Paget-Advance - Advancing Knowledge To Improve Outcome in Paget's Disease of Bone

European Commission - Research
Proposal Submission Forms

European Research Council Executive Agency
Horizon 2020
Excellent Science
Call: ERC-2017-ADG
(Call for proposals for ERC Advanced Grant)
Topic: ERC-2017-ADG
Type of action: ERC-ADG
(Advanced Grant)
Proposal number: 787270
Proposal acronym: Paget-Advance
Deadline Id: ERC-2017-ADG
Table of contents
Section Title Action
1 General information
2 Participants & contacts
3 Budget
4 Ethics
5 Call-specific questions
How to fill in the forms

The administrative forms must be filled in for each proposal using the templates available in the submission system. Some
data fields in the administrative forms are pre-filled based on the previous steps in the submission wizard.
H2020-ERC-ADG-2017.pdf Ver 1.00 20170621 Page 1 of 15 Last saved 30/08/2017 15:51:23
This proposal version was submitted by Stuart RALSTON on 30/08/2017 15:51:29 Brussels Local Time. Issued by the Participant Portal Submission Service.
Proposal ID 787270 Acronym Paget-Advance
1 - General information
Topic ERC-2017-ADG
Call Identifier ERC-2017-ADG
Type of Action ERC-ADG
Deadline Id ERC-2017-ADG
Acronym Paget-Advance
Proposal title* Advancing knowledge to improve outcome in Paget’s disease of bone
Note that for technical reasons, the following characters are not accepted in the Proposal Title and will be
removed: < > " &
Duration in months* 60
Primary ERC Review Panel* LS7
Secondary ERC Review Panel Not applicable (if applicable)
ERC Keyword 1* Pharmacology, pharmacogenomics, drug discovery and design, drug therapy
Please select, if applicable, the ERC keyword(s) that best characterise the subject of your proposal in order
of priority.
ERC Keyword 2 Health services, health care research
ERC Keyword 3 Public health and epidemiology
ERC Keyword 4 Gene therapy, cell therapy, regenerative medicine
Free keywords
Paget's disease of bone, bone disease, clinical studies
Abstract*
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.
The causes of PDB are incompletely understood. Genetic factors play a key role and some predisposing genes been
identified but many remain to be discovered as do the mechanisms by which these genes influence bone cell function.
Environmental factors also contribute but these are poorly defined, and it remains unclear how genes and environment
interact to regulate susceptibility and severity. Further research is required to permit earlier diagnosis and to define the
genetic and environmental factors that influence susceptibility to PDB so that new therapeutic strategies can be developed
to improve outcome.
The Paget-Advance programme will progress beyond state-of-the-art by identifying new genetic variants that predispose to
PDB by extended genome wide association studies and next generation sequencing of early onset familial cases where the
causal genes are unknown. A genetic profiling test will be developed to facilitate early diagnosis and treatment. The
functional mechanisms by which the variants affect bone cell function will be investigated using bioinformatics, cell culture
studies and preclinical disease models. Potential environmental triggers will be explored focusing on the intestinal
microbiome diet and biomechanical loading using preclinical disease models and prospective clinical studies.
The result will be to increase understanding of PDB and to facilitate earlier diagnosis and timely therapeutic intervention
which will improve clinical outcome for patients and those at risk of the disease. The research will also have wider
significance in advancing knowledge of the fundamental processes that regulate bone remodelling with potential spinoffs
for other bone diseases.
Remaining characters 48
In order to best review your application, do you agree that the above non-confidential proposal title
and abstract can be used, without disclosing your identity, when contacting potential reviewers? Yes No
Declarations
1) The Principal Investigator declares to have the explicit consent of all applicants on their participation and on the content of
this proposal.*
2) The Principal Investigator declares that the information contained in this proposal is correct and complete.
3) The Principal Investigator declares that this proposal complies with ethical principles (including the highest standards of
research integrity — as set out, for instance, in the European Code of Conduct for Research Integrity — and including, in
particular, avoiding fabrication, falsification, plagiarism or other research misconduct).
4) The Principal Investigator hereby declares that (please select one of the three options below):
- in case of multiple participants in the proposal, the coordinator has carried out the self-check of the financial capacity of the
organisation on http://ec.europa.eu/research/participants/portal/desktop/en/organisations/lfv.html or to be covered by a
financial viability check in an EU project for the last closed financial year.Where the result was “weak” or “insufficient”, the
Principal Investigator confirms being aware of the measures that may be imposed in accordance with the H2020 Grants
Manual (Chapter on Financial capacity check).
- in case of multiple participants in the proposal, the coordinator is exempt from the financial capacity check being a public
body including international organisations, higher or secondary education establishment or a legal entity, whose viability is
guaranteed by a Member State or associated country, as defined in
the H2020 Grants Manual (Chapter on Financial capacity check).
- in case of a sole participant in the proposal, the applicant is exempt from the financial capacity check.
5) The Principal Investigator hereby declares that each applicant has confirmed to have the financial and operational
capacity to carry out the proposed action. Where the proposal is to be retained for EU funding, each beneficiary applicant
will be required to present a formal declaration in this respect.
The Principal Investigator is only responsible for the correctness of the information relating to his/her own organisation. Each applicant
remains responsible for the correctness of the information related to him and declared above. Where the proposal to be retained for EU
funding, the coordinator and each beneficiary applicant will be required to present a formal declaration in this respect.
According to Article 131 of the Financial Regulation of 25 October 2012 on the financial rules applicable to the general budget of the Union
(Official Journal L 298 of 26.10.2012, p. 1) and Article 145 of its Rules of Application (Official Journal L 362, 31.12.2012, p.1) applicants
found guilty of misrepresentation may be subject to administrative and financial penalties under certain conditions.
Personal data protection

The assessment of your grant application will involve the collection and processing of personal data (such as your name, address and CV),
which will be performed pursuant to Regulation (EC) No 45/2001 on the protection of individuals with regard to the processing of personal
data by the Community institutions and bodies and on the free movement of such data. Unless indicated otherwise, your replies to the
questions in this form and any personal data requested are required to assess your grant application in accordance with the specifications of
the call for proposals and will be processed solely for that purpose. Details concerning the purposes and means of the processing of your
personal data as well as information on how to exercise your rights are available in the privacy statement. Applicants may lodge a complaint
about the processing of their personal data with the European Data Protection Supervisor at any time.
Your personal data may be registered in the Early Detection and Exclusion system of the European Commission (EDES), the new system
established by the Commission to reinforce the protection of the Union's financial interests and to ensure sound financial management, in
accordance with the provisions of articles 105a and 108 of the revised EU Financial Regulation (FR) (Regulation (EU, EURATOM)
2015/1929 of the European Parliament and of the Council of 28 October 2015 amending Regulation (EU, EURATOM) No 966/2012) and
articles 143 - 144 of the corresponding Rules of Application (RAP) (COMMISSION DELEGATED REGULATION (EU) 2015/2462 of 30
October 2015 amending Delegated Regulation (EU) No 1268/2012) for more information see the Privacy statement for the EDES
Database).
List of participants
# Participant Legal Name Country
1 THE UNIVERSITY OF EDINBURGH United Kingdom
Proposal ID 787270 Acronym Paget-Advance Short name UEDIN
2 - Administrative data of participating organisations

Host Institution
PIC Legal name
999974941 THE UNIVERSITY OF EDINBURGH
Short name: UEDIN
Address of the organisation
Street OLD COLLEGE, SOUTH BRIDGE
Town EDINBURGH
Postcode EH8 9YL
Country United Kingdom
Webpage www.ed.ac.uk
Legal Status of your organisation
Research and Innovation legal statuses
Public body .................................................... yes Legal person .............................. yes

Non-profit ...................................................... yes
International organisation .................................. no
International organisation of European interest ...... no
Secondary or Higher education establishment ....... yes
Research organisation ..................................... yes
Enterprise Data
SME self-declared status................................... 12/12/2008 - no
SME self-assessment ...................................... unknown
SME validation sme ......................................... 12/12/2008 - no
Based on the above details of the Beneficiary Registry the organisation is not an SME (small- and medium-sized enterprise) for the call.
Department(s) carrying out the proposed work
Department 1
Department name MRC Institute of Genetics and Molecular Medicine not applicable
Same as organisation address
Street Crewe Road South
Town Edinburgh
Postcode EH4 2XU
Country United Kingdom
Principal Investigator
The following information on the Principal Investigator is used to personalise the communications to applicants. Please make
sure that your personal information is accurate and for any ERC specific question please contact the ERC using the following
e-mail address:
For Advanced Grant Applicants: ERC-2017-AdG-applicants@ec.europa.eu
The name and e-mail of contact persons including the Principal Investigator, Host Institution contact are read-only in the administrative form, only
additional details can be edited here. To give access rights and contact details of contact persons, please save and close this form, then go back
to Step 4 of the submission wizard and save the changes.
ORCID ID
The maximum length of the identifier is 11 characters (ZZZ-9999-2010) and

Researcher ID the minimum length is 9 characters (A-1001-2010).
Other ID Please enter the type of ID here Please enter the identifier number here
Last Name* RALSTON
First Name(s)* Stuart
Title Dr.
Contact address
Current organisation name MRC Institute of Genetics and Molecular Medicine
Current Department/Faculty/Institute/
Centre for Genomic and Experimental Medicine
Laboratory name
Same as organisation address
Street Crewe Road South
Postcode/Cedex EH4 2XU Town* Edinburgh
Phone* Country* United Kingdom
Phone2 / Mobile
3 - Budget
Total eligible
Participant Number costs/€ Requested
Organisation Short Name Organisation Country
in this proposal (including 25% grant/€
indirect costs)
?
1 UEDIN UK 2 497 536 2 497 536
Total 2 497 536 2 497 536
4 - Ethics issues table

1. HUMAN EMBRYOS/FOETUSES Page
Does your research involve Human Embryonic Stem Cells (hESCs)? Yes No
Does your research involve the use of human embryos? Yes No
Does your research involve the use of human foetal tissues / cells? Yes No
2. HUMANS Page
Does your research involve human participants? Yes No 2-5
Are they volunteers for social or human sciences research? Yes No 2-5
Are they persons unable to give informed consent? Yes No
Are they vulnerable individuals or groups? Yes No
Are they children/minors? Yes No
Are they patients? Yes No
Are they healthy volunteers for medical studies? Yes No
Does your research involve physical interventions on the study participants? Yes No 9-10
Does it involve invasive techniques? Yes No
Does it involve collection of biological samples? Yes No 9-10
If your research involves processing of genetic information, see also section 4.
3. HUMAN CELLS / TISSUES Page
Does your research involve human cells or tissues (other than from Human Embryos/ Yes No
Foetuses, i.e. section 1)?
4. PERSONAL DATA Page
Does your research involve personal data collection and/or processing? Yes No 2-5
Does it involve the collection and/or processing of sensitive personal data Yes No
(e.g: health, sexual lifestyle, ethnicity, political opinion, religious or philosophical
conviction)?
Does it involve processing of genetic information? Yes No 2-5
Does it involve tracking or observation of participants? Yes No 9-10
Does your research involve further processing of previously collected personal data Yes No 2-5
(secondary use)?
5. ANIMALS Page
Does your research involve animals? Yes No 5-7
Are they vertebrates? Yes No 5-7
Are they non-human primates? Yes No
Are they genetically modified? Yes No 5-7
Are they cloned farm animals? Yes No
Are they endangered species? Yes No
Mice
6. THIRD COUNTRIES Page
In case non-EU countries are involved, do the research related activities undertaken in Yes No
these countries raise potential ethics issues?
Do you plan to use local resources (e.g. animal and/or human tissue samples, genetic Yes No
material, live animals, human remains, materials of historical value, endangered fauna or
flora samples, etc.)?
Do you plan to import any material - including personal data - from non-EU countries into Yes No
the EU?
Do you plan to export any material - including personal data - from the EU to non-EU Yes No
countries?
In case your research involves low and/or lower middle income countries, are any Yes No
benefits-sharing actions planned?
Could the situation in the country put the individuals taking part in the research at risk? Yes No
7. ENVIRONMENT & HEALTH and SAFETY

Page
Does your research involve the use of elements that may cause harm to the Yes No
environment, to animals or plants?
Does your research deal with endangered fauna and/or flora and/or protected areas? Yes No
Does your research involve the use of elements that may cause harm to humans, Yes No
including research staff?
8. DUAL USE Page
Does your research involve dual-use items in the sense of Regulation 428/2009, Yes No
or other items for which an authorisation is required?
9. EXCLUSIVE FOCUS ON CIVIL APPLICATIONS Page
Could your research raise concerns regarding the exclusive focus on civil applications? Yes No
10. MISUSE Page
Does your research have the potential for misuse of research results? Yes No
11. OTHER ETHICS ISSUES Page
Are there any other ethics issues that should be taken into consideration? Please specify Yes No
I confirm that I have taken into account all ethics issues described above and that, if any ethics issues
✖
apply, I will complete the ethics self-assessment and attach the required documents.
How to Complete your Ethics Self-Assessment
5 - Call specific questions

Eligibility
Please indicate your percentage of working time in an EU Member State or Associated Country over the
period of the grant:
100
Please note that you are expected to spend a minimum of 50% of your total working time in an EU
Member State or Associated Country.
I acknowledge that I am aware of the eligibility requirements for applying for this ERC call as specified in
the ERC Work Programme, and certify that, to the best of my knowledge my application is in compliance
with all these requirements. I understand that my proposal may be declared ineligible at any point during
the evaluation or granting process if it is found not to be compliant with these eligibility criteria.
Data-Related Questions and Data Protection

(Consent to any question below is entirely voluntary. A positive or negative answer will not affect the evaluation of your
project proposal in any form and will not be communicated to the evaluators of your project.)
For communication purposes only, the ERC asks for your permission to publish, in whatever form and
medium, your name, the proposal title, the proposal acronym, the panel, and host institution, should your Yes No
proposal be retained for funding.
Some national and regional public research funding authorities run schemes to fund ERC applicants that
score highly in the ERC's evaluation but which can not be funded by the ERC due to its limited budget. In
case your proposal could not be selected for funding by the ERC do you consent to allow the ERC to
disclose the results of your evaluation (score and ranking range) together with your name, non- Yes No
confidential proposal title and abstract, proposal acronym, host institution and your contact details to such
authorities?
The ERC is sometimes contacted for lists of ERC funded researchers by institutions that are awarding
prizes to excellent researchers. Do you consent to allow the ERC to disclose your name, non-confidential
proposal title and abstract, proposal acronym, host institution and your contact details to such Yes No
institutions?
The Scientific Council of the ERC has developed a monitoring and evaluation strategy in order to help it
fulfil its obligations to establish the ERC's overall strategy and to monitor and quality control the
programme's implementation from the scientific perspective. As provided by section 3.10 of the
ERC Rules for Submission:
a range of projects and studies may be initiated for purposes related to monitoring, study and evaluating
Yes No
the implementation of ERC actions. Do you consent to allow the third parties carrying out these projects
and studies to process the content of your proposal including your personal data and the respective
evaluation data?
The privacy statement on the processing operations of applicants and beneficiaries data for H2020
available on the Participant Portal explains further how your personal data is secured.
Ralston Part B1 Paget-Advance

 ERC Advanced Grant 2017
Research proposal [Part B1]
(Part B1 is evaluated both in Step 1 and Step 2,
Part B2 is evaluated in Step 2 only)

Advancing knowledge to improve outcome in Paget’s disease of bone

Paget-Advance

Principal Investigator: Stuart H Ralston
Host institution: University of Edinburgh
Proposed duration of funding: 60 months

Summary
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.

The causes of PDB are incompletely understood. Genetic factors play a key role and some predisposing
genes been identified but many remain to be discovered as do the mechanisms by which these genes
influence bone cell function. Environmental factors also contribute but these are poorly defined, and it
remains unclear how genes and environment interact to regulate susceptibility and severity. Further
research is required to permit earlier diagnosis and to define the genetic and environmental factors that
influence susceptibility to PDB so that new therapeutic strategies can be developed to improve outcome.

The Paget-Advance programme will progress beyond state-of-the-art by identifying new genetic variants
that predispose to PDB by extended genome wide association studies and next generation sequencing of
early onset familial cases where the causal genes are unknown. A genetic profiling test will be developed
to facilitate early diagnosis and treatment. The functional mechanisms by which the variants affect bone
cell function will be investigated using bioinformatics, cell culture studies and preclinical disease models.
Potential environmental triggers will be explored focusing on the intestinal microbiome diet and
biomechanical loading using preclinical disease models and prospective clinical studies.

The result will be to increase understanding of PDB and to facilitate earlier diagnosis and timely therapeutic
intervention which will improve clinical outcome for patients and those at risk of the disease. The research
will also have wider significance in advancing knowledge of the fundamental processes that regulate bone
remodelling with potential spinoffs for other bone diseases.

.

