysis, while femoral mechanical properties were measured using three-point bending. Results: Clcn7 F318L/+ mice exhibited significantly increased trabecular bone compared with wild-type littermates at vertebral and femoral sites across the lifespan in both sexes. The magnitude of the vertebral trabecular phenotype increased with age, whereas age-related effects in the femur were more modest and site dependent. Vertebral micro-finite element analysis predicted greater stiffness and ultimate force in F318L/+ mice irrespective of age. Cortical thickness was modestly increased, particularly in female mice, but femoral mechanical properties were largely unchanged. The relative increase in vertebral trabecular bone was similar on the FVB/NJ and C57BL/6 backgrounds in males but greater on C57BL/6 in females, whereas the femoral trabecular and cortical responses were markedly attenuated on C57BL/6 in both sexes. In G213R/+ mice, the relative trabecular phenotype was similar on the C57BL/6 and 129 backgrounds, except for a greater vertebral phenotype in 129 males. Likewise, comparison of F318L/+ and G213R/+ mice demonstrated comparable vertebral phenotypes, although mutation-specific differences emerged at the femur, particularly in males. Conclusion: Collectively, these findings demonstrate that heterozygous Clcn7 mutations produce a robust osteopetrotic phenotype characterized primarily by increased trabecular bone mass that is present regardless of sex, age, genetic background, and mutation type. Age and genetic background modify its magnitude in a site- and sex-specific manner, suggesting that local factors may contribute more strongly to phenotypic variation than the specific disease-causing Clcn7 mutation. These results provide insight into determinants of phenotypic heterogeneity in ADOII and establish a framework for future mechanistic studies of osteopetrotic bone quality and fragility.