Despite steady progress in identifying quantitative trait loci (QTLs) for bone phenotypes, relatively little progress has been made in moving from QTLs to identifying the relevant gene. We exploited the genetic structure of recombinant congenic mouse strains by performing a reciprocal intercross of the strains HcB-8 and HcB-23, phenotyped for body size, femoral biomechanical performance, and femoral diaphyseal geometry and mapped with R/qtl and QTL Cartographer. Significant QTLs are present on chromosomes 1, 2, 3, 4, 6, and 10. We found significant sex x QTL and cross-direction x QTL interactions. The chromosome 4 QTL affects multiple femoral anatomic features and biomechanical properties. The known segregating segment of chromosome 4 contains only 18 genes, among which Ece1, encoding endothelin-converting enzyme 1, stands out as a candidate. Endothelin signaling has been shown to promote the growth of osteoblastic metastases and to potentiate signaling via the Wnt pathway. The colocalizing chromosome 4 QTL Bmd7 (for bone mineral density 7) increases responsiveness to mechanical loading. By exploiting the short informative segment of chromosome 4 and the known biology, we propose that Ece1 is the gene responsible for Bmd7 and that it acts by increasing responsiveness to mechanical loading through modulation of Wnt signaling.

Skeletal fragility is an important health problem with a large genetic component. We performed a 603 animal F2 reciprocal intercross of the recombinant congenic strains HcB-8 and HcB-23 to genetically map quantitative trait loci (QTLs) for tissue-level femoral biomechanical performance. These included elastic and post-yield strain, Young’s modulus, stress and maximum strain, and toughness and were calculated from 3-point bend testing of femora by the application of standard beam equations. We mapped these with R/qtl and QTL Cartographer and established significance levels empirically by permutation testing. Significant QTLs for at least one trait are present on chromosomes 1, 6, and 10 in the full F2 population, with additional QTLs evident in subpopulations defined by sex and cross direction. On chromosome 10, we find a QTL for post-yield strain and toughness, phenotypes that have not been mapped previously. Notably, the HcB-8 allele at this QTL increases post-yield strain and toughness, but decreases bone mineral density (BMD), while the material property QTLs on chromosomes 1, 6, and at a second chromosome 10 QTL are independent of BMD. We found significant sex × QTL and cross-direction × QTL interactions. A robust, pleiotropic chromosome 4 QTL that we previously reported at the whole bone level showed no evidence of linkage at the tissue level, supporting our interpretation that modeling capacity is its primary phenotype. . Our data demonstrate an inverse relationship between femoral perimeter and Young’s modulus, with R2 = 0.27, supporting the view that geometric and material bone properties are subject to an integrated set of regulatory mechanisms. Mapping QTLs for tissue-level biomechanical performance advances understanding of the genetic basis of bone quality.

Bone biomechanical performance is a complex trait or, more properly, an ensemble of complex traits. Biomechanical performance incorporates flexibility under loading, yield and failure load, and energy to failure; all are important measures of bone function. To date, the vast majority of work has focused on yield and failure load and its surrogate, bone mineral density. We performed a reciprocal intercross of the mouse strains HcB-8 and HcB-23 to map and ultimately identify genes that contribute to differences in biomechanical performance. Mechanical testing was performed by 3-point bending of the femora. We measured femoral diaphysis cross-sectional anatomy from photographs of the fracture surfaces. We used beam equations to calculate material level mechanical properties. We performed a principal component (PC) analysis of normalized whole bone phenotypes (17 input traits). We measured distances separating mandibular landmarks from calibrated digital photographs and performed linkage analysis. Experiment-wide α = 0.05 significance thresholds were established by permutation testing. Three quantitative trait loci (QTLs) identified in these studies illustrate the advantages of the comprehensive phenotyping approach. A pleiotropic QTL on chromosome 4 affected multiple whole bone phenotypes with LOD scores as large as 17.5, encompassing size, cross-sectional ellipticity, stiffness, yield and failure load, and bone mineral density. This locus was linked to 3 of the PCs but unlinked to any of the tissue level phenotypes. From this pattern, we infer that the QTL operates by modulating the proliferative response to mechanical loading. On this basis, we successfully predicted that this locus also affects the length of a specific region of the mandible. A pleiotropic locus on chromosome 10 with LOD scores displays opposite effects on failure load and toughness with LOD scores of 4.5 and 5.5, respectively, so that the allele that increases failure load decreases toughness. A chromosome 19 QTL for PC2 with an LOD score of 4.8 was not detected with either the whole bone or tissue level phenotypes. We conclude that first, comprehensive, system-oriented phenotyping provides much information that could not be obtained by focusing on bone mineral density alone. Second, mechanical performance includes inherent trade-offs between strength and brittleness. Third, considering the aggregate phenotypic data allows prediction of novel QTLs.

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