Section a: Extended Synopsis of the scientific proposal (max. 5 pages)
Clinical importance of Paget’s disease
Paget’s disease of bone (PDB) is a common skeletal disorder that affects up to 1% of those aged 55 and
over in Europe. It is characterised by focal abnormalities of bone turnover which cause affected bones to
enlarge and become weak, leading to various complications such as pain, fractures and nerve compression
syndromes (Figure 1). These complications, when established, are irreversible but are present in about 20%
of patients when they first present (1). Bisphosphonates are a highly effective treatment for the raised
bone turnover that is characteristic of PDB (2, 3) but the response of patients with advanced disease to
bisphosphonate therapy is often disappointing (4, 5) .

Figure 1. Features and complications of PDB
A. Enlarged and deformed tibia with pseudofractures
on the anterior surface.
B Severe deformity of both tibias.
C. Skull enlargement and deafness secondary to
compression of auditory nerve.
D Increased osteoclastic bone resorption with
disruption of normal architecture
E. Pathological femoral shaft fracture occurring as the
result of minimal trauma

Current state of the art
Progress has been made in understanding the causes of PDB over recent years, but many questions remain
unanswered with regard to both the genetic variants and environmental factors that predispose to the
disease. Clinical outcomes of treatment in patients with established disease are also suboptimal (4, 5)
indicating the need for earlier detection and treatment.
Genetic determinants of PDB
Genetic factors play a key role in PDB and several susceptibility loci have now been identified through
genome wide association studies, linkage analysis and genome sequencing as recently reviewed (6) and
summarised graphically in Figure 2.

Figure 2. Genes and loci for PDB
The loci identified are colour coded
depending on their mode of
identification. The log10 p-values are
for illustration only and refer to the
results of the discovery phase of
GWAS studies. All loci shown
exceed the threshold for genome
wide significance on discovery or
replication studies. Note that some
loci have been identified through
more than one route.

Current evidence suggests that from a genetic standpoint, PDB is caused by a combination of large effect
size variants that cause the disease to be inherited in a Mendelian manner within families and more
common variants of moderate effect size that have additive effects on susceptibility (Figure 3A). The most
important large effect size variants are mutations in the SQSTM1 gene and these account for about 40% of
familial PDB cases in the UK. In PDB patients without a family history, common variants have been
estimated to account for about 13% of the population attributable risk (7). It also should be noted that
about 10% of subjects without a family history also have SQSTM1 mutations. It has been speculated that
somatic mutations in affected tissue might also play a role in localisation of the disease (8). Some studies
have been conducted to look for evidence of somatic mutations in affected tissue but analysis has been
confined the P392L mutation of SQSTM1 with results that have been conflicting (9, 10).
Genetic profiling to assess risk of PDB
People with a family history of PDB have a 7-fold increased risk of developing the disease as compared with
the general population (11). In patients without SQSTM1 mutations, the risk varies markedly between
individuals depending on the number of susceptibility alleles they inherit (12). (Figure 3) It would be highly
advantageous to be able to sub-stratify people with a family history of PDB (the easiest patients to identify
clinically) based on genetic profile so that those at highest can be identified and offered enhanced
surveillance with early treatment and those at lower risk reassured. We have already shown in a case
control study that stratification by the alleles identified so far can separate PDB cases and controls with
promising specificity and sensitivity (Figure 3). Other researchers have also explored the possibility of using
genetic profiling to identify patients with PDB (13). To date however, this has not been done in a
prospective manner.
A B
100 1.0

Figure 3. Susceptibility alleles in
AUC = 0.70
0.8 PDB cases and controls. Panel A
Relative risk of PDB
10 shows the relation between carriage

True Positive
0.6
of GWAS risk alleles and PDB in a
study of 1471 PDB cases without
0.4
1
Sensitivity = 84.4%
SQSTM1 mutations and 1671
0.2
Specificity = 30.2% controls of European (from Albagha
Accuracy = 69.5%
et al (7)) Panel B shows the receiver
0.1 0.0 operator characteristics, area under
<1 2 3 4 5 6 7 8 9 10 11+ 0.0 0.2 0.4 0.6 0.8 1.0
the curve (AUC) and sensitivity and
Risk Alleles False Positive
specificity data from this analysis

Therapeutic implications for genetic profiling
The importance of detecting individuals at high risk of PDB is that the disease is usually clinically “silent”
until at an advanced stage by which point many have already developed complications (1). When the
disease is advanced even potent bisphosphonates such as zoledronic acid have a limited impact on
complications and quality of life (4, 5). Unlike other diseases such as osteoporosis where there are a
number of well-defined risk factors that can be assessed on clinical history (14), only age, gender and family
history have been identified as risk factors for PDB (15). Since bisphosphonates are highly effective at
suppressing the elevated bone turnover that is characteristic of PDB (2) there is a real prospect that early
treatment might improve outcome. This is already being evaluated in pre-symptomatic carriers of SQSTM1
mutations in the ZiPP trial (http://www.isrctn.com/ISRCTN11616770). If we were able to extend the reach
of genetic profiling to identify patients who have risk alleles other than SQSTM1 this would be a major
advance.
Functional mechanisms by which PDB variants regulate bone turnover
Extensive functional studies have been conducted on the mechanisms by which SQSTM1 mutations affect
bone metabolism (16). It has similarly been established that variants at the OPTN locus predispose to PDB
by reducing OPTN mRNA expression, which in turn upregulates osteoclast activity through effects on the
NFkB pathway and interferon signaling (17). For the other genes and loci implicated in PDB shown in Figure
2, the functional mechanisms by which variants affect bone biology are unknown.
Environmental factors
Epidemiologic studies suggest that environmental factors are important in PDB as evidenced by a decrease
in prevalence over the recent years (18). However, very little is known about the environmental triggers of
PDB. Various factors have been suggested as possible triggers including dietary calcium and vitamin D
deficiency and exposure to environmental toxins (19, 20). Most attention has focused on chronic
paramyxovirus infection as a possible disease trigger based on the identification of nuclear inclusions in
osteoclasts that were thought to resemble measles virus (21). More recent evidence suggests that these
structures are not viral particles (22) but may instead be abnormal protein aggregates due to defects in the
autophagy pathway (23). Attempts to detect evidence of paramyxovirus nucleic acids and proteins in
patient material have yielded conflicting and inconclusive results (22, 24-30). Finally, a recent large scale
study showed no evidence of an enhanced immune response to paramyxoviruses in PDB as one would
expect if a slow virus infection was indeed the cause (31). A novel component of the present proposal will
be to investigate the role of the microbiome in PDB. Changes in the intestinal microbiome have been
implicated in oestrogen deficient bone loss (32, 33) and bone lesions in chronic multifocal osteolysis (34). It
therefore possible that changes in the microbiome could account for changes in severity of PDB over recent
decades but this has not so far been investigated. Calcium and vitamin D deficiency which have also been
linked to the risk of PDB through epidemiological studies (19, 35). The presumed mechanism is through
secondary hyperparathyroidism in a susceptible host but this has not been studied experimentally.
Anecdotal reports exist on localization of PDB with repetitive use of specific bones and/or skeletal trauma
(36, 37) suggest that biomechanical factors are important but again this has not been investigated
experimentally.
Advancing knowledge beyond current state of the art
The present programme aims to significantly advance knowledge beyond current state-of-the-art by
identifying novel genetic variants for the disease and exploring the functional effects of these variants on
bone remodelling. The programme will also examine potential environmental triggers for the disease
experimentally both in clinical studies and preclinical models and investigate how these factors interact
with genetic background to cause the disease and influence severity. The research programme is important
on several counts:
• The predisposing genetic variants for PDB will be used to develop a diagnostic test for susceptibility
to PDB which will be used to prioritise people at risk for enhanced surveillance and early
therapeutic intervention, thereby improving outcome
• The functional studies will provide new insights into the fundamental mechanisms by which bone
turnover is regulated and potentially uncover new therapeutic targets for PDB and other bone
diseases
• The studies of environmental influences will provide information on exposures and dietary changes
that could be modified in patients at risk of the disease to reduce the likelihood of it occurring.
Section 1. Identifying novel genetic variants for Paget’s disease
An extended genome wide association study will be performed. In parallel, genome sequencing will be
conducted in a subset of 100 individuals with an extreme phenotype (details below). The total sample size
of PDB cases available for the extended GWAS is now 4337 (through recruits from the ZIPP study and
collaborative centres in Australia, New Zealand, Italy, Spain, Belgium and Hungary). This is almost double
the size of cases compared with our previous GWAS (7). The number of controls will be increased to 11646,
largely by utilizing Biobank UK. Previously, we had power to detect common variants with an effect size of
about 1.4 (7) but the extended GWAS gives 82% power to detect alleles with an effect size of about 1.2 or
greater. We estimate that the extended study will double the number of associated loci to about 14. For
the GWAS we will use a custom Illumina chip available through our collaborator Prof at
Erasmus MC, Rotterdam which contains 650,000 variants optimized for GWAS and 50,000 custom variants
selected because they are predicted to cause damaging coding changes in exons or because they have
previously been identified in PDB. The custom content includes all reported mutations in SQSTM1, RIN3
and other high penetrance genes such as VCP, HNRNPA and ZNF678. The genome sequencing will be
performed in 100 “extreme” cases selected of the basis that they have an early age at onset (<55 years) and
a positive family history (in the absence of SQSTM1 mutation or mutation in a known gene). Analysis of the
sequencing data will focus on damaging variants that are over represented in the PDB cases versus four
matched controls who have previously undergone genome sequencing. This procedure is expected to be
successful in identifying rare high penetrance variants for the disease in new susceptibility genes. In order
to screen for somatic mutations whole genome sequencing will be conducted on leukocyte DNA and DNA
extracted from affected bone in patients undergoing joint replacement surgery. We will adapt methods
previously used in the analysis of somatic mutations in breast cancer (38) to determine if somatic “driver”
mutations of PDB associated genes (or other genes that regulate osteoclast activity) can account for
localisation of the disease.

Section 2. Stratifying people at risk of PDB by genetic profiling
The aim of this part of the project will to develop a genetic profiling test for susceptibility to PDB suitable
for use in routine clinical practice. The genetic profiling will comprise all common variants associated with
PDB at a genome-wide significant level from the extended GWAS, new variants associated with PDB that
are discovered through genome sequencing and known mutations in SQSTM1 and other monogenic forms
of PDB. The performance of these genetic markers in identifying patients who go onto develop PDB will be
evaluated in a prospective clinical study. We will recruit to this study by contacting a cohort of more than
1183 subjects aged between 50 and 70 with a family history of PDB identified through the ZiPP study whose
parents tested negative for SQSTM1. We will invite these individuals to take part with the aim of recruiting
at least 500 into the study. Those who consent will be invited to undergo phenotyping for PDB by
radionuclide bone scan, targeted radiographs (where bone scans show evidence suspicious of PDB) and
biochemical markers of bone turnover. Genotyping for alleles associated with PDB will be performed by
LGC Genomics using KASP assays. The same variants will be typed in SQSTM1 positive subjects from the
ZIPP study who are currently under follow-up to determine if addition of common variants to SQSTM1
mutations can enhance prediction in this very high-risk group. Associations between variants and emergent
PDB and their predictive value will be assessed by allele scoring methods and receiver-operator curve
analysis as shown in Figure 3. Further prospective validation of the role of genetic profiling in diagnosis of
PDB in the general population will utilize UK Biobank which is a population based cohort study of 500,000
people where GWAS and exome sequencing data is available. Based on current estimates (15) we expected
that about 1500 people within this cohort will develop clinical signs of PDB with age, although the number
with “silent” PDB is expected to be closer to 5000. These cohorts will provide new clinically relevant
information on the value of genetic profiling in correctly identifying emergent PDB in people at heightened
risk of the disease through family history both in the presence and absence of SQSTM1 mutations. The UK
Biobank study will provide additional information on whether it would be worthwhile to consider genetic
screening of unselected individuals from the general population.

This information will be of immediate clinical importance since when the ZIPP study reports in 2020 we will
know if zoledronic acid (ZA) therapy can prevent the development of lesions in people at high risk of PDB
because of SQSTM1 mutations. If the ZIPP study showed benefit of ZA people with SQSTM1 mutations
enhanced surveillance coupled with prophylactic ZA treatment - which has a very prolonged duration of
action (2, 3) - could be rolled out to those at high genetic risk through inheritance of other variants.
Section 3. Conducting functional analysis of PDB loci as a new paradigm to understand bone remodelling
This part of the programme will conduct functional analysis of genetic variants that are associated with PDB
to gain greater understanding of the fundamental processes bone remodelling. The methodology will
involve using bioinformatic approaches to determine if the identified variants from GWAS and sequencing
are likely to be directly pathogenic or to determine if they are acting as markers for pathogenic variants
nearby. This will entail checking to determine if variants act as eQTL for nearby genes; to determine if they
are in linkage disequilibrium (LD) with potentially damaging protein coding variants nearby and to
determine if the implicated genes represent plausible candidates for PDB. We have already done that
successfully in analysis of the OPTN locus (17) and RIN3 locus (39). In the event that no genes within regions
of interest emerge as strong candidates we will evaluate publically available Hi-C data (40) to investigate
the possibility that associations might be mediated by a long-range regulatory effect on a candidate gene
distant from the region of association. When potential candidate genes have been identified their effects
on osteoclast and osteoblast differentiation and function will be assessed by over-expression and/or
knockdown in vitro, coupled with studies in vivo using mice with targeted inactivation or point mutations of
the genes in question followed by skeletal phenotyping by microCT and histomorphometry using methods
that are established in our laboratories (17, 41-44).

Section 4. Investigating environmental triggers for PDB
This part of the programme will investigate the role of environmental factors in PDB focusing on the role of
the microbiome, dietary calcium deficiency and mechanical loading. Both preclinical and clinical approaches
will be employed.

4
The microbiome in Paget’s disease
We investigate the hypothesis is that the microbiome influences evolution of PDB by preclinical and clinical
studies. The preclinical studies will use the P394L mouse model which mimics the human disease (41). This
will involve comparing evolution of PDB-like lesions in P394L-/- mice raised under germ-free (GF) conditions
as described (45) with those raised under normal conditions and subsequently evaluating the effects of the
probiotic preparations (33, 46) on the development of bone lesions. The effects of these interventions on
lesions will be evaluated at 12 months by microCT and histological analysis of the lower limbs and spine, by
an observer blinded to treatment allocation. Each experiment will involve 20 mice per group, which, even
allowing for 10% of animals unavailable for analysis, would provide 80% power to detect a 1 standard
deviation (SD) difference in lesion severity score between groups. We will also determine if GF conditions
or probiotic treatment influences osteoclast formation in bone marrow cultures and evaluate the potential
mechanisms responsible by measurement of serum serotonin, and serum levels of cytokines and
expression of mRNA for relevant cytokines in bone marrow samples.
The clinical studies will be conducted in carriers of SQSTM1 mutations taking part in the ZIPP study and in a
subset of individuals from the cohort of 500 SQSTM1 negative subjects taking part in the genetic profiling
study described in Section 2. Participants from both of these studies will be invited to take part in this study
which aims to examine the interactions between diet, the microbiome and genotype in predicting the
presence and progression of bone lesions. We expect to be able to identify a total of about 50 subjects with
early PDB lesions in these two cohorts combined. The role of the microbiome as a determinant of lesion
development will be evaluated by matching the subjects with lesions with subjects derived from the same
cohort by age, gender, SQSTM1 mutation type and risk allele score who do not have without lesions.
Microbial gene richness will be evaluated using next generation sequencing of faecal DNA from each
subject using methods as described (47) and these gene frequencies (which represent microbial gene
profile of an individual) will then be related to presence or absence of PDB lesions with the aim of
determining if there are differences in microbial gene count between the groups. Further analysis will also
be performed using logistic regression to determine if other relevant variables such as serum 25(OH)D
levels and diet (dietary calcium and other nutrients) influence lesion development or microbial gene count.
A prospective clinical study will also be conducted to evaluate the effect of probiotics on biochemical
markers of bone turnover (ALP, PINP and CTX) in asymptomatic people with early PDB lesions. This will
involve randomizing patients with early asymptomatic PDB to receive probiotics or placebo with
measurement of changes in PINP (the most sensitive marker of activity in PDB (48)) for 3 months. The study
will involve 20 patients per group which provides 80% power to detect a 1SD difference between groups in
PINP levels. It will provide information on whether probiotics might represent a lifestyle strategy to reduce
the risk of progression of PDB for those at increased genetic risk of developing the disease.
Dietary calcium
We investigate the hypothesis is that dietary calcium deficiency influences evolution of PDB by preclinical
and clinical studies. The preclinical studies will use the P394L mouse model, but in this case the
intervention will be a low calcium diet versus normal diet between weaning and 8 weeks followed by
microCT assessment for lesion score at 8 months as described. The study will be powered to detect a 1 SD
increase in severity score and will involve 20 mice per group. The clinical studies will similarly focus on
asymptomatic people with early PDB lesions but evaluate the effects of normal diet versus high calcium
diet (1000mg daily) to determine if calcium supplementation can modify biochemical markers of bone
turnover in early PDB. Design and sample size will be similar to that for the probiotic human studies.
Mechanical loading
The role of mechanical loading on localisation of PDB lesions will be studied in the P394L-/-model (41). For
these experiments P394L-/- mice (20 per group, providing 90% power to detect a 20% increase in lesion
severity) will be undergo sub repetitive loading of the tibia as described (49) on six occasions between 8
and 20 weeks. The unloaded tibia will serve as an internal control in each animal. If mechanical loading
does indeed play a role in localizing lesions we would expect an increase in number or size of lesions in the
loaded limb versus the control (unloaded) limb.
The importance of these studies is not only in providing new insights into disease mechanisms but also in
identifying possible lifestyle strategies that would be used to favourably influence progression of early
PDB in those at increased genetic risk of the disease.
References

