Estimation of Individual Positive Anti-Islet Autoantibodies from 3 Screen ICA Titer.

The 3 Screen ICA ELISA is a novel assay capable of simultaneously measuring autoantibodies to glutamic acid decarboxylase (GADA), insulinoma-associated antigen-2 (IA-2A), and zinc transporter 8 (ZnT8A), making it a valuable tool for screening type 1 diabetes. Despite its advantages, it cannot specify which individual autoantibodies are positive or negative. This study aimed to estimate individual positive autoantibodies based on the 3 Screen ICA titer. Six hundred seventeen patients with type 1 diabetes, simultaneously measured for 3 Screen ICA and three individual autoantibodies, were divided into five groups based on their 3 Screen ICA titer. The sensitivities and contribution rates of the individual autoantibodies were then examined. The study had a cross-sectional design. Sixty-nine percent (424 of 617) of patients with type 1 diabetes had 3 Screen ICA titers exceeding the 99th percentile cut-off level (20 index). The prevalence of GADA ranged from 80% to 100% in patients with a 3 Screen ICA over 30 index and 97% of patients with a 3 Screen ICA ≥300 index. Furthermore, the prevalence of all individual autoantibodies being positive was 0% for ≤80 index and as high as 92% for ≥300 index. Significant associations were observed in specific titer groups: the 20-29.9 index group when all the individual autoantibodies were negative, the 30-79.9 index group when positive for GADA alone or IA-2A alone, the 30-299.9 index group when positive for ZnT8A alone, the 80-299.9 index group when positive for both IA-2A and ZnT8A, the 300-499.9 index group when positive for both GADA and ZnT8A, and the ≥300 index group when positive for all individual autoantibodies. These results suggest that the 3 Screen ICA titer may be helpful in estimating individual positive autoantibodies.

Islet autoantibody frequency in relatives of children with type 1 diabetes who have a type 2 diabetes diagnosis.

AIM: This study aimed to evaluate characteristics of autoimmunity in individuals who have a type 2 diagnosis and are relatives of children with type 1 diabetes. METHODS: Pre-diagnosis samples (median 17 months before onset) from relatives who were later diagnosed with type 2 diabetes were measured for autoantibodies to glutamate decarboxylase 65 (GADA), islet antigen-2 (IA-2A), zinc transporter 8 (ZnT8A) and insulin (IAA) as well as the type 1 diabetes genetic risk score (GRS2). Associations between islet autoantibodies, insulin treatment and GRS2 were analysed using Fisher's exact and t-tests. RESULTS: Among 226 relatives (64% men; mean age at sampling 41 years; mean age 54 years at diagnosis), 32 (14%) were islet autoantibody-positive for at least one autoantibody more than a decade before diagnosis. Approximately half of these (n = 15) were treated with insulin. GADA-positivity was higher in insulin-treated relatives than in non-insulin-treated relatives (12/18 [67%] vs. 6/18 [33%], p < 0.001). IAA-positivity was observed in 13/32 (41%) of relatives with autoantibodies. GRS2 scores were increased in autoantibody-positive relatives (p = 0.032), but there was no clear evidence for a difference according to treatment (p = 0.072). CONCLUSION: This study highlights the importance of measuring islet autoantibodies, including IAA, in relatives of people with type 1 diabetes to avoid misdiagnosis.

Childhood screening for type 1 diabetes comparing automated multiplex Antibody Detection by Agglutination-PCR (ADAP) with single plex islet autoantibody radiobinding assays.

BACKGROUND: Two or more autoantibodies against either insulin (IAA), glutamic acid decarboxylase (GADA), islet antigen-2 (IA-2A) or zinc transporter 8 (ZnT8A) denote stage 1 (normoglycemia) or stage 2 (dysglycemia) type 1 diabetes prior to stage 3 type 1 diabetes. Automated multiplex Antibody Detection by Agglutination-PCR (ADAP) assays in two laboratories were compared to single plex radiobinding assays (RBA) to define threshold levels for diagnostic specificity and sensitivity. METHODS: IAA, GADA, IA-2A and ZnT8A were analysed in 1504 (54% females) population based controls (PBC), 456 (55% females) doctor's office controls (DOC) and 535 (41% females) blood donor controls (BDC) as well as in 2300 (48% females) patients newly diagnosed (1-10 years of age) with stage 3 type 1 diabetes. The thresholds for autoantibody positivity were computed in 100 10-fold cross-validations to separate patients from controls either by maximizing the χ2-statistics (chisq) or using the 98th percentile of specificity (Spec98). Mean and 95% CI for threshold, sensitivity and specificity are presented. FINDINGS: The ADAP ROC curves of the four autoantibodies showed comparable AUC in the two ADAP laboratories and were higher than RBA. Detection of two or more autoantibodies using chisq showed 0.97 (0.95, 0.99) sensitivity and 0.94 (0.91, 0.97) specificity in ADAP compared to 0.90 (0.88, 0.95) sensitivity and 0.97 (0.94, 0.98) specificity in RBA. Using Spec98, ADAP showed 0.92 (0.89, 0.95) sensitivity and 0.99 (0.98, 1.00) specificity compared to 0.89 (0.77, 0.86) sensitivity and 1.00 (0.99, 1.00) specificity in the RBA. The diagnostic sensitivity and specificity were higher in PBC compared to DOC and BDC. INTERPRETATION: ADAP was comparable in two laboratories, both comparable to or better than RBA, to define threshold levels for two or more autoantibodies to stage type 1 diabetes. FUNDING: Supported by The Leona M. and Harry B. Helmsley Charitable Trust (grant number 2009-04078), the Swedish Foundation for Strategic Research (Dnr IRC15-0067) and the Swedish Research Council, Strategic Research Area (Dnr 2009-1039). AL was supported by the DiaUnion collaborative study, co-financed by EU Interreg ÖKS, Capital Region of Denmark, Region Skåne and the Novo Nordisk Foundation.

