Periodic Reporting for period 4 - Paget-Advance (Advancing knowledge to improve outcome in Paget’s disease of bone)
Período documentado: 2023-02-01 hasta 2024-07-31
Paget’s disease of bone (PDB) is a common skeletal disorder in people of European descent characterised by abnormal bone remodeling which disrupts normal bone structure causing pain, deformity, nerve compression syndromes and fractures. These complications can seriously impact on the quality of life of individuals that are affected. The causes of PDB are incompletely understood. It currently thought that susceptibility to the disease is primarily genetically determined but that the predisposing genetic variants interact with environmental influences to affect disease onset and severity. When the Paget Advance project was initiated, twelve candidate loci for susceptibility to PDB had been identified by a combination of genome wide association studies and family-based linkage or sequencing studies. The aim of the project is to progress knowledge about PDB beyond state-of-the-art by identifying new genetic variants that predispose to PDB by extended genome wide association studies coupled. This information will be used to develop a genetic profiling test that can be offered to those with a positive family history of PDB to more precisely estimate the risk of the disease developing. This will allow those who are identified to be at increased risk to be closely monitored facilitating early diagnosis and treatment. A second aim is to identify the genes that cause the disorder and investigate the functional mechanisms by which these genes affect bone cell function. These aims will be achieved by using a combination of bioinformatic studies, cell culture studies and analysis of preclinical disease models as well as investigating the hypothesis that somatic mutations may explain why some bones are affected but others are not. A third aim is to identify the environmental triggers for the disease focusing on the hypothesis that susceptibility and severity of PDB may be influenced by diet and the intestinal microbiome.
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.
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.
The work carried out so far has already progressed knowledge beyond current state-of the art.
The extended GWAS study has provisionally identified 14 new loci for PDB in the discovery sample and it is expected that the candidacy of these regions will be confirmed by genotyping of the replication cohorts within the next 3-6 months. When genome-wide significant loci have been identified this will allow us to explore the predictive value of these in identifying people with a family history of PDB who develop PDB. This will be assessed in a study called the genetic predictors for the development of Paget’s disease (GAPDPD). Ethical approval to recruit into the GAPDPD study was obtained about 15months ago but it has been impossible to enrol any participants because of the coronavirus pandemic. We are optimistic that this may improve as vaccinations roll out across the UK and other countries and we hope to be able to commence recruitment towards the end of the summer 2021.
There has been significant progress in identifying the genes that predispose to PDB and the mechanisms by which they affect bone metabolism. The work on the chromosome 1p13 locus is progressing well and is expected to be completed by the end of 2021. Work is beginning on the 7q33 locus and this is expected by the end of 2022.
Work on the role of environmental factors in PDB has been substantially delayed by the coronavirus pandemic. However, samples for analysis of the microbiome have been collected in 142 participants of the ZIPP study and we hope to be able to collect the 33 samples outstanding as soon as we can arrange the end of study visits once the pandemic has come under control.
A protocol is being prepared for an intervention study with probiotics in Individuals with PDB and this is expected to be submitted for ethical approval in the next 2-3 months. A protocol is also being prepared to collect samples of affected bone to look for evidence of somatic mutations within the same time frame. It is anticipated that recruitment to both studies will be able to commence this summer.
Progress of the project has been delayed by the coronavirus pandemic and it is likely that an extension to the duration of the project will be required for the objectives to be fulfilled.
The extended GWAS study has provisionally identified 14 new loci for PDB in the discovery sample and it is expected that the candidacy of these regions will be confirmed by genotyping of the replication cohorts within the next 3-6 months. When genome-wide significant loci have been identified this will allow us to explore the predictive value of these in identifying people with a family history of PDB who develop PDB. This will be assessed in a study called the genetic predictors for the development of Paget’s disease (GAPDPD). Ethical approval to recruit into the GAPDPD study was obtained about 15months ago but it has been impossible to enrol any participants because of the coronavirus pandemic. We are optimistic that this may improve as vaccinations roll out across the UK and other countries and we hope to be able to commence recruitment towards the end of the summer 2021.
There has been significant progress in identifying the genes that predispose to PDB and the mechanisms by which they affect bone metabolism. The work on the chromosome 1p13 locus is progressing well and is expected to be completed by the end of 2021. Work is beginning on the 7q33 locus and this is expected by the end of 2022.
Work on the role of environmental factors in PDB has been substantially delayed by the coronavirus pandemic. However, samples for analysis of the microbiome have been collected in 142 participants of the ZIPP study and we hope to be able to collect the 33 samples outstanding as soon as we can arrange the end of study visits once the pandemic has come under control.
A protocol is being prepared for an intervention study with probiotics in Individuals with PDB and this is expected to be submitted for ethical approval in the next 2-3 months. A protocol is also being prepared to collect samples of affected bone to look for evidence of somatic mutations within the same time frame. It is anticipated that recruitment to both studies will be able to commence this summer.
Progress of the project has been delayed by the coronavirus pandemic and it is likely that an extension to the duration of the project will be required for the objectives to be fulfilled.