1. Tan A, Ralston SH. Clinical Presentation of Paget's Disease: Evaluation of a Contemporary Cohort and
Systematic Review. Calcif Tissue Int. 2014;95(5):385-92.
2. Reid IR, Lyles K, Su G, Brown JP, Walsh JP, del Pino-Montes J, et al. A single infusion of zoledronic acid
produces sustained remissions in Paget disease: data to 6.5 years. J Bone Miner Res. 2011;26(9):2261-70.
3. Reid IR, Miller P, Lyles K, Fraser W, Brown JP, Saidi Y, et al. Comparison of a single infusion of
zoledronic acid with risedronate for Paget's disease. N Engl J Med. 2005;353(9):898-908.
4. Langston AL, Campbell MK, Fraser WD, MacLennan GS, Selby PL, Ralston SH. Randomised Trial of
Intensive Bisphosphonate Treatment Versus Symptomatic Management in Paget's Disease of Bone. J Bone
Miner Res. 2010;25:20-31.
5. Tan A, Goodman K, Walker A, Hudson J, MacLennan GS, Selby PL, et al. Long-Term Randomized Trial
of Intensive Versus Symptomatic Management in Paget's Disease of Bone: The PRISM-EZ Study. J Bone Miner
Res. 2017;32(6):1165-73.
6. Ralston SH, Albagha OM. Genetics of Paget's Disease of Bone. Curr Osteoporos Rep. 2014;12(3):263-
71.
7. Albagha OME, Wani S, Visconti MR, Alonso N, Goodman K, Cundy T, et al. Genome-wide association
identifies three new susceptibility loci for Paget's disease of bone. Nat Genet. 2011;43(7):685-9.
8. Ralston SH. Paget's disease of bone. Br Med J. 1993;306(6873):332-3.
9. Matthews BG, Naot D, Bava U, Callon KE, Pitto RP, McCowan SA, et al. Absence of somatic SQSTM1
mutations in Paget's disease of bone. J Clin Endocrinol Metab. 2009;94(2):691-4.
10. Merchant A, Smielewska M, Patel N, Akunowicz JD, Saria EA, Delaney JD, et al. Somatic mutations in
SQSTM1 detected in affected tissues from patients with sporadic Paget's disease of bone. J Bone Miner Res.
2009;24(3):484-94.
11. Siris ES, Ottman R, Flaster E, Kelsey JL. Familial aggregation of Paget's disease of bone. J Bone Miner
Res. 1991;6:495-500.
12. Albagha OM, Visconti MR, Alonso N, Wani S, Goodman K, Fraser WD, et al. Common susceptibility
alleles and SQSTM1 mutations predict disease extent and severity in a multinational study of patients with
Paget's disease. J Bone Miner Res. 2013;28:2238-46.
13. Guay-Belanger S, Simonyan D, Bureau A, Gagnon E, Albert C, Morissette J, et al. Development of a
molecular test of Paget's disease of bone. Bone. 2016;84:213-21.
14. Hippisley-Cox J, Coupland C. Predicting risk of osteoporotic fracture in men and women in England
and Wales: prospective derivation and validation of QFractureScores. BMJ. 2009;339:b4229.
15. van Staa TP, Selby P, Leufkens HG, Lyles K, Sprafka JM, Cooper C. Incidence and natural history of
Paget's disease of bone in England and Wales. J Bone Miner Res. 2002;17(3):465-71.
16. Vallet M, Ralston SH. Biology and Treatment of Paget's Disease of Bone. J Cell Biochem.
2016;117(2):289-99.
17. Obaid R, Wani SE, Azfer A, Hurd T, Jones R, Cohen P, et al. Optineurin Negatively Regulates Osteoclast
Differentiation by Modulating NF-kappaB and Interferon Signaling: Implications for Paget's Disease. Cell Rep.
2015;13(6):1096-102.
18. Corral-Gudino L, Borao-Cengotita-Bengoa M, Del Pino-Montes J, Ralston SH. Epidemiology of Paget's
disease of bone: A systematic review and meta-analysis of secular changes. Bone. 2013;55(2):347-52.
19. Siris ES. Epidemiological aspects of Paget's disease: family history and relationship to other medical
conditions. Semin Arth Rheum. 1994;23(4):222-5.
20. Lever JH. Paget's disease of bone in Lancashire and arsenic pesticide in cotton mill wastewater: a
speculative hypothesis. Bone. 2002;31(3):434-6.
21. Rebel A, Malkani K, Basle M, Bregeon C, Patezour A, Filmon R. Ultrastructural characteristics of
osteoclasts in Paget's disease. Rev Rhum Mal Osteoartic. 1974;41(12):767-71.
22. Helfrich MH, Hobson RP, Grabowski PS, Zurbriggen A, Cosby SL, Dickson GR, et al. A negative search
for a paramyxoviral etiology of Paget's disease of bone: molecular, immunological, and ultrastructural studies
in UK patients. J Bone Miner Res. 2000;15(12):2315-29.
23. Helfrich MH, Hocking LJ. Genetics and aetiology of Pagetic disorders of Bone. Archives Biochem
Biophys. 2014;473:172-82.
24. Matthews BG, Afzal MA, Minor PD, Bava U, Callon KE, Pitto RP, et al. Failure to detect measles virus
RNA in bone cells from patients with Paget's disease. J Clin Endocrinol Metab. 2008;93:1398-401.
6
25. Matthews D, Fry L, Powles A, Weissenbach J, Williamson R. Confirmation of genetic heterogeneity in
familial psoriasis. J Med Genet. 1995;32(7):546-8.
26. Ralston SH, Afzal MA, Helfrich MH, Fraser WD, Gallagher JA, Mee A, et al. Multicenter blinded analysis
of RT-PCR detection methods for paramyxoviruses in relation to Paget's disease of bone. Journal of bone and
mineral research : the official journal of the American Society for Bone and Mineral Research.
2007;22(4):569-77.
27. Gordon MT, Mee AP, Anderson DC, Sharpe PT. Canine distemper transcripts sequenced from pagetic
bone. Bone Miner. 1992;19:159-74.
28. Reddy SV, Singer FR, Mallette L, Roodman GD. Detection of measles virus nucleocapsid transcripts in
circulating blood cells from patients with Paget disease. J Bone Miner Res. 1996;11:1602-7.
29. Ooi CG, Walsh CA, Gallagher JA, Fraser WD. Absence of measles virus and canine distemper virus
transcripts in long- term bone marrow cultures from patients with Paget's disease of bone. Bone.
2000;27(3):417-21.
30. Birch MA, Taylor W, Fraser WD, Ralston SH, Hart CA, Gallagher JA. Absence of paramyxovirus RNA in
cultures of pagetic bone cells and in pagetic bone. J Bone Miner Res. 1994;9(1):11-6.
31. Visconti MR, Usategui-Martin R, Ralston SH. Antibody Response to Paramyxoviruses in Paget's
Disease of Bone. Calcif Tissue Int. 2017.
32. Jones RM, Mulle JG, Pacifici R. Osteomicrobiology: The influence of gut microbiota on bone in health
and disease. Bone. 2017.
33. Li JY, Chassaing B, Tyagi AM, Vaccaro C, Luo T, Adams J, et al. Sex steroid deficiency-associated bone
loss is microbiota dependent and prevented by probiotics. J Clin Invest. 2016.
34. Lukens JR, Gurung P, Vogel P, Johnson GR, Carter RA, McGoldrick DJ, et al. Dietary modulation of the
microbiome affects autoinflammatory disease. Nature. 2014;516(7530):246-9.
35. Barker DJ, Gardner MJ. Distribution of Paget's diease in England, Wales and Scotland and a possible
relationship with vitamin D deficiency in childhood. Br J Prev Soc Med. 1974;28:226-32.
36. Solomon LR. Billiard-player's fingers: an unusual case of Paget's disease of bone. British Medical
Journal. 1979;1(6168):931.
37. Hamdy R. Trauma and Paget's disease of bone. Br Med J. 1979;1(6176):1487.
38. Nik-Zainal S, Davies H, Staaf J, Ramakrishna M, Glodzik D, Zou X, et al. Landscape of somatic
mutations in 560 breast cancer whole-genome sequences. Nature. 2016;534(7605):47-54.
39. Vallet M, Soares DC, Wani S, Sophocleous A, Warner J, Salter DM, et al. Targeted sequencing of the
Paget's disease associated 14q32 locus identifies several missense coding variants in RIN3 that predispose to
Paget's disease of bone. Hum Mol Genet. 2015;24(11):3286-95.
40. Lieberman-Aiden E, van Berkum NL, Williams L, Imakaev M, Ragoczy T, Telling A, et al.
Comprehensive mapping of long-range interactions reveals folding principles of the human genome. Science.
2009;326(5950):289-93.
41. Daroszewska A, van't Hof RJ, Rojas JA, Layfield R, Landao-Basonga E, Rose L, et al. A point mutation
in the ubiquitin associated domain of SQSMT1 is sufficient to cause a Paget's disease like disorder in mice.
Hum Mol Genet. 2011;20(14):2734-44.
42. Sophocleous A, Landao-Bassonga E, van't Hof RJ, Idris AI, Ralston SH. The Type 2 Cannabinoid
Receptor Regulates Bone Mass and Ovariectomy-Induced Bone Loss by Affecting Osteoblast Differentiation
and Bone Formation. Endocrinology. 2011;152(6):2141-9.
43. Idris AI, Sophocleous A, Landao-Bassonga E, Canals M, Milligan G, Baker D, et al. Cannabinoid
receptor type 1 protects against age-related osteoporosis by regulating osteoblast and adipocyte
differentiation in marrow stromal cells. Cell Metab. 2009;10(2):139-47.
44. Idris AI, Van 't Hof RJ, Greig IR, Ridge SA, Baker D, Ross RA, et al. Regulation of bone mass, bone loss
and osteoclast activity by cannabinoid receptors. Nat Med. 2005;11(7):774-9.
45. Sjogren K, Engdahl C, Henning P, Lerner UH, Tremaroli V, Lagerquist MK, et al. The gut microbiota
regulates bone mass in mice. J Bone Miner Res. 2012;27(6):1357-67.
46. Ohlsson C, Engdahl C, Fak F, Andersson A, Windahl SH, Farman HH, et al. Probiotics protect mice from
ovariectomy-induced cortical bone loss. PLoS One. 2014;9(3):e92368.
47. Goodrich JK, Waters JL, Poole AC, Sutter JL, Koren O, Blekhman R, et al. Human genetics shape the
gut microbiome. Cell. 2014;159(4):789-99.
48. Al Nofal AA, Altayar O, BenKhadra K, Qasim Agha OQ, Asi N, Nabhan M, et al. Bone turnover markers
in Paget's disease of the bone: A Systematic review and meta-analysis. Osteoporos Int. 2015;26(7):1875-91.
7
49. De Souza RL, Matsuura M, Eckstein F, Rawlinson SC, Lanyon LE, Pitsillides AA. Non-invasive axial
loading of mouse tibiae increases cortical bone formation and modifies trabecular organization: A new model
to study cortical and cancellous compartments in a single loaded element. Bone. 2005.

Section c: Ten years track-record (max. 2 pages)

1. Key publications (from a total of 316 peer reviewed publications, H-Index 86)
1. Tan A, Goodman K, Walker A, Hudson J, MacLennan GS, Selby PL, Fraser WD, Ralston SH. Long-Term
Randomized Trial of Intensive Versus Symptomatic Management in Paget's Disease of Bone: The PRISM-EZ
Study. J Bone Miner Res 2017;32:1165-1173. (4 citations, impact factor 6.28).
2. Albagha OM, Visconti MR, Alonso N, Wani S, Goodman, K., Fraser, W. D., Gennari, L., Merlotti, D.,
Gianfrancesco, F., Esposito, T., Rendina, D., di, Stefano M., Isaia, G., Brandi, M. L., Guisti, F., Del Pino-
Montes, J., Corral-Gudino, L., Gonzalez-Sarmiento, R., Ward, L., Rea, S. L., Ratajczak, T., Walsh, J. P., and
Ralston, S. H. Common susceptibility alleles and SQSTM1 mutations predict disease extent and severity in a
multinational study of patients with Paget's disease. J Bone Miner Res. 2013;28:2238-46. (54 citations,
impact factor 6.28).
3. Albagha OME, Wani S, Visconti MR, Alonso N, Goodman K, Cundy T, Brandi ML, Chung PY, Dargie R,
Devogelaer JP, Falchetti A, Fraser WD, Gennari L, Gianfrancesco F, Hooper MJ, Van Hul W, Isaia G,
Nicholson GC, Nuti R, Del Pino MJ, Ratajczak T, Rea SL, Rendina D, Gonzalez-Sarmiento R, Di SM, Ward L,
Walsh JP, Ralston SH. Genome-wide association identifies three new susceptibility loci for Paget's disease
of bone. Nat. Genet 2011;43:685-689. (69 citations, Impact factor 27.95).
4. Daroszewska A, van't Hof RJ, Rojas JA, Layfield R, Landao-Basonga E, Rose L, Rose K, Ralston SH. A point
mutation in the ubiquitin associated domain of SQSMT1 is sufficient to cause a Paget's disease like disorder
in mice. Hum. Mol. Genet 2011;20:2734-2744, 3295 (74 citations, impact factor 5.34).
5. Sophocleous A, Landao-Bassonga E, van't Hof RJ, Idris AI, Ralston SH. The Type 2 Cannabinoid Receptor
Regulates Bone Mass and Ovariectomy-Induced Bone Loss by Affecting Osteoblast Differentiation and Bone
Formation. Endocrinology. 2011;152:2141-9. (84 citations, impact factor 5.45).
6. Albagha OM, Visconti MR, Alonso N, Langston AL, Cundy T, Dargie R, Dunlop MG, Fraser WD, Hooper MJ,
Isaia G, Nicholson GC, Del Pino MJ, Gonzalez-Sarmiento R, Di SM, Tenesa A, Walsh JP, Ralston SH (2010)
Genome-wide association study identifies variants at CSF1, OPTN and TNFRSF11A as genetic risk factors for
Paget's disease of bone. Nat. Genet 42:520-524. (126 citations, Impact factor 27.95).
7. Riches PL, McRorie E, Fraser WD, Determann C, van't Hof R, Ralston SH (2009) Osteoporosis associated
with neutralizing autoantibodies against osteoprotegerin. N Engl J Med 361:1459-1465. (70 citations, impact
factor 72.4).
Miner Res. 2010;25:20-31. (113 citations, inpact factor 6.28).
9. Idris AI, Sophocleous A, Landao-Bassonga E, Canals M, Milligan G, Baker D, van't Hof RJ, Ralston SH
(2009) Cannabinoid receptor type 1 protects against age-related osteoporosis by regulating osteoblast and
adipocyte differentiation in marrow stromal cells. Cell Metab 10:139-147 (100 citations, impact factor
18.14).
10. Ralston SH, Afzal MA, Helfrich MH, Fraser WD, Gallagher JA, Mee A, Rima B. Multicenter blinded
analysis of RT-PCR detection methods for paramyxoviruses in relation to Paget's disease of bone. J Bone
Miner Res 2007;22:569-577. (46 citations, impact factor 6.28).
Research Monographs
Ralston SH. Clinical practice. Paget's disease of bone. N. Engl. J. Med 2013;368:644-650 (84 citations, impact
factor 72.4)
Ralston SH. Pathogenesis of Paget's disease of bone. Bone 2008;43:819-825. (72 citations, Impact factor
4.14).
Scientific Leadership Profile
Professor Stuart Ralston has made numerous contributions to understanding the pathophysiology of a
range of bone diseases and in conducting clinical trials to improve the evidence base for treating bone
disease over the past 35 years. During the past 10 years, his research has focused on the genetic basis of
bone disease; the role of the endocannabinoid system in bone cell and on the pathogenesis and
management of Paget’s disease of bone

13
Genetics of Bone Disease. Ralston played a key role in founding the genetic markers for osteoporosis
consortium (GENOMOS) and in managing the Genetics for Osteoporosis consortium (GEFOS) and founded
the Genetics Determinant of Paget’s Disease consortium (GDPD). These consortia, have been instrumental
in advancing understanding of the genetic determinants of bone disease with outputs in high impact
journals including Nature, Nature Genetics, JAMA and the Annals of Internal Medicine.
Cannabinoids and bone. The Ralston laboratory were first to uncover the role of the endocannabinoid
system in bone metabolism showing that the type 1 and type 2 cannabinoid receptors play roles in
regulating osteoclast and osteoblast activity, peak bone mass and age-related bone loss as well as cancer
associated bone disease and osteoarthritis in mice. Outputs have appeared in Nature Medicine. Cell
Metabolism, Endocrinology, J Biol Chem, Osteoarthritis and Cartilage, Am J Med and Ageing Cell.
Paget’s disease of bone. Ralston has researched into Paget’s disease of bone (PDB) throughout his career,
working on the possible role of paramyxoviruses genetic determinants and treatment of PDB. Ralston’s
group pioneered the use of GWAS in PDB and have led the way in functional analysis of PDB-associated
genes including OPTN and RIN3. He is also one of the world-leading researchers into the treatment of PDB
having designed and led the Paget’s disease – Randomised trial of intensive and symptomatic management
(PRISM) trial and its extension (PRISM-EZ). These studies showed that intensive bisphosphonate therapy
has a limited impact in advanced disease provided impetus for the ZIPP trial http://www.controlled-
trials.com/ISRCTN11616770 which seeks to determine if genetic testing and targeted intervention with
bisphosphonate therapy can improve outcome in those at high risk of PDB because of SQSTM1 mutations.
Markers of esteem
Ralston was a member of the 2001 Research Assessment Exercise panel (UK), the Molecular and Cellular
Medicine Board of the MRC (2003-2008), the physiology and infections board of the MRC (1999-2001), and
the Oliver Bird Committee of the Nuffield Foundation (1992-1997). He was vice chair and member of the
research subcommittee of the Arthritis Research Campaign (2001 -2007); vice chair and member of the
Physiology & Pharmacology panel of the Wellcome Trust (2002 - 2005); and co-chair of the Career
Enhancement Awards (CEA) committee of the ASBMR (2007-2012). He also has served as a member and
currently is chair of the Commission for Human Medicines (CHM) of the Medicines and Healthcare Products
Regulatory Authority (MHRA) in the UK. Previously he served on various expert advisory groups to the MHRA
on biologicals, clinical trials and rheumatology. He also acted as chair or member of several special advisory
groups (SAG) to the European Medicines Agency.

