The study was performed in children from the BABYDIAB study, a longitudinal study examining the natural history of islet autoimmunity and type 1 diabetes in 1650 children born to a mother with type 1 diabetes or a father with type 1 diabetes
[8]. Autoantibodies against insulin (IAA), glutamic acid decarboxylase (GADA), insulinoma antigen 2 (IA-2A) and zinc transporter 8 (ZnT8-A) were measured in samples taken at all scheduled visits (at 9 months, 2, 5, 8, 11, 14, 17 and 20 years of age), and every 6 months in children with islet autoantibodies. The median follow-up time from birth to last sample was 11.1 years (range 0.75–21.7 years), and the median follow-up from islet autoantibody seroconversion to last sample was 5.4 years (range 0.1–19.6 years). Families were asked to report occurrence of symptoms of diabetes. In children with islet autoantibodies, a yearly oral glucose tolerance test was performed. Diabetes onset was defined according to ADA criteria which include unequivocal hyperglycemia with acute metabolic decompensation, or the observation on at least two occasions of a 2-hour plasma glucose >200 mg/dL after an oral glucose challenge, or a random blood glucose >200 mg/dL if accompanied by unequivocal symptoms. Since 1997, fasting blood glucose >126 mg/dL on two occasions was added to the diabetes diagnosis criteria. The BABYDIAB study was approved by the ethical committee of Bavaria, Germany (No. 95357). All families gave written informed consent to participate in the study. Investigations were carried out in accordance with the principles of the Declaration of Helsinki, as revised in 2000.
IAA, GADA, IA-2A, and ZnT8A were determined centrally by the Institute of Diabetes Research Munich using radiobinding assays as previously described
[8],
[9]. The upper limit of normal for each assay was determined using Q–Q plots and corresponded to the 99
th percentile of control children. Offspring were considered islet autoantibody-positive when two consecutive samples collected after birth were positive. Islet autoantibody assays were evaluated by the Diabetes Autoantibody Standardization Program (DASP; laboratory 121)
[10],
[11],
[12]. Sensitivity and specificity in the 2009 DASP workshop were 70% and 98% for IAA, 86% and 93% for GADA, 72% and 100% for IA-2A, 51% and 100% for the ZnT8A tryptophan variant, and 68% and 100% for the ZnT8A arginine variant, respectively.
Genotyping of the
CDKAL1 rs4712526,
CDKN2A/2B rs10811661,
FTO rs8050136,
HHEX-IDE rs5015480,
HMGA2 rs1122590,
IGF2BP2 rs4402960,
KCNJ11 rs5215,
KCNQ1 rs2237892,
MTNR1B rs1387153,
PPARG rs1801282,
SLC30A8 rs3802177 and
TCF7L2 rs7901695 SNPs was performed with the MassARRAY system using the iPLEX™ chemistry (Sequenom, San Diego, CA, USA) as previously described
[13]. The 12 loci included 9 originally reported by
[14] plus 3 that were used for genotyping of the KORA study cohort
[15]. To control for reproducibility, 16.3% of samples were genotyped in duplicate with discordance rate <0.5%. All SNPs were tested for deviation from Hardy-Weinberg equilibrium by means of chi-square or Fisher's exact test. DNA samples for genotyping were available from 1350 children. HLA-DRB1, HLA-DQA1 and HLA-DQB1 alleles were typed using PCR-amplified DNA and non-radioactive sequence-specific oligonucleotide probes as described previously
[16]. Classification into high risk HLA genotypes was based on TEDDY study inclusion genotypes for first degree relatives as previously described
[17].
Data on weight and height during follow-up at age 9 months, 2, 5, 8, 11, and 14 years of age were collected from paediatric records which were completed by trained staff at birth and by paediatricians at visits after birth. For analysis, data on height, weight and BMI at follow-up were adjusted for gender and exact age at examination and were expressed as percentiles using German reference data
[18]. Low weight was defined as BMI percentile ≤10, normal weight was defined as BMI percentile between 10 and 90, overweight was defined as BMI percentile ≥90.
The probability of islet autoantibodies was estimated by Kaplan-Meier analysis. Hazards ratios (HRs) were determined using Cox's proportional hazards model. Within islet autoantibody positive children, Kaplan-Meier analysis was used to calculate the probability of progression to diabetes where follow-up time was calculated from the age when autoantibodies were first detected to the age of type 1 diabetes diagnosis, or last contact. Based on a corrected alpha value of 0.004 and a desired statistical power of 0.8, our data allowed us to detect HRs for the probability of islet autoantibodies of ≤0.77 or ≥1.34 for TCF7L2 and of ≤0.38 or ≥5.25 for PPARG. For all other genes, the upper and lower limits of the respective detectable HRs were between these ranges. Differences of weight, height and BMI between children who developed islet autoantibodies and children who were islet autoantibody negative during follow-up at age 9 months, 2, 5, 8, 11, and 14 years were compared using Mann-Whitney-U Test. For comparison of BMI, weight, and hight at the age of seroconversion, a nested case–control population was selected from the 1650 offspring participating in BABYDIAB. These included 141 islet autoantibody positive children (cases) with weight, and height data at the age of islet autoantibody seroconversion. For each case, two controls were selected from the islet autoantibody negative children matching for gender and data of birth. In the control children, weight and height was obtained at the age corresponding to the age of seroconversion of the respective case. Seven of these control children did not have complete weight and height data resulting in a total of 275 controls. The distribution of BMI, weight, and height were compared using Chi-Square Test.
The statistical analysis was performed using the Statistical Package for the Social Sciences (SPSS 19.0; SPSS Inc., Chicago, IL, USA).
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