Autoantibody-positive Relatives Research Articles

Identification of type 1 diabetes risk phenotypes using an outcome-guided clustering analysis.

Although statistical models for predicting type 1 diabetes risk have been developed, approaches that reveal the heterogeneity of the at-risk population by identifying clinically meaningful clusters are lacking. We aimed to identify and characterise clusters of islet autoantibody-positive individuals who share similar characteristics and type 1 diabetes risk. We tested a novel outcome-guided clustering method in initially non-diabetic autoantibody-positive relatives of individuals with type 1 diabetes, using the TrialNet Pathway to Prevention study data (n=1123). The outcome of the analysis was the time to development of type 1 diabetes, and variables in the model included demographic characteristics, genetics, metabolic factors and islet autoantibodies. An independent dataset (the Diabetes Prevention Trial of Type 1 Diabetes Study) (n=706) was used for validation. The analysis revealed six clusters with varying type 1 diabetes risks, categorised into three groups based on the hierarchy of clusters. Group A comprised one cluster with high glucose levels (median for glucose mean AUC 9.48 mmol/l; IQR 9.16-10.02) and high risk (2-year diabetes-free survival probability 0.42; 95% CI 0.34, 0.51). Group B comprised one cluster with high IA-2A titres (median 287 DK units/ml; IQR 250-319) and elevated autoantibody titres (2-year diabetes-free survival probability 0.73; 95% CI 0.67, 0.80). Group C comprised four lower-risk clusters with lower autoantibody titres and glucose levels (with 2-year diabetes-free survival probability ranging from 0.84-0.99 in the four clusters). Within group C, the clusters exhibit variations in characteristics such as glucose levels, C-peptide levels and age. A decision rule for assigning individuals to clusters was developed. Use of the validation dataset confirmed that the clusters can identify individuals with similar characteristics. Demographic, metabolic, immunological and genetic markers may be used to identify clusters of distinctive characteristics and different risks of progression to type 1 diabetes among autoantibody-positive individuals with a family history of type 1 diabetes. The results also revealed the heterogeneity in the population and complex interactions between variables.

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

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. 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. 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). This study highlights the importance of measuring islet autoantibodies, including IAA, in relatives of people with type 1 diabetes to avoid misdiagnosis.

HOMA-IR and the Matsuda Index as predictors of progression to type 1 diabetes in autoantibody-positive relatives

Aim/hypothesisWe assessed whether HOMA-IR and the Matsuda Index are associated with transitions through stages of type 1 diabetes.MethodsAutoantibody (AAb)-positive relatives of individuals with type 1 diabetes (n=6256) from the TrialNet Pathway to Prevention were studied. Associations of indicators of insulin resistance (HOMA-IR) and insulin sensitivity (Matsuda Index) with BMI percentile (BMIp) and age were assessed with adjustments for measures of insulin secretion, Index60 and insulinogenic index (IGI). Cox regression was used to determine if tertiles of HOMA-IR and Matsuda Index predicted transitions from Not Staged (<2 AAbs) to Stage 1 (≥2 AAbs and normoglycaemia), from Stage 1 to Stage 2 (≥2 AAbs with dysglycaemia), and progression to Stage 3 (diabetes as defined by WHO/ADA criteria).ResultsThere were strong associations of HOMA-IR (positive) and Matsuda Index (inverse) with baseline age and BMIp (p<0.0001). After adjustments for Index60, transitioning from Stage 1 to Stage 2 was associated with higher HOMA-IR and lower Matsuda Index (HOMA-IR: HR=1.71, p<0.0001; Matsuda Index, HR=0.40, p<0.0001), as with progressing from Stages 1 or 2 to Stage 3 (HOMA-IR: HR=1.98, p<0.0001; Matsuda Index: HR=0.46, p<0.0001). Without adjustments, associations of progression to Stage 3 were inverse for HOMA-IR and positive for Matsuda Index, opposite in directionality with adjustments. When IGI was used in place of Index60, the findings were similar.Conclusions/interpretationProgression to Stages 2 and 3 of type 1 diabetes increases with HOMA-IR and decreases with the Matsuda Index after adjustments for insulin secretion. Indicators of insulin secretion appear helpful for interpreting associations of progression to type 1 diabetes with HOMA-IR or the Matsuda Index in AAb-positive relatives.Graphical

CTLA4, SH2B3, and CLEC16A diversely affect the progression of early islet autoimmunity in relatives of Type 1 diabetes patients.