Professor Ralston is regularly an invited plenary speaker at national and international symposia including
the American Society of Bone and Mineral Research; the Endocrine Society, the European Federation of
Endocrine Societies, The European League against Rheumatism, the European Human Genetics Society, The
International Bone and Mineral Society and the British Society of Rheumatology and the Bone Research
Society. In addition, he has delivered several named lectures including the Michael Mason Prize lecture of
the British Society of Rheumatology in 1997, the Alexander Fletcher Prize of the Royal College of Physicians
& Surgeons of Glasgow in 1987, the Goodall Memorial Lecture of the Royal College of Physicians and
Surgeons of Glasgow in 2002 and the Boy Frame Memorial Lecturer, at the Henry Ford hospital Detroit in
2002.

Professor Ralston is fully committed to the training of young scientists and clinicians. During his career, he
has successfully supervised 18 PhD students and 2 MD students to completion. He was lead for rheumatology
undergraduate teaching on the MB ChB course in Edinburgh between 2005 and 2012 and currently is
academic director of the online distance learning MSc in Clinical trials at Edinburgh. He was founding director
of Edinburgh Clinical Trials unit in 2009 and was academic director of the unit until October 2016.

Professor Ralston’s extensive contributions to research in the area of bone disease have been recognised by
his peers through the award of numerous prizes from national and international societies. They include
Laurence Raisz Award, of the ASBMR for translational research in bone disease; the Mike Horton Award of
the ECTS, the Harold Copp Award, of IBMS, the John B John B Johnston, of the Paget foundation; his election
to the Royal Society of Edinburgh in 2005; his election as a Fellow of the Academy of Medical Sciences in
1999 and his election as a fellow of the Royal College of Physicians of Edinburgh in 1994.
14

ERC Advanced Grant 2017
Research proposal [Part B2] 1
(not evaluated in Step 1)

Part B2: The scientific proposal (max. 15 pages)
Section a. State-of-the-art and objectives
Overview
The aim of this proposal is to advance knowledge into PDB by identifying new genes that cause the disease;
by developing a genetic profiling test to promote earlier diagnosis, by identifying the mechanisms by which
predisposing genes influence bone cell function and by investigating potential environmental triggers for the
disease. The individual components of this research programme will interact to provide new knowledge and
improve clinical outcome for patients with the PDB so that early diagnosis can be made and treatment given
before irreversible complications have developed.
1. Gaining new insights into the genetic basis of Paget’s disease
1.1 Genetics of PDB - state of the art
It is well recognised that genetic factors play a key role in Paget’s disease (1-3). Familial clustering is frequent
with a 7-fold increases risk of the disease in first degree relatives of patients as compared with controls (4,
5) and the disease is inherited in some families as an autosomal dominant trait with incomplete penetrance
(6-8). There are marked ethnic differences in susceptibility since the disease is common in the UK and many
other European countries (notably Spain and Italy) but rare in Asia and Africa (9). These differences in
susceptibility persist in migrants from UK and Europe to countries such as Australia, New Zealand and Canada
where the risk of PDB in the indigenous population is low (1, 10). At the present time twelve genes or loci
with evidence for association with PDB have been identified. However, for some of these genes, PDB is only
one component of a multisystem disease characterised by myopathy and neurodegeneration (Table 1).
Table 1. Genes and loci for PDB
Locus Syndrome Discovery Effect size Allele Likely Gene(s) Mechanisms Author
frequency* of PDB
1p13 PDB GWAS Moderate Common CSF1 Unknown Albagha (11)
1q42 PDB/ GCT Linkage Large Very rare ZNF678 Unknown Divisato (12)
5q35 PDB Linkage Large Rare SQSTM1 NFkB Laurin (13)
activation Hocking (14)
7p15 PDB/MSP NGS Large Very rare HNRNPA2B1 Unknown Kim (15)
7q33 PDB GWAS Moderate Common NUP205 Unknown Albagha (16)
8q22 PDB GWAS Moderate Common DCSTAMP Unknown Albagha (16)
9p13 IBM-PFD Linkage Large Very rare VCP Unknown Watts (17)
10p13 PDB GWAS & Moderate Common OPTN NFkB Albagha (11)
Linkage activation Obaid (18)
12q13 PDB/MSP NGS Large Very rare HNRNPA1 Unknown Kim (15)
14q32 PDB GWAS Moderate Common RIN3 Unknown Albagha (16)
Vallet (19)
15q24 PDB GWAS Moderate Common PML Unknown Albagha (16)
18q21 PDB GWAS Moderate Common TNFRSF11A Unknown Albagha (11)
GWAS- Genome wide association study; NGS – next generation sequencing; MSP- Multisystem proteinopathy; IBM-
PFD – inclusion body myopathy, Paget’s disease and frontotemporal dementia * In the general population.
Previous research into the genetic basis of PDB has been successful in providing new insights into the
molecular pathways that regulate bone remodelling. For example, discovery of mutations in SQSTM1 as a
1
Instructions for completing Part B2 can be found in the ‘Information for Applicants to the Advanced Grant 2017 Call’.
1
cause of PDB (13) uncovered a previously unsuspected role of SQSTM1 in regulating osteoclast activity
through effects on RANK induced NFkB signalling and its interaction with CYLD (20). Similarly, studies of the
10p13 locus (11, 21) identified OPTN as an important gene in the regulation of bone metabolism through
effects on the NFkB pathway and interferon signalling (18)
Other loci associated with PDB lie close to genes that play a role in osteoclast differentiation or activity. For
example, the susceptibility locus on the 1p13 locus is 87Kb upstream of CSF1 which encodes macrophage
colony stimulating factor, a protein that is essential for the early stages of osteoclast differentiation (22). The
8q22 locus lies close to the DCSTAMP gene which is essential for the formation of multinucleated osteoclasts
(23). At present, it seems likely that predisposition to PDB at these loci is mediated by variation in expression
of these genes, or by linkage disequilibrium with rare variants within the genes themselves (24) but further
research is needed to clarify this. For other loci such as 7q33 (NUP205), 14q32 (RIN3) and 15q34 (PML), the
mechanism by which PDB occurs is unclear since these genes have not previously been implicated in bone
metabolism. Mutations of the HNRNPA1/HNRNPA1B1 and VCP genes result in a multisystem proteinopathy
characterised by accumulation of abnormal protein aggregates in muscle and neurones, but the mechanisms
by which PDB occurs in these diseases remains unclear.
1.2 Genetics of PDB - advancing beyond state of the art
Progress has been made in understanding the genetics of PDB but many predisposing genes remain to be
discovered. This is because the GWAS studies that have so far been performed in PDB have had a relatively
modest sample size. The number of cases (discovery and replication) in our 2011 GWAS was 2223 (16) and
led to the identification of 7 genome wide significant loci. The GWAS sample size will be almost doubled to
4337 individuals and that is expected to double the number of loci based on previous experience in
osteoporosis (25, 26). Details of the populations that have been confirmed to take part in the extended GWAS
are shown in Table 2.
Table 2. Cohorts available for extended GWAS
Source Cases Controls Source of controls Collaborator
Discovery
PRISM study & UK Cases 749 2930 Biobank UK Various
ZIPP study 1024 4096 Biobank UK Various
Edinburgh PDB cohort 105 420 Biobank UK N/A
Subtotal 1878 7446
Replication
Belgium 246 263 Belgium (Antwerp) Dr W. Van Hul
Netherlands 85 93 Netherlands (Leiden) Dr W. van Hul
Spain 186 202 Spain (Salamanca) Dr J. Del Pino
Italy 1 354 390 Italy (Florence) Dr M.L Brandi
Italy 2 1008 1200 Italy (Siena) Dr L Gennari
Italy 3 95 102 Italy (Milan) Dr A Rubinacci
Hungary 89 100 Hungary (Budapest) Dr J Donath
Australia & New Zealand 398 1592 Biobank UK Dr M Hooper
Subtotal 2559 4200
Grand total 4337 11646
Standard methods will be used for the GWAS (16). In brief DNA samples in the discovery cohort will be
genotyped using an Illumina custom array by our collaborator at Erasmus MC,
Rotterdam. This contains 650K common markers optimised for GWAS and 50K custom variants including
damaging exonic mutations across the genome and all mutations so far identified in patients with PDB.
Genotype data from UK biobank will be used as control. Standard quality control measures will be applied as
described in the methodology section. Imputation will be performed using miniMach3 utilising the Haplotype
Reference Consortium (HRC) panel which provides better imputation accuracy than 1000 Genomes panel.
Allele frequencies will be compared between cases and controls to identify markers associated with PDB
using logistic regression. Replication of the top hits (p<1x10-5) will be sought in the Genetic Determinants of
Paget’s disease (GDPD) research consortium, a collection of PDB cases and matched controls from Australia,
Italy, Spain, Belgium and the Netherlands. Many of these centres participated in our previous GWAS but since
then we have recruited additional cohorts to GDPD from Milan and Hungary (Table 2).
Whole genome sequencing will also be employed to uncover rare variants in patients with familial PDB
where the casual gene has not yet been identified. This will complement the results of the GWAS. In the UK
about 40% of people with a family history of PDB carry a mutation in the SQSTM1 gene but in the remaining
60%, no causal gene has been identified. The sequencing effort will focus on 100 subjects selected because
there is a family history of the disease and early age at onset (<55 years) where the causal gene is unknown.
This sample size has power to detect causal variants with a frequency of about 0.005 and above. The genome
sequencing will be performed at a depth of 30x coverage using Illumina HiSeq machines in collaboration with
Edinburgh Genomics who will be employed as a subcontractor. Data from the PDB cases will be compared
with that from 400 controls derived from the Lothian Birth Cohort who have already undergone whole
genome sequencing using the same facility. After removing common SNP (>5%), and those already known to
be associated with PDB, bioinformatic studies will be performed to identify potentially functional variants
(primarily focusing on exons). The potentially functional SNP showing significant differences in frequency
between cases and controls will be further investigated by looking for evidence of transmission of PDB within
families and/or association with PDB in sporadic PDB cases from the GDPD cohort. This procedure is expected
to uncover new high penetrance variants for PDB as has been reported by other researchers using similar
technology (15, 27). Taken together, the GWAS and sequencing studies will identify new variants that
predispose to PDB. This knowledge will be further exploited in the genetic profiling experiments (section 2),
and in understanding of the biology of PDB and regulation of bone remodelling (section 3).
2. Improving outcome in PDB through genetic profiling and early intervention
2.1 Genetic profiling for PDB - state of the art
The rationale for genetic profiling is to permit earlier diagnosis in those at risk of the disease. This is urgently
needed since PDB is clinically silent for many decades before symptoms develop. By the time the patient
first presents, complications are often present (28) (Figure 1, A-C).
Figure 1. Complications of Paget’s disease at first presentation
A B C
80
ZIPP 90388-01 Panel A shows the
70
percentage of patients with
60
complications at first
50 presentation (from Tan &
Percent
40 Ralston (28)). Panel B and C

30 25%
show images from a patient
20 18% 18% in the ZIPP study who had
10 8% already developed severe
6%
cervical spine involvement
0
at recruitment.
Radionuclide X-ray cervical
bone scan spine
Symptom or complication

The importance of early detection of PDB is underscored by studies which show that many patients with
established disease have troublesome bone pain and impaired quality of life (29) which responds poorly,
even to intensive bisphosphonate therapy (29-31). Since modern bisphosphonates are highly effective in
reversing the abnormalities of bone turnover that are characteristic of PDB (32, 33), there are compelling
reasons to try and devise methods to permit earlier diagnosis so that treatment can be given at an early stage
in disease evolution.
Genetic profiling shows promise but needs further evaluation. The predisposing alleles identified by GWAS
account for about 13% of the population attributable risk of the disease but so far have only been tested
using a case control design in patients already known to have PDB (16). There is evidence that the GWAS
variants when combined with SQSTM1 mutations are associated with extent and severity of established PDB
in several populations (34, 35). It has also been reported SQSTM1 mutations when combined with GWAS
markers has a sensitivity of about 81% and specificity of 51% for detecting PDB (36) but again, this has only
been evaluated using a case control design in patients already known to have PDB.
2.2 Genetic profiling – advancing beyond state of the art

While the existing data shows promise for genetic profiling in the identification of people at risk of the disease
this needs to be confirmed by prospective studies of patients not known to have PDB.
This part of the project will determine whether genetic profiling can be used to predict the presence or
emergence of PDB in prospective studies. This will be conducted in three cohorts.
1. Carriers of SQSTM1 mutations already taking part in the ZIPP study (n=221)
2. A new cohort of subjects with a family history of the disease, but where screening of the probands
for SQSTM1 mutations was negative (n=500)
3. A population based cohort of subjects from UK Biobank (n=500,000)

2.2.1 ZIPP cohort SQSTM1 mutation carriers
Stored samples of DNA from ZIPP participants will be genotyped for new risk alleles to emerge from the
extended GWAS and genome sequencing by LGC Genomics using KASP technology. A risk allele score will be
constructed as previously described (16) and related to the presence of PDB lesions at baseline and
emergence of new PDB lesions in placebo treated subjects at the end of study (scheduled for 2018-2019).
We will evaluate the performance of the genetic risk score using a receiver-operator curve (ROC) analysis
and calculate sensitivity, specificity, positive and negative predictive value using standard methodology. This
study will determine whether GWAS risk alleles add usefully to SQSTM1 mutations in predicting the
emergence of PDB.
2.2.2 ZIPP cohort SQSTM1 negative subjects
During recruitment to the ZIPP study we screened for SQSTM1 mutations in 1431 probands with PDB who
had unaffected children. Of these, 1183 (82.7%) tested negative for SQSTM1 mutations, but consented to be
contacted for further studies. We will contact these 1183 families once again and invite the children (who
will now be aged 55 on average) to take part in a prospective study to evaluate the performance of genetic
profiling in predicting the development of PDB in SQSTM1 negative families. The aim will be to recruit 500
subjects with the expectation that about 5% may have evidence of early PDB on bone scans. Genotyping will
be performed as above, a risk allele score will be constructed (16) and related to the presence of PDB lesions,
ascertained by radionuclide bone scan and x-ray. In the longer-term participants in this study will be invited
to undergo further follow up and phenotyping after an additional 5 years, but funding will be applied
separately for this. This study will determine whether genetic profiling is of value in predicting the
development of PDB in people who are at increased risk because of a family history but who do not have
SQSTM1 mutations
2.2.3 UK Biobank
Participants of UK Biobank have already undergone GWAS and are currently undergoing genome sequencing.
These subjects can be considered representative of the general population in the UK. For this cohort, the
presence of susceptibility alleles will be related to the emergence of a clinical diagnosis of PDB ascertained
as part of routine clinical care. For this cohort of subjects, we will screen subjects in the UK Biobank database
for an emergent diagnosis of PDB. Performance of the genetic markers in predicting the onset of PDB will be
evaluated using the methods described above. Each case will be matched with at least 10 controls where
PDB has not developed. This study will determine if genomic profiling is likely to be a clinically useful
procedure in predicting the onset of PDB in the general population.
Information from these three cohorts will provide novel and clinically relevant information on the feasibility
of using genetic profiling to predict the development of PDB in patients at high, medium and low risk. If
genetic profiling is found to be of value in predicting disease, then early therapeutic intervention may be
directly implemented if the ZIPP study shows that early intervention is beneficial.

