Microbial Dysbiosis in the Pathogenesis of Rheumatoid Arthritis: Using Metagenomics to Predict Methotrexate Efficacy
Conditions: Rheumatoid Arthritis
Sponsor: Quadram Institute Bioscience

Appendix 1 - Study Protocol 
MyRA study 
Version 2 – 08/10/2018 
1 
Study protocol 
Microbial dYsbiosis in the pathogenesis 
of Rheumatoid Arthritis: using 
metagenomics to predict methotrexate 
efficacy (MyRA) 
Study Team 
Non-clinical investigators: 
Chief Investigator 
Professor Simon Carding, Quadram Institute Bioscience 
simon.carding@quadram.ac.uk (01603) 251410 
Principal Investigator 
Miss Ellie Sayers, University of East Anglia 
ellie.sayers@quadram.ac.uk (01603) 255184 
Clinical investigators: 
Principal Investigator 
Professor Alexander MacGregor, University of East Anglia 
a.macgregor@uea.ac.uk (01603) 593570
Co-investigator 
Mrs Jacqueline Chipping, University of East Anglia 
jacqueline.chipping@uea.ac.uk (01603) 597204 
Co-investigator 
Mrs Tarnya Marshall, Norfolk and Norwich University Hospital 
tarnya.marshall@nnuh.nhs.uk (01603) 597204 
Study Reference Numbers 
IRAS number:  
238047 
ClinicalTrials.gov: 
Protocol yet to be uploaded (pending ethical review) 
Sponsors number: 
QIB01/2018 
Funders number: 
No ref (Action Arthritis)  

Appendix 1 - Study Protocol 
MyRA study 
Version 2 – 08/10/2018 
2 
SIGNATURE PAGE  
Title: Microbial dysbiosis in the pathogenesis of rheumatoid arthritis: using metagenomics to predict 
methotrexate efficacy  
Sponsor’s approval: 
This protocol has been approved by the Quadram Institute Bioscience Human Research Governance 
Committee (HRGC) 
Name………………………………………………….. 
Signature…………………………………........... 
Role……………………………………………..…….. 
Date…………………………………………………… 
I have fully discussed the objectives of this study and the contents of this protocol with the Sponsor’s 
representative.  I understand that the information in this protocol is confidential and should not be 
disclosed other than to those directly involved in the execution or ethical review of the trial. 
I agree to conduct this study according to this protocol and to comply with its requirements, subject 
to ethical and safety considerations and guidelines, and to conduct the study in accordance with 
International Conference on Harmonisation (ICH) guidelines on Good Clinical Practice (GCP) and with 
the applicable regulatory requirements.   
Chief Investigator name and address: 
Prof Simon Carding 
Quadram Institute Bioscience 
Norwich Research Park 
Colney Lane 
Norwich 
NR4 7UA 
Signature……………………………………………… 
Date……………………….………... 
Dr Antonietta Melchini
QIB HRGC chair
8th October 2018
08.10.18

Appendix 1 - Study Protocol 
MyRA study 
Version 2 – 08/10/2018 
3 
 
Table of Contents 
1 
Abstract ......................................................................................................................... 5 
2 
Scientific Background .................................................................................................... 6 
2.1 
RA epidemiology and clinical treatment .................................................................. 6 
2.2 
Microbial dysbiosis and “leaky gut” in RA ................................................................ 6 
2.2.1 
The gut virome in RA ....................................................................................... 7 
2.3 
Diet and RA ............................................................................................................ 7 
 
Summary ................................................................................................................ 7 
3 
Hypothesis ..................................................................................................................... 8 
4 
Study objectives ............................................................................................................. 8 
 
Primary aim............................................................................................................. 8 
 
Secondary aims ...................................................................................................... 8 
5 
Study design and methods ............................................................................................. 8 
 
Timeline .................................................................................................................. 8 
 
Setting .................................................................................................................... 9 
 
Recruitment policy ................................................................................................ 10 
5.3.1 
Recruitment from the NOAR .......................................................................... 10 
5.3.2 
Recruitment from the participant’s GP or NNUH Rheumatology Clinician ...... 10 
 
Eligibility criteria .................................................................................................... 11 
5.4.1 
Inclusion criteria ............................................................................................. 11 
5.4.2 
Exclusion criteria ............................................................................................ 11 
 
Study procedures .................................................................................................. 11 
5.5.1 
First appointment: Early Arthritis Clinic........................................................... 11 
5.5.2 
Second appointment: Nurse Practitioner ........................................................ 13 
5.5.3 
Third and fourth appointments: Routine Treatment Review ............................ 13 
 
Withdrawal procedure ........................................................................................... 15 
 
Data collection and storage ................................................................................... 15 
5.7.1 
Collection of disease activity variables ........................................................... 15 
 
Diet and lifestyle assessment ................................................................................ 17 
 
Sample processing and analysis ........................................................................... 17 
5.9.1 
Blood samples ............................................................................................... 17 
5.9.2 
Stool samples ................................................................................................ 18 
6 
Dissemination of findings ............................................................................................. 18 
7 
Statistical methods ....................................................................................................... 19 
 
Sample size calculations ....................................................................................... 19 
 
Statistical methods for the analysis of study data .................................................. 19 
7.2.1 
Correlating microbiome parameters with treatment outcome ......................... 19 

Appendix 1 - Study Protocol 
MyRA study 
Version 2 – 08/10/2018 
4 
 
7.2.2 
Identifying microbiome alterations induced by methotrexate .......................... 19 
7.2.3 
Other statistical analyses ............................................................................... 19 
8 
Resource requirements ................................................................................................ 20 
 
Staffing requirements ............................................................................................ 20 
 
Material requirements ........................................................................................... 20 
9 
Confidentiality .............................................................................................................. 20 
10 Ethical considerations .................................................................................................. 20 
 
Informed consent .................................................................................................. 20 
 
Risks associated with study procedures ................................................................ 20 
 
Reporting of Serious Adverse Events.................................................................... 21 
 
Future research .................................................................................................... 21 
11 References .................................................................................................................. 21 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

Appendix 1 - Study Protocol 
MyRA study 
Version 2 – 08/10/2018 
5 
 
1 Abstract 
Rheumatoid arthritis (RA) is a common autoimmune disease which causes inflammation in the joints 
and resulting disability. RA has a significant impact on a person’s quality of life and carries a heavy 
financial burden both to the individual and to society. Its’ causes remain unknown, however the 
discovery of alterations in the populations of microbes that normally reside in the gut in RA patients 
has highlighted the potential of gut microbes being involved in the development of RA. This study 
will investigate this possibility further by examining the bacteria and viruses resident in the gut of 30 
patients with early rheumatoid arthritis. To evaluate the importance of gut bacteria and viruses in 
the development and successful treatment of RA with methotrexate, stool samples will be collected 
at 3 time points: a) before treatment, b) during treatment and c) following maintenance or 
escalation of treatment based on a successful or unsuccessful response, respectively. We will also 
explore if patients with RA have a leaky gut (increased intestinal permeability) and develop an 
aberrant immune response to their own gut microbes as a result of them gaining access to the 
bloodstream, and whether this is altered with methotrexate treatment. Furthermore, as diet and 
lifestyle affect RA activity and progression and can influence the activity of gut microbes, dietary and 
lifestyle data will be collected to identify potential covariants with disease onset or progression. The 
outcomes of this project will help identify microbiome-based predictive factors associated with 
response to methotrexate, and a clearer understanding of the interplay between gut microbes, 
intestinal permeability and environmental factors in the development of RA. 
 