The HLA region is the major genetic risk determinant of Type 1 diabetes. How non-HLA loci contribute to the genetic risk is incompletely understood, but there are indications that at least some impact progression of asymptomatic autoimmunity. We examined whether SNPs in 7 susceptibility loci (INS, SH2B3, PTPN2, PTPN22, CTLA4, CLEC16A, and IL2RA) could improve prediction of the progression from single to multiple autoantibody positivity, and from there on to diagnosis. SNPs were genotyped in persistently autoantibody positive relatives by allelic discrimination qPCR and disease progression was studied by multivariate Cox regression analysis. In our cohort, only the CTLA4 GA genotype (rs3087243, P = 0.002) and the CLEC16A AA genotype (rs12708716, P = 0.021) were associated with accelerated progression from single to multiple autoantibody positivity, but their effects were restricted to presence of HLA-DQ2/DQ8, and IAA as first autoantibody, respectively. The interaction of CTLA4 and HLA-DQ2/DQ8 overruled the effect of DQ2/DQ8 alone. The HLA-DQ2/DQ8-mediated risk of progression to multiple autoantibodies nearly entirely depended on heterozygosity for CTLA4. The SH2B3 TT genotype (rs3184504) was protective for HLA-DQ8 positive subjects (P = 0.003). At the stage of multiple autoantibodies, only the CTLA4 GA genotype was a minor independent risk factor for progression towards clinical diabetes (P = 0.034). Our study shows that non-HLA polymorphisms impact progression of islet autoimmunity in a subgroup-, stage- and SNP-specific way, suggesting distinct mechanisms. If confirmed, these findings may help refine risk assessment, follow-up, and prevention trials in risk groups.

TCF7L2 Genetic Variants Do Not Influence Insulin Sensitivity or Secretion Indices in Autoantibody-Positive Individuals at Risk for Type 1 Diabetes.

We aimed to test whether type 2 diabetes (T2D)-associated TCF7L2 genetic variants affect insulin sensitivity or secretion in autoantibody-positive relatives at risk for type 1 diabetes (T1D). We studied autoantibody-positive TrialNet Pathway to Prevention study participants (N = 1,061) (mean age 16.3 years) with TCF7L2 single nucleotide polymorphism (SNP) information and baseline oral glucose tolerance test (OGTT) to calculate indices of insulin sensitivity and secretion. With Bonferroni correction for multiple comparisons, P values < 0.0086 were considered statistically significant. None, one, and two T2D-linked TCF7L2 alleles were present in 48.1%, 43.9%, and 8.0% of the participants, respectively. Insulin sensitivity (as reflected by 1/fasting insulin [1/IF]) decreased with increasing BMI z score and was lower in Hispanics. Insulin secretion (as measured by 30-min C-peptide index) positively correlated with age and BMI z score. Oral disposition index was negatively correlated with age, BMI z score, and Hispanic ethnicity. None of the indices were associated with TCF7L2 SNPs. In multivariable analysis models with age, BMI z score, ethnicity, sex, and TCF7L2 alleles as independent variables, C-peptide index increased with age, while BMI z score was associated with higher insulin secretion (C-peptide index), lower insulin sensitivity (1/IF), and lower disposition index; there was no significant effect of TCF7L2 SNPs on any of these indices. When restricting the analyses to participants with a normal OGTT (n = 743; 70%), the results were similar. In nondiabetic autoantibody-positive individuals, TCF7L2 SNPs were not related to insulin sensitivity or secretion indices after accounting for BMI z score, age, sex, and ethnicity.

Excess BMI Accelerates Islet Autoimmunity in Older Children and Adolescents.

