B. Van der Auwera

Seasonality in clinical onset of Type 1 diabetes in Belgian patients above the age of 10 is restricted to HLA-DQ2/DQ8-negative males, which explains the male to female excess in incidence

Aims/hypothesisType 1 diabetes arises from an interplay between environmental and genetic factors. The reported seasonality at diagnosis supports the hypothesis that currently unknown external triggers play a role in the onset of the disease. We investigated whether a seasonal pattern is observed at diagnosis in Belgian Type 1 diabetic patients, and if so whether seasonality varies according to age, sex and genetic risk, all known to affect the incidence of Type 1 diabetes.MethodsThe seasonal pattern at clinical diagnosis was assessed in 2176 islet antibody-positive diabetic patients aged 0 to 39 years diagnosed between 1989 and 2000. Additional stratification was performed for age, sex and HLA-DQ genotype.ResultsOverall, a significant seasonal pattern at clinical diagnosis of diabetes was observed (p<0.001). More subjects were diagnosed in the period of November to February (n=829) than during the period of June to September (n=619) characterised by higher averages of maximal daily temperature and daily hours of sunshine. However, the seasonal pattern was restricted to patients diagnosed above the age of 10 (0–9 years: p=0.398; 10–19 years: p<0.001; 20–29 years: p=0.003; 30–39 years: p=0.015). Since older age at diagnosis is associated with a male to female excess and a lower prevalence of the genetic accelerator HLA-DQ2/DQ8, we further stratified the patients aged 10 to 39 years (n=1675) according to HLA-DQ genotype and sex, and we found that the seasonal pattern was largely restricted to male subjects lacking DQ2/DQ8 (n=748; p<0.001 vs all others: n=927; p=0.031).Conclusions/interpretationIn a subgroup of male patients diagnosed over the age of 10, the later stages of the subclinical disease process may be more driven by sex- and season-dependent external factors than in younger, female and genetically more susceptible subjects. These factors may explain the male to female excess in diabetes diagnosed in early adulthood.

Combined positivity for HLA DQ2/DQ8 and IA-2 antibodies defines population at high risk of developing type 1 diabetes

Aims/hypothesisPrevention trials in first-degree relatives of type 1 diabetic patients are hampered by large interindividual differences in progression rate to diabetes. We investigated whether specific combinations of immune and genetic markers can identify subgroups with more homogeneous progression to clinical onset.MethodsAntibodies against islet cell cytoplasm (ICA), insulin (IAA), glutamate decarboxylase (GADA) and IA-2 protein (IA-2A) were measured in 790 non-diabetic control subjects and 4,589 first-degree relatives under age 40.ResultsOn first sampling, 11.1% of the siblings presented at least one antibody type (p<0.001 vs other relatives). During follow-up (median 52 months) 43 subjects developed type 1 diabetes (31 siblings, ten offspring of a diabetic father, two offspring of a diabetic mother). Using Kaplan–Meier survival analysis and Cox regression, IA-2A conferred the highest 5-year diabetes risk (>50%) irrespective of the number of antibodies present. In initially IA-2A-positive relatives (n=58) progression to hyperglycaemia depended more on HLA DQ status than on type of kinship (84% progression in the presence of DQ2/DQ8 vs 32% in its absence; p<0.003). In IA-2A-negative relatives (n=4,531) 5-year progression to diabetes increased with the number of other antibodies (ICA, GADA and/or IAA) (p<0.001) but overall did not exceed 10% even for two or more antibodies. Among relatives initially positive for one or more antibody type other than IA-2A (n=315), there was significantly more progression to diabetes (overall still <10%) in carriers of DQ2 (p<0.001 vs no DQ2), regardless of DQ8 status.Conclusions/interpretationThese observations suggest that the HLA-DQ-inferred risk of diabetes can proceed through two distinct pathways distinguished by IA-2A status. Combined positivity for DQ2/DQ8 and IA-2A defines a more homogeneous high-risk population for prevention trials than those used so far.

A retroviral long terminal repeat adjacent to the HLA DQB1 gene (DQ-LTR13) modifies Type I diabetes susceptibility on high risk DQ haplotypes

