Minna Kiviniemi

Effect of HLA Class I and Class II Alleles on Progression from Autoantibody Positivity to Overt Type 1 Diabetes in Children with Risk-Associated Class II Genotypes

OBJECTIVE Class II alleles define the main HLA effect on type 1 diabetes, but there is an independent effect of certain class I alleles. Class II and class I molecules are differently involved in the initiation and effector phases of the immune response, suggesting that class I alleles would be important determinants in the rate of β-cell destruction. To test this hypothesis we analyzed the role of HLA class I and class II gene polymorphisms in the progression from diabetes-associated autoimmunity to clinical disease. RESEARCH DESIGN AND METHODS The effect of HLA-DR-DQ haplotypes and a panel of class I HLA-A and -B alleles on the progression from autoantibody seroconversion to clinical diabetes was studied in 249 children persistently positive for at least one biochemical diabetes-associated autoantibody in addition to islet cell autoantibody. RESULTS The progression to clinical disease was separately analyzed after the appearance of the first and the second persistent biochemical autoantibody using Cox regression. Multivariate analysis demonstrated a significant protective effect of the A*03 allele (odds ratio [OR] 0.61, P = 0.042 after the first and OR 0.55, P = 0.027 after the second autoantibody), whereas the B*39 allele had a promoting effect after seroconversion for the second autoantibody (OR 2.4, P = 0.014). When children with the DR3/DR4 genotype were separately analyzed, HLA-B*39 had a strong effect (OR 6.6, P = 0.004 and OR 7.5, P = 0.007, after the appearance of the first and the second autoantibody, respectively). The protective effect of A*03 was seen only among children without the DR3/DR4 combination. CONCLUSIONS These results confirm that class I alleles affect the progression of diabetes-associated autoimmunity and demonstrate interactions between class I and class II alleles.

Th1/Th17 Plasticity Is a Marker of Advanced β Cell Autoimmunity and Impaired Glucose Tolerance in Humans

Upregulation of IL-17 immunity and detrimental effects of IL-17 on human islets have been implicated in human type 1 diabetes. In animal models, the plasticity of Th1/Th17 cells contributes to the development of autoimmune diabetes. In this study, we demonstrate that the upregulation of the IL-17 pathway and Th1/Th17 plasticity in peripheral blood are markers of advanced β cell autoimmunity and impaired β cell function in human type 1 diabetes. Activated Th17 immunity was observed in the late stage of preclinical diabetes in children with β cell autoimmunity and impaired glucose tolerance, but not in children with early β cell autoimmunity. We found an increased ratio of IFN-γ/IL-17 expression in Th17 cells in children with advanced β cell autoimmunity, which correlated with HbA1c and plasma glucose concentrations in an oral glucose tolerance test, and thus impaired β cell function. Low expression of Helios was seen in Th17 cells, suggesting that Th1/Th17 cells are not converted thymus-derived regulatory T cells. Our results suggest that the development of Th1/Th17 plasticity may serve as a biomarker of disease progression from β cell autoantibody positivity to type 1 diabetes. These data in human type 1 diabetes emphasize the role of Th1/Th17 plasticity as a potential contributor to tissue destruction in autoimmune conditions.

A homogeneous high-throughput genotyping method based on competitive hybridization

OBJECTIVES A reliable high-throughput assay system is necessary for the analysis of the ever-increasing numbers of single-nucleotide polymorphisms (SNP) relevant to genetic screening studies. We describe an assay suitable also for large-scale screening programs. DESIGN AND METHODS The one-step assay is based on asymmetric PCR amplification of the target sequence and subsequent time-resolved fluorescence measurement. Asymmetric amplification results in a single-stranded PCR product that is detected in the amplification vessel with a highly sensitive, homogeneous hybridization method. RESULTS A dual label, homogeneous high-throughput platform for nucleic acid sequence analysis was developed and validated using a C/T single-nucleotide polymorphism in the insulin gene as a model analyte and applied also to two other SNP-assays (poliovirus receptor A/G-polymorphism and CD86-gene exon 2 A/G-polymorphism). CONCLUSIONS The described high-throughput genotyping technology is very competitive in price, simple in design and easily applied to any analyte sequence.

