EXtremely Early-onset Type 1 Diabetes EXtremely Early-onset Type 1 Diabetes (A Musketeers' Memorandum Study)

Type 1 diabetes (T1D) is a common autoimmune disease that causes destruction of pancreatic,
insulin producing beta cells, leading to high blood glucose. T1D is regarded as a childhood
disease with an average age of diagnosis of 13 years, but the age presentation is very
variable from young infants until late adulthood.

In Exeter, a group of rare children who have developed T1D in the first year of life (Patel)
is described as having Extremely Early-onset Type 1 Diabetes (EET1D). Studying these rare
patients is important because they are presenting with autoimmunity right at the beginning of
life when the immune system is not yet fully developed and at a time when pancreatic
autoimmunity first emerges (Krisher) and so this study may give novel insights into the cause
of T1D.

The Exeter Molecular Genetics Laboratory is a world referral centre (www.diabetesgenes.org)
for Neonatal Diabetes (NDM). Most cases of diabetes diagnosed under 6 months do not have
EET1D but have genetic mutations in beta cell genes that lead to impaired insulin production
(NDM)(Ellard; De Franco). Exeter is able to identify the remaining <20% without a mutation in
a beta cell gene who actually have EET1D. Exeter uses a novel measure of T1D risk genes,
called the T1D Genetic Risk Score (T1D GRS), showing that a proportion of the remaining
patients have very high T1D risk and therefore EET1D(Patel). Understanding the mechanism for
very early presentation could be highly important as immune strategies to intervene before or
after people get T1D may differ by age of onset.

The results may focus the research community on events that occur before birth and may then
inform new efforts to prevent or intervene in the underlying destruction of beta cells in
T1D.

Hypotheses:

i) Extreme early-onset T1D (EET1D) is associated with classic biomarkers of T1D, such as
islet specific autoantibodies, autoreactive islet specific CD8 T cells, and loss of beta cell
function, whereas monogenic neonatal diabetes will not be associated with these markers.

ii) EET1D will be associated with more rapid beta cell loss than T1D presenting at older
ages.

iii) The mechanisms for EET1D will be due to rare changes in immune genes or due to a
particularly potent, early response of the immune system to beta cells, as measured by
autoreactive T cells or immune gene expression when compared to older onset T1D.

Study Aim: The EXE-T1D study will take people with T1D diagnosed before the age of 24 months
and compare them to people with T1D diagnosed at more typical ages (1-20 years) and people
diagnosed with non-autoimmune diabetes at a similar very young age (children with neonatal
diabetes [NDM]).

EXE-T1D is an observational study organised into two sub-studies:

Study 1: Cross-sectional study of existing patients with EET1D (n=100 v 100): Assess islet
autoantibodies, islet T cell autoimmunity, C-peptide, RNAseq, genetics and clinical features
of EET1D compared to T1D in selected patients referred over the last 15 years to the Exeter
genetics team or Dr Oram who are of varying ages and durations of diabetes.

Study 2: Newly referred patients (n=20 v 20): Recruit newly diagnosed patients with EET1D who
are referred to Exeter or Dr Oram for diagnostic testing to allow assessment of immune
phenotype in patients close to diagnosis(Abreu; Unger; Velthuis). Assess immune function
longitudinally by collecting a blood sample for serum and peripheral lymphocytes, islet
autoantibodies and C-peptide assessment shortly after referral, and approximately 2 years
later.

Through promotion of the study we may be approached by patients' GPs and by patients
themselves, particularly because EET1D is rare. Recruitment to the study in this setting will
be by our team, including our Paediatric Diabetes Specialist Nurse, who will provide
information about the study and feedback inclusion of the participant in the study to the GP
and diabetes clinician as appropriate.

UK participants will be recruited under UK wide ethics. Exeter will recruit patients from
international centres in collaboration with local clinicians that have specific Institutional
Review Board (IRB) approval.

The Exeter Clinical Laboratories encompass the Exeter Blood Sciences Laboratory and Exeter
Molecular Genetics Laboratory at the Royal Devon and Exeter NHS Foundation Trust and will
perform C-peptide, islet autoantibody and genetic tests.

Peripheral lymphocyte (PBMC) analysis for autoreactive CD4 and CD8 T cells will be performed
by Bart Roep (City of Hope, CA, USA) and Tim Tree (King's College London). RNAseq will be
performed by Cate Speake and the Benaroya Research Institute, Seattle (USA).