Abstract.Aims/hypothesis:HLA-DQ genes, located in the human leukocyte antigen region on chromosome 6 p, are the main inherited factors predisposing to Type I (insulin-dependent) diabetes mellitus. Endogenous retroviral long-terminal repeats are integrated at several sites within this region, one of which is known to enhance susceptibility for Type I diabetes. We examined another LTR within the HLA-region as an additional genetic risk marker. Methods: We investigated the segregation of one long-terminal repeat (DQ-LTR13), located 1.3 kb upstream of HLA DQB1 with different HLA-DQ haplotypes, and its transmission to patients. A total of 284 Caucasian families (203 German and 81 Belgian) with at least one diabetic offspring were genotyped for DQA1, DQB1 and DQ-LTR13. Results:DQ8/LTR13+ was preferentially transmitted (139 transmitted vs 28 not transmitted; PTDT = 1.67 × 10–14) whereas no deviation from expected transmission frequencies was observed for DQ8/LTR13– (20 transmitted vs 17 not transmitted; PTDT = 1.00). DQ8/LTR13+ alleles conferred a significantly higher risk for Type I diabetes than DQ8/LTR13– alleles (pχ2 = 2.58 × 10–14). This difference remained significant even after DRB1 subtyping (pχ2 = 0.02). Also, there was a significant difference when comparing the transmission of DQ2/LTR13+ and DQ2/LTR13– alleles (pχ2 = 0.01), the latter conferring an increased risk. The transmission of DQ-LTR13+ haplotypes did not show any differences regarding paternal, maternal or gender-related stratification. However, DQ8/LTR13– was significantly more often transmitted from mothers (pχ2 = 0.01) and to female patients (pχ2 = 0.04). Conclusion/interpretation: We conclude that DQ-LTR13 marks additional genetic risk for Type I diabetes on predisposing DRB1*0401-DQ8 and DQ2 haplotypes and will help to further define susceptibility in this gene region. [Diabetologia (2002) 45: 443–447]

Validation of an enzyme-linked immunosorbent assay for C-peptide analysis in Cameroon

AIMS To validate an ELISA method for C-peptide analysis in Cameroon. METHODS We evaluated the linearity, detection limit, functional sensitivity, precision and accuracy, and further investigated for cross-reactivity by proinsulin, and interferences by lipids, bilirubin and hemoglobin. This method was compared with the Roche electrochemiluminescence immunoassay. C-peptide stability was assessed following a series of freeze-thaw cycles, and after storage at room temperature. The C-peptide reference range was determined by analyzing fifty plasma samples of Cameroonians without diabetes. RESULTS The ELISA was linear at least up to 7.09 μg/L, and had a detection limit of 0.09 μg/L, and a functional sensitivity of 0.32 μg/L. The inter- and intraassay %CV were 2.9-9.9%, and 5.2-9.4%, respectively. Recoveries were 81-94% in serum, and 93-98% in buffer. Comparison with the ECLIA yielded a good correlation coefficient (R(2)=0.98). There was no cross-reactivity with proinsulin, and no interference with lipids, bilirubin and hemoglobin. C-peptide was stable at room temperature for 24 h and up to 7 freeze-thaw cycles for medium (1-6 μg/L) and high (>6 μg/L) levels (<-15°C and <-70°C). The reference range for C-peptide was 0.38-3.63 μg/L. CONCLUSIONS This method is suitable for C-peptide analysis in low-income countries like Cameroon.

Proinsulin and Its Conversion Intermediates in Human Pancreas and Isolated Islet Tissue: Kinetics and Steady-State Analysis

In non-insulin-dependent diabetes, circulating insulin-related immunoreactivity (IRI) is often composed of a higher fraction of the incompletely converted forms proinsulin and des-31, 32 proinsulin. The present study describes an immunoadsorption method for measuring the proportions of proinsulin, its two split products, and insulin in human pancreatic tissue and for determining their rates of formation in human isolated islets. The method uses two junction-specific monoclonal proinsulin antibodies in a protein G fractionation; it is validated by ≥90% specificity and recovery. The peptide contents measured in tissue extracts were comparable to those determined in a previously developed immunoradiometric assay. In the nine tissue extracts from nondiabetic donor organs, 97% of IRI corresponded to insulin, 1% to proinsulin, 2% to the des-31, 32 proinsulin conversion product, and 0.1% to des-64, 65 proinsulin. Two samples from non-insulin-dependent diabetics under sulfonylurea treatment contained a fourfold lower content of IRI but the peptide distribution was comparable except for a low percentage (0.3) of proinsulin in one case. In pulse-chase experiments on three preparations of human islets isolated from nondiabetic donors, proinsulin represented the major (>90%) IRI that was synthesized at the end of the 30-min pulse; a subsequent 90-min chase at either 2.5 or 10 mM glucose resulted in conversion of 75% of proinsulin to des-31, 32 (20%) and des-64, 65 (2%) intermediates and to insulin (50%); after a 180-min chase, 88% of proinsulin was converted to insulin, but 10% remained present as proinsulin. In a pulse-chase experiment on islets isolated from tissue with a high proportion of des-31, 32 intermediate (5% instead of 2%), the conversion process was slower (45% after 90 min and 70% after 180 min) and resulted in a higher fraction of des-31, 32 intermediate, suggesting that the elevated tissue content in this intermediate is caused by a reduced PC2 converting activity. These data confirm that des-31, 32 proinsulin represents the major conversion intermediate in normal human islets and indicate the existence of slow converters, possibly as a result of decreased enzymatic processing of the prohormones AC junction.