Biomechanical properties of HcB-8 and HcB-23 have been summarized in detail [Saless et al., 2010b
]. Briefly, HcB-8 bones are smaller and more cylindrical, and they have lower yield and maximum loads but absorb more energy prior to fracture and have a higher Young's modulus. Therefore, it is not the case that one strain has a better biomechanical performance than the other but rather that their mechanical performances differ, with HcB-23 bones being stronger but achieving greater strength at the expense of greater brittleness.
We chose these strains for the genetic experiments in part because of their contrasting biomechanical behavior but also because, as members of the same recombinant congenic strain series, they share alleles over approximately 75% of the genome [Groot et al., 1992
]. This impacts the genetic analysis in 2 important ways. First, it reduces the significance threshold from an LOD score of approximately 4.3 for a genome-wide intercross to one of approximately 2.8. This is simply a reflection of the shared genetic information, resulting in the performance of fewer statistical tests. Second, the strains’ construction allowed crossovers to occur between the ancestral strains. Recombination breakpoints serve to constrain the possible locations of QTLs mapped in a cross between HcB strains in a way that is not possible in an intercross between ordinary inbred strains.
Chromosome 4 contains only a short informative region between HcB-8 and HcB-23, spanning approximately 6 Mb of physical length and about 1 cM of genetic length. It harbors the single most robust QTL found in the entire experiment (fig. ). It encompasses 10 whole bone level phenotypes and displays LOD scores as high as 17.5 (shape factor), as well as double digit LOD scores for maximum load, outer major axis length, and cross-sectional area [Saless et al., 2009
]. It is also linked to 3 of the 4 PC we constructed from the raw data, which together account for 80% of the overall phenotypic variance observed in the experiment [Saless et al., 2010a
]. However, it is not linked to any material property phenotypes [Saless et al., 2010b
]. Thus, this locus is pleiotropic at the level of whole bone structure and mechanical performance and also at the level of PCs, which by definition are mutually orthogonal.
Fig. 1. Chromosome 4 linkage. The x-axis shows the map position and the y-axis shows the LOD score. The map includes only 1 cM. The longer line shows the α = 0.05 significance threshold. CSA = Cross-sectional area; OutMajAx = outer major axis; InMinAx (more ...)
On the basis of these data, particularly the negative result of the material property mapping, the primary effect of the QTL was on bone size. Further, because the linkage signal was stronger for the shape factor than for any other included trait, we further inferred that the QTL affected not size per se but rather modeling in response to mechanical loading. Notably, prior work has demonstrated that at least one gene mediating responsiveness to mechanical loading is present on chromosome 4 [Robling et al., 2003
To test this interpretation, we performed linkage analysis of mandible length dividing the mandible into 3 segments separated by anatomical landmarks. The anterior segment extends from the mandibular symphysis to the first molar (M-M), the middle segment extends from the first molar to the coronoid process (M-C), and the posterior segment extends from the coronoid process to the condylar process. The M-M segment does not contain any insertions for muscles of mastication, while the M-C segment contains the insertion of the masseter. We predicted that M-C length would be linked to chromosome 4, while M-M length would not be. This is exactly what we found, as shown in figure . M-M length is linked to chromosome 6, and a second QTL for M-C length is also present on chromosome 1. We did not map any QTLs for C-C length.
Linkage mapping of mandibular length. The x-axis shows the location and the y-axis shows the LOD score. The α = 0.05 significance levels are LOD = 2.8 for M-M and 2.9 for M-C.
The parental strains display opposite behavior with regard to strength, as expressed by maximum load, and ductility, as expressed by energy to failure or toughness. A QTL on chromosome 10 displays the same behavior at the level of a single pleiotropic QTL as illustrated in figure . For this QTL, the HcB-8 allele increases toughness and postyield strain while simultaneously decreasing maximum load and BMD [Saless et al., 2009
]. This behavior is precisely that which was previously shown to maximize the statistical power of multiple phenotype mapping [Allison et al., 1998
Effect plots for D10Mit106. Top left = Maximum load; top right = toughness; bottom left = BMD; bottom right = postyield strain.
We performed a PC analysis of the whole femur phenotypes and performed linkage mapping of the 4 whose eigenvalues exceeded 1 [Saless et al., 2010a
]. We found a QTL for PC2 on chromosome 19 that was undetected in either of the whole bone properties. Inspection of the eigenvector suggests that PC2 represents size without mechanical performance, including large negative contributions from yield load, maximum load, stiffness, energy to failure, and BMD and large positive contributions from inner minor axis, inner major axis, outer major axis, and shape factor. The QTL has an LOD score of 4.7 and accounts for 3.5% of the phenotypic variance, and HcB-8 contributes the high phenotype allele. The informative segment of chromosome 19 is short, spanning approximately 10 Mb. Among the genes in the interval, Ostf1
, encoding osteoclast stimulating factor 1, is an obvious positional candidate gene. Ostf1
, an intracellular signaling protein that promotes osteoclast maturation and activity [Reddy et al., 1998
] was originally isolated from mouse embryo cDNA. This gene is an attractive candidate because the loading of PC2 is heavily weighted by inner minor and inner major axis lengths, phenotypes that reflect the size of the marrow space.