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

Study Overview
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Brief Summary
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Type 1 diabetes (T1D) results from destruction of insulin-producing beta cells in the pancreas by the body's own immune system (autoimmunity). It is not fully understood what causes this type of diabetes and why there is variation in age of onset and severity between people who develop the disease. The aim of this work is to study very unusual people who develop T1D extremely young, as babies under 2 years of age (EET1D). The investigators think that, for the condition to have developed that early, they must have an unusual or extreme form of autoimmunity. Studying people with EET1D will enable us to look at exactly what goes wrong with the immune system because they have one of the most extreme forms of the disease. We may be able to learn a lot about the disease from a small number of rare individuals. We aim to confirm that they have autoimmune type 1 diabetes and then try to understand how they have developed diabetes so young by studying their immune system genes, the function of their immune system, and environmental factors (such as maternal genetics) that may play a role in their development of the disease. People with diabetes diagnosed under 12 months are very rare, live all over the world. and are usually referred to Exeter for genetic testing. As part of their wider clinical team, we will contact them via their clinician to ask for more information about their diabetes and their family history. We will collect samples to study whether they still make any of their own insulin and whether they make specific antibodies against their beta cells in the pancreas. Separately, we will study their immune system in depth using immune cells isolated from a blood sample. We will then study these cells using cutting edge techniques by Dr Tim Tree at King's College London, by Professor Bart Roep at the Diabetes Metabolism Research Institute Faculty, City of Hope National Medical Center, California (USA), and Dr Cate Speake, Benaroya Research Institute, Seattle (USA). Some of these tests have never been used in people of young ages around the world, so an aim of this project will be to develop methods that can be used to study people even if they live far away. Additional funding extends the study for a further 3 years (Phase 2) 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.

Detailed Description
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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 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 referred over the last 15 years to the Exeter genetics team/Prof 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/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, including a 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 may 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 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. 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 Funders 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.

Official Title
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Understanding Beta-cell Destruction Through the Study of EXtremely Early-onset Type 1 Diabetes (A Musketeers' Memorandum Study)

Conditions
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Type1 Diabetes Mellitus

Intervention / Treatment
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* Diagnostic Test: Beta Cell Loss and Immune Function
* Other: Immune Function with RNAseq


Participation Criteria
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Eligibility Criteria
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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. Monogenic / NDM controls Diagnosis of diabetes <12 months Diagnosis of monogenic / NDM (confirmed by Exeter Molecular Genetics Laboratory). 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. Monogenic/NDM controls Diagnosis of diabetes <24 months Age 0 to 18 months at recruitment Diagnosis of monogenic/NDM (confirmed by Exeter Molecular Genetics Laboratory). Non-diabetic controls Aged 0-6 years Attending specified participating hospital sites for elective surgery, including but not limited to: inguinal hernia repair, umbilical/midline hernia repair, orchidopexy, gastrostomy insertion/change, hypospadias repair, cleft palate repair, excision of accessory digit, laryngoscopy, adenoidectomy, tonsillectomy, MRI under general anaesthesia, eye surgery. 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). Non-diabetic controls: Aged >6 years Diagnosis of diabetes or other autoimmune condition Known immunological disorder On immunosuppressive medication Ongoing infections/sepsis Major congenital abnormality or significant systemic illness that may affect the immune system, e.g. metabolic disease, 22q deletion syndrome Recent (within two weeks) febrile illness Renal failure. 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).