3. Role of somatic mutations in PDB
3.1 Somatic mutations in PDB - state of the art
The focal distribution of PDB has puzzled clinicians for decades. One theory, supported by anecdote is that
repetitive mechanical loading or traumatic events early in life may determine the sites of involvement (37,
38). This possibility will be addressed in Section 5 of the proposal. Another possibility to be addressed here
that was suggested by the applicant many years ago (39), is that PDB lesions develop because of somatic
mutations in affected bone, in effect, the Knudsen two hit hypothesis (40). This possibility of somatic
mutations in PDB has been investigated by two research groups. In both cases, the analysis was limited to
determining if the P392L mutation of SQSTM1 is present in affected bone. The results were conflicting with
positive results in one study in 2 out of 5 patients (41) whereas another study involving 28 samples from 22
patients, found no evidence of somatic mutations (42).
3.2 Somatic mutations in PDB- advancing beyond state of the art
The objectives of this component of the research programme will be to determine if somatic mutations are
present in genes that might be expected to cause osteoclast activation in affected tissue. This analysis will
not only focus on candidate genes implicated in PDB (as previously) but rather will be conducted on a genome
wide basis. The study will involve obtaining paired blood samples, and samples of affected bone in PDB
patients undergoing orthopaedic procedures as part of normal clinical care. We already have affected tissue
and leukocyte DNA available from 8 PDB subjects recruited locally but the numbers will be increased aiming
for a total of 20 subjects by new recruitment during the course of the project. We will determine if bone is
affected primarily by the radiographic appearances prior to surgery but samples will also be obtained at
surgery and sent for histology to confirm affection status. We will extract DNA from the tissues using standard
methods and perform whole genome sequencing to catalogue the mutational spectrum in affected tissue
and peripheral blood. The techniques to be used will be adapted from those that have been employed to
identify somatic mutations in breast cancer (43). In essence, the procedure involves using filtering tools to
identify variants (as well as chromosomal duplication or deletions) are present in affected tissue but not in
leukocyte DNA. The variants and regions of interest will then be prioritised for further analysis using
bioinformatic techniques to determine if the variants are likely candidates for local increases in bone
remodelling (by focusing on variants within genes implicated in PDB or those in pathways that influence
osteoclast differentiation or function). Subsequently, variants of interest will be evaluated using various
bioinformatic tools (19). Those considered to be potentially pathogenic on the basis of these initial screens
will be functionally assessed for effects on osteoclast and osteoblast activity using the methods described in
section 4.
4. Functional analysis of PDB loci as a new paradigm to understand bone remodelling
4.1 Functional analysis of PDB genes - state of the art
The mechanisms by which SQSTM1 and OPTN variants regulate bone metabolism have been investigated.
While the pathways by which mutations in HNRNPA2B1, HNRNPA1, VCP and ZNF678 affect bone cell function
are less well understood these are under investigation by other researchers and will not be considered
further here. Instead, the functional analysis will focus on the GWAS loci already identified and the new loci
which will be uncovered as the result of the extended GWAS and sequencing efforts. For the 1p13 locus, the
region of strongest association lies in a putative enhancer region for CSF1 suggesting that the predisposing
variants might regulate CSF1 mRNA expression, but this remains to be established. For the 18q21 locus,
TNFRSF11A (which encodes the Receptor Activator of NFkB – RANK) is the most likely candidate but the
mechanisms by which variants regulate RANK activity are not well understood. In one study the pA192V
coding variant of TNFRSF11A was found to increased NFkB signalling in HEK cells but only when co-
transfected with SQSTM1 expressing constructs (44). In another study, no difference in NFkB signalling was
observed following transfection of constructs with different variants at rs35211496 (encoding pH141Y) and
rs1805034 (encoding pA192V) into HEK cells (45). The functional mechanisms by which variants at the
DCSTAMP locus predispose to PDB have not been explored, but a gain of function mechanism that promotes
fusion of osteoclast precursors seem likely (23). For the 14q32 locus, there is compelling evidence that RIN3
is the candidate (19) . The RIN3 gene product has been shown to be expressed at an mRNA and protein level
in osteoclasts and various missense mutations identified which are associated with PDB (19). However, the
functional consequences of these mutations are not yet clear and the signalling mechanisms by which RIN3
regulates bone cell function are unknown. For the 15q24 locus we have evidence that PML is the candidate
(rather than other genes in the locus) but the mechanism by which PML regulates bone cell function remains
to be identified.
4.2 Functional analysis of PDB genes – advancing beyond state of the art
The objectives of this component of the research programme are to define the mechanisms by which variants
at known PDB-associated loci and the new loci which we will discover as the result of the extended GWAS
and sequencing studies influence bone remodelling. An overview of the approach is illustrated graphically in
Figure 2 with more detailed discussion below and in the methodology section.

Figure 2. Approaches to be used for functional analysis of PDB loci
Identify variants in
Identify potentially
genome wide GWAS NGS pathogenic variants
significant loci
Test variants for Variants associated with PDB
association with PDB in and/or transmitted in
case control analysis families?
Variants associated with Bioinformatic analysis to prioritise

Yes Yes No
PDB? candidate variants for further study
No
Is candidate gene linked to Deprioritise
variant known to have role in or discard
Deprioritise bone metabolism?
or discard No
Yes No
• Gene expression (eQTL) • Expression in bone cells

• Testing of regulatory variants Evaluate mechanisms by Evaluate role of • Over-expression &
• Over-expression of protein which variant(s) alter gene candidate gene in knockdown in bone cells
coding variants expression or function bone • Phenotype mice with gene
• Mouse models knockout
Yes
Is there evidence that
gene plays a role in No
Outcome bone?
Advances in understanding
PDB and role of associated Deprioritise
genes in bone metabolism or discard

4.2.1 Identifying candidate genes in the regions of interest
The first step in analysis of new loci will be to perform association studies with known variants within the
region identified through resources such as the 1000 genomes project, ExaC and the genome sequencing in
PBD cases. Those that are confirmed to be over-represented in PDB cases versus controls will be evaluated
for possible functionality by bioinformatic techniques such as SuRFR (46) which integrates information from
the ENCODE and FANTOM projects and additional tools such as SIFT, PolyPhen-2, Condel, MutationTaster
(19). In addition to the eQTL data that are publically available we will determine if variants of potential
interest are eQTL for expression of genes within the locus in an expression database that we have generated
in-house based on GWAS and RNA sequencing of short-term bone marrow cultures from 165 healthy
individuals. This is an important resource since data on gene expression in bone cells and bone tissue is sparse
on publically available databases. The outcome of this procedure will be to identify variants and candidate
genes linked to these variants that contribute to the PDB phenotype. For genes not known to influence bone
metabolism (for example those in the 7q33 locus), further steps are described in 4.2.2. For genes that are
known to regulate bone metabolism (for example CSF1, TNFRSF11A) further steps are described in 4.2.3
4.2.2 Defining the effects of novel candidate genes on bone metabolism
If the initial analysis suggests that the potential candidate gene is not known to play a role in bone
metabolism then the first step will be to define whether or not the gene of interest affects bone cell function.
This will involve looking for evidence that the gene is expressed in bone or bone cells at the RNA or protein
level. This will be done by quantitative PCR of cDNA prepared from whole bone, bone marrow and cultured
osteoclasts and osteoblasts and also by immunohistochemistry (19). If the gene is expressed in bone the
next step will be to create an expression construct and over express the gene of interest in RAW 247.1 cells
(to assess effects on osteoclast differentiation) or MC3T3-E1 cells (to assess effects on osteoblast
differentiation or function). In parallel, studies will be conducted to determine if knockdown of expression of
the target gene in the same cell types by siRNA affects osteoclast or osteoblast function. If these experiments
point to a significant effect on bone cell function the gene will be prioritised for in vivo studies by examining
mice with targeted inactivation of the gene of interest for abnormalities in bone metabolism. In most cases
it is expected that mutant mice will be available through ongoing initiatives such as the international mouse
phenotyping consortium. In the event that genetically modified mice are not available we would be able to
generate them in house using the gene targeting facility at our institute. Phenotyping will include analysis of
RANKL and M-CSF induced bone marrow cultures (47, 48) in wild type and mutant mice to determine if
inactivation of the gene affects osteoclast differentiation or bone resorption in vitro and performing
osteoblast cultures to determine if differentiation and function are affected. Depending on the results of
these studies, bone marrow co-cultures (49) may also be performed to determine if any effects observed
reside in the osteoclast or osteoblast lineage. All of these techniques are in routine use within our
laboratories (18, 50). Finally, skeletal phenotyping of the mutant mice will be performed using microCT and
bone histomorphometry of wild type and mutant mice at 52 weeks to determine if there are any differences
in bone mass and bone turnover between wild type and mutant mice or if focal PDB-like bone lesions develop
in mutant mice with ageing (51).
4.2.3 Defining the mechanisms by which predisposing variants affect gene function
This part of the programme will investigate the mechanisms by which variants within or close to candidate
genes modulate gene function to cause PDB. These approaches will be used as a first step for genes known
to play a role in bone metabolism (such as CSF1 and TNFRSF11A) and as a second step for genes where effects
on bone metabolism have been demonstrated by the procedures described in 4.2.2. Candidate variants will
be evaluated to determine if they are located in genomic regions with features that suggest a role in gene
regulation (using SuRFR and ENCODE resources) and also to examine whether the variants are eQTL for
expression of candidate genes within the locus (or trans-eQTL for other genes). If evidence is gained to
support an effect on gene regulation, further mechanistic studies will be conducted. Several complementary
approaches will be used. One will be to use genome editing with CRISPR/Cas in a relevant cell line to
introduce the variant of interest into the genome (or to delete the region encompassing the variant). If
changes in gene expression are demonstrated, this will provide proof of principle that the variants have a
regulatory function. Additional approaches that may be used to assess regulatory variants will include Chip-
Seq, gel shift assays and reporter assays in vitro to evaluate the effects of specific variants on transcription
factor binding and gene regulation and quantitation of gene expression in cultured bone cells (either
osteoclast like cells or osteoblast like cells) from patients of different genotype. Allele specific expression will
be assessed in heterozygotes for the variants of interest in a suitable cell type. We have previous experience
in all of these techniques (52, 53).
If there is no evidence to support a regulatory effect of variants and bioinformatic evidence suggests that the
associated variants directly alter protein sequence or are in linkage disequilibrium with variants that alter
protein sequence a different series of experiments will be performed. Missense variants will be evaluated
by generating an expression construct in a suitable vector. The effect of the variant allele will be compared
with over expression of the wild type allele using readouts such as generation of multinucleated osteoclast
like cells (RAW 247.1) or bone nodule formation under mineralising conditions (MC3T3-E1). Depending on
the identity of the gene, further experiments may be performed, such as to assess the effect of wild type and
mutant variants on NFkB signalling following RANK stimulation using reporter assays. Additional experiments
may be required (depending on the gene that is being assessed) to identify interacting molecules and to
evaluate the signalling mechanisms by which variants of interest affect bone cell function. Precise details will
depend on the nature of the interaction and the function of the interacting protein but will follow the general
approaches used in previous studies from our laboratory (48, 54, 55).
5. Exploring Environmental determinants of Paget’s disease
5.1 Environmental determinants of PDB - state of the art
Although susceptibility to PDB is strongly influenced by genetic background, environmental exposures also
exert an important influence. Evidence of this comes from epidemiological studies which have shown
significant decreases in disease prevalence and severity in most countries over the past 25 years (9, 56-59).
It is of interest, however, that in the Campania region of Italy, the severity has increased (60) and in some
regions of Spain, no major changes in prevalence or severity of PDB have been observed (61). The reasons
for this are unclear. Various factors have been suggested as possible triggers for PDB including dietary
calcium or vitamin D deficiency and exposure to environmental toxins (62, 63), repetitive biomechanical
loading of affected bones and local trauma (38, 64). The only environmental factor that has been studied
experimentally is chronic paramyxovirus infection. This was suggested as a possible disease trigger based
on the identification of nuclear inclusions in osteoclasts that were thought to resemble measles virus (65).
However, more recent work has shown that these structures are morphologically distinct from measles
virus particles on ultrastructural analysis (66) and experimental evidence has been gained to suggest that
they may be abnormal protein aggregates most likely due to defects in the autophagy pathway (67).
Furthermore, attempts to detect evidence of paramyxovirus nucleic acids and proteins in patient material
have yielded inconclusive results (66, 68-74) and a recent large scale study showed no evidence of an
enhanced immune response to paramyxoviruses in PDB as one would expect if a slow virus infection was
the cause (75). Although experimental studies have shown that targeted over expression of measles virus
nucleocapsids protein to osteoclasts can result in abnormalities of osteoclast function and bone turnover
(76) over-expression of other viral proteins can do the same (77). Given the fact that there have been
repeated unsuccessful attempts to detect viral mRNA in patient material using highly sensitive quantitative
PCR assays by several independent research groups and that only one group has detected measles virus, I
believe that further research along these lines is unlikely to be fruitful.
5.2 Environmental triggers – advancing beyond state of the part
The studies in this section of the grant proposal will address the novel hypothesis that diet associated
changes in the intestinal microbiome influence the development and severity of PDB as well as conducting
experimental studies to investigate the effects of dietary calcium deficiency and biomechanical loading on
evolution of the disease. There has recently been interest in the role of the microbiome in the regulation of
bone loss associated with oestrogen deficiency in animal models (78, 79). Sjögren and colleagues found
that germ free mice had higher bone mass and reduced bone resorption compared with mice on a normal
diet and that the animals on a normal diet had increase expression of pro-inflammatory cytokines in the
bone marrow (80). In another study by Li and colleagues (79), oestrogen deficiency was found to increase
gut permeability, increase numbers of Th17 cells and upregulate production of the osteoclastogenic
cytokines IL-17, TNF, and RANKL in the gut and bone marrow. These changes were abrogated by
sterilisation of the gut and by administration of probiotics. Of relevance to PDB, diet induced changes in
the microbiome have also been shown to influence development of osteolytic lesions in mice with chronic
multifocal osteolysis (CMO) a focal bone disease caused by mutations in the Pstpip2 gene (81). These mice
develop an inflammatory bone disease resembling osteomyelitis, driven by increases in production of IL-1b.
The bone lesions in these mice were substantially ameliorated by antibiotic therapy or by introduction of a
diet rich in saturated fats and cholesterol which altered the gut flora. A key component of the present
proposal, discussed in more detail below will be to study the novel hypothesis that the intestinal
microbiome may play a role in PDB.
5.2.1 Role of the microbiome in a mouse model of PDB
This will be investigated in the P394L-/- model of PDB (51) where we will seek to determine if manipulating
the intestinal microbiome by antibiotics or probiotics can influence the development of bone lesions. We
have already reported that about 95% of these mice develop characteristic PDB like bone lesions by 12
months of age which can be detected by microCT of the lower limbs (51). The experiment will involve
raising P394L-/- mice under germ-free (GF) conditions and comparing the evolution of lesions with those
raised under normal conditions using a protocol similar to that described by Sjögren and colleagues (80).
Female P394L-/- mice will be followed for 12 months, at which point the experiment will be terminated and
lesions evaluated by microCT and histology by an observer blinded to treatment allocation. The experiment
will involve 20 mice per group which, even allowing for 10% animals lost or unavailable for analysis, would
provide 80% power to detect a 1 standard deviation reduction in lesion severity score (equivalent to an
absolute difference in lesion score of 30%) between GF and control groups. In addition to the microCT and
histology, RANKL and M-CSF stimulated bone marrow cultures will be prepared from both groups of mice at
termination of the experiment to determine if the GF environment can modify the osteoclastogenic
potential of bone marrow cells. To identify the potential mediators responsible, we will also measure
serotonin, TNF, IL-1, IL-6, MCSF, IL-17, RANKL and OPG on serum obtained by cardiac puncture and study
expression of mRNA in bone marrow for relevant cytokines (TNF, IL-1, IL-6, IL-17, RANKL, OPG and MCSF). If
this experiment confirms that GF conditions can ameliorate the development of PDB like lesions, additional
experiments will be performed to determine if administration of probiotics to mice reared on a normal diet
can also modify the number or severity of lesions in the same model. The probiotic intervention experiment
will involve randomising P394L-/- mice to a diet supplemented with the VSL#3 which contains 8 different
strains of live bacteria (79). Heat sterilised VSL#3 supplements will be used in the control group. The
experiment will be powered to detect a 1 standard deviation reduction in bone lesions score in the
intervention versus control group and will require 20 mice per group followed for 12 months. The outcome
of this experiment will be to determine of the microbiome influences the development of lesions in this
model and if this can be modified by probiotics which would have potential therapeutic consequences
5.2.2 The microbiome as a modifying factor in humans at risk of PDB
The human studies will be conducted in carriers of SQSTM1 mutations taking part in the ZIPP study as well
as in a subset of individuals from the cohort of SQSTM1 negative subjects taking part in the genetic profiling
study described in section 2.2. Participants from both of these studies will be used to examine the
interactions between diet, the microbiome and genotype in predicting the presence and progression of
bone lesions. We already know that 15% of the individuals in ZIPP had PDB lesions at baseline (n=22) and
expect this to increase to 30% by end of the study in untreated patients. We expect that between 5%-10%
of the SQSTM1 negative subjects with a family history of PDB will have early PDB lesions by the time they
are assessed providing a total sample of about 50 subjects with lesions. We will match the subjects with
lesions and those without lesions by age, gender, SQSTM1 mutation type and risk allele score to examine
the role of diet and intestinal microbial profile on presence of lesions. Diet will be evaluated by food
frequency questionnaires. Microbial gene richness in all subjects will be evaluated using next generation
sequencing of faecal DNA (82). The reads will be mapped onto reference catalogues of microbial genes and
abundance of each gene normalized by dividing the number of reads by then genes nucleotide length. The
normalized gene abundances will be transformed into relative frequencies of each gene by dividing by the
total number of unique reads for that individual as described (82). These gene frequencies (which represent
microbial gene profile of an individual) will then be related to presence or absence of PDB lesions to
address the hypothesis that microbial diversity and/or colonisation with specific microbes are related to the
presence of PDB lesions. Further analysis will also be performed using logistic regression to determine if
other relevant variables such as age, gender, SQSTM1 mutation type, carriage of other PDB risk alleles,
serum 25(OH)D levels and diet (dietary calcium and other nutrients) influence lesion development. This will
provide a unique insight into the role of diet and the microbiome as modulators of susceptibility to the
emergence of PDB in those at increased genetic risk of the disease.

