Bone Abstracts

Introduction: Paget’s disease of bone (PDB) is a common disorder characterised by increased and disorganised bone remodelling. Previous genome wide association studies identified a locus for susceptibility to PDB on chromosome 1p13, tagged by rs484959 which lies 87 kb upstream of the CSF1 gene. This is a strong candidate for PDB since it encodes macrophage colony-stimulating factor (M-CSF) a critical cytokine for osteoclast formation and survival.

Purpose: To conduct bioinformatic analysis and fine mapping of the chromosome 1p13 susceptibility locus to identify the mechanisms by which genetic variants in this region predispose to PDB.

Methods: Bioinformatic analysis was conducted using the ENCODE database to identify potential regulatory motifs in the region of strongest association and we conducted fine mapping of the region using a combination of next-generation sequencing and Sanger sequencing in 272 PDB cases and 110 controls to identify potentially causal variants.

Results: Bioinformatic analysis showed that two of the three strongest hits on GWAS were within an H3K27Ac mark and close to DNAase1 hypersensitivity sites, both of which suggest that the mechanism of association might be through an effect on gene regulation Mutation screening of the coding regions of CSF1 gene identified three missense mutations but at equal frequency in cases and controls. Sequencing of the elements in a 5 kb region surrounding the top hit revealed variants within several potential regulatory elements that were greatly enriched in PDB cases versus controls. The most strong association was with a SNP within a region predicted to bind AP1 and CEBP and POLR2 site 46.7 vs 12%, P=1.44×10−10) which have key roles in regulating bone turnover.

Conclusion(s): These observations are consistent with the hypothesis that common genetic variants located upstream of CSF1 predispose to the PDB by modulatory effects on regulation of CSF1. Further studies are in progress using CRISPR/Cas to disrupt the region and investigate the effects of CSF1 mRNA expression in cell lines and to examine the relation between variants at the 1p13 locus and circulating levels of MCSF in patients with PDB and controls.