Sustained excess BMI increases the risk of type 1 diabetes (T1D) in autoantibody-positive relatives without diabetes of patients. We tested whether elevated BMI also accelerates the progression of islet autoimmunity before T1D diagnosis. We studied 706 single autoantibody-positive pediatric TrialNet participants (ages 1.6-18.6 years at baseline). Cumulative excess BMI (ceBMI) was calculated for each participant based on longitudinally accumulated BMI ≥85th age- and sex-adjusted percentile. Recursive partitioning analysis and multivariable modeling defined the age cut point differentiating the risk for progression to multiple positive autoantibodies. At baseline, 175 children (25%) had a BMI ≥85th percentile. ceBMI range was -9.2 to 15.6 kg/m2 (median -1.91), with ceBMI ≥0 kg/m2 corresponding to persistently elevated BMI ≥85th percentile. Younger age increased the progression to multiple autoantibodies, with age cutoff of 9 years defined by recursive partitioning analysis. Although ceBMI was not significantly associated with progression from single to multiple autoantibodies overall, there was an interaction with ceBMI ≥0 kg/m2, age, and HLA (P = 0.009). Among children ≥9 years old without HLA DR3-DQ2 and DR4-DQ8, ceBMI ≥0 kg/m2 increased the rate of progression from single to multiple positive autoantibodies (hazard ratio 7.32, P = 0.004) and conferred a risk similar to that in those with T1D-associated HLA haplotypes. In participants <9 years old, the effect of ceBMI on progression to multiple autoantibodies was not significant regardless of HLA type. These data support that elevated BMI may exacerbate islet autoimmunity prior to clinical T1D, particularly in children with lower risk based on age and HLA. Interventions to maintain normal BMI may prevent or delay the progression of islet autoimmunity.

A Type 1 Diabetes Genetic Risk Score Predicts Progression of Islet Autoimmunity and Development of Type 1 Diabetes in Individuals at Risk.

OBJECTIVEWe tested the ability of a type 1 diabetes (T1D) genetic risk score (GRS) to predict progression of islet autoimmunity and T1D in at-risk individuals.RESEARCH DESIGN AND METHODSWe studied the 1,244 TrialNet Pathway to Prevention study participants (T1D patients’ relatives without diabetes and with one or more positive autoantibodies) who were genotyped with Illumina ImmunoChip (median [range] age at initial autoantibody determination 11.1 years [1.2–51.8], 48% male, 80.5% non-Hispanic white, median follow-up 5.4 years). Of 291 participants with a single positive autoantibody at screening, 157 converted to multiple autoantibody positivity and 55 developed diabetes. Of 953 participants with multiple positive autoantibodies at screening, 419 developed diabetes. We calculated the T1D GRS from 30 T1D-associated single nucleotide polymorphisms. We used multivariable Cox regression models, time-dependent receiver operating characteristic curves, and area under the curve (AUC) measures to evaluate prognostic utility of T1D GRS, age, sex, Diabetes Prevention Trial–Type 1 (DPT-1) Risk Score, positive autoantibody number or type, HLA DR3/DR4-DQ8 status, and race/ethnicity. We used recursive partitioning analyses to identify cut points in continuous variables.RESULTSHigher T1D GRS significantly increased the rate of progression to T1D adjusting for DPT-1 Risk Score, age, number of positive autoantibodies, sex, and ethnicity (hazard ratio [HR] 1.29 for a 0.05 increase, 95% CI 1.06–1.6; P = 0.011). Progression to T1D was best predicted by a combined model with GRS, number of positive autoantibodies, DPT-1 Risk Score, and age (7-year time-integrated AUC = 0.79, 5-year AUC = 0.73). Higher GRS was significantly associated with increased progression rate from single to multiple positive autoantibodies after adjusting for age, autoantibody type, ethnicity, and sex (HR 2.27 for GRS >0.295, 95% CI 1.47–3.51; P = 0.0002).CONCLUSIONSThe T1D GRS independently predicts progression to T1D and improves prediction along T1D stages in autoantibody-positive relatives.

Ethnic differences in progression of islet autoimmunity and type 1 diabetes in relatives at risk.