Prevalence, diagnostic utility, and clinical characteristics of ZnT8 antibody in children with type 1 diabetes in Northern Taiwan.

BACKGROUND: The ZnT8 autoantibody is used to independently diagnose type 1 diabetes (T1D) and as a prediction factor in high-risk populations. This is the first report in Taiwan on the prevalence, diagnostic utility, and clinical characteristics of zinc transporter 8 autoantibody (ZnT8A) in children with T1D. METHODS: We performed a retrospective analysis of 268 children (130 boys, 138 girls) newly diagnosed with T1D at three hospitals in North Taiwan from February 1994 to August 2021. RESULTS: ZnT8A was detected in 117 patients (43.7 %). The combined diagnostic rate of the four antibodies, including glutamic acid decarboxylase autoantibody (GADA), islet antigen 2 autoantibody (IA2A), insulin autoantibody (IAA), and ZnT8A, can reach 86.19 % while that of the original three antibodies is 84.3 %. IA2A (64.9 %) showed the highest positive rate, followed by GADA (64.2 %), ZnT8A (43.7 %), and IAA (22.0 %). Of the 268 patients, five (1.9 %) were only ZnT8A+. All antibodies were positive in 19 (7.1 %) people, whereas 37 others (13.8 %) had all antibodies negative. ZnT8A has the strongest relationship with IA2A. 5 patients had ZnT8A positive only. 5/(37 + 5) (about 12 %) T1D patients were diagnosed by ZnT8A testing. CONCLUSIONS: ZnT8A testing can diagnose up to 12 % more patients with T1D along with three other antibodies. Furthermore, since the ZnT8A titer decreased over time, it should be tested within six months of onset in Taiwanese patients with T1D.

The Unfavorable Impact of DR9/DR9 Genotype on the Frequency and Quality of Partial Remission in Type 1 Diabetes.

CONTEXT: Partial remission (PR) is a specific stage in type 1 diabetes (T1D). Although human leukocyte antigen (HLA) class II loci are the strongest genetic determinants in T1D, the relationship between PR and HLA remains unclear. OBJECTIVE: To investigate the association between PR status and HLA genotypes in patients with T1D. METHODS: A total of 237 patients with T1D were included. PR was defined according to C-peptide ≥300 pmol/L. The frequency of PR and peak C-peptide levels during remission phase were compared according to HLA status. Clinical characteristics including age of onset and diabetes autoantibodies were collected. All analyses were duplicated when subjects were divided into childhood- and adult-onset T1D. RESULTS: The median follow-up time was 24 months, 65.8% (156/237) of patients with T1D went into PR. DR9/DR9 carriers had a lower PR rate (44.2% vs 70.6%, P = .001) and were less likely to enter PR (OR = 0.218, 95% CI 0.098-0.487, P < .001) than the non-DR9/DR9 carriers, observed in both childhood- and adult-onset T1D. Besides, the peak C-peptide level during PR phase was also lower in DR9/DR9 carriers, and more notable in adult-onset T1D. When compared with non-DR9/DR9 carriers, T1D with DR9/DR9 genotype presented an older age of onset and a lower positivity of zinc transporter 8 antibody (ZnT8A), and the lower trend of ZnT8A was only found in adult-onset T1D (P = .049). CONCLUSION: Patients with T1D carrying susceptible DR9/DR9 are less prone to undergo PR. Additionally, the recovery extent of ß-cell function during the PR phase tends to be lower in adults carrying DR9/DR9, which might be associated with ZnT8A.

Genetic variation at ERBB3/IKZF4 and sexual dimorphism in epitope spreading in single autoantibody-positive relatives.

