Coxsackievirus B1 infections are associated with the initiation of insulin-driven autoimmunity that progresses to type 1 diabetes

Aims/hypothesis Islet autoimmunity usually starts with the appearance of autoantibodies against either insulin (IAA) or GAD65 (GADA). This categorises children with preclinical type 1 diabetes into two immune phenotypes, which differ in their genetic background and may have different aetiology. The aim was to study whether Coxsackievirus group B (CVB) infections, which have been linked to the initiation of islet autoimmunity, are associated with either of these two phenotypes in children with HLA-conferred susceptibility to type 1 diabetes. Methods All samples were from children in the Finnish Type 1 Diabetes Prediction and Prevention (DIPP) study. Individuals are recruited to the DIPP study from the general population of new-born infants who carry defined HLA genotypes associated with susceptibility to type 1 diabetes. Our study cohort included 91 children who developed IAA and 78 children who developed GADA as their first appearing single autoantibody and remained persistently seropositive for islet autoantibodies, along with 181 and 151 individually matched autoantibody negative control children, respectively. Seroconversion to positivity for neutralising antibodies was detected as the surrogate marker of CVB infections in serial follow-up serum samples collected before and at the appearance of islet autoantibodies in each individual. Results CVB1 infections were associated with the appearance of IAA as the first autoantibody (OR 2.4 [95% CI 1.4, 4.2], corrected p = 0.018). CVB5 infection also tended to be associated with the appearance of IAA, however, this did not reach statistical significance (OR 2.3, [0.7, 7.5], p = 0.163); no other CVB types were associated with increased risk of IAA. Children who had signs of a CVB1 infection either alone or prior to infections by other CVBs were at the highest risk for developing IAA (OR 5.3 [95% CI 2.4, 11.7], p <0.001). None of the CVBs were associated with the appearance of GADA. Conclusions/interpretation CVB1 infections may contribute to the initiation of islet autoimmunity being particularly important in the insulin-driven autoimmune process.Peer reviewe

Permutation-based significance analysis reduces the type 1 error rate in bisulfite sequencing data analysis of human umbilical cord blood samples

DNA methylation patterns are largely established in-utero and might mediate the impacts of in-utero conditions on later health outcomes. Associations between perinatal DNA methylation marks and pregnancy-related variables, such as maternal age and gestational weight gain, have been earlier studied with methylation microarrays, which typically cover less than 2% of human CpG sites. To detect such associations outside these regions, we chose the bisulphite sequencing approach. We collected and curated clinical data on 200 newborn infants; whose umbilical cord blood samples were analysed with the reduced representation bisulphite sequencing (RRBS) method. A generalized linear mixed-effects model was fit for each high coverage CpG site, followed by spatial and multiple testing adjustment of P values to identify differentially methylated cytosines (DMCs) and regions (DMRs) associated with clinical variables, such as maternal age, mode of delivery, and birth weight. Type 1 error rate was then evaluated with a permutation analysis. We discovered a strong inflation of spatially adjusted P values through the permutation analysis, which we then applied for empirical type 1 error control. The inflation of P values was caused by a common method for spatial adjustment and DMR detection, implemented in tools comb-p and RADMeth. Based on empirically estimated significance thresholds, very little differential methylation was associated with any of the studied clinical variables, other than sex. With this analysis workflow, the sex-associated differentially methylated regions were highly reproducible across studies, technologies, and statistical models.Peer reviewe

Serum 25-Hydroxyvitamin D Concentrations at Birth in Children Screened for HLA-DQB1 Conferred Risk for Type 1 Diabetes

Context: Vitamin D has several effects on the immune system that might be of relevance for the pathogenesis of type 1 diabetes (T1D).Objective: To evaluate whether umbilical cord serum concentrations of 25-hydroxy-vitamin D (25[OH]D) differ in children developing either islet autoimmunity (IA) or overt T1D during childhood and adolescence.Design: Umbilical cord serum samples from 764 children born from 1994 to 2004 with HLA-DQB1 conferred risk for T1 D participating in the Type 1 Diabetes Prediction and Prevention Study were analyzed for 25(OH)D using an enzyme immunoassay.Setting: DIPP clinics in Turku, Oulu, and Tampere University Hospitals, Finland.Participants: Two hundred fifty children who developed T1D diabetes at a median age of 6.7 years (interquartile range [IQR] 4.0 to 10.1 years) and 132 additional case children who developed IA, i.e., positivity for multiple islet autoantibodies. Cases were matched for date of birth, gender, and area of birth with 382 control children who remained autoantibody negative. The median duration of follow up was 9.8 years (IQR 5.7 to 13.1 years).Main Outcome Measure: The median 25(OH)D concentrations.Results: The median 25(OH)D concentration in cord serum was low [31.1 nmol/L (IQR 24.0 to 41.8); 88% Conclusions: The 25(OH)D concentrations at birth are not associated with the development of T1D during childhood.</div

Serum 25-Hydroxyvitamin D Concentrations at Birth in Children Screened for HLA-DQB1 Conferred Risk for Type 1 Diabetes

