Bmd8 exhibits additive inheritance.
Candidate Genes
The Authors present evidence suggesting that Mouse Chromosome 6 gene Pparg may be a likely candidate for QTL Bmd8. The B6.C3H-6T (6T) congenic mouse was used to study this QTL wherein the donor C3H ranges from D6Mit274 to D6Mit216 including Pparg.
Previously QTL, Bmd8, for the phenotype of total femoral volumetric bone mineral density (BMD) and Igf1sl1, for serum IGF-1 were mapped to a mid-dsital region of Chromosome 6 in a cross between C3H/HeJ (C3H) and C57BL/6J (B6) inbred mouse strains [J:78754 and J:66640, respectively].
In the current study a congenic strain, B6.C3H-(D6Mit93-D6Mit216)/J, (B6.C3H-6T), was developed for gaining insight into the biology underlying these QTL by generating mice that were homozygous for B6 alleles for the entire genome, except for the introgressed segment which was homozygous for C3H alleles.Female B6.C3H-6T congenic mice have lower femoral volumetric bone mineral density (vBMD), smaller periosteal circumference, slightly shorter femurs and lower serum IGF-1 than either B6 or C3H mice. The congenic mice also exhibit a decrease in trabecular bone volume fraction of the distal femur that is coincident with an increase in marrow adipocytes and an impairment in osteoblast differentiation. During the development of the congenic strain it was observed that no recombination events occurred between D6Mit124 and D6Mit150. Further experimentation determined that there was a 25 cM paracentric inversion on mid-distal Chr 6 in the C3H/HeJ strain and that foundation stocks were homozygous for the inversion. Both the D6Mit124 and the D6Mit150 markers were within the inverted region but D6Mit93 was not. The upper inversion breakpoint was further resolved to be distal to rs13478767 (55.2 Mb) but proximal to D6Mit124 (71.3 Mb) and the lower inversion breakpoint was distal to D6Mit150 (116.1 Mb) but proximal to D6Mit254 (125.3 Mb) To identify candidate genes related to the QTL of interest the question of whether the inversion per se, or alleles within the QTL, or both, were responsible for the developmentof a unique skeletal and metabolic phenotype that included a profound change in stromal cell allocation from preosteoblasts into adipocytes was explored. Disruption in the organization and order of genes within the genome, without disturbing the structure of a gene unit, can cause a variety of diseases.Liver was collected from three 8-week-old B6 and three B6.C3H-6T female mice. Images were quantified using GeneChipTM Operating Software(GCOS) v1.2. Probe level data were imported into the R software environment and expression values were summarized using the RobustMultiChip Average (RMA) function in the R/affy package. The data discussed in this publication have been deposited in NCBIs Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo/) and are accessible through GEO Series accession number GSE5959. The pattern of differential gene expression on Chr 6 in the liver of B6.C3H-6T and B6 mice was examined by microarray. At approximately 72 Mb and 116 Mb a clear pattern of gene expression emerged. At the approximate breakpoints of the inversion the majority of genes are clearly down regulated in liver of B6.C3H-6T congenic mice. [Fig 2.]To determine if the low BMD and low serum IGF-1 phenotypes of the B6.C3H-6T congenic mousewere a function of allelic differences from C3H, or due entirely to the Chr 6 inversion, a new congenic strain, B6.BXH6-(D6Mit102-rs3727110)/J, (B.H-6) was generated by introgressing an app 30 Mb region from BXH6 RI line onto a B6 background. Mice were typed at each generation and backcrossing continued for 8 generations. N8F1 mice were intercrossed producing N8F2 progeny. Mice homozygous for the C3H-like alleles were intercrossed for 2 more generations, yielding N8F4 and creating B.H-6.Areal BMD and body composition was assessed; all bones for density and architecture analysis were collected from 16 week old female mice; femur lengths were measured for density; total vBMD values were calculated; femurs were scanned to evaluate trabecular bone volume fraction and microarchitecture in the secondary spongiosa of the distal femur; statistical evaluation of bone and body composition was conducted.At 16 weeks of age, female B.H-6 congenic mice weighed more than both B6 and the B6.C3H-6T congenic mice. B.H-6 mice also had higher absolute fat mass and a higher percentage of total body fat that either B6 or B6.C3H-6T mice. In addition the strain had a lower lean mass. Unlike the B6.C3H-6T mice, the B.H-6 female mice had a similar total femoral vBMD compared to B6 mice. Most striking, the B.H-6 mice had an increase in trabecular bone volume compared to either B6 or B6.C3H-6T.Results show that the B6.C3H-6T and B.H-6 congenic strains, despite having the same C3H/HeJ alleles on distal Chr 6 and identical B6 backgrounds, have a drastically different anthropomorphic and skeletal phneotype. The B6.C3H-6T mouse is a small lean mouse with very low BMD but marrow adipogenesis, where B.H-6 is fatter with higher BMD. These data support the thesis that the chromosomal inversion, not the allelic differences between C3H and B6, is responsible for the metabolic and skeletal phenotypes of B6.C3H-6T.The most likely candidate gene to be disrupted by this inversion because of its genomic location is Pparg, a nuclear receptor that is essential for adipogenesis, and also a negative regulator of osteoblastogenesis when activated by specific ligands.
Mapping and Phenotype information for this QTL,
its variants and associated markers
Linkage analysis was performed on 986 (C57BL/6J x C3H/HeJ)F2 female animals using 107 microsatellite markers to identify QTLs associated with bone mineral density (BMD). Parental strain C3H/HeJ exhibits higher bone mineral density values compared to C57BL/6J. Bmd8 mapped to 51 cM on mouse Chromosome 6 with a peak LOD=4.56 at D6Mit150 in linkage to femoral BMD. C3H/HeJ-derived alleles confer an additively-inherited decrease in femoral BMD at Bmd8.
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