 
 
 
Figure 1. Proposed mechanism of the gut-joint axis in inflammatory arthritis (Jethwa, 20171) 

Appendix 1 - Study Protocol 
MyRA study 
Version 2 – 08/10/2018 
6 
 
2 Scientific Background 
2.1 
RA epidemiology and clinical treatment  
Rheumatoid arthritis (RA) is a chronic autoimmune condition characterized by inflammation of the 
joints that affects approximately 0.3-1% of the world population2. Its causes are unclear. Although 
linked with genetic predisposition, low concordance of RA among genetically identical twins suggests 
a significant contribution of environmental factors3.  RA may be either seropositive or seronegative, 
based on the respective presence or absence of the autoantibodies anti-cyclic citrullinated peptide 
(anti-CCP; directed against various joint components) and/or rheumatoid factor (RF; directed against 
immunoglobulin G).   
Oral methotrexate (MTX) is the standard initial treatment provided for early RA.  It is one of several 
disease-modifying anti-rheumatic drugs (DMARDs) which may be used in combination if MTX 
monotherapy is ineffective.  The disease-activity lowering effects of MTX are only seen at a clinically 
important level (i.e. a reduction of ≥1.2 in the Disease Activity Score [DAS]) in approximately 34% of 
patients4.  MTX; which has various side effects including increased risk of infection, is therefore 
sometimes used with little or no clinical benefit to the patient.  There are no reliable predictors of 
MTX response, although those concomitantly using steroid injections (prednisolone) and of the male 
sex are more likely to respond well4.  As some gut bacteria are able to assimilate and chemically alter 
MTX in vitro, it is possible that the composition of the gut microbiome might be involved in MTX 
response5–7.   
2.2 
Microbial dysbiosis and “leaky gut” in RA 
The vast population of microbes that inhabit the human gut, i.e. the microbiota, includes a wide 
variety of bacteria, fungi and viruses. The composition of these microbes is altered (microbial 
dysbiosis) in some chronic conditions including RA.  A gut-joint axis (Figure 1) has been proposed for 
RA pathogenesis whereby microorganisms in the gut are able to influence disease initiation and 
progression, potentially via increased intestinal permeability (“leaky gut”) and microbial dysbiosis1.  
However, although several studies have identified microbial dysbiosis in RA patients8–10, it is not 
clear how these changes contribute to disease outcomes.   
The microbiota alterations identified so far in RA have generally consisted of a reduced diversity of 
bacteria, coupled with increased or decreased abundance of individual microbial taxa.  For instance, 
abundance of Prevotella copri is associated with new-onset RA, and Collinsella; which has the ability 
to alter intestinal permeability possibly allowing microbial/dietary components in the gut lumen to 
access host immune structures, is also increased in abundance RA8–11.  On the contrary, potentially 
beneficial bacteria such as Faecalibacterium are depleted in some RA patients10.  The relevance of 
these alterations to RA treatment is unknown, although some differences in the abundance of 
specific bacteria between responders and non-responders to MTX and herbal medications have 
been observed9.  This suggests the involvement of gut microbes in response to MTX, potentiating the 
use of gut microbiota profiles as a predictive tool for response, or the use of microbiota-altering 
strategies to improve treatment outcome.    
The gut-joint axis theory encompasses the possibility that the autoimmune response seen in RA may 
arise as a result of immune responses against antigens in the gut (microbial or dietary), which are 
cross-reactive with antigens expressed by human cells (molecular mimicry)9,12.  These immune 
responses may be enabled by an increase in intestinal permeability.  There is variable evidence of 
increased intestinal permeability in RA patients, which may also be influenced by gut microbes such 
as Collinsella10,13,14.  However, little is known about any specific immune responses against gut 
microbes, or how immune cells respond to the expanded subsets of microbes in RA.  Elucidating if 

Appendix 1 - Study Protocol 
MyRA study 
Version 2 – 08/10/2018 
7 
 
microbial dysbiosis and/or an immune response against gut microbes effects disease severity and 
response to treatment is important to make microbiome research translatable and identify targets 
for the improvement of treatment strategies.   
2.2.1 The gut virome in RA 
Gut viruses (the virome) are a largely overlooked component of the human gut microbiome which is 
surprising as they represent its largest component15.  A major factor in our limited understanding of 
the gut virome is the lack of suitable tools and analytical methods. The virome consists mainly of 
viruses that infect bacteria (phages), which most likely control the structure and function of bacterial 
populations in the gut16,17. Like bacterial populations, the composition of viruses in the gut varies 
according to environmental factors such as diet and antibiotics15,18.  
A study of the virome in inflammatory bowel disease (IBD) demonstrated changes to the intestinal 
virome in a disease state, with an increase in the number of different viruses present and an 
associated decrease in bacterial populations, suggesting a predator-prey relationship between the 
two19. The intestinal virome has not yet been characterized in patients with RA, though as phages 
are able to significantly alter bacterial populations it is possible they may be initiating factors for 
microbial dysbiosis or alter the abundance of MTX response-associated bacteria.  Alternatively, the 
ability of viruses to cross the intestinal barrier, gain entry to the body and induce host immune 
responses could provide a trigger for the generation of cross-reactive antibodies in RA19,20.   
2.3 
Diet and RA 
Environmental factors such as smoking, diet, and infection are associated with risk of RA21–23.  Many 
of these factors also influence the composition of the human gut microbiome24.  Microbiome studies 
therefore need to account for environmental factors to provide a more comprehensive and 
insightful analysis, although this is not always done.  For instance, dietary analysis has been absent 
from microbiome studies in RA, despite diet being a potential causal factor in disease-associated 
microbial dysbiosis25.  RA patients often feel that their symptoms are associated with dietary 
fluctuations26, and diet represents an easily modifiable risk factor for RA.  However, little reliable 
evidence exists for the benefits of using dietary intervention to improve RA disease activity27, and no 
studies have been carried out to assess whether diet is associated with RA treatment response or 
response-associated microbiota profiles.  Identifying dietary components associated with microbial 
dysbiosis in RA could enable the generation of personalized treatment programs incorporating diet 
recommendations, which would be a simple and cost-effective way of enhancing treatment 
response in RA.     
 Summary  
Microbial dysbiosis has been established in RA in several studies, although the cause of dysbiosis and 
its relevance to clinical management of RA is unknown.  Additionally, the ability of microbes in the 
gut to initiate immune responses contributing to RA; possibly via increased intestinal permeability, is 
not well understood.  This study will primarily investigate whether gut microbial dysbiosis; or more 
specifically microbial diversity (Shannon index), is associated with MTX treatment response in RA.  
This will build on a previous study9 in which differences in the abundance of specific bacterial groups 
were identified between responders and non-responders to MTX, by defining a more specific early 
RA cohort on MTX monotherapy, characterizing changes to the viral as well as bacterial components 
of the microbiome, investigating intestinal permeability in these participants and exploring 
microbiome associations with diet and lifestyle factors.  We will investigate longitudinal changes to 
the microbiota induced by MTX over 6 months and attempt to correlate a continuous microbiome 
parameter (α-diversity/Shannon index) with change in DAS28-C-reactive protein (DAS28-CRP).  