Vitamin D has several effects on the immune system that might be of relevance for the pathogenesis of type 1 diabetes (T1D).To evaluate whether umbilical cord serum concentrations of 25-hydroxy-vitamin D (25[OH]D) differ in children developing either islet autoimmunity (IA) or overt T1D during childhood and adolescence.Umbilical cord serum samples from 764 children born from 1994 to 2004 with HLA-DQB1 conferred risk for T1D participating in the Type 1 Diabetes Prediction and Prevention Study were analyzed for 25(OH)D using an enzyme immunoassay.DIPP clinics in Turku, Oulu, and Tampere University Hospitals, Finland.Two hundred fifty children who developed T1D diabetes at a median age of 6.7 years (interquartile range [IQR] 4.0 to 10.1 years) and 132 additional case children who developed IA, i.e., positivity for multiple islet autoantibodies. Cases were matched for date of birth, gender, and area of birth with 382 control children who remained autoantibody negative. The median duration of follow up was 9.8 years (IQR 5.7 to 13.1 years).The median 25(OH)D concentrations.The median 25(OH)D concentration in cord serum was low [31.1 nmol/L (IQR 24.0 to 41.8); 88% <50 nmol/L], but not statistically different between children who developed T1D or IA and their control groups (P = 0.70). The levels were associated mainly with geographical location, year and month of birth, age of the mother, and maternal intake of vitamin D during pregnancy.The 25(OH)D concentrations at birth are not associated with the development of T1D during childhood.Peer reviewe

Permutation-based significance analysis reduces the type 1 error rate in bisulfite sequencing data analysis of human umbilical cord blood samples

DNA methylation patterns are largely established in-utero and might mediate the impacts of in-utero conditions on later health outcomes. Associations between perinatal DNA methylation marks and pregnancy-related variables, such as maternal age and gestational weight gain, have been earlier studied with methylation microarrays, which typically cover less than 2% of human CpG sites. To detect such associations outside these regions, we chose the bisulphite sequencing approach. We collected and curated clinical data on 200 newborn infants; whose umbilical cord blood samples were analysed with the reduced representation bisulphite sequencing (RRBS) method. A generalized linear mixed-effects model was fit for each high coverage CpG site, followed by spatial and multiple testing adjustment of P values to identify differentially methylated cytosines (DMCs) and regions (DMRs) associated with clinical variables, such as maternal age, mode of delivery, and birth weight. Type 1 error rate was then evaluated with a permutation analysis. We discovered a strong inflation of spatially adjusted P values through the permutation analysis, which we then applied for empirical type 1 error control. The inflation of P values was caused by a common method for spatial adjustment and DMR detection, implemented in tools comb-p and RADMeth. Based on empirically estimated significance thresholds, very little differential methylation was associated with any of the studied clinical variables, other than sex. With this analysis workflow, the sex-associated differentially methylated regions were highly reproducible across studies, technologies, and statistical models.</p

Umbilical cord blood DNA methylation in children who later develop type 1 diabetes

Aims/hypothesis: Distinct DNA methylation patterns have recently been observed to precede type 1 diabetes in whole blood collected from young children. Our aim was to determine whether perinatal DNA methylation is associated with later progression to type 1 diabetes.Methods: Reduced representation bisulphite sequencing (RRBS) analysis was performed on umbilical cord blood samples collected within the Finnish Type 1 Diabetes Prediction and Prevention (DIPP) Study. Children later diagnosed with type 1 diabetes and/or who tested positive for multiple islet autoantibodies (n = 43) were compared with control individuals (n = 79) who remained autoantibody-negative throughout the DIPP follow-up until 15 years of age. Potential confounding factors related to the pregnancy and the mother were included in the analysis.Results: No differences in the umbilical cord blood methylation patterns were observed between the cases and controls at a false discovery rate Conclusions/interpretation: Based on our results, differences between children who progress to type 1 diabetes and those who remain healthy throughout childhood are not yet present in the perinatal DNA methylome. However, we cannot exclude the possibility that such differences would be found in a larger dataset.</p

Serum APOC1 levels are decreased in young autoantibody positive children who rapidly progress to type 1 diabetes

Abstract Better understanding of the early events in the development of type 1 diabetes is needed to improve prediction and monitoring of the disease progression during the substantially heterogeneous presymptomatic period of the beta cell damaging process. To address this concern, we used mass spectrometry-based proteomics to analyse longitudinal pre-onset plasma sample series from children positive for multiple islet autoantibodies who had rapidly progressed to type 1 diabetes before 4 years of age (n = 10) and compared these with similar measurements from matched children who were either positive for a single autoantibody (n = 10) or autoantibody negative (n = 10). Following statistical analysis of the longitudinal data, targeted serum proteomics was used to verify 11 proteins putatively associated with the disease development in a similar yet independent and larger cohort of children who progressed to the disease within 5 years of age (n = 31) and matched autoantibody negative children (n = 31). These data reiterated extensive age-related trends for protein levels in young children. Further, these analyses demonstrated that the serum levels of two peptides unique for apolipoprotein C1 (APOC1) were decreased after the appearance of the first islet autoantibody and remained relatively less abundant in children who progressed to type 1 diabetes, in comparison to autoantibody negative children