We hypothesised that progression of islet autoimmunity and type 1 diabetes mellitus differs among races/ethnicities in at-risk individuals. In this study, we analysed the data from the Type 1 Diabetes TrialNet Pathway to Prevention Study. We studied 4873 non-diabetic, autoantibody-positive relatives of individuals with type 1 diabetes followed prospectively (11% Hispanic, 80.9% non-Hispanic white [NHW], 2.9% non-Hispanic black [NHB] and 5.2% non-Hispanic other [NHO]). Primary outcomes were time from single autoantibody positivity confirmation to multiple autoantibody positivity, and time from multiple autoantibody positivity to type 1 diabetes mellitus diagnosis. Conversion from single to multiple autoantibody positivity was less common in Hispanic individuals than in NHW individuals (HR 0.66 [95% CI 0.46, 0.96], p = 0.028) adjusting for autoantibody type, age, sex, Diabetes Prevention Trial Type 1 Risk Score and HLA-DR3-DQ2/DR4-DQ8 genotype. In participants who screened positive for multiple autoantibodies (n = 2834), time to type 1 diabetes did not differ by race/ethnicity overall (p = 0.91). In children who were <12years old when multiple autoantibody positivity was determined, being overweight/obese had differential effects by ethnicity: type 1 diabetes risk was increased by 36% in NHW children (HR 1.36 [95% CI 1.04, 1.77], p = 0.024) and was nearly quadrupled in Hispanic children (HR 3.8 [95% CI 1.6, 9.1], p = 0.0026). We did not observe this interaction in participants who were ≥12 years old at determination of autoantibody positivity, although this group size was limited. No significant differential risks were observed between individuals of NHB and NHW ethnicity. The risk and rate of progression of islet autoimmunity were lower in Hispanic compared with NHW at-risk individuals, while significant differences in the development of type 1 diabetes were limited to children <12years old and were modified by BMI.

Effect of Oral Insulin on Prevention of Diabetes in Relatives of Patients With Type 1 Diabetes

Type 1 diabetes requires major lifestyle changes and carries increased morbidity and mortality. Prevention or delay of diabetes would have major clinical effect. To determine whether oral insulin delays onset of type 1 diabetes in autoantibody-positive relatives of patients with type 1 diabetes. Between March 2, 2007, and December 21, 2015, relatives with at least 2 autoantibodies, including insulin autoantibodies and normal glucose tolerance, were enrolled in Canada, the United States, Australia, New Zealand, the United Kingdom, Italy, Sweden, Finland, and Germany. The main study group (n = 389) had first-phase insulin release on an intravenous glucose tolerance test that was higher than the threshold. The 55 patients in the secondary stratum 1 had an identical antibody profile as the main study group except they had first-phase insulin release that was lower than the threshold. Secondary strata 2 (n = 114) and strata 3 (n = 3) had different autoantibody profiles and first-phase insulin release threshold combinations. Follow-up continued through December 31, 2016. Randomization to receive 7.5 mg/d of oral insulin (n = 283) or placebo (n = 277), including participants in the main study group who received oral insulin (n = 203) or placebo (n = 186). The primary outcome was time to diabetes in the main study group. Significance was based on a 1-sided threshold of .05, and 1-sided 95% CIs are reported. Of a total of 560 randomized participants (median enrollment age, 8.2 years; interquartile range [IQR], 5.7-12.1 years; 170 boys [60%]; 90.7% white non-Hispanic; 57.6% with a sibling with type 1 diabetes), 550 completed the trial including 389 participants (median age, 8.4 years; 245 boys [63%]), 382 (96%) in the main study group. During a median follow-up of 2.7 years (IQR, 1.5-4.6 years) in the main study group, diabetes was diagnosed in 58 participants (28.5%) in the oral insulin group and 62 (33%) in the placebo group. Time to diabetes was not significantly different between the 2 groups (hazard ratio [HR], 0.87; 95% CI, 0-1.2; P = .21). In secondary stratum 1 (n = 55), diabetes was diagnosed in 13 participants (48.1%) in the oral insulin group and in 19 participants (70.3%) in the placebo group. The time to diabetes was significantly longer with oral insulin (HR, 0.45; 95% CI, 0-0.82; P = .006). The HR for time to diabetes for the between-group comparisons for the 116 participants in the other secondary stratum was 1.03 (95% CI, 0-2.11; P = .53) and for the entire cohort of 560 participants was 0.83 (95% CI, 0-1.07; P = .11), which were not significantly different. The most common adverse event was infection (n = 254), with 134 events in the oral insulin group and 120 events in the placebo group, but no significant study-related adverse events occurred. Among autoantibody-positive relatives of patients with type 1 diabetes, oral insulin at a dose of 7.5 mg/d, compared with placebo, did not delay or prevent the development of type 1 diabetes over 2.7 years. These findings do not support oral insulin as used in this study for diabetes prevention. clinicaltrials.gov Identifier: NCT00419562.