Appendix 1 - Study Protocol 
MyRA study 
Version 2 – 08/10/2018 
8 
 
Correlations between specific microbial abundances; including that of viruses, and treatment 
response will also be identified.  Together, these analyses will help identify response-associated 
microbiota alterations prior to and during MTX treatment, which could be targeted to improve MTX 
treatment response.  Any response-associated microbiota alterations will be tested for association 
with diet and lifestyle variables, to provide insight into whether these alterations could be caused by 
environmental factors affecting the microbiota.  Finally, we will investigate whether systemic 
immune responses against gut microbes are present in RA, which could suggest increased intestinal 
permeability and a possible “gut origin” for RA.   
3 Hypothesis  
A single microbiome parameter (α-diversity, microbial diversity measured by Shannon index) in 
treatment-naïve early RA patients positively correlates with MTX response at 3 months (measured 
by change in DAS28- CRP) and can be used to predict treatment response in these patients.   
4 Study objectives  
This study has been designed primarily to provide information about changes in gut bacteria and 
viruses of RA patients undergoing treatment with MTX.  The primary outcome in this study is DAS28-
CRP (a measure of disease activity recorded at every routine clinical appointment) studied as a 
continuous variable.  More information on the collection of disease activity variables including 
DAS28-CRP can be found in Section 5.7. 
 Primary aim  
To determine alterations in gut bacteria and viruses of early RA patients and if specific changes are 
associated with the outcome of MTX treatment, by estimating the correlation between microbial 
diversity (α-diversity measured by Shannon index) and response to MTX measured by change in 
DAS28-CRP.   
 Secondary aims  
1. To explore more broadly the correlations between baseline gut microbiome parameters; 
such as compositional and functional profiles, and MTX outcome or disease severity 
measured by DAS28-CRP, Simplified Disease Activity Index (SDAI), CRP, ESR or autoantibody 
titer. 
2. To explore longitudinal alterations to the composition of gut bacteria and viruses in early RA 
patients throughout 6 months of MTX treatment. 
3. To explore if environmental factors such as diet and lifestyle can be used to explain 
response-associated alterations in bacterial and/or viral populations in early RA patients. 
4. To determine if RA patients generate autoimmune responses to their own intestinal 
microbes (indicative of increased intestinal permeability) and if this changes with treatment, 
by testing the humoral (antibody) and cellular (peripheral blood mononuclear cell [PBMC]) 
reactivity against commensal microbes. 
5 Study design and methods  
 Timeline 
A prospective cohort of 30 early RA patients will be recruited to complete this study, with data 
collection taking place at 3 time points: a) before treatment, b) during treatment (3 months) and c) 
following treatment maintenance or escalation depending on successful or unsuccessful response (6 

Appendix 1 - Study Protocol 
MyRA study 
Version 2 – 08/10/2018 
9 
 
months).  An outline of the study is provided in Figure 2, study flowchart describing the involvement 
of participants in the study in Figure 3, and data collection time points in Figure 4. 
 Setting  
Sample processing and data analysis will take place using facilities available at the Norwich Research 
Park (NRP) in Norwich, including the Quadram Institute Bioscience (QIB) and University of East Anglia 
(UEA) including the Bob Champion Research and Education Building (BCREB).  Identification of 
participants will take place mainly at the Norfolk Arthritis Register (NOAR) office at UEA, and 
recruitment of further participants may take place at the Rheumatology Department at Norfolk and 
Norwich University Hospital (NNUH) or at participating General Practitioner (GP) clinics around 
Norfolk.  Study visits will coincide with routine appointments and take place at the Rheumatology 
Department at NNUH. 
 

Appendix 1 - Study Protocol 
MyRA study 
Version 2 – 08/10/2018 
10 
 
 Recruitment policy  
A total of 30 participants will be recruited to complete this study, although the recruitment aim will 
be at least 35 to account for drop out.  Recruitment will run for 11 months from October 2018 
through August 2019, following successful ethical approval and funding applications.  This will allow 
for preparation and sequencing of final samples and analysis of data before September 2020, with 
the final study appointments finishing in April 2020. 
5.3.1 Recruitment from the NOAR  
Potential participants will be identified primarily via the incoming stream of GP referrals to the Early 
Arthritis Clinic at NNUH received by the Norfolk Arthritis Register (NOAR); an ongoing inception 
cohort of inflammatory arthritis patients in Norfolk, by Jacqueline Chipping (NOAR Clinical Manager 
and Research Nurse).  NOAR was established in 1989 and investigates the development and 
treatment of inflammatory polyarthritis along its natural course.  More than 4,000 patients are 
registered in NOAR, which involves longitudinal 12-monthly study visits where the Research Nurse 
collects information on historical and current health and joint symptoms and takes a blood sample.  
Further information on NOAR can be found in the NOAR protocol (Appendix 13) and at the NNUH 
website: http://www.nnuh.nhs.uk/departments/rheumatology/norfolk-arthritis-register/  
5.3.1.1 NOAR participants meeting ACR 2010 criteria for RA  
NOAR recruits approximately 18 participants per month, of which approximately 5 would potentially 
be suitable for this study as they meet ACR criteria for RA at baseline.  Participants will be invited via 
the ongoing stream of referrals to NOAR, allowing around 55 participants to be invited to this study 
over the 11 month recruitment period.  Existing NOAR members will also be invited to this study.  As 
the NNUH appointments are part of an 18 week pathway, it will be possible to invite existing NOAR 
participants meeting the study criteria and recruited to NOAR within the last 4-5 months 
(approximately 25 potential participants), as they will not have had their initial appointment yet.  
This provides a minimum of 80 participants who can be invited to this study, which should be 
sufficient; alongside recruitment from GPs and Rheumatology Clinicians, to obtain 35 participants. 
5.3.1.2 Recruitment procedure from NOAR 
Figures 2 and 3 shows representations of the study timeline from the Study Team and participants 
point of view, respectively.  A patient presenting to their GP with inflammatory arthritis and being 
referred to the Early Arthritis Clinic at NNUH will be notified to NOAR as part of their normal 
recruitment procedure, and at the same time as sending out the NOAR participant information sheet 
and consent form (Appendix 14 and 15); if they meet the eligibility criteria for this study, the NOAR 
Research Nurse will also send out an invitation letter (Appendix 2), Participant Information Sheet 
(PIS) (Appendix 3) containing a response slip, watermarked versions of the consent forms (Appendix 
24 and 25) and prepaid envelope for the MyRA study.  If a positive response slip is received, the 
participant will be contacted via telephone by Ellie Sayers or Jacqueline Chipping to discuss the study 
further and explain what will happen at their first Early Arthritis Clinic appointment.  Existing NOAR 
members suitable for this study; who have not yet had their initial NNUH Early Arthritis Clinic 
appointment, will also be identified by the NOAR Research Nurse and a copy of the MyRA invitation 
letter (Appendix 2) and PIS (Appendix 3) will be sent to them inviting them to the study, along with 
watermarked versions of the consent forms (Appendix 24 and 25) so they may review these prior to 
their first study visit if they chose to take part.   
5.3.2 Recruitment from the participant’s GP or NNUH Rheumatology Clinician 
In addition to NOAR, MyRA participants will be recruited directly via the patient’s own GP or NNUH 
Rheumatology Clinician.  GPs and clinicians will be provided with study information to give to 

