Linkage disequilibrium between TAP2 variants and HLA class II alleles; no primary association between TAP2 variants and insulin-dependent diabetes mellitus

The TAP1 and TAP2 genes, located in the HLA class II region, encode subunits of a peptide transporter. Both genes display limited genetic variability; four different nucleotide substitutions have been found in theTAP2 gene. Here studies on linkage disequilibrium between TAP2 variants and HLA class II alleles are reported, in an attempt to evaluate whether TAP2 variants are associated with insulin-dependent diabetes mellitus (IDDM). As reported previously, a significant decrease of homozygosity for TAP2 alleles encoding alanine at residue 665 (665 Ala) and glut amine at 687 (687 Gin) paralleled by an increase in homozygosity for TAP2 alleles encoding threonine at residue 665 (665 Thr) and a stop codon at 687 (687 Stop), was found in both Finnish and Norwegian IDDM patients compared to random controls. However, a strong linkage disequilibrium between these TAP2 polymorphisms and given HLA-DR and -DQ genes was observed among healthy controls. The frequent 665 Thr and 687 Stop variants were in linkage disequilibrium both with the DR4-DQ8 and the DR3-DQ2 haplotypes, haplotypes which are strongly associated with IDDM. In contrast, the DR1-DQ5 and DR13-DQ6 (e.g. DQB1*0603) haplotypes, which are decreased among IDDM patients, were associated with the 665 Ala and 687 Gin variants. Thus, when DR- and DQ-matched patients and controls were compared, associations of the investigated TAP2 variants and IDDM were no longer detectable. These data, therefore, indicate that the associations previously found between certain TAP2 variants and IDDM are secondary to a primary association between this disease and particular DQαβ heterodimers.     

No independent association between a tumor necrosis factor-α promotor region polymorphism and insulin-dependent diabetes mellitus

Several studies have implicated tumor necrosis factor (TNF)-α in the pathogenesis of insulin-dependent diabetes mellitus (IDDM). In the present study we analyzed the first reported TNF-α gene polymorphism in relation to IDDM. We have made frequence analysis and tested in vitro lipopolysaccharide (LPS)-induced TNF-α secretion. A significant difference in allele frequency was observed between patients and controls (p = 0.03). However, a very strong association of the uncommonTNF2 allele was observed with the HLA-B8, –DR3 alleles. The relative risk (RR) of TNF2 was 2.2 compared to a RRof 3.1 for DR3. One reason for this difference was the identification of the TNF1 allele on the otherwise strongly IDDM-associated HLA-DR3 haplotype: DQB1*0201, DQA1*0501, DRB1*0301, TNFc2, TNFB*2, TNFal, TNFb5, B18. Thus, the IDDM-associated TNF2 allele had no DR3-independent value as a disease marker. The LPS-induced TNF-α production by human monocytes in relation to genotypes demonstrated that TNF1/2 heterozygous individuals had higher, though not statistically significantly (p = 0.08) levels than TNF1-homozygous subjects. However, this difference was rather small, unlikely to be of biological significance and based on the present material we cannot establish the functional importance of this polymorphism.     

Association of tumor necrosis factor (TNF) and class II major histocompatibility complex alleles with the secretion of TNF-a and TNF-0 by human mononuclear cells: a possible link to insulin-dependent diabetes mellitus

We have investigated the correlation between different tumor necrosis factor (TNF) and class II major histocompatibility complex alleles in the lipopolysaccharide- or phytohemagglutinin-induced secretion of TNF-α and TNF-β by human monocytes and peripheral blood mononuclear cells in 87 unrelated Danish male individuals. Significant differences in TNF-α secretory capacity between TNF Ncol restriction fragment length polymorphisms, TNFa and TNFc micro-satellite alleles and DR alleles were identified. No correlation with TNF-β secretory capacity was found for any of the markers studied. TNF genotyping allowed us to define four extended HLA haplotypes which correlate with TNF-α secretory capacity. Two of these are DR4 positive: DQw8, DR4, TNFB*1, TNFa6, B44, A2 and DQw8, DR4, TNFB*2, TNFa2, B15, A2. Individuals carrying the TNFB*2, TNFa2 haplotype had a higher TNF-α secretory capacity than those carrying the TNFB*1, TNFa6 haplotype. In a group of DR3/DR4 heterozygous patients with insulin-dependent diabetes mellitus (IDDM), the frequency of the TNFa2 allele was higher than in HLA-DR matched controls, whereas theTNFa6 allele was more frequent in control individuals. In the DR3/DR4 heterozygous diabetic group 12/26 had the alleles combination DQw8, DR4 (Dw4), C4A3, TNFB*2, TNFa2, B15, whereas only 1/18 controls had this haplotype. This diabetogenic haplotype is identical to the DR4 haplotype which correlates with a higher TNF-α response. These observations suggest a direct role for the TNF locus in the pathogenesis of IDDM.     

