The work carried out so far has been summarised below organised according to the headings outlined in Annex 1 of the grant application.
Section 1. Identifying novel genetic variants for Paget’s disease
An extended genome wide association study (GWAS) has already been performed involving 2330 PDB patients and 15,000 controls resulting in identification of fourteen new regions of susceptibility in the discovery phase. Replication studies are in progress involving a further 1350 PDB cases and 1600 controls from different European countries to confirm the candidacy of these regions as genetic risk factors for PDB. The replication studies are expected to be completed and analysed within the coming 3-6 months.
Section 2. Stratifying people at risk of PDB by genetic profiling
An epigenome wide association study (EWAS) has been performed, analysed and submitted for publication. This has been successful in identifying fourteen differentially methylated sites that were associated with PDB and which discriminate cases and controls with high sensitivity and specificity. Interestingly, many of these markers were located close to different genes from those identified by the GWAS studies. These observations confirm the importance of genetic and epigenetic factors in PDB and illustrate that both may be useful as disease markers and also may provide complementary information. The utility of this approach will be combined with genetic profiling once validated susceptibility loci have been identified as the result of the extended GWAS study
Section 3 Conducting functional analysis of PDB loci as a new paradigm to understand bone remodelling
Very significant advances have been made in this domain. Our research has indicated that the candidate gene on 1p13 is CSF1 which encodes macrophage colony stimulating factor (M-CSF), a cytokine that plays a critical role in osteoclast differentiation. Serum concentrations of M-CSF were found to be 4-fold higher in PDB patients than controls and the deletion of region of strongest association about 87Kb upstream of M-CSF in vitro using CRISPR/Cas has been shown to influence CSF1 expression. This provides evidence to suggest that the predisposing variants cause PDB by increasing M-CSF production promoting osteoclast differentiation. The likely predisposing gene at the 14q32 locus has been identified as Rab and Ras interacting factor 3 (RIN3). Analysis of preclinical models have suggested that RIN3 regulates osteoclastic bone resorption and bone formation. It has also been found that the predisposing gene at the 14q24 locus is PML. Bioinformatic analysis showed that the predisposing was an expression quantitative trait locus for the PML gene with reduced expression for the PDB-predisposing allele. To investigate the candidacy of PML we conducted skeletal phenotyping of mice with targeted inactivation of this gene. The Pml deficient mice had evidence of high bone remodelling with increased osteoclast and osteoblast activity both in vitro and in vivo indicating the importance of this gene as a regulator of bone remodelling. These findings indicate that the 14q24 variant down-regulates PML expression which in turn stimulates osteoclast and osteoblast activity. A manuscript describing the results of these experiments is under revision for the Journal of Bone and Mineral Research. The TNFRSF11A gene, which encodes receptor activator of nuclear factor kappa B (RANK) is the most likely candidate gene at the 18q21 locus. Functional studies were conducted on a model of severe early onset PDB caused by an insertion mutation in the RANK signal peptide. These studies showed that when present in heterozygous form, the insertion mutation increased osteoclast formation and extended survival but in a manner independent of RANKL-stimulation since the insertion mutation blunted response to RANKL. Even more surprisingly, when the same mutation was present in homozygous form, this resulted in complete absence of osteoclasts and a phenotype consistent with osteopetrosis where there is failure of osteoclast formation.
Investigating environmental triggers for PDB
Research is now being initiated on the environmental triggers for PDB focusing on the hypothesis that alterations in the intestinal microbiome may act as a modifying factor for onset and severity of PDB. This research is being carried out in collaboration scientists and researchers from Probi, a global company focused on probiotics for supplements and functional food. The aim will be to determine whether therapeutic interventions with probiotic solutions may favourably influence disease activity and symptoms in clinical studies of Paget’s disease as well as in preclinical models.
