Gene expression profile in osteoclasts from patients with Paget's disease of bone

Paget's disease of bone (PDB) is a common metabolic bone disorder with a significant genetic component. To date, only one gene associated with PDB has been identified, the p62-Sequestosome1 gene (SQSTM1), and more than 20 mutations of this gene have been reported in PDB, the most common being the P392L substitution. In order to search for differentially expressed genes in PDB, we investigated the relative gene expression profile of candidate genes in osteoclast (OCL) cultures from 12 PDB patients and six unmatched healthy controls with known genetic status regarding p62, including healthy carriers of the P392L mutation. We selected 48 OCL-expressed candidate genes that may be involved in relevant pathways of PDB pathogenesis, such as OCL signaling, survival, bone resorption activity, or adhesion. In OCL cultures derived from peripheral blood mononuclear cells, total RNA extraction was performed, followed by real-time PCR experiments. Relative quantification analysis utilized the qBase method where relative expression levels were normalized with respect to a set of reference primer pairs for three housekeeping genes. When compared to non-mutated healthy controls, OCL cultures from PDB patients displayed a significant down-regulation in genes involved in apoptosis (CASP3 and TNFRSF10A), in cell signaling (TNFRSF11A), in the OCL bone resorbing function (ACP5 and CTSK) and in the gene coding for Tau protein (MAPT) (all comparisons, p<0.0001). Comparison of relative gene expression in PDB patients with P392L mutation versus PDB patients without SQSTM1 mutation did not provide significant differential gene expression. However, we observed a non-significant decrease in the expression of several genes such as IL6ST, HIF1A, OSTM1, TNFRSF-10B and -10D, PDK1, MAPT and CASP3 in healthy carriers of the P392L mutation. These results provide important information about the mis-regulated activities of pagetic OCL, and highlight the role of altered apoptosis pathways in these cells. They also suggest that the SQSTM1 P392L mutation plays a role in PDB pathogenesis, even at early preclinical stages in healthy carriers of the P392L mutation.