All participants (or their legal guardian) recruited to the study will be required to give
written informed consent and will be informed of their right to withdraw from the study at
any time without prejudice or jeopardy to any future clinical care.

Patients identified and screened as being suitable for this study will have a blood sample
and optional urine sample collected by the clinical team at a time and location suitable for
the patient, clinical and study teams.

Study 2: In addition to the first visit, another blood sample for PBMC, C-peptide and
Autoantibody analysis will be collected in an identical manner to the first sample,
approximately 2 years (+/-6 months) later.

Non-UK samples will be collected by collaborating international centres with their own IRB
approval. The local team will spin and freeze the EDTA plasma sample and store it on site
while the PBMCs are extracted as per Exeter's SOP. All tubes will then be couriered to
Exeter. If no local team is available to extract PBMCs, all tubes will be couriered to Exeter
for analysis. For some centres, it may be possible to arrange for the samples to be flown
directly to the UK for PBMC extraction.

End of Study Definition: last participant's final study visit plus 6 months to enable
follow-up data capture.

Sample Receipt/Chain of Custody/Accountability The Exeter Clinical Laboratories have an
established pipeline for receiving and processing all research samples, including
documentation of chain of custody. Surplus samples will be processed, logged and frozen at
-80°C within 24 hours of receipt. All samples will be appropriately labelled in accordance
with the 1998 Data Protection Act. Biological samples collected from participants will be
transported, stored, accessed and processed in accordance with national legislation relating
to the use and storage of human tissue for research purposes.Participants will have the
opportunity to consent to gift samples at the end of the study for future research.

Results of analyses undertaken by the Exeter Clinical Laboratories will be electronically
uploaded directly to the study database and linked to Study IDs.

Safety, Definitions and Reporting Risks Blood samples will be collected by staff trained in
venepuncture. Any potential discomfort or side-effects will be equivalent to that experienced
in routine clinical care.

Benefits The C-peptide and autoantibody results may help to confirm a diagnosis of T1D so
will be reported back to clinicians responsible for the patient's diabetes care. Decisions
about ongoing clinical care and treatment will be made externally to the research study but
treatment will be recorded.

Adverse effects Should any unforeseen adverse events arise that are possibly, probably, or
definitely related to a study procedure, they will be reported to the Sponsor and CI/central
coordinating team within 24 hours of the CI or PI or co-investigators becoming aware of the
event.

Confidentiality All information related to study participants will be kept confidential and
managed in accordance with the Data Protection Act, NHS Caldicott Guardian, The Research
Governance Framework for Health and Social Care and Research Ethics Committee Approval.

Participant data will be held in a link-anonymised format, with personal identifiable data
only accessible to personnel with training in data protection who require this information to
perform their study role.

Record Retention and Archiving When the research study is complete, it is a requirement of
the Research Governance Framework and Sponsor Trust Policy that the records are kept for a
further 15 years.

Local investigator site files must be archived at the external site according to local R&D
requirements. They will not be stored at the coordinating centre's archiving facility.

Statistical Considerations Sample Size Total recruitment target is 240: Study 1: 100 with
EET1D plus 100 controls (N=200); Study 2: 20 EET1D plus 20 controls (N=40) Feasibility: The
sample size has been selected to assess feasibility rather than on the basis of statistical
power. In reality with these extremely rare but potentially very interesting patients, every
single patient recruited could contribute on their own to a novel discovery. The immune, beta
cell or autoantibody differences the study may reveal are unknown but a group of 20 v 20
gives an 80% power (alpha 0.05) to detect a difference of 10% v 50% in a proportion between
the two groups and a power of 85% (alpha 0.05) to detect a 1 SD difference in a continuous
variable.

Statistical analysis: The EET1D as described are unique and findings in the various studies
are difficult to predict given the novel nature of this study. The study using 100 EET1D v
100 controls gives a 90% power (alpha 0.05) to detect a difference in proportions of a binary
variable of 50% v 30% and a 0.6SD difference in a continuous variable, and similarly a group
of 20 v 20 gives an 80% power (alpha 0.05) to detect a difference of 10% v 50% in the two
groups and a power of 85% (alpha 0.05) to detect a 1 SD difference in a continuous variable.