Appendix 1 - Study Protocol 
MyRA study 
Version 2 – 08/10/2018 
11 
 
potential participants entering their clinics who might fit the eligibility criteria.  Participating GPs and 
clinicians will be provided with this Study Protocol so they may answer brief questions about the 
study, but potential participants will still be asked to return the response slip to QIB and will receive 
a telephone call from Ellie Sayers or Jacqueline Chipping prior to visiting NNUH for their initial Early 
Arthritis Clinic appointment.  If a potential participant is recruited from their initial Early Arthritis 
Clinic appointment at NNUH and responds positively using the response slip, they will receive the 
telephone call prior to their Nurse Practitioner appointment at NNUH.   
 Eligibility criteria  
5.4.1 Inclusion criteria  
• 
18-65 years of age 
• 
RA diagnosis based on ACR 2010 classification criteria with symptoms starting within the last 
2 years 
• 
Referred by GP to the Early Arthritis Clinic at NNUH  
• 
Commencing methotrexate monotherapy for the first time 
5.4.2 Exclusion criteria  
• 
Initially commencing combination therapy (prior to first stool sample) rather than 
methotrexate monotherapy i.e. MTX combined with another DMARD or prednisolone 
• 
Commencement of MTX therapy prior to first stool sample or cessation of MTX therapy at 
any point during the study 
• 
History of psoriasis  
• 
Those currently suffering from, or have ever suffered from, any diagnosed gastrointestinal 
disease, gastrointestinal disorders including regular diarrhoea and constipation (excluding 
hiatus hernia unless symptomatic) and/or have undergone gastrointestinal surgery.  
• 
Those regularly (3+ times/week) taking self-prescribed over the counter medications for 
digestive/gastrointestinal conditions 
• 
Use of laxatives within 7 days prior to sampling unless these have been used on a regular 
basis (3+ times/week) for more than one month prior to the study and will continue to be 
used throughout the study period 
• 
The use of over-the-counter medications or food/drinks containing pre and/or probiotics 
within 7 days prior to sampling, unless these have been used on a regular basis (3+ 
times/week) for more than one month prior to the study and will continue to be used 
throughout the study period 
• 
Significant alteration of the participant’s normal diet at any point during the study (e.g. 
adoption of the 5:2 fasting diet) 
• 
Regular (3+ times/week) or recent (within 3 months) use of colonic irrigation or other bowel 
cleansing techniques 
• 
Recently returned to the UK following a period abroad, and who have suffered gastric 
symptoms during the period abroad or on return to the UK. These will be assessed on an 
individual basis 
• 
Currently taking or finished a course of antibiotics within the last 3 months  
• 
Currently pregnant or lactating  
• 
Living with or related to any member of the Study Team 
• 
Those who have limited or no understanding of spoken and written English 
 Study procedures  
5.5.1 First appointment: Early Arthritis Clinic  

Appendix 1 - Study Protocol 
MyRA study 
Version 2 – 08/10/2018 
12 
 
5.5.1.1 Informed consent  
At the participant’s first Early Arthritis Clinic appointment, any member of the Study Team 
appropriately trained in taking Informed Consent will be present to ensure the participant knows 
what the study involves and has read and understood the PIS before asking for written Informed 
Consent, both for participation in the study and for long-term storage of samples at the Norwich 
Research Park (NRP) Biorepository (Appendix 4 and 5).  The consent to store samples long-term in 
the NRP Biorepository is completely optional.  Alternatively, the Rheumatology Clinician in charge of 
the participant’s care may take written Informed Consent on behalf of the study team.  The clinicians 
will have copies of this protocol and will have completed mandatory General Clinical Practice (GCP) 
training which involves guidelines for taking Informed Consent.  Consent will not be sought from 
potential participants who heard about the study for the first time on the same day.  
If the participant is identified by their Rheumatology Clinician at their initial Early Arthritis Clinic 
appointment and responds positively to the invitation letter, they will not provide informed consent 
at this stage.  Therefore, informed consent for these participants will be taken at their Nurse 
Practitioner (NP) appointment by an appropriately trained member of the study team before taking 
the first study samples.  Watermarked versions of the consent forms (Appendix 24 and 25) will be 
posted to the participant prior to this appointment for them to review and they will have had an 
initial telephone call with a member of the study team.   
After consent, a letter to the participant’s GP will be generated to inform them of their patient’s 
involvement with MyRA (Appendix 6).  The participant will have agreed to this and provided their GP 
details as part of the consent process.   
5.5.1.2 Providing initial study materials  
The participant will then be provided with a stool collection kit containing a home stool collection 
device (Fecotainer), a clear plastic zip-lock bag to place the Fecotainer in and instructions for taking 
the stool sample (Appendix 7).  A 7-day food diary (Appendix 8) will be provided to be completed the 
week prior to the participant’s Nurse Practitioner (NP) appointment (approximately 3 weeks after 
their first Early Arthritis Clinic appointment), where they will commence methotrexate (MTX) 
treatment as part of their normal clinical care.  Participants will be asked to bring a fresh stool 
sample to the NP appointment (collected within 2 hours of the appointment), or if they are unable 
to do this they may wish to organize collection of the stool sample with Ellie Sayers or Jacqueline 
Chipping either the day before their NP appointment, or the day after provided they have not yet 
begun MTX treatment at the time.    
5.5.1.3 Collection of samples and data if the participant is started on methotrexate at their 
first Early Arthritis Clinic appointment  
Should the Rheumatology Clinician begin their patient on MTX at this first appointment; without 
arranging an NP appointment, stool and blood samples may be collected at this time point (if the 
clinician agrees it is suitable to do this prior to commencing the first dose of MTX).   Collection of 
samples and data would be as per Section 5.5.2.1, 5.5.2.2 and 5.5.2.3, except the participant would 
be asked to fill in the 7-day food diary from this point forward and bring it to their next Routine 
Treatment Review where the study team will be present.  In this case, the participant would then 
only be seen at their 3 and 6-month Routine Treatment Review appointments (Section 5.5.3) and 
not at an NP appointment.  No samples or data will be collected before the participant has provided 
written informed consent to take part in this study.   