The Role of Age and Excess Body Mass Index in Progression to Type 1 Diabetes in At-Risk Adults.

Given the global rise in both type 1 diabetes incidence and obesity, the role of body mass index (BMI) on type 1 diabetes pathophysiology has gained great interest. Sustained excess BMI in pediatric participants of the TrialNet Pathway to Prevention (PTP) cohort increased risk for progression to type 1 diabetes, but the effects of age and obesity in adults remain largely unknown. To determine the effect of age and sustained obesity on the risk for type 1 diabetes in adult participants in the TrialNet PTP cohort (i.e., nondiabetic autoantibody-positive relatives of patients with type 1 diabetes). Longitudinally accumulated BMI >25 kg/m2 was calculated to generate a cumulative excess BMI (ceBMI) for each participant, with ceBMI values ≥0 kg/m2 and ≥5 kg/m2 representing sustained overweight or obese status, respectively. Recursive partitioning analysis yielded sex- and age-specific thresholds for ceBMI that confer the greatest risk for type 1 diabetes progression. In this cohort of 665 adults (age 20 to 50 years; median follow-up, 3.9 years), 49 participants developed type 1 diabetes. Age was an independent protective factor for type 1 diabetes progression (hazard ratio, 0.95; P = 0.008), with a threshold of >35 years that reduced risk for type 1 diabetes. In men age >35 years and women age <35 years, sustained obesity (ceBMI ≥5 kg/m2) increased the risk for type 1 diabetes. Age is an important factor for type 1 diabetes progression in adults and influences the impact of elevated BMI, indicating an interplay of excess weight, age, and sex in adult type 1 diabetes pathophysiology.

Glucokinase Gene May Be a More Suitable Target Than the Insulin Gene for Detection of β Cell Death.

Detection and quantification of unmethylated circulating insulin (INS) DNA presumably released from β cells has been previously used for assessing their destruction. As the targets within the INS gene suffer from suboptimal specificity, we sought to improve the assay parameters by using the glucokinase gene (GCK) tissue-specific pancreatic promoter. The amount of methylated and unmethylated GCK DNA was measured using a droplet polymerase chain reaction assay and compared with the previously published INS-targeted assay. The method was tested using synthetic target sequences and DNA from pancreatic islets, blood, brain, kidney, large intestine, liver, lung, small intestine, and stomach. Circulating serum DNA was obtained from children with recent-onset type 1 diabetes (T1D) (n = 25), autoantibody-positive first-degree relatives of T1D patients (n = 14), and healthy controls (n = 20). The unmethylated GCK DNA was found to be more islet specific than unmethylated INS DNA. The proportion of the unmethylated GCK DNA was lower than INS in all tested extrapancreatic tissues, except kidney. Although the amounts of methylated DNA measured by the two assays were similar, the INS assay detected considerably more unmethylated DNA. Whereas none of the assays showed significant increase in the amount of unmethylated DNA, the ratio of unmethylated/methylated GCK DNA was borderline significantly increased in autoantibody-positive relatives compared with T1D patients (P = 0.04) and controls (P = 0.06). Targeting the assay into the GCK gene improved analytical parameters of the assay. As the amount of unmethylated target DNA in properly treated samples is very low, the clinical utility of this method remains to be evaluated.

Association of serum microRNAs with islet autoimmunity, disease progression and metabolic impairment in relatives at risk of type 1 diabetes.