Genetic susceptibility to type 1 diabetes in childhood - estimation of HLA class II associated disease risk and class II effect in various phases of islet autoimmunity.

The HLA‐DR/DQ region remains the major determinant of susceptibility to type 1 diabetes (T1D) despite the more than 50 risk affecting loci outside human leukocyte antigen (HLA) region that have been identified. We aimed at developing a simple risk estimation based on HLA class II genotyping, which was also tested by analyzing HLA class II effect on the autoantibody seroconversion and further progression to diabetes.

The HLA-B∗39 allele increases type 1 diabetes risk conferred by HLA-DRB1∗04:04-DQB1∗03:02 and HLA-DRB1∗08-DQB1∗04 class II haplotypes

To further characterise the effect of the HLA-B*39 allele on type 1 diabetes risk we assessed its role in different HLA-DR/DQ haplotypes and genotypes using 1764 nuclear families with a diabetic child collected in the framework of the Finnish Paediatric Diabetes Register. HLA assays were based on sequence specific hybridization using lanthanide labelled oligonucleotide probes. Transmissions of major HLA-DR/DQ haplotypes with and without the HLA-B*39 allele to diabetic index cases were analysed by direct haplotype and allele counting. The HLA-B*39 allele significantly increased the disease risk conferred by DRB1*04:04-DQA1*03-DQB1*03:02 and (DR8)-DQB1*04 haplotypes. The same effect was observed on genotype level as disease association for the HLA-B*39 allele was observed in multiple genotypes containing DRB1*04:04-DQA1*03-DQB1*03:02 or (DR8)-DQB1*04 haplotypes. Finally we considered the two common subtypes of the HLA-B*39 allele, B*39:01 and B*39:06 and observed their unequal distribution when stratified for specific DR-DQ haplotypes. The risk for type 1 diabetes conferred by certain DR/DQ haplotypes is modified by the presence of the HLA-B*39 and this confirms the independent disease predisposing effect of the HLA-B*39 allele. The results can be applied in enhancing the sensitivity and specificity of DR/DQ based screening programs for subjects at disease risk.

Two insulin gene single nucleotide polymorphisms associated with type 1 diabetes risk in the Finnish and Swedish populations

We have developed high-throughput tests for the detection of the insulin gene region SNPs −23HphI and −2221MspI. The potential of these markers to enhance the efficiency of type 1 diabetes risk screening was then evaluated by analyzing them in Finnish and Swedish populations. Blood spots on filter paper were analyzed using PCR followed by sequence-specific hybridization and time-resolved fluorometry reading. Distribution of the genotypes at both positions differed significantly among the affected children compared to the controls. The risk genotypes (CC, AA) were significantly more common in Finland than in Sweden, both among patients and controls. The VNTR genotype homozygous for the protective class III alleles showed a significantly stronger protective effect than the heterozygote (p = 0.02). Analyzing both SNPs enabled the detection of VNTR class III subclasses IIIA and IIIB. The observed significance between effects of the protective genotypes was due to the strong protective effect of the IIIA/IIIA genotype. IIIA/IIIA was the only genotype with significant discrepancy between protective effects compared to the other class III genotypes. These observations suggest that heterogeneity between the protective IDDM2 lineages could exist, and analyzing both −23HphI and −2221MspI would thus potentially enhance the sensitivity and specificity of type 1 diabetes risk estimation.