Ages Eligible for Study
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Maximum Age: 70 Years

Sexes Eligible for Study
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All

Accepts Healthy Volunteers
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No

Study Plan
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How is the study designed?
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Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Study 1: Existing EET1D (Case)<br>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 (e.g. STAT3 or FOXP3 mutation). | Diagnostic Test: Beta Cell Loss and Immune Function<br>* Beta cell loss (measured by serum/urine C-peptide), islet-specific autoantibodies, T1D risk genes and autoreactive CD8 T cells.<br>Other: Immune Function with RNAseq<br>* Immune function (measuring autoantibodies, autoreactive CD8 T cells and RNAseq of immune genes).<br>|
| Study 1: T1D (Control)<br>Age 0-70 years (matched to above) Clinical diagnosis of T1D (diagnosed age 1-20 years) Insulin treated from diagnosis. | Diagnostic Test: Beta Cell Loss and Immune Function<br>* Beta cell loss (measured by serum/urine C-peptide), islet-specific autoantibodies, T1D risk genes and autoreactive CD8 T cells.<br>Other: Immune Function with RNAseq<br>* Immune function (measuring autoantibodies, autoreactive CD8 T cells and RNAseq of immune genes).<br>|
| Study 2: Newly diagnosed EET1D (Case)<br>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 (e.g. STAT3 or FOXP3 mutation) | Diagnostic Test: Beta Cell Loss and Immune Function<br>* Beta cell loss (measured by serum/urine C-peptide), islet-specific autoantibodies, T1D risk genes and autoreactive CD8 T cells.<br>Other: Immune Function with RNAseq<br>* Immune function (measuring autoantibodies, autoreactive CD8 T cells and RNAseq of immune genes).<br>|
| Monogenic / NDM (Control)<br>Diagnosis of diabetes <24 months Age 0 to 24 months at recruitment Diagnosis of Monogenic / NDM (confirmed by Exeter Molecular Genetics Laboratory). | Diagnostic Test: Beta Cell Loss and Immune Function<br>* Beta cell loss (measured by serum/urine C-peptide), islet-specific autoantibodies, T1D risk genes and autoreactive CD8 T cells.<br>Other: Immune Function with RNAseq<br>* Immune function (measuring autoantibodies, autoreactive CD8 T cells and RNAseq of immune genes).<br>|
| Without diabetes (Control)<br>Aged 0-6 years Attending specified participating hospital sites for elective surgery, including but not limited to: inguinal hernia repair, umbilical/midline hernia repair, orchidopexy, gastrostomy insertion/change, hypospadias repair, cleft palate repair, excision of accessory digit, laryngoscopy, adenoidectomy, tonsillectomy, MRI under general anaesthesia, eye surgery. Should recruitment be slower than anticipated, we would recruit children with congenital non-immune thyroid disease when they attend paediatric clinic for blood draw. | Diagnostic Test: Beta Cell Loss and Immune Function<br>* Beta cell loss (measured by serum/urine C-peptide), islet-specific autoantibodies, T1D risk genes and autoreactive CD8 T cells.<br>Other: Immune Function with RNAseq<br>* Immune function (measuring autoantibodies, autoreactive CD8 T cells and RNAseq of immune genes).<br>|

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Measure beta cell function in EET1D compared to T1D, NDM and non-diabetic controls. | C-peptide and GAD, IA2, ZnT8 autoantibody measurement | Within 12 months of last participant's final visit. | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Immune phenotyping in EET1D compared to T1D, NDM and non-diabetic controls. | Presence/quantity of autoreactive CD8 and Treg; T cells; RNAseq; HLA alleles | Within 12 months of last participant's final visit. | 
| Difference in immune gene expression | Difference in immune gene expression, as measured by RNAseq in newly diagnosed EET1D v NDM | Within 12 months of last participant's final visit. | 
| Association of maternal and paternal non-inherited HLA alleles with EET1D | Association of maternal and paternal non-inherited HLA alleles with EET1D v older onset T1D and NDM | Within 12 months of last participant's final visit. | 

 Terms related to the study
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Keywords Provided by Centre Hospitalier Valida
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type 1 diabetes, monogenic diabetes, autoimmune diabetes, early-onset autoimmune diabetes, beta cell (β-cell) destruction, type 1 diabetes genetic risk, extremely early-onset Type 1 diabetes, neonatal diabetes