Patients and controls
We studied 301 white unrelated Spanish type 1 diabetes patients (150 women, 151 men) diagnosed according to the criteria of the American Diabetes Association (ADA) and 646 healthy controls, who were recruited from among blood donors in the Madrid area (therefore with an age ranging from 18 to 60 years).
The age at onset for the consecutively recruited type 1 diabetes patients ranged from 1 to 55 years old (mean: 17.3 ± 10.0 years; median age at onset 15 years). All patients were insulin-dependent at the time of the study. Informed consent was obtained from all participants included in the study, which was approved by the Ethics Committee of the Hospital Clínico San Carlos.
As cut-off age for stratified analysis, we decided to choose patients younger than 16 years, because 15 years is our median and in non-normalised variables that is the best statistical parameter to subdivide a cohort in order to maximise the statistical power.
In order to increase the statistical power, we included an additional Dutch cohort of 429 type 1 diabetes patients (median age at diagnosis 8.7 years, range 1–16 years) and 720 healthy unrelated controls. The diagnosis was made according to International Society of Paediatric and Adolescent Diabetes (ISPAD) criteria. All patients gave their informed consent and the Medical Ethics Committee of Leiden University Medical Centre approved this study.
Patients and controls were genotyped for the CAPSL SNPs rs1445898 and rs1010601 and for IL7R SNPs rs6891932, rs987106 and rs3194051. Genotyping was done using TaqMan assays in a 7900HT fast real-time PCR System (Applied Biosystems, Foster City, CA, USA). All assays (identification numbers: C__8811801_1, C___2025977_10, C__8811858_10, C___8811808_10 and C__25616805_10, respectively) were performed as recommended by the manufacturer. Call rate success was over 97%; genotyping was repeated in 10% of the samples and the results were consistent.
Differences in allele and genotype frequencies were calculated by χ
2 or Fisher’s exact test when necessary. Associations were estimated by the OR with 95% CI. Statistical analysis used Epi Info v. 6.02 (CDC, Atlanta, GA, USA). No statistically significant deviations from Hardy–Weinberg equilibrium were observed for genotypes of these polymorphisms in our sample.
Maximum-likelihood haplotype frequencies were estimated by applying the expectation maximisation (EM) and partition ligation (PL) algorithms, both implemented by Haploview 4.0 software . Linkage disequilibrium was calculated using the algorithm from Gabriel et al. implemented in Haploview 4.0 software .
The Mann–Whitney Test was performed by SPSS 13.0 software (SPSS, Chicago, IL, USA).
The Mantel–Haenszel method was performed by EpiDat 3.1 software (http://dxsp.sergas.es)