Experiment
The HcB/Dem recombinant congenic panel included 27 parallel strains established by the full-sibling inbreeding pairs of C3H/DiSnA (C3H) x C57BL/10ScSnA (B10) N3 backcross animals to fixation. Each HcB strain derives 7/8 ths of its alleles from the C3H background strain and and the remainder from the donor B10 strain.
The current study reports on the application of linkage mapping strategy in a reciprocal cross between the recombinant congenic strains HcB8/Dem (HcB-8) and HcB23/Dem (HcB-23) seeking QTL linked to variation in bone biochemical performance. The two strains differ in tissue level biochemical performance. 603 F2 animals (124 HcB-8 x HcB-23 females, 167 HcB-8 x HcB-23 males, 163 HcB-23 x HcB-8 females and 147 HcB-23 x HcB-8 males) were analyzed.
At approximately 17 weeks of age (the age at which mice achieve peak bone mass) areal BMD of isolated femora was measured by dual X-ray absorptometry (DXA). Femoral diaphysis biochemical performance was tested by quasi-static 3-point bending under displacement control. Digital photographs of the fracture plane of each bone were analyzed. F2 progeny were genotyped at 41 microsatellite markers that were informative in the cross. Genetic and physical locations of markers and genes were from the Mouse Genome Database Build 37.
Primary linkage analysis was performed by interval mapping using R/qtl using the Haley-Knott regression method, sex and cross-direction were used as either additive or interactive covariates. Secondary analysis was performed by composite interval mapping and multiple trait linkage analysis was performed using QTL Cartographer with phenotype adjusted for sex and cross direction. Experiment wide significance threshold LOD scores were between 2.7 and 2.9.
Table 2: Significant QTL for at least one phenotype were detected on Chromosomes 1, 2, 3, 4, 6, and 10 in the F2 population. Additional significant linkages were present in subgroups. The Chromosome 4 QTL had the largest linkage signal in the cross.
QTL Bntq18, bone traits QTL 18, mapped to peak marker D4Mit54 (66 cM) on Chromosome 4 in analysis of all F2 mice with significant linkage to the following bone traits:
body mass (LOD=4.8), yield load (LOD=9.5), maximum load (LOD= 13.1), stiffness (LOD=6.5), femoral BMD (LOD=8.8), cross-sectioned area (LOD=13.4), perimeter (LOD= 7.8), inner most axis (LOD=3.5), outer major axis (LOD=12.5), shape factor (LOD=17.5) cross-sectional inertia (LOD=4.1), femoral length (LOD=4.1), and slenderness (LOD-9.2). These traits were neither statistically nor biologically independent.
Table 3: For body mass and femoral length, Bntq18 has a small or negligible effect additive effect, whereas the dominance effect leads to heterozygotes being heavier and having longer femora than either homozygote. For the remaining traits the HcB-23 allele, derived from the C3H progenitor, confered the greater phenotypic value. Genotypes with at least one copy of the HcB-23 allele resulted in larger, more elliptical, stiffer, stronger, denser bone.
Recombination events that gave rise to the HcB-8 strain resulted in the fraction of a donor segment spanning at most 6Mb. Of the genes present in the donor segment, Ece1, encoding endothelia-converting enzyme 1, stood out as a strong candidate gene for Bntq18. Endothelin signaling has been shown to promote growth of osteoblastic metastases and to potentiate signaling via the Wnt pathway.
Curator Note: The authors note that QTL Bmd7, a QTL for bone mineral density, colocalizes with the QTL identified here on Chr 7. However, Bmd7 was originally mapped in a C57BL/6J x C3H/HeJ F2 cross which differs from the mapping population used here. We have assigned the QTL identified here, in the current study, with novel nomenclature.
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