The BABYDIAB study examined the natural history of autoimmunity to islet antigens from birth in children of parents with type 1 diabetes . Families were eligible to participate if one or both parents had type 1 diabetes. Recruitment into the study began in 1989 and ended in 2000. Recruitment was facilitated through advertisements in paediatric and patient journals, and in paediatric and neonatal clinics. Participation was voluntary. The study was coordinated by the Diabetes Research Institute in Munich through direct contact with the families and the family paediatrician. Cord blood was obtained in obstetric departments from eligible families who had consented to participation. Venous blood samples from the child during follow-up were obtained at paediatric clinics at age 9 months and at 2, 5, 8, 11, 14 and 17 years. The median follow-up time from birth to last sample was 8 years (range 0.75–18.5 years). Questionnaires were completed at birth and at each paediatric visit. The cumulative dropout rate was 16.0% by age 5 years and 20.9% by age 8 years. All families gave written informed consent to participate in the BABYDIAB study. The study was approved by the Ethics Committee of Bavaria, Germany (Bayerische Landesärztekammer [Bavarian Medical Council] no. 95357).
For the present study, all children who were followed until at least 2 years of age were included (n = 1,489). Islet autoantibodies and antibodies to tissue transglutaminase C (tTG-Abs) were measured in samples taken at all scheduled visits. Antibodies to thyroid peroxidase (TPO-Abs) were measured in the last available blood sample from all these children. In 1,474 children there was also sufficient serum for measurement of antibodies to 21-hydroxylase (21-OH-Abs) in the last sample. In children found positive for TPO-Abs or 21-OH-Abs, all earlier samples were also measured to determine the age of appearance of these antibodies. Children who developed type 1 diabetes (n = 36) were not followed after diagnosis.
TPO-Abs were measured by a direct radiobinding assay according to the manufacturer’s instructions (CentAK anti-TPO; Medipan, Dahlewitz/Berlin, Germany). Briefly, 50 µl serum was incubated with 125I-labelled recombinant human TPO in assay buffer with 50 µl protein A suspension for 30 min. Subsequently 1 ml assay buffer was added. Pellets were washed and counted in a gamma counter (Packard, Meridan, CT, USA). TPO-Abs were expressed as U/ml. Samples were defined as TPO-Abs-positive above a threshold of 50 U/ml as suggested by the manufacturer and confirmed using QQ-plot analysis, which plots the distribution of the variable (autoantibody) against the autoantibody concentration, allowing assessment of where distribution deviates from normal . The inter-assay coefficient of variation was 11% at 660 U/ml and 23% at 34 U/ml.
21-OH-Abs were measured by radiobinding assay similar to those previously described for glutamic acid decarboxylase antibodies (GAD-Abs) and islet antigen 2 antibodies (IA-2-Abs) . Briefly, 2 µl serum was incubated with 20,000 cpm of [35S]methionine-labelled, in vitro-transcribed/translated recombinant human 21-OH in 50 mmol/l Tris buffered saline containing 1% Tween 20 (TBST). Antibody-bound 21-OH was recovered with protein-A sepharose beads (GE Healthcare Life Sciences, Amersham, UK), beads washed five times in TBST and scintillation counted in a Top Count Microplate Scintillation Counter; Packard). Relative antibody concentration was expressed as an index defined as: (cpm in the unknown sample—cpm in the negative standard)/(cpm in the positive standard—cpm in the negative standard)×100. The threshold was determined using QQ plots and was set at 6 index units, which also corresponded to the 99th percentile of 100 control children (mean age 7.5 years; interquartile range 5.1–9.1). The inter-assay coefficient of variation was 16% at 41 index units.
IgA antibodies to tissue transglutaminase (tTG-Abs) were measured by ELISA according to the manufacturer’s instructions (Eurospital, Trieste, Italy) and by radiobinding assay with [35S]methionine-labelled, in vitro-transcribed/translated recombinant human tTG as previously described . Thresholds for positivity were determined using QQ plots and corresponded to the 95th percentile of control children without diabetes or coeliac disease for the ELISA and the 99th percentile of control samples for the radiobinding assay. Samples were defined as positive if they were detected above these thresholds in both assays.
Insulin autoantibodies (IAA), GAD autoantibodies (GAD-Abs) and IA-2 autoantibodies (IA2-Abs) were measured by radiobinding assays, as previously described [11, 20]. The upper limits of normal were determined using Q-Q plots and corresponded to the 99th percentile of control children, i.e. 1.5 local units/ml for IAA, 25 WHO units/ml for GAD-Abs and 4 WHO units/ml for IA2-Abs. Using these thresholds for positivity, the assays had sensitivities and specificities of 70 and 99% (IAA), 86 and 93% (GAD-Abs), 72 and 100% (IA2-Abs) and 84 and 100% for multiple islet autoantibodies in the Third Diabetes Autoantibodies Standardization Program Proficiency Workshop . The inter-assay coefficient of variation for samples with low autoantibody titre was 11% for IAA, 18% for GAD-Abs and 16% for IA2-Abs.
All antibody measurements were performed on coded samples that were blinded to the operator.
HLA DR and DQ genotypes were determined in 1,413 children of parents with type 1 diabetes. The remaining 76 children did not provide a suitable sample for HLA typing. HLA-DRB1, HLA-DQA1 and HLA-DQB1 alleles were typed using PCR-amplified DNA and non-radioactive sequence-specific oligonucleotide probes as described previously .
Notification of clinical disease
Thyroid and other autoimmune disease status of the child and other family members was reported by response to questionnaires received at each study visit. None of the children were reported to have congenital hypothyroidism. In addition, as previously described , children who were persistently positive for tTG-Abs were recommended to have intestinal biopsies for diagnosis of coeliac disease. Families were not notified of thyroid autoantibody status and no recommendation with respect to thyroid function was provided to families of children positive for TPO-Abs.
Time-to-event methods were used to calculate risks (life table analysis) and to compare autoantibody outcome for participants with different covariate categories (life table analysis and Cox proportional hazards model). In children with a positive autoantibody outcome, the age at the first positive sample was used as the event time. Analysis considered censoring in losses to follow-up and in participants with antibody-negative status at the follow-up visit age of their last autoantibody-negative sample. The log rank test was used for comparisons of covariate categories in life table analysis. HRs were calculated using Cox proportional hazards model. Variables that were significant (p < 0.05) in the univariate model were tested in the multivariate model using forward conditional analysis. The proportional hazards assumption in the Cox model was tested by examining the log minus log plot of each covariate for parallel curves and by using a time-dependent Cox regression that included the covariate in question and the interaction between time and the covariate. The interaction was not significant for all covariates indicating that the hazards were proportional.
TPO antibody incidence was determined by calculating the incremental increase in risk at 2, 5, 8, 11 and 14 years corrected for the time interval between visits, and expressed as cases per 100 participants per year. For all analyses, a two-tailed p value of 0.05 was considered significant. All statistical analyses were performed using the Statistical Package for Social Science (SPSS 15.0; SPSS, Chicago, IL, USA).