Appendix 1 - Study Protocol 
MyRA study 
Version 2 – 08/10/2018 
13 
 
5.5.2 Second appointment: Nurse Practitioner  
As part of their normal care pathway, the participant will commence MTX treatment after this 
appointment.  Should the participant or their clinician decide for any reason not to commence MTX 
treatment, they would be excluded from the study from this point.  If continuing the study, 
participants will be provided with another stool collection kit at the end of this appointment 
(Fecotainer, instructions for use [Appendix 7], clear plastic zip-lock bag).  No further 7-day food 
diaries will be provided.   
5.5.2.1 Collection of stool sample 
At the participant’s NP appointment their stool sample will be taken from them by a member of the 
study team, if they were able to produce this sample within the 2 hours prior to this appointment.  If 
this was not possible, collection of a fresh stool sample from the participant’s location may be 
organized between the participant and Ellie Sayers or Jacqueline Chipping for the following day, 
provided the sample is produced prior to the participant taking their first dose of methotrexate.  If 
the participant was consented at their NP appointment, they will be asked to produce a stool sample 
before taking their first dose of methotrexate.  The participant will be asked to bring another fresh 
stool sample (taken within 2 hours) to their next appointment in 3 months’ time, or to arrange 
collection of this sample at a time more suitable to them, using the stool collection kit provided.    
5.5.2.2 Collection of blood sample 
The on-duty Healthcare Assistant (HCA) or Phlebotomist at the NNUH; who will be taking 
approximately 4ml blood as part of the participant’s routine clinical care (to test for inflammatory 
biomarkers), will be asked to obtain an additional 40 mL blood sample during the same procedure to 
minimize inconvenience to the participant.  This additional 40 mL blood sample will be collected 
solely for the purpose of this study, and in the form of 2 x 10 mL whole blood (20 mL in Vacutainer 
EDTA tubes) and 2 x 10 mL serum (20 mL in Vacutainer SST tubes).   
5.5.2.3 Collection of 7-day food diary, baseline health and lifestyle questionnaire and 
clinical data 
The participant’s completed 7-day food diary will be collected, and any member of the Study Team 
will go through a baseline health and lifestyle questionnaire (Appendix 9) with the participant at this 
stage.  No further 7-day food diaries will be provided to the participant for the purpose of this study.  
A methotrexate treatment record will be provided to the participant (Appendix 23) where they will 
record the dates and dosages of methotrexate taken.  Participants will need to keep this document 
until they have completed the study, so it will be retrieved at the final study visit (6 month routine 
treatment review).  
After the appointment, a member of the study team with appropriate access to NNUH hospital 
systems will obtain clinical data including DAS28-CRP (recorded by the clinician) using the Case 
Report Form (Appendix 11). 
5.5.3 Third and fourth appointments: Routine Treatment Review  
The following two data collection points will coincide with routine treatment review appointments 
organized as part of the participant’s normal care pathway (at approximately 3 and 6 months from 
the NP appointment or time of commencing MTX/post-MTX).   
The 3-month appointment will reflect the NP appointment in that another 40 mL blood sample will 
be taken for the purpose of this study by the on-duty HCA/Phlebotomist; as per Section 5.5.2.2, and 
the participant’s stool sample will be collected from them as per Section 5.5.2.1.  Clinical data will be 
recorded by a member of the study team with appropriate access to NNUH hospital systems using 
the Case Report Form (Appendix 11).  Any member of the Study Team will go through another health 

Appendix 1 - Study Protocol 
MyRA study 
Version 2 – 08/10/2018 
14 
 
and lifestyle questionnaire with the participant, although this will be a shortened partial version of 
the questionnaire aimed at identifying changes (if any) from the NP appointment (Appendix 10).   
The participant will be provided with another stool collection kit for the final stool sample to be 

Appendix 1 - Study Protocol 
MyRA study 
Version 2 – 08/10/2018 
15 
 
brought to their next treatment review (at 6 months post-MTX, approximately 3 months after their 
3-month treatment review).   
At the 6-month treatment review, the participant’s final stool sample will be collected as previously, 
any member of the Study Team will go through a final follow-up health and lifestyle questionnaire 
(Appendix 10) with the participant and their methotrexate treatment record (Appendix 23) will be 
collected.  Clinical data will be recorded by a member of the study team with appropriate access to 
NNUH hospital systems using the Case Report Form (Appendix 11).  No further blood samples will be 
taken for the purpose of this study at this 6-month treatment review.  Following this final 
appointment, no further information will be required from the participant and their involvement 
with this study will end.  Their routine care will continue as normal.  
 Withdrawal procedure  
If at any point during the study the participant wishes to withdraw, they may do so without giving a 
reason and their clinical care and participation in future studies at QIB, UEA or NNUH will not be 
affected.  The participant will be sent a withdrawal letter (Appendix 12) explaining this and thanking 
them for their participation so far.  Any samples or data collected up to the point of withdrawal will 
be kept and used in the study.   
The withdrawal letter (Appendix 12) will also be sent to participants if they need to be withdrawn 
from the study by a member of the study team due to meeting exclusion criteria; for instance, due 
to cessation of MTX therapy or starting MTX prior to collection of the first stool sample.  
 Data collection and storage 
Data will be collected in the form of a health and lifestyle questionnaire (Appendix 9 and 10) and 7-
day food diary (Appendix 8), in addition to clinical metadata collected by any member of the Study 
Team with the necessary access to hospital systems (Ellie Sayers, Jacqueline Chipping, Alexander 
MacGregor).  Clinical data including DAS28-CRP, CRP, erythrocyte sedimentation rate (ESR), anti-
cyclic citrullinated peptide (anti-CCP), rheumatoid factor, height/weight and Bristol stool score will 
be collected by means of a study-specific Case Report Form (Appendix 11).   
All potentially patient-identifiable and other physical data (e.g. forms) will be stored in a locked file 
cabinet at QIB, with access restricted to study scientists.  Patients will be provided with a unique 
patient identifier consisting of an unrelated sequence of numbers which does not contain any 
personal information (MyRA_year_month of consent_participant number [MyRA_18_10_01), and 
samples will be anonymized at the point of receipt using a suffix to this identifier (_time point and 
sample type [stool or blood] e.g. S1 or B1 _date sample taken [MyRA_18_10_01_S1_251018]).  
Linked information will be kept in separate files from the personal information pertaining to each 
participant, and if the participant is also taking part in the NOAR study this information will include 
their unique NOAR identifier.  The location of each sample in QIB (or later the NRP Biorepository) will 
be recorded.  Any data electronically inputted or generated in relation to forms and samples will be 
stored in a secure (password-protected or encrypted) database on QIB computers.   
5.7.1 Collection of disease activity variables 
5.7.1.1 DAS28-CRP 
The DAS28 is a composite score made up of counts of the number of swollen (SJC) and tender (TJC) 
joints out of a total 28 measured, erythrocyte sedimentation rate (DAS28-ESR) or C-reactive protein 
(DAS28-CRP), and a health assessment questionnaire (patient global assessment of disease activity 
[PtGA], measured via a visual analogue scale [VAS]) filled in by the patient.  While DAS28-ESR and 

Appendix 1 - Study Protocol 
MyRA study 
Version 2 – 08/10/2018 
16 
 
DAS28-CRP are often used interchangeably in clinical practice, DAS28-CRP will be used for this study; 
as ESR is not always measured in clinical laboratories.   
DAS28-CRP is used by clinicians to monitor patient progress and inform treatment strategies, and 
will be recorded by the clinician at each routine appointment.  DAS28-CRP in the participant’s 
electronic or paper notes will be recorded by a member of the study team with appropriate access 
to hospital systems via the Case Report Form (Appendix 11).  Should the DAS28-CRP not be recorded 
during a routine clinical appointment, each component of the DAS28-CRP is listed individually to 
enable calculation by the study team using the below equation28:   
0.56 × √𝑇𝐽𝐶+ 0.28 × √𝑆𝐽𝐶+ 0.36 × ln(𝐶𝑅𝑃+ 1) + 0.014 × 𝑃𝑡𝐺𝐴 + 0.96 
5.7.1.2 Other measures of disease activity 
Although DAS28-CRP is the standard measure used in medicine to measure RA disease activity, other 
measures may be used by some clinicians.  Therefore another disease activity variable which we will 
record in this study via the Case Report Form (Appendix 11) is the Simplified Disease Activity Index 
(SDAI), which uses the same variables as DAS28-CRP but with the addition of provider global 
assessment of disease activity (PrGA).  PrGA is a similar measure to PtGA and is also measured on a 
VAS, but is an indicator of the clinician’s opinion on their patient’s disease activity.  The SDAI is 
calculated as a cumulative sum of SJC, TJC, PrGA, PtGA and CRP. 
Activity  
Pre-
study 
Week 0 
(Early 
Arthritis 
Clinic) 
Week 2-3 
(Nurse 
Practitioner) 
Week 12-15 
(3 month 
Routine 
Treatment 
Review) 
Week 24-27 
(6 month 
Routine 
Treatment 
Review) 
Identify suitable participant 
and send PIS and response 
slip 
✓ 
 