AIMS/HYPOTHESIS: We examined whether the non-HLA susceptibility locus ERBB3/IKZF4 influences progression of type 1 diabetes stage specifically according to sex. METHODS: SNPs of ERBB3 (rs2292239 T/G) and IKZF4 (rs1701704 G/T) were screened by allelic discrimination quantitative PCR assay in first-degree relatives of type 1 diabetes patients who had developed at least one circulating autoantibody. The effect of ERBB3/IKZF4 genotypes and sex, on the progression of single autoantibody positivity to multiple autoantibody positivity and from multiple autoantibody positivity to diabetes, was studied by Kaplan-Meier analysis and multivariate Cox regression. RESULTS: In the cohort of autoantibody-positive first-degree relatives, the risk allele frequencies for ERBB3 rs2292239 (T) and IKZF4 rs1701704 (G) were increased. There was a significant male excess at the stage of multiple autoantibody positivity (p = 0.021). In Kaplan-Meier survival analysis, progression from single to multiple antibody positivity was delayed in female participants with genotype ERBB3 GG (p = 0.018, vs ERBB3 TG+TT) or IKZF4 TT (p = 0.023, vs IKZF4 GT+GG), but not in male participants. In multivariate Cox regression models, the interaction effects between female sex and ERBB3 GG (p = 0.012; HR = 0.305 [95% CI 0.120, 0.773]) or between female sex and IKZF4 TT (p = 0.011; HR = 0.329 [95% CI 0.140, 0.777]) emerged as potential determinants of delayed progression to multiple autoantibodies. The progression from multiple autoantibody positivity to type 1 diabetes appeared not to be influenced by ERBB3/IKZF4. CONCLUSIONS/INTERPRETATION: In siblings and offspring of type 1 diabetes patients, polymorphism in region ERBB3/IKZF4 may affect disease progression at the level of epitope spreading in female individuals. Our findings suggest that interaction between sex and ERBB3/IKZF4 may contribute to the post-pubertal male excess in type 1 diabetes.

Simplifying prediction of disease progression in pre-symptomatic type 1 diabetes using a single blood sample.

AIMS/HYPOTHESIS: Accurate prediction of disease progression in individuals with pre-symptomatic type 1 diabetes has potential to prevent ketoacidosis and accelerate development of disease-modifying therapies. Current tools for predicting risk require multiple blood samples taken during an OGTT. Our aim was to develop and validate a simpler tool based on a single blood draw. METHODS: Models to predict disease progression using a single OGTT time point (0, 30, 60, 90 or 120 min) were developed using TrialNet data collected from relatives with type 1 diabetes and validated in independent populations at high genetic risk of type 1 diabetes (TrialNet, Diabetes Prevention Trial-Type 1, The Environmental Determinants of Diabetes in the Young [1]) and in a general population of Bavarian children who participated in Fr1da. RESULTS: Cox proportional hazards models combining plasma glucose, C-peptide, sex, age, BMI, HbA1c and insulinoma antigen-2 autoantibody status predicted disease progression in all populations. In TrialNet, the AUC for receiver operating characteristic curves for models named M60, M90 and M120, based on sampling at 60, 90 and 120 min, was 0.760, 0.761 and 0.745, respectively. These were not significantly different from the AUC of 0.760 for the gold standard Diabetes Prevention Trial Risk Score, which requires five OGTT blood samples. In TEDDY, where only 120 min blood sampling had been performed, the M120 AUC was 0.865. In Fr1da, the M120 AUC of 0.742 was significantly greater than the M60 AUC of 0.615. CONCLUSIONS/INTERPRETATION: Prediction models based on a single OGTT blood draw accurately predict disease progression from stage 1 or 2 to stage 3 type 1 diabetes. The operational simplicity of M120, its validity across different at-risk populations and the requirement for 120 min sampling to stage type 1 diabetes suggest M120 could be readily applied to decrease the cost and complexity of risk stratification.

High-affinity ZnT8 Autoantibodies by Electrochemiluminescence Assay Improve Risk Prediction for Type 1 Diabetes.