5.2.3 Intervention with probiotics in early PDB
Should the results of these cross-sectional studies described in 5.2.2 show an association between diet, the
microbiome and evolution of PDB, a prospective clinical study will be designed to evaluate the effect of a
probiotic manipulation using the commercially available product VSL#3 on the microbiota and biochemical
markers of bone turnover in subjects with early, asymptomatic PDB lesions and/or elevations in serum total
alkaline phosphatase (ALP). The aim of this study would be to determine if a probiotic intervention can
modify bone turnover in people with very early PDB. Participants from the ZIPP study that have already
taken part in the genetic profiling study (section 2.2) will be invited to participate in this prospective study.
At baseline, fasting samples will be obtained for measurements of serum total ALP, bone specific ALP, PINP
(a marker of bone formation) and NTX (a marker of bone resorption). The participants will be randomised
to receive active VSL#3 over a period of 3 months or a matched preparation in which the bacteria have
been killed by heat treatment (placebo). Measurements of biochemical markers will be repeated after 7
days, then monthly until 3 months when the experiment will be terminated. The primary outcome of the
study will be to determine if VSL#3 reduces PINP concentrations (the most sensitive markers of bone
turnover in PDB) by 20% or more (approximately 1 standard deviation) as compared with placebo. Changes
in bone ALP and NTX will be evaluated as secondary outcomes. A sample size of 17 individuals per group
will provide 80% power to detect a 1SD fall in markers but the sample will be inflated to 20 individuals to
account for missing data and patients lost to follow up. The importance of this study will be to determine if
probiotics might represent a lifestyle strategy to reduce the risk of PDB in those genetically at increased
risk
5.2.4 Dietary calcium deficiency in a model of PDB
It has been hypothesised that dietary calcium or vitamin D deficiency influence the evolution and severity
of PDB. This will be tested in the P394L mouse model of PDB. We will randomise P394L-/- mice to receive a
normal or low calcium diet from weaning until 6 weeks of age and investigate the effects on bone lesion
score in both groups at 8 months of age. This experiment will be designed to provide 80% power to detect a
1SD increase in lesion severity score (approximately a 30% increase). To achieve this power, 20 female mice
per group will be selected and randomised to a normal or low calcium diet. At termination of the
experiment screening for lesions will be conducted by microCT as described in section 5.2.1. In order to
assess potential mechanisms blood will be obtained by cardiac puncture and analysed for PTH, calcium,
PINP and NTX and bone marrow cultures prepared stimulated by RANK and MCSF to determine if the
dietary manipulation alters the osteoclastogenic potential of bone marrow. Also bone marrow will be
analysed for expression of relevant genes including MCSF, RANKL, OPG and RANK.
5.2.5 Dietary calcium in humans with early PDB
For this study, we will evaluate the effect of dietary calcium on bone turnover in early PDB in humans.
Rather than investigate dietary calcium deficiency (which may be considered unethical) we will invite
patients with early, asymptomatic PDB to enter a study in which they will be randomised to receive a
normal diet (standard care) or a diet enriched in calcium aiming to provide more than 1000mg daily. The
primary outcome of the study will be to determine if the calcium supplementation reduces PINP
concentrations (the most sensitive markers of bone turnover in PDB) by 20% or more (approximately 1
standard deviation) as compared with placebo. Changes in bone ALP and NTX will be evaluated as
secondary outcomes. A sample size of 17 individuals per group will provide 80% power to detect a 1SD fall
in markers but the sample will be inflated to 20 individuals to account for missing data and patients lost to
follow up. Measurements of biochemical markers will be repeated after 7 days, then monthly until 3
months when the experiment will be terminated. The importance of this study will be to determine if
enhancing dietary calcium might represent a lifestyle strategy to reduce the risk of PDB in those
genetically at increased risk of the disease.
5.2.5 Mechanical loading
The role of mechanical loading on localisation of PDB lesions will be studied in heterozygous (P394L+/-) mice
to determine if repetitive mechanical loading of the tibia accelerates the progression of lesions using the
mechanical loading rig developed by Lanyon and colleagues (83). The aim will be to determine if loading
causes PDB-like lesions to localise preferentially to the loaded as opposed to contralateral tibia. The
loading will be conducted at two weekly intervals between the age of 8 and 20 weeks in female mice. The
mice will then be observed and the experiment terminated when the mice are 8 months of age. The
unloaded tibia will be used as an internal control. If mechanical loading does indeed play a role in localizing
lesions we would expect an increase in number or size of lesions in the loaded limb. We have previously
shown that about 25% of P394L+/- mice develop bone lesions in the tibia by 8 months of age. The study will
be powered to detect a 1 SD (30%) increase in lesion number in the loaded limb which will require 20 mice
(assuming that lesion number in the contralateral limb remains unchanged). The lesions will be detected
and quantitated by microCT and histology as detailed in 5.2.1.
Taken together, the experiments described will provide new information on the possible role of the
microbiome and dietary calcium in a mouse model of PDB and in humans. The mechanical loading studies
will also provide new information on the role of this factors as potential mechanisms for disease
localisation. Their clinical importance will be to give an indication if lifestyle modifications (such as diet or
probiotic supplementation) could be advised to favourably influence progression of the disease in those at
risk.
Study Timelines
10
Section b. Methodology
Genome wide association
Genotyping for the GWAS will be performed using an Illumina custom array available through our
collaborator Prof at the genetic laboratory, Erasmus MC. This array contains 650K
variants optimised for GWAS and 50K additional variants including all those previously identified in the
pathogenesis of PDB. Standard quality control measures will be applied, excluding SNPs with a low call rate
(<95%), those that deviate from Hardy-Weinberg equilibrium (p < 10-7), those with minor allele frequency
(MAF) < 0.01. Samples with low quality (call rate < 97.5%), those with excess heterozygosity, gender
mismatch, non-European ancestry and cryptic family relatedness will also be removed from the final
analysis. Genotyping cluster plots for significant and suggestive SNPs (p < 5 x 10-5) will be visually inspected
and only the SNPs with high quality genotype data used in the analysis. Imputation will be performed using
miniMach3 utilising the Haplotype Reference Consortium (HRC) panel which provides better imputation
accuracy than 1000 Genomes. Quality control will be repeated after imputation to exclude markers with
MAF < 1%, and SNPs with poor imputation quality (r2 < 0.3). Following these measures individual variants
will be tested for association using PLINK software (1-degree-of-freedom). Logistic regression models will
also be used to test for association using four principal components as covariates. Variants with a p-value of
1x 10-5 or below will be selected for replication in the GDPD consortium. Each variant of interest will be
subject to meta-analysis in the replication cohorts using METAL software.
Whole genome sequencing
Whole genome sequencing will be employed to identify novel rare PDB-associated variants as opposed to
exomes since coverage is better and the costs are now similar. The sequencing will be outsourced and
performed at 30x depth using the Illumina HiSeq X platforms at Edinburgh Genomics
(https//genomics.ed.ac.uk) who will perform the genotyping, QC and initial bioinformatic analysis. Briefly
the DNA will be sheared and subjected to Illumina paired-end DNA library preparation and then sequenced.
Short insert paired-end reads will be aligned to the reference human genome (GRCh37) using the Burrows-
Wheeler Aligner, BWA (v0.5.9). The BAM files will be further processed to realign around known INDELs,
base quality score recalibration, addition of BAQ tags using GATK. The SNV calls will be completed using
samtools/bcftools (version 0.1.18-r579), and will be called to produce a VCF file. Sites will be called using
the Variant Quality Score Recalibration (VQSR) and GATK Unified Genotyper to recall the sites and alleles
discovered by samtools. The VariantRecalibrator within GATK will be used to model the variants, then GATK
ApplyRecalibration will be applied to assign VQSLOD scores. The VQSLOD score threshold will be set at -
0.6804 since that has been shown to improve site concordance with duplicate samples sequenced using
high-depth exome sequence methods. Variants with HWE of (p<1x10-6) will be removed.
Screening for somatic mutations
Screening for somatic mutations will be achieved by whole genome sequencing of DNA extracted from
bone lesions and leukocyte DNA from the same patient. The sequencing will again be subcontracted to
Edinburgh Genomics. The CaVEMan programme (http://cancerit.github.io/CaVEMan/) will be used to call
somatic substitutions. This employs an expectation maximization (EM) algorithm to call variants in second-
generation sequencing reads generating a probability score for potential genotypes at each genomic
position. A putative ‘somatic’ genotype probability of 80% and above will be applied as a cut off to define
the presence of somatic mutations. Insertions and deletions in the affected tissue and leukocyte DNA will
be modified Pindel version 2.0. (http://cancerit.github.io/cgpPindel/) on the GRCh37 genome build. Indels
will be required to be present in 5 or more reads in the affected tissue and not in the leukocyte DNA.
Structural variants will be screened for using the first BRASS algorithm (https://github.com/cancerit/BRASS)
through discordantly mapping paired-end reads from short insert data by using BWA alignments.
Discordantly mapping read pairs that are likely to span breakpoints, as well as a selection of nearby
properly-paired reads, will be grouped for each region of interest. Using the Velvet de novo assembler.
Copy number variants will be screened for using Nexus Copy Number software (version 9.0) on whole
genome sequencing data.
Osteoclast cultures
The effects of PDB candidate genes on osteoclast formation will be evaluated by over-expression of
constructs in RAW 267.1 cells and/or in primary bone marrow cultures prepared from wild type mice and
mice with mutations in the genes of interest. The cultures will be stimulated with MCSF and RANKL and
11
effects on osteoclast differentiation assessed by counting the number of TRAP positive multinucleated cells.
In some experiments bone resorption will be assessed by plating the cells into wells containing dentine
discs and measuring resorption area using reflected light microscopy as described (84).
Osteoblast cultures and bone nodule cultures
The effects of PDB candidate genes on osteoblast activity will be performed by over-expression of
constructs in MC3T3-E1 cells and/or primary osteoblast cultures prepared from mice with mutations in the
genes of interest and wild type littermates. Effects on osteoblast proliferation will be studied after 3-5 days
by cell counting using Alamar blue and on differentiation by measuring alkaline phosphatase in cell lysates.
Studies of osteoblast function in mice with mutations in genes of interest will employ primary osteoblasts
isolated from the calvarial bones (85). Effects on proliferation will be assessed as described for the
MC3T3E1 cultures. Effects on differentiation will be assessed in long term cultures (10-21 days) of calvarial
osteoblasts in osteogenic medium with quantitation of mineralised nodule numbers as described (86).
Osteoblast bone marrow co-cultures
These cultures will be used to examine cross-talk between osteoblast like cells and osteoclast
differentiation in cultured derived from mice with inactivation or mutations in genes implicated in PDB. The
osteoblast like cells will be derived from collagenase digestion of calvarial bones and the osteoclast
precursors from bone marrow flushed from the long bones of 3-5-month-old mice. The cells will be
cultured together in the presence of 10 nM 1,25-dihydroxyvitamin D3 and at the end of the culture period,
osteoclasts will be counted following TRAP staining and resorption pits quantified by reflected light
microscopy as described (87)
microCT
Assessment of bone structure and bone density will be performed by microCT using a Skyscan 1172 system
on the vertebral and limb bones of mice. To screen for the presence of PDB-like lesions, the entire limb or
lumbar spine will be scanned at 16.9 µm resolution with more detailed scans of bones with lesions at a
resolution of 5 µm. The presence and extent of lesions will be assessed by an observer blinded to treatment
allocation or genotype and scored according to their size as mild (score=1), moderate (score=2), or severe
(score=3). A total severity score will be calculated for each animal by adding scores for individual lesions.
Groups will be compared by t-test following log transformation of lesion score data. In selected
experiments, assessment of bone density and structure at sites unaffected by lesions will be evaluated by
microCT of the proximal tibial metaphysis and/or distal femoral metaphysis scanning at a resolution of 5
µm. The reconstruction will be performed using the Skyscan NRecon package. Trabecular bone parameters
will be measured using Skyscan CTAn software in a stack of 200 slices immediately distal from the growth
plate as described (87).
Bone histology and histomorphometry
Bone histology and histomorphometry will be performed following intraperitoneal injections of Calcein
(20mg/kg), on day 6 and day 2 prior to the mice being sacrificed using standard methods (51, 87, 88). The
samples will be embedded in methyl methacrylate, 5 µm sections cut using a tungsten steel knife and
sections will be stained for tartrate resistant acid phosphatase (TRAP) to visualise osteoclasts and then
counterstained with haematoxylin. Calcein double labelling will be visualised on a fluorescence microscope
fitted with a digital camera. Quantitative histomorphometry will be performed with the aid of software
developed in-house based on the Aphelion Image Analysis toolkit (ADCIS, France).
Mechanical loading
This will be performed in collaboration with Dr at the Royal Dick Vet School who has
previous experience in this area. In brief, the tibias will be loaded at 2 weekly intervals for 10 minutes daily
over 3 days to generate compressive strains of 5000 microstrain at the tibial midshaft in anaesthetised mice
using the loading rig developed by Lanyon (89).
Section c. Resources (including project costs)

