| Download datasets and supplementary data files |
|---|
Summary
Significant advances have been made in the discovery of genes affecting bone mineral density (BMD); however, our understanding of its genetic basis remains incomplete. In the current study, genome-wide association (GWA) and co-expression network analysis was used in the recently described Hybrid Mouse Diversity Panel (HMDP) to identify and functionally characterize novel BMD genes. In the HMDP, a GWA of total body, spinal and femoral BMD revealed four significant associations (-log10P > 5.39) affecting at least one BMD trait on chromosomes (Chrs.) 7, 11, 12 and 17. The associations implicated a total of 163 genes with each association harboring between 14 and 112 genes. This list was reduced to 26 functional candidates by identifying those genes that were regulated by local eQTL in bone or harbored potentially functional non-synonymous (NS) SNPs. This analysis revealed that the most significant BMD SNP on Chr. 12 was a NS SNP in the additional sex combs like-2 (Asxl2) gene that was predicted to be functional. The involvement of Asxl2 in the regulation of bone mass was confirmed by the observation that Asxl2 knockout mice had reduced BMD. To begin to unravel the mechanism though which Asxl2 influenced BMD, a gene co-expression network was created using cortical bone gene expression microarray data from the HMDP strains. Asxl2 was identified as a member of a co-expression gene module enriched for genes involved in the differentiation of myeloid cells. In bone, osteoclasts are bone-resorbing cell of myeloid origin, suggesting that Asxl2 may play a role in osteoclast differentiation. In agreement, the knockdown of Asxl2 in bone marrow macrophages impaired their ability to form osteoclasts. This study identifies a new regulator of BMD and osteoclastogenesis and highlights the power of GWA and systems genetics in the mouse for dissecting complex genetic traits.
Experiment design
RNA from cortical bone (femoral diaphysis free of marrow) were profiled from 99 Hybrid Mouse Diversity Panel strains were profiled. Sixteen-week old male mice were used in this study. A total of 1-3 mice per strain were arrayed.
About platform
Illumina mouse-6 v1.1 expression beadchip
About data processing
The expression values were transformed using the Variance Stabilizing Transformation (VST), and normalized with the Robust Spline Normalization (RSN) algorithm using the LumiR R package. After normalization, the ComBat software was used to adjust for batch effects using an empirical Bayes method.
Citation
Farber CR, Bennett BJ, Orozco L, Zou W et al. Mouse genome-wide association and systems genetics identify Asxl2 as a regulator of bone mineral density and osteoclastogenesis. PLoS Genet 2011 Apr;7(4):e1002038. PMID: 21490954 Calabrese G, Bennett BJ, Orozco L, Kang HM et al. Systems genetic analysis of osteoblast-lineage cells. PLoS Genet 2012 Dec;8(12):e1003150. PMID: 23300464.
Acknowledgment
Calabrese G, Bennett BJ, Orozco L, Kang HM, Eskin E, Dombret C, De Backer O, Lusis AJ, Farber CR.