 
 
 
Telephone discussion to 
discuss study and confirm 
date and time of next 
appointment (on receipt of 
positive response) 
✓ 
✓* 
 
 
 
Informed consent 
 
✓ 
✓* 
 
 
Provide Fecotainer to 
participant 
 
✓ 
✓ 
✓ 
 
Provide 7-day food diary to 
participant 
 
✓ 
✓* 
 
 
Provide methotrexate 
treatment record to 
participant 
 
 
✓ 
 
 
Stool sample and health 
questionnaire collected 
 
 
✓ 
✓ 
✓ 
Figure 4. Overview of data collection time points 

Appendix 1 - Study Protocol 
MyRA study 
Version 2 – 08/10/2018 
17 
 
*If participant is identified at the Early Arthritis Clinic, these activities will be carried out at this later 
stage.   
  Diet and lifestyle assessment  
7-day food diaries have been provided by the European Prospective Investigation of Cancer (EPIC) 
study, are validated and approved for re-use in this study.  Ellie Sayers will be responsible for the 
data entry and analysis of these food diaries, for which expertise will be drawn from diet and 
nutrition researchers at UEA.  Dietary analysis software such as Tinuviel (WISP) will be used for 
determination of micro/macronutrient intakes from food diaries, as it has been in previous studies29–
31.  The specific diet components to be analyzed will be based on current known dietary risk factors 
for RA, including red meat, fatty fish (omega 3), and vitamin C intake32–34.  Other components to be 
investigated will be determined in an analysis of epidemiological data from the EPIC/NOAR link 
(participants in EPIC who are also registered with NOAR, for which there is historical 7-day food diary 
information available).  Additional dietary components identified in association with methotrexate 
response from the EPIC/NOAR link data will be drivers for the analysis of food diaries in this study.   
The health and lifestyle questionnaires are taken from the Microbiome of the Ageing Gut and its 
Effect on Cognition (MOTION) study, and have been approved for re-use in this study.  The IRAS 
number for the MOTION study is 241617, and the ethical application is still in progress.  The health 
and lifestyle questionnaire was reviewed by Patient Participation Groups (PPG) from four Norfolk GP 
sites. Twelve members from the four PPG groups returned comments on the questionnaire. 
Comments were noted and amendments made.  The questionnaire consists of several questions 
related to factors which can affect microbiome composition, such as the use of over-the-counter or 
prescription drugs, living arrangements and recent travel.  Recording these factors at baseline and 
changes at each subsequent time point will help account for confounding variables during the 
analysis of microbiome data. 
 Sample processing and analysis 
5.9.1 Blood samples 
5.9.1.1 Collection and storage 
Blood samples will be collected at the second and third appointments by the on-duty HCA or 
Phlebotomist at the NNUH Rheumatology Department who will be taking blood samples as part of 
the participant’s routine clinical care.  A total of 80 mL blood will be taken for the purpose of this 
study at the second and third appointment (pre- and post-MTX), in the form of 2 x 10 mL Vacutainer 
SSTs and 2 x 10 mL Vacutainer EDTA tubes.  Serum samples will be aliquoted and frozen in a -20°C 
freezer, whereas whole blood samples will be processed to isolate PBMCs which will be stored in 
cryovials in liquid nitrogen until further analysis can be carried out.   Both samples will be stored at 
QIB (within a maximum of 1 hour after receipt) for the duration of the study and will be transferred 
to the NRP Biorepository at the end of the study for long-term storage. 
7-day food diary collected 
 
 
✓ 
✓* 
 
Methotrexate treatment 
record collected  
 
 
 
 
✓ 
Clinical data collected via 
case report form 
 
 
✓ 
✓ 
✓ 
40 mL blood sample (20 mL 
serum, 20 mL whole blood) 
 
 
✓ 
✓ 
 

Appendix 1 - Study Protocol 
MyRA study 
Version 2 – 08/10/2018 
18 
 
5.9.1.2 Immunological analyses  
Serum and whole blood samples will be used in immunoassays currently being developed at QIB.  
The serum assay will involve enzyme-linked immunosorbent assays (ELISA) in which systemic 
antibodies against components of the participant’s own microbiota (derived from their stool sample) 
are detected.  This analysis will identify if the participant has systemic immunity against their own 
gut microbiota, which could be an indicator of a “leaky gut”.  PBMCs will be isolated from whole 
blood samples and frozen for future use in cellular assays for immune reactivity to commensal 
microbes; for instance, using proliferation assays and cytokine production assays.  Together, these 
assays will provide information on systemic immune reactivity towards components of commensal 
microbes, giving an indicator of intestinal permeability and the ability of these microbes to elicit 
immune responses in the host.  Changes to the participant’s immune reactivity against their own gut 
microbes may occur with treatment (for instance if there was increased intestinal permeability prior 
to treatment which resolved with MTX), which will also be assessed in this study.   
5.9.2 Stool samples 
5.9.2.1 Collection and storage 
Stool samples will be collected by the participant and received by any member of the Study Team at 
the second, third and fourth appointments.  These samples will either be delivered to the NNUH by 
the participant at their routine appointment or collected by a member of the study team from their 
home a day either side of this appointment.  Collection by the study team from the participant’s 
location will have been organized between the participant and study team to arrange a suitable 
time.  Each stool sample will be separated into aliquots and frozen at -80°C in a QIB freezer until 
further analysis can be carried out.  This will be done as soon as possible after collection of the fresh 
sample (within a maximum 1 hour after receiving the sample), to avoid unwanted changes to the 
microbial communities present in the sample.  Any remaining original sample will be discarded, and 
at the end of the study stool samples left over will be transferred to the NRP Biorepository for long-
term storage.   
5.9.2.2 Shotgun metagenomic sequencing  
Total microbial DNA will be extracted from stool samples, quantified and sent to Novogene (Hong 
Kong) for shotgun metagenomic sequencing on the Illumina platform (insert size, 300bp; paired-end 
read length, 150bp; depth, 10GB data per sample).  The resulting sequencing data received from 
Novogene will be used to identify the α-diversity (Shannon Index) and bacterial composition and 
functional profiles in the stool samples.  The raw sequences will also be used to identify viral 
sequences, using a pipeline currently being developed at QIB.  Correlations will be drawn between α-
diversity (Shannon Index) and change in DAS28-CRP to answer the primary research aim.  Various 
statistical analyses (see Statistical Methods) will be carried out to determine the diversity (e.g. 
Shannon index, Bray-Curtis dissimilarity) and differential abundance of microbial genera between 
responders and non-responders to MTX; which will inform on the reliability of microbiome 
parameters as predictors of MTX response.  This analysis will be carried out by Ellie Sayers with the 
support of QIB Bioinformaticians.   
6 Dissemination of findings  
Data generated from this study will be anonymized and published as part of a postgraduate thesis 
and research journal articles.  The data may also be presented in anonymized form at conferences.  
At the end of the study, a brief, lay summary of the findings of the study will be posted to 
participants on QIB headed paper, meeting the HRA transparency requirements under the new 

