Bone Abstracts

Paget’s disease of bone (PDB) is a common disease characterised by focal abnormalities in bone turnover. Previous GWAS identified a susceptibility locus for PDB on chromosome 15q24 tagged by a coding SNP rs5742915 (p.Phe645Leu) located in PML. The aim of this study was to fine map this locus to identify functional genetic variants that predispose to PDB using targeted DNA sequencing and functional analysis in bone cells.

A 200 kb region surrounding the rs5742915 was captured and sequenced on illumina HiSeq2000 platform in 138 PDB cases and 50 controls. Potential regulatory variants were assessed using the ENCODE database. Expression analysis was performed using western blot in cultured bone cells.

DNA sequencing results identified 22 missense variants (five in LOXL1, 13 in PML, three in ISLR and one in ISLR2). However, only three missense variants (all located within PML) showed significant association with PDB (P<0.05). Additionally, 33 potentially regulatory variants were identified in this region, of which two variants located in PML promoter showed significant association with PDB (P<0.003).

We next investigated PML gene expression in bone cells and found that PML was expressed in RAW 264.7 cells as well as bone marrow derived macrophages and at all stages during their differentiation into osteoclasts. PML was also expressed at all stages of osteoblast differentiation in cultured cells derived from mouse calvaria. PML is a tumour suppressor gene that is involved in multiple cellular functions including cell growth, apoptosis, and antiviral responses but has never been implicated in bone metabolism. It may be involved by controlling maturation of myeloid cells, proliferation and osteogenic differentiation of human mesenchymal stem cells. Our results suggest that PML is the susceptibility gene for PDB in this region. However, further work is in progress to confirm our findings in larger cohorts and to investigate the role of PML in bone metabolism using knockdown and over-expression in bone cells.