CONTEXT: Single ZnT8 autoantibody (ZnT8A) positivity by standard radiobinding assay (RBA) is commonly seen in nondiabetes population-based screening and the risk of progression to type 1 diabetes (T1D) in subjects with single ZnT8A is unknown. OBJECTIVE: Identify the risk of progression to T1D in individuals positive only for ZnT8A. METHODS: We developed an electrochemiluminescence (ECL) assay to detect high-affinity ZnT8A and validated it in 3 populations: 302 patients newly diagnosed with T1D, 135 nondiabetic children positive for ZnT8A by RBA among 23 400 children screened by the Autoimmunity Screening for Kids (ASK) study, and 123 nondiabetic children multiple autoantibody positive or single ZnT8A positive by RBA participating in the Diabetes Autoimmunity Study in the Young (DAISY). RESULTS: In 302 patients with T1D at diagnosis, the positivity for ZnT8A was 62% both in RBA and ECL. Among ASK 135 participants positive for RBA-ZnT8A, 64 were detected ZnT8A as the only islet autoantibody. Of these 64, only 9 were confirmed by ECL-ZnT8A, found to be of high affinity with increased T1D risk. The overall positive predictive value of ECL-ZnT8A for T1D risk was 87.1%, significantly higher than that of RBA-ZnT8A (53.5%, P < .001). In DAISY, 11 of 2547 children who had no positivity previously detected for other islet autoantibodies were identified as single ZnT8A by RBA; of these, 3 were confirmed positive by ECL-ZnT8A and all 3 progressed to clinical T1D. CONCLUSION: A large proportion of ZnT8A by RBA are single ZnT8A with low T1D risk, whereas ZnT8A by ECL was of high affinity and high prediction for T1D development.

Zinc transporter 8 (ZnT8) autoantibody prevalence in black South African participants with type 1 diabetes.

BACKGROUND: Autoantibodies to ß-cell specific antigens are markers of type 1 diabetes. The most recently identified autoantibodies are targeted to the zinc transporter 8 (ZnT8) protein located in the membrane of ß-cell insulin secretory granules. The prevalence of ZnT8 autoantibodies in newly diagnosed participants with type 1 diabetes has been found to range from 33 to 80 %. Due to the lack of data on the immunological aetiology of type 1 diabetes in African populations, this study aimed to determine the prevalence of ZnT8 autoantibodies in black South Africans with type 1 diabetes and whether ZnT8 autoantibody positivity was associated with age at diagnosis and disease duration. METHODS: Participants with type 1 diabetes and controls were recruited from the greater Johannesburg area, South Africa. Positivity for ZnT8, GAD65 and IA2 autoantibodies was determined by ELISA. RESULTS: Participants with type 1 diabetes (n = 183) and controls (n = 49) were matched for age (29.1 ± 9.53 vs. 27.3 ± 7.29, respectively; p = 0.248). The mean age at diagnosis for participants with type 1 diabetes was 20.8 ± 8.46 years. The prevalence of ZnT8 autoantibody positivity was 17.5 % (32 of 183) in participants with type 1 diabetes with a median disease duration of 7.00 [2.00; 11.0] years. ZnT8 autoantibody prevalence in newly diagnosed participants (< 1 year duration) was 27.3 % (6 of 22). Logistic regression analysis found an association between ZnT8 autoantibody positivity and shorter disease duration (OR: 0.9 (0.81-1.00); p = 0.042). In addition, ZnT8 autoantibody positivity was significantly associated with an increased chance of being GAD65 (OR: 3.37 (1.10-10.3)) and IA2 (OR: 8.63 (2.82-26.4)) autoantibody positive. Multiple regression analysis found no association between ZnT8 autoantibody positivity and age at diagnosis. However, the presence of ≥ 2 autoantibodies was associated with a younger age at diagnosis of type 1 diabetes when compared to participants with ≤ 1 autoantibody (B = -5.270; p = 0.002). CONCLUSIONS: The presence of ZnT8 autoantibodies was not related to a younger age at diagnosis in black South African patients with type 1 diabetes. However, the greater the numbers of autoantibodies present in an individual the earlier the age at diagnosis. ZnT8 autoantibodies decline with disease duration in the black South African population.

Novel autoantibodies to the ß-cell surface epitopes of ZnT8 in patients progressing to type-1 diabetes.

Type 1 diabetes (T1D) is a chronic autoimmune disease characterized by autoimmune destruction of insulin-producing ß-cells in pancreatic islets. Seroconversions to islet autoantibodies (IAbs) precede the disease onset by many years, but the role of humoral autoimmunity in the disease initiation and progression are unclear. In the present study, we identified a new IAb directed to the extracellular epitopes of ZnT8 (ZnT8ec) in newly diagnosed patients with T1D, and demonstrated immunofluorescence staining of the surface of human ß-cells by autoantibodies to ZnT8ec (ZnT8ecA). With the assay specificity set on 99th percentile of 336 healthy controls, the ZnT8ecA positivity rate was 23.6% (74/313) in patients with T1D. Moreover, 30 children in a longitudinal follow up of clinical T1D development were selected for sequential expression of four major IAbs (IAA, GADA, IA-2A and ZnT8icA). Among them, 10 children were ZnT8ecA positive. Remarkably, ZnT8ecA was the earliest IAb to appear in all 10 children. The identification of ZnT8ec as a cell surface target of humoral autoimmunity in the earliest phase of IAb responses opens a new avenue of investigation into the role of IAbs in the development of ß-cell autoimmunity.