Appendix 1 - Study Protocol 
MyRA study 
Version 2 – 08/10/2018 
19 
 
General Data Protection Regulation (GDPR) legislation. Anonymized datasets will be kept indefinitely 
and available to other researchers to comply with journal requirements when required. 
7 Statistical methods  
 Sample size calculations  
Sample size calculations were carried out by Dr. George Savva, a QIB statistician.  Previous studies 
suggest that change in DAS28 score in response to treatment has a standard deviation of 1 to 1.3 
points35,36.  The minimum clinically relevant difference in DAS28 has been reported in two different 
studies as 1-1.2 points36,37. 
Hence, to detect a clinically relevant difference in DAS28 we will aim to recruit enough participants 
to estimate an effect size of one standard deviation in the outcome per two standard deviations in 
Shannon index, corresponding to a correlation coefficient of 0.5, typically considered a large effect.   
This will require 30 subjects to complete the study (true power=82.5%, size=0.05), hence our target 
sample size for recruitment is 35, allowing for 5 participants to drop-out of the study.  Using the 
change score from baseline to the mean of two follow-up responses as the primary outcome will 
slightly improve the power of the study but it is difficult to know by how much without prior 
information on the longitudinal variation of DAS28.  Power calculations were conducted using the 
pwr package in R. 
Multivariate analyses linking detail of the structure and composition of the microbiome to outcomes 
are purely exploratory and will be reported as such. 
 Statistical methods for the analysis of study data  
7.2.1 Correlating microbiome parameters with treatment outcome  
Spearmans correlation coefficient will be used to identify statistically significant associations 
between (continuous variables) Shannon index at baseline and change in DAS28-CRP from baseline 
to 3 months post-MTX.  In addition, Shannon index and other microbiome characteristics such as 
taxa and gene abundance will be analysed for covariation with other clinical metadata (CRP, anti-
CCP, RF), also using Spearmans correlation coefficient.  The predictive capability of the microbiome 
for MTX response will be assessed by generation of a cross-validated predictive model, using a 
machine-learning R statistical package such as randomForest. 
7.2.2 Identifying microbiome alterations induced by methotrexate 
Longitudinal changes to the microbiome during MTX treatment will be studied using a regression 
model.  Alterations to the microbiome throughout methotrexate treatment will also be visualized 
using principle coordinates analysis (PCoA) and β-diversity (Bray-Curtis dissimilarity; between-
sample diversity).  This analysis will be done using an R package such as vegan. 
7.2.3 Other statistical analyses  
Multivariable models will be developed to explore whether diet and lifestyle factors can help 
account for any alterations seen in the microbiome associated with treatment response.  Blood work 
will mainly consist of ELISA, for which parametric tests will be used to determine the signal strength 
of fluorescence from each sample, corresponding to the titer of antibodies against enteric microbes. 
All analyses will be conducted in R, GraphPad Prism or similar statistical programs. 

Appendix 1 - Study Protocol 
MyRA study 
Version 2 – 08/10/2018 
20 
 
8 Resource requirements 
 Staffing requirements 
Processing and analysis of samples and data will be carried out primarily by Ellie Sayers, a PhD 
Student at UEA for whom a maintenance stipend is provided by a Faculty of Medicine and Health 
Science Studentship, and for which this study will form the main part of a PhD.  In addition, 
Jacqueline Chipping or another NOAR Research Nurse may be required to take blood samples, assist 
with consenting participants and collecting stool samples.  Novogene (Hong Kong) services will be 
used for shotgun metagenomic sequencing.    
 Material requirements 
A successful funding application to the local Norfolk charity Action Arthritis (Registered Charity 
Number 292569) has provided £18,629.02 for lab consumables and study materials (Appendix 16).  
The 7-day food diaries are already available for use at the BCREB.  A short general health 
questionnaire has been generated by Simon Carding for use in a larger project (the MOTION study, 
IRAS no. 241617, ethical application still in progress) to encompass factors affecting the intestinal 
microbiome, such as exercise, medications and lifestyle.   
9 Confidentiality 
All members of the Study Team will be fully compliant with the requirements of the EU General Data 
Protection Regulation (GDPR) 2016/679 and the UK Data Protection Act (DPA) 2018 with regards to 
the collection, storage, processing and disclosure of personal information and will uphold the GDPR 
and DPA core principles to maintain confidentiality.  Personal information will be anonymized in a 
linked form on receipt, by using a unique identifier consisting of an unrelated sequence of characters 
and numbers, and stored securely in a locked file cabinet at QIB with keys available only to members 
of the study team.  Linked data will be also only be accessible by the study team and will stored in an 
encrypted or password-protected database on UEA or QIB computers.  The data will be stored for 15 
years in case it is required for further research, and the data custodian will be Ellie Sayers. 
10 Ethical considerations  
As this study involves the recruitment and participation of human subjects from the National Health 
Service (NHS), appropriate Research Ethics Committee (REC) approval will be obtained by applying to 
the Health Research Authority (HRA) via the Integrated Research Application System (IRAS).   
 Informed consent 
Written informed consent will be obtained from participants entering into the study by an 
appropriately trained member of the Study Team or Rheumatology Clinician, and members of the 
Study Team will continuously ensure the participants are happy to continue with the study and 
provide samples, in accordance with National Institute for Health Research Good Clinical Practice 
(GCP) guidelines.   
 Risks associated with study procedures  
The risks associated with venipuncture such as discomfort and bruising will be minimized as only an 
appropriately trained HCA/Phlebotomist at NNUH will carry out this procedure, unless they are 
unable to do so for any reason in which case an appropriately trained member of the Study Team 
(e.g. the NOAR Research Nurse) will carry out the procedure.   

