Investigation of PML locus on chr15 for susceptibility to Paget’s disease of bone

Abstract

Paget’s disease of bone (PDB) is a common bone disorder. The disease is characterised by localised abnormalities in bone turnover (Ralston et al. 2008). Genetic factors are important for PDB. Genome-wide association studies (GWAS) identified a susceptibility locus for PDB on chromosome 15q24.1 in addition to six other loci (Albagha et al. 2010; Albagha et al. 2011). The strongest signal for disease association from this locus is located within Promyelocytic Leukemia (PML). PML is a tumour suppressor gene involved in chromosomal translocation leading to acute promyelocytic leukaemia (APL). PML is involved in multiple cellular functions like cell growth, senescence, DNA repair, antiviral response and apoptosis but has never been implicated directly in bone metabolism. The aim of my PhD was to explore role of PML in bone biology. Fine mapping of the 15q locus was performed using targeted next generation sequencing. In chapter 4, Targeted DNA sequencing of the PDB susceptibility locus on chr15 identified variants within PML that are associated with the disease. In chapter 5, expression analysis revealed expression of Pml in bone cells from murine and human tissue and its expression varies during the development and differentiation of these cells. PML expression was found to be downregulated in patients with PDB. In chapters 6 and 7, experiments with RAW 264.7 cell line and Pml knock out mice point to a negative regulatory role of PML in osteoclast differentiation. Osteoclasts from Pml knockout mice show increased formation, fusion and activity in response to RANKL compared to wt mice. The expression levels of osteoclast related genes such as Ctsk (Cathepsin K), Dcstamp and Nfatc1 were also higher in Pml knockout mice. Osteoblasts differentiated from these mice show increased mineralised nodule formation in vitro compared to wt mice. Skeletal phenotyping by micro CT revealed no significant differences in structure and bone density between Pml knockout and wt mice irrespective of age. Interferon gamma (IFN-γ) is a cytokine which is known to induce Interferon Regulatory Factor 8 (IRF-8) and PML in mouse peritoneal macrophages (Dror et al. 2007). IRF-8 is a transcription factor downregulated during RANKL mediated osteoclastogenesis (Zhao et al. 2009) and an essential regulator of PML gene in activated macrophages (Dror et al. 2007). In chapter 8, it was found that IFN-γ inhibited osteoclastogenesis in wt mice but failed to have a pronounced effect on osteoclasts in mice lacking Pml. Thus, lack of PML possibly renders IFN-γ and IRF-8, ineffective in suppressing osteoclastogenesis. Thus, PML controls osteoclast development probably by modulating response of IRF-8 and IFN-γ in association with direct or indirect regulation of osteoclast related genes such as Ctsk (encoding Cathepsin K), Dcstamp and Nfatc1. Osteoclasts are already primed for increased formation, differentiation, fusion and activity due to upregulation of these factors in absence of PML. My doctoral study therefore identifies PML as a novel regulator of bone metabolism. Functional genetic variants within PML gene possibly contribute to reduced PML expression thereby predisposing to PDB by increasing osteoclast differentiation and activity with an increase in osteoblast mineralisation possibly to counteract increased bone resorption.