MicroRNAs (miRNAs) are key regulators of gene expression and novel biomarkers for many diseases. We investigated the hypothesis that serum levels of some miRNAs would be associated with islet autoimmunity and/or progression to type 1 diabetes. We measured levels of 93 miRNAs most commonly detected in serum. This retrospective cohort study included 150 autoantibody-positive and 150 autoantibody-negative family-matched siblings enrolled in the TrialNet Pathway to Prevention Study. This was a young cohort (mean age=11years), and most autoantibody-positive relatives were at high risk because they had multiple autoantibodies, with 39/150 (26%, progressors) developing type 1 diabetes within an average 8.7months of follow-up. We analysed miRNA levels in relation to autoantibody status, future development of diabetes and OGTT C-peptide and glucose indices of disease progression. Fifteen miRNAs were differentially expressed when comparing autoantibody-positive/negative siblings (range -2.5 to 1.3-fold). But receiver operating characteristic (ROC) analysis indicated low specificity and sensitivity. Seven additional miRNAs were differentially expressed among autoantibody-positive relatives according to disease progression; ROC returned significant AUC values and identified miRNA cut-off levels associated with an increased risk of disease in both cross-sectional and survival analyses. Levels of several miRNAs showed significant correlations (r values range 0.22-0.55) with OGTT outcomes. miR-21-3p, miR-29a-3p and miR-424-5p had the most robust associations. Serum levels of selected miRNAs are associated with disease progression and confer additional risk of the development of type 1 diabetes in young autoantibody-positive relatives. Further studies, including longitudinal assessments, are warranted to further define miRNA biomarkers for prediction of disease risk and progression.

Excess BMI in Childhood: A Modifiable Risk Factor for Type 1 Diabetes Development?

OBJECTIVEWe aimed to determine the effect of elevated BMI over time on the progression to type 1 diabetes in youth.RESEARCH DESIGN AND METHODSWe studied 1,117 children in the TrialNet Pathway to Prevention cohort (autoantibody-positive relatives of patients with type 1 diabetes). Longitudinally accumulated BMI above the 85th age- and sex-adjusted percentile generated a cumulative excess BMI (ceBMI) index. Recursive partitioning and multivariate analyses yielded sex- and age-specific ceBMI thresholds for greatest type 1 diabetes risk.RESULTSHigher ceBMI conferred significantly greater risk of progressing to type 1 diabetes. The increased diabetes risk occurred at lower ceBMI values in children <12 years of age compared with older subjects and in females versus males.CONCLUSIONSElevated BMI is associated with increased risk of diabetes progression in pediatric autoantibody-positive relatives, but the effect varies by sex and age.

Do Electrochemiluminescence Assays Improve Prediction of Time to Type 1 Diabetes in Autoantibody-Positive TrialNet Subjects?

To explore whether electrochemiluminescence (ECL) assays can help improve prediction of time to type 1 diabetes in the TrialNet autoantibody-positive population. TrialNet subjects who were positive for one or more autoantibodies (microinsulin autoantibody, GAD65 autoantibody [GADA], IA-2A, and ZnT8A) with available ECL-insulin autoantibody (IAA) and ECL-GADA data at their initial visit were analyzed; after a median follow-up of 24 months, 177 of these 1,287 subjects developed diabetes. Univariate analyses showed that autoantibodies by radioimmunoassays (RIAs), ECL-IAA, ECL-GADA, age, sex, number of positive autoantibodies, presence of HLA DR3/4-DQ8 genotype, HbA1c, and oral glucose tolerance test (OGTT) measurements were all significantly associated with progression to diabetes. Subjects who were ECL positive had a risk of progression to diabetes within 6 years of 58% compared with 5% for the ECL-negative subjects (P < 0.0001). Multivariate Cox proportional hazards models were compared, with the base model including age, sex, OGTT measurements, and number of positive autoantibodies by RIAs. The model with positivity for ECL-GADA and/or ECL-IAA was the best, and factors that remained significantly associated with time to diabetes were area under the curve (AUC) C-peptide, fasting C-peptide, AUC glucose, number of positive autoantibodies by RIAs, and ECL positivity. Adding ECL to the Diabetes Prevention Trial risk score (DPTRS) improved the receiver operating characteristic curves with AUC of 0.83 (P < 0.0001). ECL assays improved the ability to predict time to diabetes in these autoantibody-positive relatives at risk for developing diabetes. These findings might be helpful in the design and eligibility criteria for prevention trials in the future.

