Identification of novel genetic loci for bone size and mechanosensitivity in an ENU mutant exhibiting decreased bone size.
Using a dominant ENU mutagenesis screen in C57BL/6J (B6) mice to reveal gene function, we identified a mutant, 917M, with a reduced bone size phenotype, which is expressed only in males. We show that mutation results in osteoblasts with reduced proliferation, increased apoptosis, and an impaired response to in vitro mechanical load. The mutation is mapped to a novel locus (LOD score of 7.9 at 10.5 cM) on chromosome 4.


BACKGROUND
Using a dominant ENU mutagenesis screen in C57BL/6J (B6) mice to reveal gene function, we identified a mutant, 917M, with a reduced bone size phenotype, which is expressed only in males. In this report, we show the chromosomal location of this mutation using linkage analysis and cellular characterization of the mutant phenotype.


METHODS
The mutant mouse was bred to wildtype B6 to produce progeny for characterization of the bone size phenotype. Periosteal osteoblasts isolated from the tibia and femur of mutant and wildtype mice were studied for proliferation, differentiation, and apoptosis potential. To determine the chromosomal location of the mutation, a low-resolution linkage map was established by completing a genome-wide scan in B6C3H F2 male mice generated from intercross breeding of mutant mice.


CONCLUSIONS
Mutant progeny (16 weeks old) displayed a total body bone area that was 10-13% lower and a periosteal circumference that was 5-8% lower at the femur and tibia midshaft compared with wildtype B6 mice. Periosteal osteoblasts from mutant mice showed 17-27% reduced cell proliferation and 23% increased apoptosis compared with wildtype controls. In addition, osteoblasts from mutant mice showed an impaired response to shear stress-induced proliferation rate, an in vitro model for mechanical loading. Interval mapping in B6C3H F2 males (n = 69) indicated two major loci affecting bone size on chromosome 1 at 45 cM (LOD 4.9) and chromosome 4 at 10.5 cM (LOD 7.9, genome-wide p < 0.01). Interval mapping using body weight as covariate revealed only one significant interval at chromosome 4 (LOD 6.8). Alleles of the chromosome 4 interval inherited from the B6 mutant strain contributed to a significantly lower bone size than those inherited from C3H. A pairwise interaction analysis showed evidence for a significant interaction between loci on chromosome 1 with the chromosome 4 quantitative trait loci. The 917M locus on chromosome 4 seems to be novel because it does not correspond with those loci previously associated with bone size on chromosome 4 in B6 and C3H/HeJ mice or other crosses.
Srivastava
Apurva K
AK
Musculoskeletal Disease Center, Jerry L. Pettis Veterans Administration Medical Center, Loma Linda, California 92357, USA.
Kapur
Sanjay
S
Mohan
Suburaman
S
Yu
Hongrun
H
Kapur
Sonia
S
Wergedal
Jon
J
Baylink
David J
DJ
D000477:Alkylating Agents; D000483:Alleles; D000818:Animals; D017209:Apoptosis; D001862:Bone Resorption; D001842:Bone and Bones / Q000033:anatomy & histology / Q000187:drug effects* / Q000502:physiology; D002454:Cell Differentiation; D049109:Cell Proliferation; D002874:Chromosome Mapping; D003433:Crosses, Genetic; D005038:Ethylnitrosourea; D005260:Female; D016678:Genome; D008126:Lod Score; D008297:Male; D051379:Mice; D008809:Mice, Inbred C3H; D008810:Mice, Inbred C57BL; D008957:Models, Genetic; D016296:Mutagenesis; D009153:Mutagens; D009154:Mutation; D010006:Osteoblasts / Q000166:cytology / Q000378:metabolism; D010641:Phenotype; D040641:Quantitative Trait Loci; D012737:Sex Factors; D013314:Stress, Mechanical
D000477:Alkylating Agents; D009153:Mutagens; D005038:Ethylnitrosourea