Autoimmunity in type I (insulin-dependent) diabetes mellitus in North India

Pancreatic islet cell, thyroid, gastric parietal cell, and adrenal autoantibodies were studied in 110 young insulin-dependent diabetics (Type I; IDDM), 12 non-insulin-dependent diabetics (NIDDM), 26 patients with pancreatic diabetes, and 123 age- and sex-matched healthy controls. All the patients were aged 30 years or under at the onset of diabetes. Islet cell antibody was found in 31% of the patients with IDDM, but in only one patient with NIDDM, one patient with pancreatic diabetes, and one healthy control subject. Thyroid, parietal cell, and/or adrenal antibodies were present in 26% of the IDDM patients, 17% of the NIDDM patients, 12% of the patients with pancreatic diabetes, and 19% of the control subjects. There was no association between the presence of islet cell antibody and other organ-specific autoantibodies and any particular HLA phenotype. Data from the North Indian study have been compared with those from other populations of the world, similarities and differences have been brought out, and their significance has been discussed. The relative contribution of the autoimmune component in the etiopathogenesis of different forms of diabetes mellitus varies among the different populations of the world, accounting to some extent for the observed differences in incidence and clinical profiles.     

HLA-associated inverse correlation between T cell and antibody responsiveness to islet autoantigen in recent-onset insulin-dependent diabetes mellitus

Insulin-dependent diabetes mellitus (IDDM) is a T cell-dependent immune-mediated disease. Recently, a novel islet cell antigen (ICA69) recognized by autoantibodies was described. We tested T cell responsiveness to ICA69 in peripheral blood of patients with recent onset IDDM (n = 46), patients with long-standing IDDM (n = 44), non-diabetic age-matched, islet cell autoantibody- and glutamic acid decarboxylase (GAD)65 antibody-negative first-degree relatives of IDDM patients (n = 15) and rheumatoid arthritis patients (n = 22). T cell responsiveness was significantly higher in recent onset IDDM patients, compared to IDDM patients post-disease onset, non-diabetic first degree relatives and rheumatoid arthritis patients (p < 0.001). In responding IDDM patients a significant inverse correlation between T cell and autoantibody responsiveness to ICA69 was observed (p < 0.0005). Immunogenetic evaluation revealed an association of HLA-DR3 with T cell responsiveness to ICA69 (p < 0.02) and absence of ICA69-reactive autoantibodies (p < 0.04). The increased T cell reactivity to ICA69 in the absence of antibody reactivity at onset of IDMM is associated with an HLA class II immune response gene, and therefore suggestive of a genetically controlled selective activation of T helper subsets to a specific autoantigen in humans.     

HLA class II associations with Type 1 diabetes mellitus: a multivariate approach

The association of HLA class II phenotype with the development of insulin-dependent (Type 1) diabetes mellitus (IDDM) is well established but the contribution of the various HLA-DR and -DQ alleles and haplotypes to disease predisposition is not fully understood. We have determined haplotype and genotype odds ratios, and further employed multivariate tree analysis to explore the contribution of individual HLA-DRDQ haplotypes to the genetic risk for developing IDDM in the Dutch population. Next to haplotype and genotype odds ratios, multivariate tree analysis techniques provide overall risk calculations for each modeled parameter, and offer insight in the interaction of the model parameters via tree-shaped reports, in which subsequent stratifications on the data can easily be followed. We compared 206 Dutch IDDM patients with 840 serologically typed random healthy unrelated Dutch Caucasoid controls. The multivariate tree analysis showed that the HLA-DR7DQ9 and DR15DQ6 haplotype were strongly associated with disease protection (OR = 0.04, P = 0.0003, and OR = 0.07, P < or = 0.0001, respectively). The highest ORs were found for the DR4DQ8/DR8DQ4 genotype (OR = 21.04, P = 0.001), followed by DR4DQ8/DR17DQ2 (OR = 12.45, P < 0.0001) and DR9DQ9/DR17DQ2 (OR = 10.87, P = 0.02). DR4DQ8 homozygous and DR17DQ2 homozygous individuals have a disease OR of 9.0 and 3.0 (P = 0.01 and 0.03), respectively. In conclusion, the results from haplotype, genotype, and tree analyses provide insight into the disease associations for combinations of HLA-DRDQ haplotypes. We confirm that the DR9DQ9/DR17DQ2 genotype is associated with susceptibility in the Dutch population, which has previously been noticed as a HLA risk genotypes in Asian populations only.     