Diagnostic value of combined islet antigen-reactive T cells and autoantibodies assays for type 1 diabetes mellitus.

AIMS/INTRODUCTION: Type 1 diabetes mellitus is a T cell-mediated autoimmune disease. However, the determination of the autoimmune status of type 1 diabetes mellitus relies on islet autoantibodies (Abs), as T-cell assay is not routinely carried out. This study aimed to investigate the diagnostic value of combined assay of islet antigen-specific T cells and Abs in type 1 diabetes mellitus patients. MATERIALS AND METHODS: A total of 54 patients with type 1 diabetes mellitus and 56 healthy controls were enrolled. Abs against glutamic acid decarboxylase (GAD), islet antigen-2 and zinc transporter 8 were detected by radioligand assay. Interferon-γ-secreting T cells responding to glutamic acid decarboxylase 65 and C-peptide (CP) were measured by enzyme-linked immunospot. RESULTS: The positive rate for T-cell responses was significantly higher in patients with type 1 diabetes mellitus than that in controls (P < 0.001). The combined positive rate of Abs and T-cell assay was significantly higher than that of Abs assay alone (85.2% vs 64.8%, P = 0.015). A significant difference in fasting CP level was found between the T+ and T- groups (0.07 ± 0.05 vs 0.11 ± 0.09 nmol/L, P = 0.033). Furthermore, levels of fasting CP and postprandial CP were both lower in the Ab- T+ group than the Ab- T- group (fasting CP 0.06 ± 0.05 vs 0.16 ± 0.12 nmol/L, P = 0.041; postprandial CP 0.12 ± 0.13 vs 0.27 ± 0.12 nmol/L, P = 0.024). CONCLUSIONS: Enzyme-linked immunospot assays in combination with Abs detection could improve the diagnostic sensitivity of autoimmune diabetes.

Autoantibodies against zinc transporter 8 further stratify the autoantibody-defined risk for type 1 diabetes in a general population of schoolchildren and have distinctive isoform binding patterns in different forms of autoimmune diabetes: results from the Karlsburg Type 1 Diabetes Risk Study.

AIMS: To evaluate the diagnostic relevance of autoantibodies against zinc transporter 8 (ZnT8) in schoolchildren from the general population as well as in people with autoimmune diabetes. METHODS: A total of 137 schoolchildren positive for at least one of the three major diabetes-associated autoantibodies, without diabetes heredity or preselection on HLA typing, from the Karlsburg Type 1 Diabetes Risk Study, as well as 102 people at type 1 diabetes onset, 88 people with latent autoimmune diabetes in adults and 119 people with type 2 diabetes, were analysed for different ZnT8 autoantibody variants. RESULTS: Zinc transporter 8 autoantibody positivity was found in 18% of autoantibody-positive schoolchildren, with a noticeable association with other autoantibodies associated with type 1 diabetes and disease progression. Furthermore, ZnT8 autoantibody positivity was associated with diabetes progression in schoolchildren positive for autoantibodies against insulinoma-associated antigen-2 (IA-2) and, importantly, in seven IA-2 autoantibody-negative schoolchildren. Additionally, ZnT8 autoantibodies were found in 56% of people with type 1 diabetes, predominantly directed against all three ZnT8 variants and comparable to schoolchildren with multiple autoantibodies. In contrast, ZnT8 autoantibodies were detected in 10% of people with latent autoimmune diabetes in adults, none of them with reactivity to all three isoforms. CONCLUSION: Zinc transporter 8 autoantibodies are useful markers for prediction of type 1 diabetes in a general population, further stratifying the risk of progression in autoantibody-positive children. ZnT8 autoantibodies are also important markers in adult-onset diabetes, with a completely different reaction pattern in type 1 diabetes in comparison to latent autoimmune diabetes in adults, and may therefore help to differentiate between the two forms.

Different interaction of onset age and duration of type 1 diabetes on the dynamics of autoantibodies to insulinoma-associated antigen-2 and zinc transporter 8.