Tetraspanin 7 autoantibodies in type 1 diabetes.

Autoantibodies to pancreatic beta cell proteins are markers of asymptomatic type 1 diabetes. The aim was to determine whether autoantibodies to the beta cell protein tetraspanin 7 would improve the ability to identify autoimmunity against pancreatic beta cells. Full length and external domain fragments of tetraspanin 7 were expressed as luciferase-tagged fusion proteins and used in immunoprecipitation assays to measure autoantibodies in samples from 363 patients with type 1 diabetes at onset of disease, 503 beta cell autoantibody negative first-degree relatives of patients, and 212 relatives with autoantibodies to insulin, glutamic acid decarboxylase, insulinoma antigen 2 or zinc transporter 8. Antibody binding was observed against the full length and external domains of tetraspanin 7, and was strongest against the full length protein. Autoantibodies that could be inhibited by untagged tetraspanin 7 were detected in 5 (1%) of 503 autoantibody negative relatives, 3 (3.2%) of 94 autoantibody negative patients, 95 (35.3%) of 269 autoantibody positive patients, 1 (1%) of 98 single autoantibody positive relatives and 25 (21.9%) of 114 multiple autoantibody positive relatives. Progression to diabetes did not differ between multiple autoantibody positive relatives with and without tetraspanin 7 autoantibodies. Tetraspanin 7 is an autoantigen in type 1 diabetes. Tetraspanin 7 autoantibodies are a marker of type 1 diabetes, but provide minor additional value to existing autoantibodies in identifying beta cell autoimmunity.

ECL-IAA and ECL-GADA Can Identify High-Risk Single Autoantibody-Positive Relatives in the TrialNet Pathway to Prevention Study.

Relatives with single positive islet autoantibodies have a much lower risk of progression to diabetes than those with multiple autoantibodies. TrialNet subjects positive for single autoantibody to insulin (mIAA) (n = 50) or single autoantibody to glutamic acid decarboxylase (GADA) (n = 50) were analyzed using new electrochemiluminescence (ECL) assays (ECL-IAA and ECL-GADA, respectively) at their initial visit and longitudinally over time. Affinity assays were performed on a subset of single autoantibody-positive subjects at initial and most recent visits. After a mean follow-up of 5.3 years, 20 subjects developed type 1 diabetes. Among either single GADA or single mIAA subjects, those who were positive in the ECL assay showed higher affinity at the initial visit, and affinity results stayed consistent over time. No converting events from low to high or high to low affinity were seen over time. Confirmed positivity for ECL is associated with high affinity and can help staging of risk for type 1 diabetes in single autoantibody-positive subjects.

HLA-DRB1*15:01-DQA1*01:02-DQB1*06:02 Haplotype Protects Autoantibody-Positive Relatives From Type 1 Diabetes Throughout the Stages of Disease Progression.

The HLA-DRB1*15:01-DQA1*01:02-DQB1*06:02 haplotype is linked to protection from the development of type 1 diabetes (T1D). However, it is not known at which stages in the natural history of T1D development this haplotype affords protection. We examined a cohort of 3,358 autoantibody-positive relatives of T1D patients in the Pathway to Prevention (PTP) Study of the Type 1 Diabetes TrialNet. The PTP study examines risk factors for T1D and disease progression in relatives. HLA typing revealed that 155 relatives carried this protective haplotype. A comparison with 60 autoantibody-negative relatives suggested protection from autoantibody development. Moreover, the relatives with DRB1*15:01-DQA1*01:02-DQB1*06:02 less frequently expressed autoantibodies associated with higher T1D risk, were less likely to have multiple autoantibodies at baseline, and rarely converted from single to multiple autoantibody positivity on follow-up. These relatives also had lower frequencies of metabolic abnormalities at baseline and exhibited no overall metabolic worsening on follow-up. Ultimately, they had a very low 5-year cumulative incidence of T1D. In conclusion, the protective influence of DRB1*15:01-DQA1*01:02-DQB1*06:02 spans from autoantibody development through all stages of progression, and relatives with this allele only rarely develop T1D.