Human leukocyte antigen (DR1)-DQB1*0501 and (DR15)-DQB1*0602 haplotypes are associated with humoral responses to early food allergens in children

Background: Infants’ immunological responses to cow’s milk (CM) proteins, which in 2–3% result in allergy, may partially depend on genetic factors. We evaluated whether genes with immunological functions, i.e. human leukocyte antigen (HLA) II, the protein tyrosine phosphatase, non-receptor type 22 (PTPN22) and filaggrin, modulate immune responses to dietary antigens. Methods: We analyzed 14 HLA class II haplotypes, the PTPN22 1858 SNP (R620W allele) and 5 known filaggrin null mutations from blood samples of 87 patients with CM allergy (CMA) and 76 control subjects (age 8.0–9.3 years). Serum levels of IgA, IgG, IgG1 and IgG4 antibodies to β-lactoglobulin, α-casein and ovalbumin were measured with enzyme-linked immunosorbent assay, levels of IgE antibodies to CM, ovalbumin and birch with UniCap (Phadia, Uppsala, Sweden). Results: In children with CMA, the HLA (DR15)-DQB1*0602 haplotype was associated with high levels of β-lactoglobulin-specific total IgG (p < 0.001) and IgG4 (p < 0.001) and α-casein-specific total IgG (p = 0.003) and IgG4 (p = 0.002), but not among control subjects. (DR1/10)-DQB1*0501 was associated with lower levels of β-lactoglobulin-specific total IgG (p < 0.001) and IgG4 (p < 0.001), ovalbumin-specific total IgG (p = 0.002) and IgG4 (p < 0.001), particularly in control subjects (p < 0.001). Six children with eczema (3 with CMA) had the filaggrin mutation del22824. PTPN22 was not associated with specific antibody responses or CMA. Conclusion: The HLA II, but not PTPN22 or filaggrin, genotype modulates humoral responses to early food allergens, whereas none of these genes was associated with CMA.

A homogeneous HLA-B*27 genotyping assay using dried reagent mixtures

The presence of HLA-B*27 allele with patients suspected with ankylosing spondylitis can be used in the diagnostic process. We have developed an assay for typing for the HLA-B*27 in whole blood dried on sample collection cards using pre-dried reagent wells and homogeneous time-resolved fluorescence based PCR approach. Essentially only the sample needs to be added to the dry ready-to-use reaction well in order to start the homogenous amplification assay. The method was validated with 229 samples also typed with an existing DELFIA-based method and results of both assays were 100% concordant. The dried reagents were shown to be stable at least up to eight weeks at room temperature without any decline in their performance.

Exploring the risk factors for differences in the cumulative incidence of coeliac disease in two neighboring countries: the prospective DIABIMMUNE study

BACKGROUND During the last several decades the prevalence of coeliac disease (CD) has increased worldwide. AIM To compare the cumulative incidence of CD between Estonian and Finnish children and to identify the risk factors. MATERIALS AND METHODS Children were recruited as part of the DIABIMMUNE Study. In the birth cohort (BC) 258 children from Estonia and 305 from Finland, and in the young childrens cohort (YCC) 1363 and 1384 children were followed up, respectively. The diagnosis of CD was made in accordance with the ESPGHAN guidelines-the presence of IgA-tTG antibodies and small bowel villous atrophy. RESULTS During the study period 29 children developed CD. The cumulative incidence of CD was significantly higher in Finland (0.77% vs 0.27%; P=0.01). No difference was seen between the children with CD and the controls in the duration of breastfeeding or the age at cereal introduction. The BC children with CD had had significantly more episodes of infections with fever by the age of 12 months compared to the controls (3.4 vs 1.4; P=0.04). CONCLUSION The 5-year cumulative incidence of childhood CD is significantly higher in Finland than in Estonia. Sequential infections early in life may increase the risk for developing CD.

Risk genes and autoantibodies in Egyptian children with type 1 diabetes – low frequency of autoantibodies in carriers of the HLA‐DRB1*04:05‐DQA1*03‐DQB1*02 risk haplotype

The study aimed to define the frequencies of type 1 diabetes‐associated gene polymorphisms and their associations with various diabetes‐associated autoantibodies in Egyptian children.