AIMS/INTRODUCTION: This study aimed to investigate the dynamics associated with autoantibodies to insulinoma-associated antigen-2 (IA-2A) and zinc transporter 8 (ZnT8A) relating to the onset age and disease duration in patients with type 1 diabetes. METHODS: Using bridging-type enzyme-linked immunosorbent assay, IA-2A, ZnT8A and glutamic acid decarboxylase autoantibodies were evaluated in 269 patients with type 1 diabetes (median onset age 18.2 years, range 0.8-86 years; median diabetes duration 7 years, range 0-58 years). We then compared the prevalence of these autoantibodies among the different age groups, along with the duration of diabetes using the Cochran-Armitage trend test and multivariate logistic regression analysis. RESULTS: The prevalence of IA-2A, ZnT8A and glutamic acid decarboxylase autoantibodies in patients with duration of ≤3 years was 41.1, 36.7 and 72.2%, respectively, with 80.0% expressing one or more of these autoantibodies. This prevalence declined according to the disease duration (P < 0.005). Both IA-2A and ZnT8A were more frequently observed in younger patients, whereas glutamic acid decarboxylase autoantibodies was more common in older patients. Multivariate logistic regression analysis showed that there was a significant interaction between the onset age and duration of diabetes in patients diagnosed when aged ≤10 years regarding all anti-islet autoantibodies (P < 0.05). However, for patients diagnosed in the middle tertile (aged 11-30 years), the interaction was significant only for ZnT8A, and for those with late-onset diabetes (aged ≥31 years) only for IA-2A. CONCLUSIONS: The current study showed that the rate of disappearance of anti-islet autoantibodies is faster in patients aged ≤10 years, and that even though both proteins are localized in the insulin granule membrane, humoral autoimmunity to IA-2 and ZnT8 differs according to the age of onset.

Islet autoantibody profiles associated with higher diabetes risk in Lithuanian compared with English schoolchildren.

During a 15-year period, the incidence of type 1 diabetes has doubled in Lithuania, while increasing by a third in England; however, England still has a higher incidence. Analysis of sera collected from non-diabetic schoolchildren from Lithuania and England more than 20 years ago showed a similar number of multiple autoantibody-positive schoolchildren between the populations, but a higher prevalence of islet antigen-2 autoantibodies (IA-2A) in English schoolchildren. We aimed to use recently developed, more specific islet autoantibody tests to characterize differences in humoral autoimmunity between these two general population cohorts in greater detail. Samples from 88 Lithuanian and 133 English schoolchildren previously found islet autoantibody-positive were selected for measurement of additional islet autoantibodies by radioimmunoassay. Samples were tested for autoantibodies to zinc transporter 8 (ZnT8A), GAD (96-585), the protein tyrosine phosphatase region of islet antigen-2 (PTPA) and the related IA-2ßA, while autoantibodies to IA-2A were reassayed using the current harmonized method. IA-2-related autoantibodies PTPA (0·13 versus 0·45%, P = 0·027) and IA-2ßA (0 versus 0·35%, P < 0·001), but not IA-2A measured using the harmonized method, were less common in Lithuanian compared to English schoolchildren. Lithuanian schoolchildren who were islet autoantibody-positive were positive for fewer biochemical autoantibodies compared with English schoolchildren (P = 0·043). Background rates of islet autoimmunity in childhood differ subtly between countries, which have different incidences of type 1 diabetes. The optimal screening strategy (age and combination of markers) for detection of islet autoimmunity may vary between countries, dependent upon the pattern of autoantibodies found in the general population.

Prevalence of Zinc Transporter 8 Auto Antibodies among Newly Diagnosed Type 1 Diabetic Cases Admitted to Assiut University Children Hospital.

Type 1 diabetes mellitus (T1DM) remains the most common form of diabetes in childhood. The incidence of type 1 diabetes is continuously increased. Zinc transporter protein 8 antibodies (ZnT8A) measurement can be helpful in detection of suspected new cases of type 1diabetes when other islet auto antibodies are negative. We evaluated the role of ZnT8A in diagnosis of new cases of T1DM in comparison to islet cell antibody (ICA), and assessed its prediction value among siblings. 31 of newly diagnosed T1DM patients and 55 age and sex matched healthy siblings were included. Measurements of ZnT8A and ICA was carried out by ELISA. ZnT8A had 45% sensitivity and 69% specificity while ICA had 64.5% sensitivity and 83.64% specificity. 22.6% of diabetic patients had high level of ZnT8A as compared to 20% of siblings (P < 0.001 and P < 0.001, respectively). 28.6% of diabetic patients with high titer ZnT8A had positive ICA (P < 0.04) as compared to 63.6% in sibling group (P < 0.001). It is concluded that ZnT8A and ICA play an important role in diagnosis and prediction of T1DM cases.

Expression of zinc transporter 8 in thyroid tissues from patients with immune and non-immune thyroid diseases.

