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 at the beginning of life when the immune system is not yet fully developed and at a time when pancreatic autoimmunity first emerges (Krisher), 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.

Additional funding extended the study to include recruitment of infants without diabetes, aged 0-6 years, as controls to enable assessment of how the abnormalities found in autoimmune and non-autoimmune diabetes compare to normal early life development of the immune system.

Hypotheses:

1. 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 children with monogenic neonatal diabetes or without diabetes will not show abnormalities in 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]), and infants without diabetes matched for age.

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 of varying ages and durations of diabetes referred over the last 15 years to the Exeter genetics team/Prof Oram.

Study 2: Newly referred patients (n=20 v 20): Recruit newly diagnosed patients with EET1D who are referred to Exeter/Prof 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.

The investigators may also be approached by patients' GPs and by patients themselves. Recruitment to the study in this setting will be by the investigator's team 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 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, a repeat blood sample for PBMC, C-peptide and Autoantibody analysis may be collected, 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.

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 at least 240: Study 1: 100 with EET1D plus 100 controls (N=200); Study 2: EET1D v Monogenic / NDM controls (N=30+); Non-diabetic controls: (N=20+) 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 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 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.

Funders: Diabetes UK; The Leona M. and Harry B. Helmsley Charitable Trust. Publication Policy: On completion of the study, the data will be analysed and a Final Study Report will be prepared and submitted to the Funders, Sponsor and REC.