Appendix 1 - Study Protocol 
MyRA study 
Version 2 – 08/10/2018 
21 
 
 Reporting of Serious Adverse Events 
As this study does not involve any intervention or procedure which will not be part of the 
participant’s routine clinical care, we will not officially record any Serious Adverse Events (SAEs).  
 Future research 
Samples and data obtained from this study may be used in future research projects, where those 
projects have obtained the appropriate ethical approval to do so.  No samples or data from this 
study will be used by other projects without the appropriate ethical approval.   
11 References  
1. 
Jethwa, H. & Abraham, S. The evidence for microbiome manipulation in inflammatory 
arthritis. Rheumatology (Oxford, England) 56, 1452–1460 (2017). 
2. 
WHO. Chronic rheumatic conditions. WHO (2016). Available at: 
http://www.who.int/chp/topics/rheumatic/en/. (Accessed: 16th March 2018) 
3. 
MacGregor, A. J. et al. Characterizing the quantitative genetic contribution to rheumatoid 
arthritis using data from twins. Arthritis Rheumatol. 43, 30–37 (2000). 
4. 
Saevarsdottir, S. et al. Predictors of response to methotrexate in early DMARD naive 
rheumatoid arthritis: Results from the initial open-label phase of the SWEFOT trial. Ann. 
Rheum. Dis. 70, 469–475 (2011). 
5. 
Valerino, D., Johns, D., Zaharko, D. & Oliverio, V. Studies of the metabolism of methotrexate 
by intestinal flora—I: Identification and study of biological properties of the metabolite 4-
amino-4-deoxy-N10-methylpteroic acid. Biochem. Pharmacol. 21, 821–831 (1972). 
6. 
Webb, M. Inactivation of analogues of folic acid by certain non-exacting bacteria. BBA - 
Biochim. Biophys. Acta 17, 212–225 (1955). 
7. 
Levy, C. C. & Goldman, P. The enzymatic hydrolysis of methotrexate. J. Biol. Chem. 242, 2933–
2938 (1967). 
8. 
Scher, J. U. et al. Expansion of intestinal Prevotella copri correlates with enhanced 
susceptibility to arthritis. Elife 2, e01202 (2013). 
9. 
Zhang, X. et al. The oral and gut microbiomes are perturbed in rheumatoid arthritis and partly 
normalized after treatment. Nat Med 21, 895–905 (2015). 
10. 
Chen, J. et al. An expansion of rare lineage intestinal microbes characterizes rheumatoid 
arthritis. Genome Med. 8, (2016). 
11. 
Maeda, Y. et al. Dysbiosis Contributes to Arthritis Development via Activation of Autoreactive 
T Cells in the Intestine. Arthritis Rheumatol 68, 2646–2661 (2016). 
12. 
Tsuda, R. et al. Monoclonal antibody against citrullinated peptides obtained from rheumatoid 
arthritis patients reacts with numerous citrullinated microbial and food proteins. Arthritis 
Rheumatol. 67, 2020–2031 (2015). 
13. 
Mielants, H. et al. Intestinal mucosal permeability in inflammatory rheumatic diseases. I. Role 
of antiinflammatory drugs. J. Rheumatol. 18, 389–393 (1991). 
14. 
Bjarnason, I. et al. Intestinal permeability and inflammation in rheumatoid arthritis: effects of 
non-steroidal anti-inflammatory drugs. Lancet 324, 1171–1174 (1984). 
15. 
Carding, S. R., Davis, N. & Hoyles, L. Review article: the human intestinal virome in health and 

Appendix 1 - Study Protocol 
MyRA study 
Version 2 – 08/10/2018 
22 
 
disease. Alimentary Pharmacology and Therapeutics 46, 800–815 (2017). 
16. 
Reyes, A. et al. Viruses in the fecal microbiota of monozygotic twins and their mothers. 
Nature 466, 334–338 (2010). 
17. 
Breitbart, M. et al. Viral diversity and dynamics in an infant gut. Res. Microbiol. 159, 367–373 
(2008). 
18. 
Minot, S. et al. The human gut virome: inter-individual variation and dynamic response to 
diet. Genome Res 21, 1616–1625 (2011). 
19. 
Norman, J. M. et al. Disease-specific alterations in the enteric virome in inflammatory bowel 
disease. Cell 160, 447–460 (2015). 
20. 
Nguyen, S. et al. Bacteriophage Transcytosis Provides a Mechanism To Cross Epithelial Cell 
Layers. MBio 8, e01874-17 (2017). 
21. 
Di Giuseppe, D., Discacciati, A., Orsini, N. & Wolk, A. Cigarette smoking and risk of rheumatoid 
arthritis: a dose-response meta-analysis. Arthritis Res Ther 16, R61 (2014). 
22. 
Hayashi, H. et al. Nutritional status in relation to adipokines and oxidative stress is associated 
with disease activity in patients with rheumatoid arthritis. Nutrition 28, 1109–14 (2012). 
23. 
Kharlamova, N. et al. Antibodies to Porphyromonas gingivalis Indicate Interaction between 
Oral Infection, Smoking, and Risk Genes in Rheumatoid Arthritis Etiology. Arthritis Rheumatol. 
68, 604–613 (2016). 
24. 
Debelius, J. et al. Tiny microbes, enormous impacts: What matters in gut microbiome 
studies? Genome Biol. 17, (2016). 
25. 
Chan, Y. K., Estaki, M. & Gibson, D. L. Clinical consequences of diet-induced dysbiosis. Ann. 
Nutr. Metab. 63, 28–40 (2013). 
26. 
Tedeschi, S. K. et al. Diet and Rheumatoid Arthritis Symptoms: Survey Results From a 
Rheumatoid Arthritis Registry. Arthritis Care Res. 69, 1920–1925 (2017). 
27. 
Hagen, K. B., Byfuglien, M. G., Falzon, L., Olsen, S. U. & Smedslund, G. Dietary interventions 
for rheumatoid arthritis. Cochrane Database Syst. Rev. CD006400 (2009). 
doi:10.1002/14651858.CD006400.pub2 
28. 
Anderson, J. et al. Rheumatoid Arthritis Disease Activity Measures: American College of 
Rheumatology Recommendations for Use in Clinical Practice. (2012). 
29. 
Jennings, A., Cassidy, A., Van Sluijs, E. M. F., Griffin, S. J. & Welch, A. A. Associations between 
eating frequency, adiposity, diet, and activity in 9-10 year old healthy-weight and centrally 
obese children. Obesity 20, 1462–1468 (2012). 
30. 
Harrison, F. et al. Food and drink consumption at school lunchtime: The impact of lunch type 
and contribution to overall intake in British 9-10-year-old children. Public Health Nutr. 16, 
1132–1139 (2013). 
31. 
Davidson, R. et al. Isothiocyanates are detected in human synovial fluid following broccoli 
consumption and can affect the tissues of the knee joint. Sci. Rep. 7, (2017). 
32. 
Pattison, D. J. et al. Dietary risk factors for the development of inflammatory polyarthritis: 
Evidence for a role of high level of red meat consumption. Arthritis Rheum. 50, 3804–3812 
(2004). 
33. 
Pattison, D. J. et al. Vitamin C and the risk of developing inflammatory polyarthritis: 

Appendix 1 - Study Protocol 
MyRA study 
Version 2 – 08/10/2018 
23 
 
prospective nested case-control study. Ann Rheum Dis 63, 843–847 (2004). 
34. 
Di Giuseppe, D., Crippa, A., Orsini, N. & Wolk, A. Fish consumption and risk of rheumatoid 
arthritis: a dose-response meta-analysis. Arthritis Res Ther 16, 446 (2014). 
35. 
Fransen, J., Bernelot Moens, H., Speyer, I. & C M van Riel, P. L. Effectiveness of systematic 
monitoring of rheumatoid arthritis disease activity in daily practice: a multicentre, cluster 
randomised controlled trial. Ann Rheum Dis 64, 1294–1298 (2005). 
36. 
Ward, M. M., Guthrie, L. C. & Alba, M. I. Clinically important changes in individual and 
composite measures of rheumatoid arthritis activity: Thresholds applicable in clinical trials. 
Ann. Rheum. Dis. 74, 1691–1696 (2015). 
37. 
Aletaha, D., Funovits, J., Ward, M. M., Smolen, J. S. & Kvien, T. K. Perception of improvement 
in patients with rheumatoid arthritis varies with disease activity levels at baseline. Arthritis 
Rheum. 61, 313–320 (2009).