Comprehensive Skeletal Phenotyping and Linkage Mapping in an Intercross of Recombinant Congenic Mouse Strains HcB-8 and HcB-23

Abstract

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.

Genetic perturbations that impair functional trait interactions lead to reduced bone strength and increased fragility in mice

“…Ct.Ar and TMD were adjusted for body size but not robustness in the prior study and so these outcomes are not comparable. We and others identified QTLs regulating the interaction among traits (4,5,9,18,30,44,45). However, these studies were not conducted explicitly in the context of functional adaptations and thus it is difficult to compare the QTLs identified previously with the outcomes reported here.…”

“…This outcome provides a novel biomechanical mechanism linking genotype with phenotype. Thus, efforts aimed at identifying genes regulating fracture resistance may benefit from targeting the functional interaction among traits (7–9,38) and thus how the emergent set of traits defines the repertoire of fracture resistance properties.…”

“…Functional adaptations may complicate the choice of phenotype used in genetic studies, because genetic variants affecting one trait are sometimes compensated by coordinated changes in other traits (7–9). Because the skeletal system shows a particular pattern in the way traits are coordinately regulated (i.e., a network of trait interactions), individuals can achieve similar functional outcomes by assembling different sets of traits (10).…”

Endothelin signaling regulates mineralization and posttranscriptionally regulates SOST in TMOb cells via miR 126-3p

“…Previous work in our laboratory using the recombinant congenic mice HcB‐8 and HcB‐23 identified ECE‐1 , the gene encoding ECE‐1, as a positional candidate for a pleiotropic quantitative trait locus influencing bone strength and size in skeletally mature mice (Saless et al. , , ,b; Kristianto et al. ).…”

Combined exposure to big endothelin-1 and mechanical loading in bovine sternal cores promotes osteogenesis

“…We identified endothelin converting enzyme 1 ( Ece1 ) as a candidate gene within the quantitative trait loci bmd7 on mouse chromosome 4 which is responsible for 40% of the variation in bone size, strength and density between the recombinant congenic mice strains HCB8 and HCB23 [26–28]. …”