Monitoring of this study will ensure compliance with Good Clinical Practice. The
Investigators will permit monitoring, audits, REC review, and regulatory inspections by
providing the Sponsor(s), Regulators and REC direct access to source data and other
documents.

No financial and other competing interests to disclose for the Chief Investigator, PIs at
each site and committee members for the overall study management.

NHS Indemnity will apply to UK participants and UK public liability insurance is provided by
the University of Exeter.

Accidental protocol deviations can happen at any time and must be adequately documented and
reported to the Chief Investigator and Sponsor immediately.

Access to the final study dataset The Exeter study team will have access to the final
dataset. Of the multiple analyses done during the study, relevant co-investigators for each
analysis (e.g. RNAseq for Cate Speake) will have access to the datasets they have contributed
to.

Public and Patient Involvement The CI's direct contact with patients with a diagnosis of
diabetes in the first 12 months of life led to the study design. Patients, relatives and
clinicians agree there would be significant benefit to knowing why and how T1D can present so
young and whether understanding this could help with treatment or prevention.

This work is funded by a patient-focused charity, Diabetes UK. Publication Policy On
completion of the study, the data will be analysed and a Final Study Report will be prepared
and submitted to the Funder, Sponsor and REC. Results will be written up and submitted for
publication in a peer-reviewed journal(s). Abstracts will be submitted to national and
international conferences. Written information in the form of a letter/newsletter outlining
the key findings of the study will be posted on the study website.

- The University of Exeter owns the data arising from the study.

- There are no time limits or review requirements on the publications.

- The Funder will be acknowledged within publications but does not have review or
publication rights.

- After the Final Study Report has been compiled, participants may specifically request
results from their PI.

Inclusion Criteria:

Study 1:

EET1D

- Aged 0 to 70 years

- Clinical diagnosis of diabetes <24 months (+ evidence of WHO diabetes criteria)

- Negative genetic test for mutations causing non-autoimmune neonatal diabetes if
diagnosed <12 months

- Type 1 diabetes genetic risk score >50th centile of T1D reference group, or monogenic
cause of T1D.

T1D Controls

- Age 0-70 years (matched to above)

- Clinical diagnosis of T1D (diagnosed age 1-20 years)

- Insulin treated from diagnosis.

Study 2:

EET1D

- Aged 0 to 24 months at recruitment

- Clinical diagnosis of diabetes <24 months (+ evidence of WHO diabetes criteria)

- Negative genetic test for mutations causing non-autoimmune neonatal diabetes

- Type 1 diabetes genetic risk score >50th centile of T1D reference group, or monogenic
cause of T1D.

NDM controls

- Diagnosis of diabetes <24 months

- Age 0 to 18 months at recruitment

- Diagnosis of NDM (confirmed by Exeter Molecular Genetics Laboratory).

Exclusion Criteria:

Study 1:

- Aged >70 years

- No diagnosis of diabetes

- MODY (e.g. caused by HNF1A/HNF4A/HNF1B/GCK mutations), type 2 diabetes or diabetes
related to pancreatic insufficiency or syndromic diabetes

- Intercurrent illness at time of sampling for PBMCs (see below).

Study 2:

- Aged >24 months

- Clinical diagnosis of diabetes >24 months

- Intercurrent illness at time of sampling for PBMCs or RNA (see below).

For PBMC and RNA sampling: Exclusion for factors that may alter T cell function and RNAseq

Review the following exclusion criteria carefully at time of appointment as some details
may have changed since initial contact:

- Recreational drug use (excluding cannabis use more than 1 week prior to blood
sampling) - drug abuse may alter T cell function

- Alcohol related illness (excessive alcohol consumption may alter T cell function)

- Renal failure: Creatinine >200 (as may alter T cell function)

- Any other medical condition which, in the opinion of the investigator, would affect
the safety of the subject's participation.

Factors that if temporary would lead to rearrangement of study visit but if long duration,
may lead to exclusion subject to the CI's discretion:

- Pregnant or lactating (as this may limit blood sampling and affect T cell function)

- Any infectious illness within the last 2 weeks if it was a febrile illness, or within
2-3 days if it was non-febrile (as this may activate T cells non-specifically)

- Taking steroids or other immunosuppressive medications (as these may alter T cell
function)

- Received any immunoglobulin treatments or blood products in the last 3 months (as
these may alter T cell function).