INTRODUCTION: Recent studies have revealed the presence of zinc and the expression of zinc transporter (ZnT) family members in most endocrine cell types. It was demonstrated that ZnT family plays an important role in the synthesis and secretion of many hormones. Moreover, recently ZnT8 was described as a newly islet autoantigen in type 1 diabetes. MATERIALS AND METHODS: We studied the expression of ZnT8 transporter in thyroid tissues from patients with immune and non-immune thyroid diseases. The study was performed in thyroid tissues after thyroidectomy from patients with thyroid non-toxic nodular goitre (NTNG; n = 17, mean age 15.8 ± 2.2 years) and cases with Graves' disease (n = 20, mean age 15.6 ± 2.8). In our study we investigated the expression of ZnT8 in human thyroid tissues from patients with immune and non-immune thyroid diseases using immunohistochemistry, Western Blot as well as immunofluorescence analyses. To the best of our knowledge, this is the first investigation which identified ZnT8 protein expression in human thyroid tissues, moreover, confirmed by three different laboratory techniques. Results and Conclusions Expression of ZnT8 transporter was identified by immunohistochemistry in the thyroid tissues from paediatric patients with Graves' disease (on +++) and non-toxic nodular goitre (on ++). ZnT8 transporter expression was found both in thyroid follicular cells (within the cytoplasm and cytoplasmic membrane in follicular cells) and C cells (membrane-cytoplasmic reaction) in fluorescence. Predominant expression of ZnT8 in band 41 kDa in immune than in non-immune thyroid disorders may suggest potential role of ZnT8 as a new thyroid autoanitgen but it requires further study on a larger cohort.

Dynamics of Islet Autoantibodies During Prospective Follow-Up From Birth to Age 15 Years.

CONTEXT: We set out to characterize the dynamics of islet autoantibodies over the first 15 years of life in children carrying genetic susceptibility to type 1 diabetes (T1D). We also assessed systematically the role of zinc transporter 8 autoantibodies (ZnT8A) in this context. DESIGN: HLA-predisposed children (N = 1006, 53.0% boys) recruited from the general population during 1994 to 1997 were observed from birth over a median time of 14.9 years (range, 1.9-15.5 years) for ZnT8A, islet cell (ICA), insulin (IAA), glutamate decarboxylase (GADA), and islet antigen-2 (IA-2A) antibodies, and for T1D. RESULTS: By age 15.5 years, 35 (3.5%) children had progressed to T1D. Islet autoimmunity developed in 275 (27.3%) children at a median age of 7.4 years (range, 0.3-15.1 years). The ICA seroconversion rate increased toward puberty, but the biochemically defined autoantibodies peaked at a young age. Before age 2 years, ZnT8A and IAA appeared commonly as the first autoantibody, but in the preschool years IA-2A- and especially GADA-initiated autoimmunity increased. Thereafter, GADA-positive seroconversions continued to appear steadily until ages 10 to 15 years. Inverse IAA seroconversions occurred frequently (49.3% turned negative) and marked a prolonged delay from seroconversion to diagnosis compared to persistent IAA (8.2 vs 3.4 years; P = .01). CONCLUSIONS: In HLA-predisposed children, the primary autoantibody is characteristic of age and might reflect the events driving the disease process toward clinical T1D. Autoantibody persistence affects the risk of T1D. These findings provide a framework for identifying disease subpopulations and for personalizing the efforts to predict and prevent T1D.

Type 1 diabetes in Africa: an immunogenetic study in the Amhara of North-West Ethiopia.

AIMS/HYPOTHESIS: We aimed to characterise the immunogenic background of insulin-dependent diabetes in a resource-poor rural African community. The study was initiated because reports of low autoantibody prevalence and phenotypic differences from European-origin cases with type 1 diabetes have raised doubts as to the role of autoimmunity in this and similar populations. METHODS: A study of consecutive, unselected cases of recently diagnosed, insulin-dependent diabetes (n = 236, ≤35 years) and control participants (n = 200) was carried out in the ethnic Amhara of rural North-West Ethiopia. We assessed their demographic and socioeconomic characteristics, and measured non-fasting C-peptide, diabetes-associated autoantibodies and HLA-DRB1 alleles. Leveraging genome-wide genotyping, we performed both a principal component analysis and, given the relatively modest sample size, a provisional genome-wide association study. Type 1 diabetes genetic risk scores were calculated to compare their genetic background with known European type 1 diabetes determinants. RESULTS: Patients presented with stunted growth and low BMI, and were insulin sensitive; only 15.3% had diabetes onset at ≤15 years. C-peptide levels were low but not absent. With clinical diabetes onset at ≤15, 16-25 and 26-35 years, 86.1%, 59.7% and 50.0% were autoantibody positive, respectively. Most had autoantibodies to GAD (GADA) as a single antibody; the prevalence of positivity for autoantibodies to IA-2 (IA-2A) and ZnT8 (ZnT8A) was low in all age groups. Principal component analysis showed that the Amhara genomes were distinct from modern European and other African genomes. HLA-DRB1*03:01 (p = 0.0014) and HLA-DRB1*04 (p = 0.0001) were positively associated with this form of diabetes, while HLA-DRB1*15 was protective (p < 0.0001). The mean type 1 diabetes genetic risk score (derived from European data) was higher in patients than control participants (p = 1.60 × 10-7). Interestingly, despite the modest sample size, autoantibody-positive patients revealed evidence of association with SNPs in the well-characterised MHC region, already known to explain half of type 1 diabetes heritability in Europeans. CONCLUSIONS/INTERPRETATION: The majority of patients with insulin-dependent diabetes in rural North-West Ethiopia have the immunogenetic characteristics of autoimmune type 1 diabetes. Phenotypic differences between type 1 diabetes in rural North-West Ethiopia and the industrialised world remain unexplained.

