Experiment
Mouse strains C57BL/10ScSnA and C3H/DiSnA mice have different bone geometry and biomechanical performance.
A pleiotropic quantitative trait locus QTL, Bntq18, for bone geometry and mechanical performance in mice was mapped to distal chromosome 4 via an intercross of recombinant congenic mice HcB8/Dem and HcB23/Dem [J:150531]. To study the QTL in isolation, C3.B10-(rs6355453-rs13478087) (C.B.4.3) and C3.B10-(rs6369860-D4Mit170) (C.B.4.2) congenic strains were generated that harbor ~20 Mb and ~3 Mb, respectively, of chromosome 4 overlapping segments from C57BL/10ScSnA (B10) within the locus on a C3H/DiSnA (C3H) background.
The parental mice in this study were the recombinant congenic strains HcB8/Dem, HcB13/Dem, and the recipient progenitor of those strains was, C3H/DiSnA. The congenic strains were constructed by backcrossing the donor strains to the recipient strain C3H/DiSnA to yield the long congenic strain C.B.4.3 and the short congenic strain C.B.4.2, respectively.
Male congenic mice were intercrossed with C3H/DiSnA female mice to yield an F2 cohort for phenotyping. The animals were maintained to an age of 17 1 weeks, the age at which mice achieve peak bone mass. Following euthanasia, animals were weighed and measured (rostro-anal length), and femora and humeri were dissected.
For the long chromosome 4 congenic strain (C.B.4.3) mice, a total of 52 mice were studied. Mechanical testing was performed on 9 C3H/C3H males, 8 C3H/C3H females, 8 C3H/B10 males, 9 C3H/B10 females, 10 B10/B10 males and 8 B10/B10 females. For the short chromosome 4 congenic strain (C.B.4.2), a total of 54 mice were studied. Mechanical testing was performed on 10 C3H/C3H males, 4 C3H/C3H females, 17 C3H/B10 males, 8 C3H/B10 females, 9 B10/B10 males and 6 B10/B10 females.
The F2 progenies were genotyped at microsatellite and SNP markers. Genetic and physical locations of markers and genes are from the Mouse Genome Database, MGI 6.0 (http://www.informatics.jax.org/genes.shtml). Only non-recombinant mice were held for phenotyping. Areal BMD was measured from isolated femora by dual X-ray absorptometry (DXA). Femoral diaphysis biomechanical performance was measured by quasi-static 3-point bending under displacement. Comparisons of intercross subgroups were by 2-way ANOVA, with genotype and sex as factors, with post hoc evaluation of significant differences between groups by the Holm-Sidak test.
A total of 52 chromosome 4 long congenic segment (C3H x C.B.4.3) F2 mice were studied, including 9 C3H/C3H males, 8 C3H/C3H females, 8 C3H/B10 males, 9 C3/B10 females, 10 B10/B10 males and 8 B10/B10 females. A total of 54 short chromosome 4 congenic segment (C3 x C.B.4.2) F2 mice were also studies, including 10 C3H/C3H males, 4 C3H/C3H females, 17 C3H/B10 males, 8 C3H/B10 females, 9 B10/B10 males and 6 B10/B10 females.
In the C.B.4.3 strain, homozygous B10/B10 male mice had smaller cross sectional area (CSA) and reduced total displacement than homozygous C3H/C3H mice. Sex by genotype interaction was also observed for maximum load and stiffness for C3H/C3H and B10/B10 mice, respectively. In the C.B.4.2 strain, homozygous B10/B10 mice had lower total displacement, post-yield displacement (PYD), stiffness, yield load and maximum load than mice harboring C3H allele. Sex by genotype interaction was observed in B10/B10 mice for perimeter, outer minor axis (OMA) and CSA. There were no significant differences in tissue level mechanical performance, which suggested that the QTL, Bntq18, acts primarily on circumferential bone size.
The data confirm the prior QTL mapping data and support other work demonstrating the importance of chromosome 4 QTL on bone modeling and bone responses to mechanical loading.
Ece1 lies within both the C.B.4.3 and the much smaller C.B.4.2 congenic segments, and thus is strengthened as a candidate gene by the findings here. Until an in vivo demonstration of the effect of Ece1 on important bone phenotypes is provided, it must be considered as a candidate gene within the various chromosome 4 congenic segments, rather than as the primary gene underlying the Bntq18 bone QTL in the region.
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