Resources
The studies will be carried out at the MRC institute of Genetics and Molecular Medicine (IGMM) where we
have access to all the facilities necessary for successful completion of the project. The GWAS genotyping
and will be externally contracted and performed in collaboration with the genomics laboratory at Erasmus
MC, with our longstanding collaborator Professor . The genome sequencing will similarly
12
be outsourced to Edinburgh Genomics. The genotyping for the GWAS replication and genetic profiling will
be outsourced to LGC Genomics. The genome sequencing results will be analysed in collaboration with the
highly expert bioinformatics section at IGMM who have expertise in supporting a wide variety of large scale
genome sequencing. The GWAS data will be analysed in-house.
Research team
The research team will be led by Stuart Ralston (PI) who will devote 40% of his time to the proposal.
will also contribute to the proposal will advise technical aspects of the proposal, relating to
the genomic studies (10% FTE).
with a remit to support studies on the genetics of musculoskeletal disease. Funding is requested for two
postdoctoral research assistants (PDRA). One will be to focus the genetic studies including GWAS, genome
sequencing, detection of somatic mutations and development of a genetic profiling test for PDB. This post
will be required for 36 months. Bioinformatics support will be provided through the core resources of the
MRC institute of Genetics and Molecular Medicine and through Edinburgh Genomics. The second will
conduct functional analysis of the genes identified as predisposing to PDB and will be involved in the P392L
studies. This post will be required for 60 months. The PDRA will be supported by a research technician
employed for 60 months to assist with routine tasks such as animal husbandry, microCT scanning, cell
cultures, retrieving tissues from experimental animals and preparing them for sectioning and histology,
immunostaining and quantitative PCR.
A full-time research nurses supported by an administrative assistant will be required mainly during year 1
to re-contact participants of the ZIPP study and to approach them and consent them for inclusion into the
genomic profiling studies described in section 2 of the proposal. Thereafter nurse support will be required
but this will be funded at an hourly rate (€65) to cover time involved in study visits. A part time physician
(20% FTE) physician to assist with design and supervision of the clinical studies. The clinical studies will
require a part time data programmer (20% FTE) to set up and maintain a secure database to hold relevant
clinical data for participants that will take part in the studies and a part time trial manager (20%) FTE to
assist with the ethics submissions and regulatory approvals.
Materials and consumables
Genotyping: The genotyping will be subcontracted to external providers. Costs for the Illumina Custom
array genotyping are €32 per sample. Genotyping for the GWAS replication and for SNP in the genetic
profiling study will be subcontracted to LCG genomics at an estimated cost of approximately €8 per sample,
including set up which assumes that up to 100 SNP may need to be tested. Costs for the whole genome
sequencing in familial cases and for the detection of somatic mutations are €1040 per sample. The
metagenomic sequencing in the microbiome studies will be outsourced to LCG who have quoted a cost of
€210 per sample.
Laboratory consumables: Bioinformatics support will be required for cleaning and processing the whole
genome sequencing data (€17,000) and for data storage (€1250). Laboratory consumables costs are
requested at a rate of €17,500 per annum for the PDRA involved in the functional studies which are
standard cell and molecular biology running costs in the UK. For the PDRA involved in the genomic studies
costs of €7,500 per annum are requested during years 1-3.
Animals. These costs are expected to be incurred during the whole course of the study following the
identification of PDB associated genes where studies are required to investigate the effects on bone
structure and bone metabolism in vivo. We estimate that 3 mouse strains will require maintenance and
phenotyping during the course of the project, in addition to the studies of the microbiome, dietary calcium
and biomechanical loading in P394L mice. The average cost per cage per week (occupancy 4 mice) is €25.
Over 4 years the maintenance costs (4416 cage weeks) are €110,400. Additional costs are requested for
experimental procedures including microCT (236 scans @ €50), special diets and probiotics (€4000), other
procedures (236 @ €5) and mechanical loading (60 @ €150)
Clinical studies: Support is requested for bone scans and confirmatory x-rays in the studies of genetic
profiling, and measurement of biochemical markers. Support for biochemical markers will also be required
for the intervention studies (320 time points @ €40). Costs for the study visit in the genomic profiling study
are estimated as €200 per participant (research nurse time at €65 per hour, including consumables). The
intervention studies (microbiome and dietary calcium studies) will require four clinic visits will be required.
Total costs for this study (research nurse time and consumables) are estimated as €325 per subject.
13
A breakdown of materials and consumable costs for individual components of the programme are
illustrated below.
Item Year 1 Year 2 Year 3 Year 4 Year 5 Total
Bioinfomatics support € 7,500 € 7,500 € 2,000 € 17,000
Animal maintenance and procedures € 28,076 € 28,076 € 28,076 € 28,076 € 28,076 € 140,380
Consumables postdoc 1 € 7,500 € 7,500 € 7,500 € 22,500
Consumables postdoc 2 € 17,500 € 17,500 € 17,500 € 17,500 € 17,500 € 87,500
Radionuclide bone scans cross sectional study (500 scans @ €150) € 75,000 € 75,000
X-rays cross sectional study (50 @ €100) € 5,000 € 5,000
Biochemical markers genomic profiling study (500 patients @ €40) € 17,500 € 2,500 € 20,000
Study visits genomic profiling (500 patients @ €280) € 125,000 € 125,000
Study visits microbiome study (100 patient visits @ €280) € 28,000 € 28,000
Telephone € 500 € 500 € 1,000
Participant Travel (500 patient visits @ €30) € 5,000 € 5,000 € 2,500 € 2,500 € 15,000
Biochemical markers probiotic and dietary studies (80 @ €40) € 3,200 € 3,200
Study visits probiotic and dietary studies (80 visits @ €325) € 26,000 € 26,000
Meetings travel € 3,000 € 3,000 € 3,000 € 3,000 € 500 € 12,500
Open access publication costs € 2,000 € 2,000 € 2,000 € 2,000 € 8,000
Stationary, printing, postage € 500 € 250 € 100 € 100 € 100 € 1,050
Data storage € 250 € 250 € 250 € 250 € 250 € 1,250
Computers € 3,500 € 3,500
Audit costs € 4,500 € 4,500
Total € 72,826 € 294,076 € 67,926 € 84,626 € 76,926 € 596,380

Genotyping and sequencing (externally contracted) Year 1 Year 2 Year 3 Year 4 Year 5 Total
GWAS cases (1129 @ €32) € 36,128 € 36,128
Replication cases & Controls (5167 @ €8) € 41,336 € 41,336
Genomic profiling (500 @ €8) € 3,500 € 3,500
Genome sequencing cases (100 @ €1040) € 104,000 € 104,000
Genome sequencing somatic mutations (40 @ 1040) € 41,600 € 41,600
Metagenomic sequencing microbiome study (100 @ €210) € 21,000 € 21,000
Total € 181,464 € 3,500 € 41,600 € 21,000 € - € 247,564
Cost Category Total in euro

3
PI 0
Senior Staff 0
Personnel Postdocs €936,889
Students 0
Other €266,698
Direct i. Total Direct costs for Personnel (in euro) €1,203,598
Costs2 Travel €27,500
Equipment €0
Other Consumables €360,580
goods and Publications (including Open Access fees) €8000
services Other: Animals & Procedures €140,380
ii. Total Other Direct Costs (in euro) €596,380
A – Total Direct Costs (i + ii) (in euro) €1,799,978
B – Indirect Costs (overheads) 25% of Direct Costs4 (in euro) €449,994
C1 – Subcontracting Costs (no overheads) (in euro) €247,564
C2 – Other Direct Costs with no overheads5 (in euro)
Total Estimated Eligible Costs (A + B + C) (in euro) €2,497,545
Total Requested Grant (in euro) €2,497,545
2
An additional cost category 'Direct costing for Large Research Infrastructures' applicable to H2020 can be added to this
table (below ‘Other Goods and services’) for PIs who are hosted by institutions with Large Research Infrastructures of a
value of at least EUR 20 million and only after having received a positive ex-ante assessment from the Commission's
services (see ‘Information for Applicants to the Advanced Grant 2017 Call’ for more details).
3
When calculating the salary, please take into account the percentage of your dedicated working time to run the ERC
funded project (i.e. minimum 30% of your total working time).
4
Please note that the overheads are fixed to a flat rate of exactly 25%.
5
Such as the costs of resources made available by third parties which are not used on the premises of the beneficiary (see
‘Information for Applicants to the Advanced Grant 2017 Call’ for details).
14
Request for additional funding above Justification

EUR 2 500 000
Funding is requested for a replacement microCT scanner

(b) Purchase of major equipment (SkyScan 1294) to replace the existing instrument in our
. laboratory (Skyscan 1172) which is now 12 years old.
The scanner has faster throughput and a higher
resolution than the existing machine. Total cost is
€351,000

Please indicate the duration of the project in months:6 60
Please indicate the % of working time the PI dedicates to the project over the period of
40%
the grant:

The principal investigator Professor Ralston confirms that he will devote 40% of his time to the research
project over the period of the grant. He will be responsible for supervising all members of the research team
and will be involved in design, execution and analysis of data from the studies as well as interpreting the
results and supervising write up of the results for publication.

6
The maximum award is reduced pro rata temporis for projects of a shorter duration (e.g. for a project of 48 months
duration the maximum requested EU contribution allowed is EUR 2 million). Additional funding to cover major one-off
costs is not subject to pro-rata temporis reduction for projects of shorter duration (e.g. with additional funding it is possible
to request a maximum EU contribution of EUR 3 million for a project of 48 months duration).
15
References