Histological validation of a type 1 diabetes clinical diagnostic model for classification of diabetes.

AIMS: Misclassification of diabetes is common due to an overlap in the clinical features of type 1 and type 2 diabetes. Combined diagnostic models incorporating clinical and biomarker information have recently been developed that can aid classification, but they have not been validated using pancreatic pathology. We evaluated a clinical diagnostic model against histologically defined type 1 diabetes. METHODS: We classified cases from the Network for Pancreatic Organ donors with Diabetes (nPOD) biobank as type 1 (n = 111) or non-type 1 (n = 42) diabetes using histopathology. Type 1 diabetes was defined by lobular loss of insulin-containing islets along with multiple insulin-deficient islets. We assessed the discriminative performance of previously described type 1 diabetes diagnostic models, based on clinical features (age at diagnosis, BMI) and biomarker data [autoantibodies, type 1 diabetes genetic risk score (T1D-GRS)], and singular features for identifying type 1 diabetes by the area under the curve of the receiver operator characteristic (AUC-ROC). RESULTS: Diagnostic models validated well against histologically defined type 1 diabetes. The model combining clinical features, islet autoantibodies and T1D-GRS was strongly discriminative of type 1 diabetes, and performed better than clinical features alone (AUC-ROC 0.97 vs. 0.95; P = 0.03). Histological classification of type 1 diabetes was concordant with serum C-peptide [median < 17 pmol/l (limit of detection) vs. 1037 pmol/l in non-type 1 diabetes; P < 0.0001]. CONCLUSIONS: Our study provides robust histological evidence that a clinical diagnostic model, combining clinical features and biomarkers, could improve diabetes classification. Our study also provides reassurance that a C-peptide-based definition of type 1 diabetes is an appropriate surrogate outcome that can be used in large clinical studies where histological definition is impossible. Parts of this study were presented in abstract form at the Network for Pancreatic Organ Donors Conference, Florida, USA, 19-22 February 2019 and Diabetes UK Professional Conference, Liverpool, UK, 6-8 March 2019.

Hierarchical Order of Distinct Autoantibody Spreading and Progression to Type 1 Diabetes in the TEDDY Study.

OBJECTIVE: The first-appearing ß-cell autoantibody has been shown to influence risk of type 1 diabetes (T1D). Here, we assessed the risk of autoantibody spreading to the second-appearing autoantibody and further progression to clinical disease in The Environmental Determinants of Diabetes in the Young (TEDDY) study. RESEARCH DESIGN AND METHODS: Eligible children with increased HLA-DR-DQ genetic risk for T1D were followed quarterly from age 3 months up to 15 years for development of a single first-appearing autoantibody (GAD antibody [GADA], insulin autoantibody [IAA], or insulinoma antigen-2 autoantibody [IA-2A]) and subsequent development of a single second-appearing autoantibody and progression to T1D. Autoantibody positivity was defined as positivity for a specific autoantibody at two consecutive visits confirmed in two laboratories. Zinc transporter 8 autoantibody (ZnT8A) was measured in children who developed another autoantibody. RESULTS: There were 608 children who developed a single first-appearing autoantibody (IAA, n = 282, or GADA, n = 326) with a median follow-up of 12.5 years from birth. The risk of a second-appearing autoantibody was independent of GADA versus IAA as a first-appearing autoantibody (adjusted hazard ratio [HR] 1.12; 95% CI 0.88-1.42; P = 0.36). Second-appearing GADA, IAA, IA-2A, or ZnT8A conferred an increased risk of T1D compared with children who remained positive for a single autoantibody, e.g., IAA or GADA second (adjusted HR 6.44; 95% CI 3.78-10.98), IA-2A second (adjusted HR 16.33; 95% CI 9.10-29.29; P < 0.0001), or ZnT8A second (adjusted HR 5.35; 95% CI 2.61-10.95; P < 0.0001). In children who developed a distinct second autoantibody, IA-2A (adjusted HR 3.08; 95% CI 2.04-4.65; P < 0.0001) conferred a greater risk of progression to T1D as compared with GADA or IAA. Additionally, both a younger initial age at seroconversion and shorter time to the development of the second-appearing autoantibody increased the risk for T1D. CONCLUSIONS: The hierarchical order of distinct autoantibody spreading was independent of the first-appearing autoantibody type and was age-dependent and augmented the risk of progression to T1D.