The implications of autoantibodies to a single islet antigen in relatives with normal glucose tolerance: development of other autoantibodies and progression to type 1 diabetes.

Aims/hypothesisAutoantibodies directed at single islet autoantigens are associated with lower overall risk of type 1 diabetes than multiple autoantibodies, but individuals with one autoantibody may progress to higher risk categories. We examined the characteristics of this progression in relatives followed prospectively in the TrialNet Pathway to Prevention.MethodsThe study population comprised 983 relatives who were single autoantibody positive with normal baseline glucose tolerance (median age 16.2 years). Samples were screened for antibodies to GAD, insulinoma-associated antigen 2 (IA-2) and insulin, and all positive samples tested for antibodies to zinc transporter 8 and islet cell antibodies.ResultsAntibodies to at least one additional islet autoantigen appeared in 118 of 983 relatives (overall 5 year risk 22%, 95% CI [17.9, 26.1]). At baseline, antibodies to GAD alone (68%) were more frequent than antibodies to insulin (26%) or IA-2 (6%), but all were associated with a similar risk of developing additional autoantibodies. Risk was associated with younger age (p = 0.002) and HLA class II genotype, but was similar in high and intermediate genetic risk groups (p = 0.65). Relatives who became multiple autoantibody positive during the follow-up had increased risk of developing diabetes comparable with the risk in relatives with multiple autoantibodies at study entry.Conclusions/interpretationProgression of islet autoimmunity in single autoantibody positive relatives in late childhood/adult life is associated with a predominance of autoantibodies to GAD and a distinct HLA risk profile. This heterogeneity in type 1 diabetes autoimmunity has potentially important implications for disease prevention.Electronic supplementary materialThe online version of this article (doi:10.1007/s00125-015-3830-2) contains peer-reviewed but unedited supplementary material, which is available to authorised users.

Use of Dried Capillary Blood Sampling for Islet Autoantibody Screening in Relatives: A Feasibility Study.

Islet autoantibody testing provides the basis for assessment of risk of progression to type 1 diabetes. We set out to determine the feasibility and acceptability of dried capillary blood spot-based screening to identify islet autoantibody-positive relatives potentially eligible for inclusion in prevention trials. Dried blood spot (DBS) and venous samples were collected from 229 relatives participating in the TrialNet Pathway to Prevention Study. Both samples were tested for glutamic acid decarboxylase, islet antigen 2, and zinc transporter 8 autoantibodies, and venous samples were additionally tested for insulin autoantibodies and islet cell antibodies. We defined multiple autoantibody positive as two or more autoantibodies in venous serum and DBS screen positive if one or more autoantibodies were detected. Participant questionnaires compared the sample collection methods. Of 44 relatives who were multiple autoantibody positive in venous samples, 42 (95.5%) were DBS screen positive, and DBS accurately detected 145 of 147 autoantibody-negative relatives (98.6%). Capillary blood sampling was perceived as more painful than venous blood draw, but 60% of participants would prefer initial screening using home fingerstick with clinic visits only required if autoantibodies were found. Capillary blood sampling could facilitate screening for type 1 diabetes prevention studies.

The development and utility of a novel scale that quantifies the glycemic progression toward type 1 diabetes over 6 months.

We developed a scale to serve as a potential end point for 6-month glycemic progression (PS6M) toward type 1 diabetes (T1D) in autoantibody-positive relatives of individuals with T1D. The PS6M was developed from Diabetes Prevention Trial-Type 1 (DPT-1) data and tested in the TrialNet Pathway to Prevention Study (PTP). It is the difference between 6-month glucose sum values (30-120 min oral glucose tolerance test values) and values predicted for nonprogressors. The PS6M predicted T1D in the PTP (P < 0.001). The area under the receiver operating chacteristic curve was greater (P < 0.001) for the PS6M than for the baseline-to-6-month difference. PS6M values were higher in those with two or more autoantibodies, 30-0 min C-peptide values <2.00 ng/mL, or DPT-1 Risk Scores >7.00 (P < 0.001 for all). The PS6M is an indicator of short-term glycemic progression to T1D that could be a useful tool for assessing preventive treatments and biomarkers.