1. Ralston SH, Albagha OM. Genetics of Paget's Disease of Bone. Curr Osteoporos Rep. 2014;12(3):263-
71.
2. Cundy T. Paget's disease of bone. Metabolism. 2017.
3. Singer FR. Paget's disease of bone-genetic and environmental factors. Nat Rev Endocrinol.
2015;11(11):662-71.
4. Siris ES, Ottman R, Flaster E, Kelsey JL. Familial aggregation of Paget's disease of bone. J Bone Miner
Res. 1991;6:495-500.
5. Sofaer JA, Holloway SM, Emery AE. A family study of Paget's disease of bone. J Epidemiol Community
Health. 1983;37:226-31.
6. Morissette J, Laurin N, Brown JP. Sequestosome 1: Mutation Frequencies, Haplotypes, and
Phenotypes in Familial Paget's Disease of Bone. J Bone Miner Res. 2006;21 Suppl 2:38-44.
7. Hocking L, Slee F, Haslam SI, Cundy T, Nicholson G, van Hul W, et al. Familial Paget's disease of bone:
patterns of inheritance and frequency of linkage to chromosome 18q. Bone. 2000;26(6):577-80.
8. Morales-Piga AA, Rey-Rey JS, Corres-Gonzalez J, Garcia-Sagredo JM, Lopez-Abente G. Frequency and
characteristics of familial aggregation of Paget's disease of bone. J Bone Miner Res. 1995;10:663-70.
9. Corral-Gudino L, Borao-Cengotita-Bengoa M, Del Pino-Montes J, Ralston SH. Epidemiology of Paget's
disease of bone: A systematic review and meta-analysis of secular changes. Bone. 2013;55(2):347-52.
10. Gardner MJ, Guyer PB, Barker DJP. Radiological prevalance of Paget's disease of bone in British
migrants to Australia. Br Med J. 1978;1:1655-7.
11. Albagha OM, Visconti MR, Alonso N, Langston AL, Cundy T, Dargie R, et al. Genome-wide association
study identifies variants at CSF1, OPTN and TNFRSF11A as genetic risk factors for Paget's disease of bone. Nat
Genet. 2010;42:520-4.
12. Divisato G, Formicola D, Esposito T, Merlotti D, Pazzaglia L, Del Fattore A, et al. ZNF687 Mutations in
Severe Paget Disease of Bone Associated with Giant Cell Tumor. Am J Hum Genet. 2016;98(2):275-86.
13. Laurin N, Brown JP, Morissette J, Raymond V. Recurrent Mutation of the Gene Encoding
sequestosome 1 (SQSTM1/p62) in Paget Disease of Bone. Am J Hum Genet. 2002;70(6):1582-8.
14. Hocking LJ, Lucas GJA, Daroszewska A, Mangion J, Olavesen M, Nicholson GC, et al. Domain specific
mutations in Sequestosome 1 (SQSTM1) cause familial and sporadic Paget's disease. Hum Mol Genet.
2002;11(22):2735-9.
15. Kim HJ, Kim NC, Wang YD, Scarborough EA, Moore J, Diaz Z, et al. Mutations in prion-like domains in
hnRNPA2B1 and hnRNPA1 cause multisystem proteinopathy and ALS. Nature. 2013;495(7442):467-73.
16. Albagha OME, Wani S, Visconti MR, Alonso N, Goodman K, Cundy T, et al. Genome-wide association
identifies three new susceptibility loci for Paget's disease of bone. Nat Genet. 2011;43(7):685-9.
17. Watts GD, Wymer J, Kovach MJ, Mehta SG, Mumm S, Darvish D, et al. Inclusion body myopathy
associated with Paget disease of bone and frontotemporal dementia is caused by mutant valosin-containing
protein. Nat Genet. 2004;36(4):377-81.
18. Obaid R, Wani SE, Azfer A, Hurd T, Jones R, Cohen P, et al. Optineurin Negatively Regulates Osteoclast
Differentiation by Modulating NF-kappaB and Interferon Signaling: Implications for Paget's Disease. Cell Rep.
2015;13(6):1096-102.
19. Vallet M, Soares DC, Wani S, Sophocleous A, Warner J, Salter DM, et al. Targeted sequencing of the
Paget's disease associated 14q32 locus identifies several missense coding variants in RIN3 that predispose to
Paget's disease of bone. Hum Mol Genet. 2015;24(11):3286-95.
20. Jin W, Chang M, Paul EM, Babu G, Lee AJ, Reiley W, et al. Deubiquitinating enzyme CYLD negatively
regulates RANK signaling and osteoclastogenesis in mice. J Clin Invest. 2008;118(5):1858-66.
21. Lucas G, Riches P, Hocking L, Cundy T, Nicholson G, Walsh J, et al. Identification of a Major Locus for
Paget Disease on Chromosome 10p13 in Families of British Descent. J Bone Miner Res. 2008;23(1):58-63.
22. Morohashi T, Corboz VA, Fleisch H, Cecchini MG, Felix R. Macrophage colony-stimulating factor
restores bone resorption in op/op bone in vitro in conjunction with parathyroid hormone or 1, 25-
dihydroxyvitamin D3. J Bone Miner Res. 1994;9:401-7.
23. Yagi M, Miyamoto T, Sawatani Y, Iwamoto K, Hosogane N, Fujita N, et al. DC-STAMP is essential for
cell-cell fusion in osteoclasts and foreign body giant cells. J Exp Med. 2005;202(3):345-51.
24. Beauregard M, Gagnon E, Guay-Belanger S, Morissette J, Brown JP, Michou L. Identification of rare
genetic variants in novel loci associated with Paget's disease of bone. Hum Genet. 2013.
16
25. Richards JB, Rivadeneira F, Inouye M, Pastinen TM, Soranzo N, Wilson SG, et al. Bone mineral density,
osteoporosis, and osteoporotic fractures: a genome-wide association study. Lancet. 2008;371(9623):1505-
12.
26. Rivadeneira F, Styrkarsdottir U, Estrada K, Halldorsson BV, Hsu YH, Richards JB, et al. Twenty bone-
mineral-density loci identified by large-scale meta-analysis of genome-wide association studies. Nat Genet.
2009;41:1119-206.
27. Divisato G, Formicola D, Esposito T, Merlotti D, Pazzaglia L, Del FA, et al. ZNF687 Mutations in Severe
Paget Disease of Bone Associated with Giant Cell Tumor. Am J Hum Genet. 2016;98(2):275-86.
28. Tan A, Ralston SH. Clinical Presentation of Paget's Disease: Evaluation of a Contemporary Cohort and
Systematic Review. Calcif Tissue Int. 2014;95(5):385-92.
29. Langston AL, Campbell MK, Fraser WD, Maclennan G, Selby P, Ralston SH. Clinical Determinants of
Quality of Life in Paget's Disease of Bone. Calcif Tissue Int. 2007;80(1):1-9.
30. Tan A, Goodman K, Walker A, Hudson J, MacLennan GS, Selby PL, et al. Long-Term Randomized Trial
of Intensive Versus Symptomatic Management in Paget's Disease of Bone: The PRISM-EZ Study. J Bone Miner
Res. 2017;32(6):1165-73.
Miner Res. 2010;25:20-31.
32. Hosking D, Lyles K, Brown JP, Fraser WD, Miller P, Curiel MD, et al. Long-term control of bone
turnover in Paget's disease with zoledronic acid and risedronate. J Bone Miner Res. 2007;22(1):142-8.
33. Reid IR, Miller P, Lyles K, Fraser W, Brown JP, Saidi Y, et al. Comparison of a single infusion of
zoledronic acid with risedronate for Paget's disease. N Engl J Med. 2005;353(9):898-908.
34. Albagha OM, Visconti MR, Alonso N, Wani S, Goodman K, Fraser WD, et al. Common susceptibility
alleles and SQSTM1 mutations predict disease extent and severity in a multinational study of patients with
Paget's disease. J Bone Miner Res. 2013;28:2238-46.
35. Visconti MR, Langston AL, Alonso N, Goodman K, Selby PL, Fraser WD, et al. Mutations of SQSTM1
are associated with severity and clinical outcome in Paget's disease of bone. J Bone Miner Res.
2010;25(11):2368-73.
36. Guay-Belanger S, Simonyan D, Bureau A, Gagnon E, Albert C, Morissette J, et al. Development of a
molecular test of Paget's disease of bone. Bone. 2016;84:213-21.
37. Hamdy R. Trauma and Paget's disease of bone [letter]. British Medical Journal. 1979;1(6176):1487.
38. Solomon LR. Billiard-player's fingers: an unusual case of Paget's disease of bone. British Medical
Journal. 1979;1(6168):931.
39. Ralston SH. Paget's disease of bone. Br Med J. 1993;306(6873):332-3.
40. Knudsen AG. Hereditary cancer, oncogenes and anti-oncogenes. Cancer Res. 1985;45:1437-43.
41. Merchant A, Smielewska M, Patel N, Akunowicz JD, Saria EA, Delaney JD, et al. Somatic mutations in
SQSTM1 detected in affected tissues from patients with sporadic Paget's disease of bone. J Bone Miner Res.
2009;24(3):484-94.
42. Matthews BG, Naot D, Bava U, Callon KE, Pitto RP, McCowan SA, et al. Absence of somatic SQSTM1
mutations in Paget's disease of bone. J Clin Endocrinol Metab. 2009;94(2):691-4.
43. Nik-Zainal S, Davies H, Staaf J, Ramakrishna M, Glodzik D, Zou X, et al. Landscape of somatic mutations
in 560 breast cancer whole-genome sequences. Nature. 2016;534(7605):47-54.
44. Gianfrancesco F, Rendina D, Di SM, Mingione A, Esposito T, Merlotti D, et al. A nonsynonymous
TNFRSF11A variation increases NFkappaB activity and the severity of Paget's disease. J Bone Miner Res.
2012;27(2):443-52.
45. Chung PY, Beyens G, Riches PL, Van WL, de FF, Jennes K, et al. Genetic variation in the TNFRSF11A
gene encoding RANK is associated with susceptibility to Paget's disease of bone. J Bone Miner Res.
2010;25:2316-29.
46. Ryan NM, Morris SW, Porteous DJ, Taylor MS, Evans KL. SuRFing the genomics wave: an R package
for prioritising SNPs by functionality. Genome Med. 2014;6(10):79.
47. Sophocleous A, Landao-Bassonga E, van't Hof RJ, Idris AI, Ralston SH. The Type 2 Cannabinoid
Receptor Regulates Bone Mass and Ovariectomy-Induced Bone Loss by Affecting Osteoblast Differentiation
and Bone Formation. Endocrinology. 2011;152(6):2141-9.
17
48. Idris AI, Sophocleous A, Landao-Bassonga E, Canals M, Milligan G, Baker D, et al. Cannabinoid
receptor type 1 protects against age-related osteoporosis by regulating osteoblast and adipocyte
differentiation in marrow stromal cells. Cell Metab. 2009;10(2):139-47.
49. van't Hof RJ, Armour KJ, Smith LM, Armour KE, Wei XQ, Liew FY, et al. Requirement of the inducible
nitric oxide synthase pathway for IL-1-induced osteoclastic bone resorption. Proceedings of the National
Academy of Sciences of the United States of America. 2000;97(14):7993-8.
50. Albagha OME, Lakshminarayan R, Ralston SH. A Novel VCP Mutation in a Patient with Paget's Disease
of Bone without Myopathy and Neurological Involvement. Journal of Bone & Mineral Research.
2014;Supplement 1:FR0010-(abstract).
51. Daroszewska A, van't Hof RJ, Rojas JA, Layfield R, Landao-Basonga E, Rose L, et al. A point mutation
in the ubiquitin associated domain of SQSMT1 is sufficient to cause a Paget's disease like disorder in mice.
Hum Mol Genet. 2011;20(14):2734-44.
52. Jin H, Ralston SH. Analysis of transcriptional regulation in bone cells. Methods Mol Biol.
2012;816:233-47.
53. Mann V, Hobson EE, Li B, Stewart TL, Grant SF, Robins SP, et al. A COL1A1 Sp1 binding site
polymorphism predisposes to osteoporotic fracture by affecting bone density and quality. J Clin Invest.
2001;107(7):899-907.
54. Idris AI, Libouban H, Nyangoga H, Landao-Bassonga E, Chappard D, Ralston SH. Pharmacologic
inhibitors of IkappaB kinase suppress growth and migration of mammary carcinosarcoma cells in vitro and
prevent osteolytic bone metastasis in vivo. Mol Cancer Ther. 2009;8(8):2339-47.
55. Idris A, Mrak E, Greig I, Guidobono F, Ralston SH, van 't HR. ABD56 causes osteoclast apoptosis by
inhibiting the NFkappaB and ERK pathways. Biochem Biophys Res Commun. 2008;371(1):94-8.
56. Bolland MJ, Tong PC, Naot D, Callon KE, Wattie DJ, Gamble GD, et al. Delayed Development of Paget's
Disease in Offspring Inheriting SQSTM1 Mutations. J Bone Miner Res. 2007;22(3):411-5.
57. Cundy HR, Gamble G, Wattie D, Rutland M, Cundy T. Paget's disease of bone in New Zealand:
continued decline in disease severity. Calcif Tissue Int. 2004;75(5):358-64.
58. Doyle T, Gunn J, Anderson G, Gill M, Cundy T. Paget's disease in New Zealand: evidence for declining
prevalence. Bone. 2002;31(5):616-9.
59. Poor G, Donath J, Fornet B, Cooper C. Epidemiology of Paget's disease in Europe: the prevalence is
decreasing. J Bone Miner Res. 2006;21(10):1545-9.
60. Rendina D, Gennari L, De Filippo G, Merlotti D, de Campora E, Fazioli F, et al. Evidence for increased
clinical severity of familial and sporadic Paget's disease of bone in Campania, southern Italy. J Bone Miner
Res. 2006;21(12):1828-35.
61. Corral-Gudino L, Garcia-Aparicio J, Sanchez-Gonzalez MD, Miron-Canelo JA, Blanco JF, Ralston SH, et
al. Secular changes in Paget's disease: contrasting changes in the number of new referrals and in disease
severity in two neighboring regions of Spain. Osteoporos Int. 2013;24(2):443-50.
62. Siris ES. Epidemiological aspects of Paget's disease: family history and relationship to other medical
conditions. Semin Arth Rheum. 1994;23(4):222-5.
63. Lever JH. Paget's disease of bone in Lancashire and arsenic pesticide in cotton mill wastewater: a
speculative hypothesis. Bone. 2002;31(3):434-6.
64. Hamdy R. Trauma and Paget's disease of bone. Br Med J. 1979;1(6176):1487.
65. Rebel A, Malkani K, Basle M, Bregeon C, Patezour A, Filmon R. Ultrastructural characteristics of
osteoclasts in Paget's disease. Rev Rhum Mal Osteoartic. 1974;41(12):767-71.
66. Helfrich MH, Hobson RP, Grabowski PS, Zurbriggen A, Cosby SL, Dickson GR, et al. A negative search
for a paramyxoviral etiology of Paget's disease of bone: molecular, immunological, and ultrastructural studies
in UK patients. J Bone Miner Res. 2000;15(12):2315-29.
67. Helfrich MH, Hocking LJ. Genetics and aetiology of Pagetic disorders of Bone. Archives Biochem
Biophys. 2014;473:172-82.
68. Matthews BG, Afzal MA, Minor PD, Bava U, Callon KE, Pitto RP, et al. Failure to detect measles virus
RNA in bone cells from patients with Paget's disease. J Clin Endocrinol Metab. 2008;93:1398-401.
69. Matthews D, Fry L, Powles A, Weissenbach J, Williamson R. Confirmation of genetic heterogeneity in
familial psoriasis. J Med Genet. 1995;32(7):546-8.
70. Ralston SH, Afzal MA, Helfrich MH, Fraser WD, Gallagher JA, Mee A, et al. Multicenter blinded analysis
of RT-PCR detection methods for paramyxoviruses in relation to Paget's disease of bone. Journal of bone and
18
mineral research : the official journal of the American Society for Bone and Mineral Research.
2007;22(4):569-77.
71. Gordon MT, Mee AP, Anderson DC, Sharpe PT. Canine distemper transcripts sequenced from pagetic
bone. Bone Miner. 1992;19:159-74.
72. Reddy SV, Singer FR, Mallette L, Roodman GD. Detection of measles virus nucleocapsid transcripts in
circulating blood cells from patients with Paget disease. J Bone Miner Res. 1996;11:1602-7.
73. Ooi CG, Walsh CA, Gallagher JA, Fraser WD. Absence of measles virus and canine distemper virus
transcripts in long- term bone marrow cultures from patients with Paget's disease of bone. Bone.
2000;27(3):417-21.
74. Birch MA, Taylor W, Fraser WD, Ralston SH, Hart CA, Gallagher JA. Absence of paramyxovirus RNA in
cultures of pagetic bone cells and in pagetic bone. J Bone Miner Res. 1994;9(1):11-6.
75. Visconti MR, Usategui-Martin R, Ralston SH. Antibody Response to Paramyxoviruses in Paget's
Disease of Bone. Calcif Tissue Int. 2017.
76. Kurihara N, Hiruma Y, Yamana K, Michou L, Rousseau C, Morissette J, et al. Contributions of the
measles virus nucleocapsid gene and the SQSTM1/p62(P392L) mutation to Paget's disease. Cell Metab.
2011;13(1):23-34.
77. Ruddle NH, Li CB, Horne WC, Santiago P, Troiano N, Jay G, et al. Mice transgenic for HTLV-I LTR-tax
exhibit tax expression in bone, skeletal alterations, and high bone turnover. Virology. 1993;197(1):196-204.
78. Jones RM, Mulle JG, Pacifici R. Osteomicrobiology: The influence of gut microbiota on bone in health
and disease. Bone. 2017.
79. Li JY, Chassaing B, Tyagi AM, Vaccaro C, Luo T, Adams J, et al. Sex steroid deficiency-associated bone
loss is microbiota dependent and prevented by probiotics. J Clin Invest. 2016.
80. Sjogren K, Engdahl C, Henning P, Lerner UH, Tremaroli V, Lagerquist MK, et al. The gut microbiota
regulates bone mass in mice. J Bone Miner Res. 2012;27(6):1357-67.
81. Lukens JR, Gurung P, Vogel P, Johnson GR, Carter RA, McGoldrick DJ, et al. Dietary modulation of the
microbiome affects autoinflammatory disease. Nature. 2014;516(7530):246-9.
82. Cotillard A, Kennedy SP, Kong LC, Prifti E, Pons N, Le CE, et al. Dietary intervention impact on gut
microbial gene richness. Nature. 2013;500(7464):585-8.
83. De Souza RL, Matsuura M, Eckstein F, Rawlinson SC, Lanyon LE, Pitsillides AA. Non-invasive axial
loading of mouse tibiae increases cortical bone formation and modifies trabecular organization: A new model
to study cortical and cancellous compartments in a single loaded element. Bone. 2005.
84. van't Hof RJ, Idris AI, Ridge SA, Dunford J, Greig IR, Ralston SH. Identification of biphenylcarboxylic
acid derivatives as a novel class of bone resorption inhibitors. J Bone Miner Res. 2004;19(10):1651-60.
85. Idris AI, Rojas J, Greig IR, van't Hof RJ, Ralston SH. Aminobisphosphonates cause osteoblast apoptosis
and inhibit bone nodule formation in vitro. Calcif Tissue Int. 2008;82(3):191-201.
86. Stewart TL, Roschger P, Misof BM, Mann V, Fratzl P, Klaushofer K, et al. Association of COLIA1 Sp1
alleles with defective bone nodule formation in vitro and abnormal bone mineralisation in vivo. Calcif Tissue
Int. 2005;77(2):113-8.
87. Idris AI, Sophocleous A, Landao-Bassonga E, van't Hof RJ, Ralston SH. Regulation of bone mass,
osteoclast function and ovariectomy-induced bone loss by the type 2 cannabinoid receptor. Endocrinology.
2008;149(11):5619-26.
88. Erben RG, Glosmann M. Histomorphometry in rodents. Methods Mol Biol. 2012;816:279-303.
89. Ehrlich PJ, Noble BS, Jessop HL, Stevens HY, Mosley JR, Lanyon LE. The effect of in vivo mechanical
loading on estrogen receptor alpha expression in rat ulnar osteocytes. J Bone Miner Res. 2002;17(9):1646-
55.

19
Ralston Paget-Advance
 ERC Advanced Grant 2017

Advancing knowledge to improve outcome in Paget’s disease of bone
Paget-Advance

Annex 1 Ethical Self-Assessment

Ethical issues relating to this project are summarised in the table below and further discussed

Informed consent:
The genome wide association studies and genome sequencing studies described in section 1 will be performed on patient
cohorts from the genetics determinants of Paget’s disease consortium (GDPD) where biological samples and DNA have
already been obtained with written informed consent. This applies to both the PDB cases and the controls. In all cases
assembly of the cohorts was obtained following approval by the relevant research ethics committees in participating
centres. Ethical approval for analysis of this multicentre study has already been obtained and the approval letter has been
uploaded with the application.
The studies described in section 2 of the programme will involve re-contacting patients who took part in the ZIPP study.
These subjects have given written informed consent to be re-contacted for future research studies relevant to Paget’s
disease of bone. The ethics approval letter for the ZIPP study has been uploaded with the application
For the prospective studies on genomic profiling, the microbiome studies and the intervention studies, ethical approval
will be sought and further informed consent will be obtained from all subjects. While genome wide sequencing will be
performed from some subjects the focus of the studies will focus only on Paget’s disease. We will not evaluate gene
variants implicated in other disease states, thereby avoiding issues relating to health insurance or incidental findings
relevant to diseases other than PDB. For the prospective studies, all patients will be informed that they have the ability to
withdraw from participation at any time. These studies will be performed according to the principles of the international
conference on harmonisation of good clinical practice (ICH-GCP) and the declaration of Helsinki.
Data protection issues:

The cohorts of individuals taking part in the GWAS studies and genome sequencing studies will be identifiable to the
research team only by a participant ID number which will be linked to relevant phenotypic and demographic data. Details
of these subjects will be held along with copied of the signed informed consent forms at the participating centres but these
will not be made available to the study team. All of the data generated as the result of the studies will be held on password
protected secure servers at the University of Edinburgh. Access to the data will be restricted to bona fide members of the
research team approved by the principal investigator.
Use of animals:
Some animal experimentation is necessary as part of this study since evaluation of the effects of candidate genes on
bone requires that the whole animal is studied to look at effects on bone structure and bone turnover in vivo. Many of the
experiments can however be performed in cultured cells and cell lines and this will be done whenever it is feasible to do
so. The animal experimentation will be carried out according to the UK Home Office Animals (scientific procedures) act
which promotes the principles of the 3Rs (Replace, Reduce, Refine). Prior to gaining home office approval for a
programme of work, all projects require to undergo ethical review at the University of Edinburgh. Such a review has
already been performed for the animal experiments proposed in this study and the procedures and experiments have
been approved as being appropriate. A copy of the UK Home Office approval for the animal studies has been uploaded
with the application For each experiment power calculations will be performed to gain as accurate an estimate of the
number of animals required to provide sufficient power to detect the effects sought. Following experimentation ad
termination of the experiments the animals will be promptly and humanely killed.
Human embryonic stem cells:

Not relevant for this application

Paget-Advance - Advancing Knowledge To Improve Outcome in Paget's Disease of Bone

Uploaded by

Copyright:

Available Formats

You might also like

Paget-Advance - Advancing Knowledge To Improve Outcome in Paget's Disease of Bone

Uploaded by

Document Information

Original Description:

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Paget-Advance - Advancing Knowledge To Improve Outcome in Paget's Disease of Bone

Uploaded by

Copyright:

Available Formats

European Commission - Research

Proposal Submission Forms

Section Title Action

2 Participants & contacts

How to fill in the forms

H2020-ERC-ADG-2017.pdf Ver 1.00 20170621 Page 1 of 15 Last saved 30/08/2017 15:51:23

Proposal ID 787270 Acronym Paget-Advance

Call Identifier ERC-2017-ADG

Type of Action ERC-ADG

Proposal title* Advancing knowledge to improve outcome in Paget’s disease of bone

Primary ERC Review Panel* LS7

Secondary ERC Review Panel Not applicable (if applicable)

ERC Keyword 2 Health services, health care research

ERC Keyword 3 Public health and epidemiology

ERC Keyword 4 Gene therapy, cell therapy, regenerative medicine

H2020-ERC-ADG-2017.pdf Ver 1.00 20170621 Page 2 of 15 Last saved 30/08/2017 15:51:23

Proposal ID 787270 Acronym Paget-Advance

H2020-ERC-ADG-2017.pdf Ver 1.00 20170621 Page 3 of 15 Last saved 30/08/2017 15:51:23

Proposal ID 787270 Acronym Paget-Advance

Personal data protection

H2020-ERC-ADG-2017.pdf Ver 1.00 20170621 Page 4 of 15 Last saved 30/08/2017 15:51:23

Proposal ID 787270 Acronym Paget-Advance

1 THE UNIVERSITY OF EDINBURGH United Kingdom

H2020-ERC-ADG-2017.pdf Ver 1.00 20170621 Page 5 of 15 Last saved 30/08/2017 15:51:23

Proposal ID 787270 Acronym Paget-Advance Short name UEDIN

2 - Administrative data of participating organisations

Short name: UEDIN

Address of the organisation

Street OLD COLLEGE, SOUTH BRIDGE

Postcode EH8 9YL

Country United Kingdom

Legal Status of your organisation

Research and Innovation legal statuses

Public body .................................................... yes Legal person .............................. yes

SME self-declared status................................... 12/12/2008 - no

SME self-assessment ...................................... unknown

SME validation sme ......................................... 12/12/2008 - no

H2020-ERC-ADG-2017.pdf Ver 1.00 20170621 Page 6 of 15 Last saved 30/08/2017 15:51:23

Proposal ID 787270 Acronym Paget-Advance Short name UEDIN

Department(s) carrying out the proposed work

Same as organisation address

Street Crewe Road South

Postcode EH4 2XU

Country United Kingdom

H2020-ERC-ADG-2017.pdf Ver 1.00 20170621 Page 7 of 15 Last saved 30/08/2017 15:51:23

Proposal ID 787270 Acronym Paget-Advance Short name UEDIN

The maximum length of the identifier is 11 characters (ZZZ-9999-2010) and

Last Name* RALSTON

First Name(s)* Stuart

Same as organisation address

Street Crewe Road South

Postcode/Cedex EH4 2XU Town* Edinburgh

Phone* Country* United Kingdom

H2020-ERC-ADG-2017.pdf Ver 1.00 20170621 Page 8 of 15 Last saved 30/08/2017 15:51:23

Proposal ID 787270 Acronym Paget-Advance

1 UEDIN UK 2 497 536 2 497 536

Total 2 497 536 2 497 536

 ERC Advanced Grant 2017