QTL analysis of macrophages from an AKR/JxDBA/2J intercross identified the Gpnmb gene as a modifier of lysosome function

Our prior studies found differences in the AKR/J and DBA/2J strains in regard to atherosclerosis and macrophage phenotypes including cholesterol ester loading, cholesterol efflux, and autolysosome formation. The goal of this study was to determine if there were differences in macrophage lysosome function, and if so to use quantitative trait locus (QTL) analysis to identify the causal gene. Lysosome function was measured by incubation with an exogenous double-labeled ovalbumin indicator sensitive to proteolysis. DBA/2J vs. AKR/J bone marrow macrophages had significantly decreased lysosome function. Macrophages were cultured from 120 mice derived from an AKR/JxDBA/2J F intercross. We measured lysosome function and performed a high density genome scan. QTL analysis yielded two genome wide significant loci on chromosomes 6 and 17, called macrophage lysosome function modifier () loci and . After adjusting for , two additional loci were identified. Based on proximity to the peak, macrophage mRNA expression differences with AKR/J >> DBA/2J, and a protein coding nonsense variant in DBA/2J, the gene, encoding a lysosomal membrane protein, was our top candidate. To test this candidate, expression was knocked down with siRNA in AKR/J macrophages; and, to express the wildtype in DBA/2J macrophages, we obtained a DBA/2 substrain, DBA/2J-Gpnmb+/SjJ, which was isolated from the parental strain prior to its acquiring the nonsense mutation, and subsequently back crossed to the modern DBA/2J background. Knockdown of in AKR/J macrophages decreased lysosome function, while restoration of the wildtype allele in DBA/2J macrophages increased lysosome function. However, this modifier of lysosome function was not responsible for the strain differences in macrophage cholesterol ester loading or cholesterol efflux. In conclusion, we identified the gene as the major modifier of lysosome function and we showed that the ‘QTL in a dish’ strategy is efficient in identifying modifier genes.