Genetic heterogeneity between type 1 a and type lb insulin-dependent diabetes mellitus: HLA Class II and TAP gene analysis

The aim of this study was to compare the genetic susceptibility linked to the HLA Class II region genes of the Major Histocompatibility Complex in isolated insulin-dependent diabetes mellitus (la-IDDM) and insulin-dependent diabetes mellitus associated with another autoimmune endocrinopathy (lb-IDDM). HLA genes DRB1, DQA1 and DQB1 were studied at the genomic level, as well as genes TAP1 and TAP2. One hundred and seventy-nine la-IDDM diabetic patients were compared with 83 lb-IDDM patients. While it appeared that common genetic traits characterize diabetes regardless of the subtype (la or lb), certain features differentiate the two forms of IDDM. Extending the analysis of risk haplotypes DRB1*03 and DRB1*04 to TAP genes elicited a difference between la-IDDM and lb-IDDM patients. Haplo-type DRB1*03 was thus characterized in la-IDDM patients by a lower frequency of alleles TAP1-B (13.5%) and TAP2-B (16.2%), not found in lb-IDDM patients (33.3% for each allele). Likewise, haplotype DRB1*04 is characterized in lb-IDDM patients by a lower frequency of alleles TAP1-C (4.0%) and TAP2-B (8.0%) than in la-IDDM patients (22.2% and 25.9%, respectively). In total, this study showed that extending the characterization of HLA Class II haplotypes to TAP genes discriminates between the forms of diabetes restricted to a specific pancreatic affection and those reflecting a wider autoimmune disorder affecting several organs.     

Distribution of HLA-DRB1, -DQA1 and -DQB1 alleles and DQA1-DQB1 genotypes among Norwegian patients with insulin-dependent diabetes mellitus

We have studied 87 unrelated Caucasian insulin-dependent diabetes mellitus (IDDM) patients and 181 healthy controls by oligotyping for 20 DRB1, eight DQA1 and 13 DQB1 alleles, and established their DR-DQ haplotypes and DQ genotypes. An increase of DRB1 alleles encoding DR4 was found among IDDM patients, but the distribution of DR4 subtypes did not differ among DR4-positive IDDM patients and controls. The frequency of certain DRB1-DQA1-DQB1 haplotypes and DQA1-DQB1 genotypes was significantly increased among IDDM patients. Taken together, the data suggest that IDDM is primarily associated with several (at least five) different DQ alpha beta heterodimers.     

Restriction of self-antigen presentation to cytolytic T lymphocytes by mouse peptide pumps

Transport of an immunogenic self-peptide from the second domain of the mouse major histocompatibility complex (MHC) H-2K^d class I molecule is blocked at the TAP1-TAP2 peptide pump level due to its amino acid sequence and is not presented to cytolytic T lymphocytes (CTL). We demonstrate that first, TAP1-TAP2 pumps can restrict antigen presentation by selecting against internal peptide motifs which are not involved in peptide binding to MHC class I molecules. Second, some molecules targeted to the endoplasmic reticulum are processed for MHC class I presentation in the cytosol. Third, some abundantly expressed immunogenic self-peptides are cytosolically sequestered. The advantage for the host, in terms of the peripheral T cell repertoire is that the spared CTL can be used to recognize foreign antigens. It is, however, anticipated that this advantage will be exploited by pathogens to evade immune surveillance by similar strategies.     

Jejuna of patients with insulin-dependent diabetes mellitus (IDDM) show signs of immune activation

The roles of enteric viruses and food antigens as possible triggers in human insulin-dependent diabetes mellitus and the evidence that mucosal-associated homing receptors are important in both human and experimental diabetes prompted us to undertake an immunohistochemical study of intestinal specimens from patients with IDDM. We studied jejunal morphology and immunohistochemistry in 26 patients with IDDM, 13 of whom had the HLA-DQB1*0201 gene and therefore a higher risk of coeliac disease. The findings were compared with those in specimens from age-matched controls. Villous structure and the density of the intraepithelial lymphocytes were normal in every biopsy specimen. The extent of positivity with anti-DR and -DP antibodies in the villous epithelium was significantly greater in the specimens from patients than in those from controls (P = 0.0002 in both comparisons). The crypts were also more positive: for DR P = 0.0001, and for DP P = 0.002. The densities of T cells, CD4+, CD8+, and T cell receptor alpha/beta+ and gamma/delta+ cells in the epithelium and lamina propria were similar in patients and controls, but the patients had significantly more alpha 4/beta 7 integrin+ cells in the lamina propria (P = 0.006). No difference was seen between HLA-DQB1*0201-positive and -negative patients. These findings reflect a stage of inflammation in the structurally normal intestines of patients with IDDM and suggest secretion of inflammatory Th1-type cytokines in the intestine.     

DNA methylation and mRNA expression of HLA‐DQA1 alleles in type 1 diabetes mellitus

Type 1 diabetes (T1D) belongs among polygenic multifactorial autoimmune diseases. The highest risk is associated with human leucocyte antigen (HLA) class II genes, including HLA‐DQA1 gene. Our aim was to investigate DNA methylation of HLA‐DQA1 promoter alleles (QAP) and correlate methylation status with individual HLA‐DQA1 allele expression of patients with T1D and healthy controls. DNA methylation is one of the epigenetic modifications that regulate gene expression and is known to be shaped by the environment.Sixty one patients with T1D and 39 healthy controls were involved in this study. Isolated DNA was treated with sodium bisulphite and HLA‐DQA1 promoter sequence was amplified using nested PCR. After sequencing, DNA methylation of HLA‐DQA1 promoter alleles was analysed. Individual mRNA HLA‐DQA1 relative allele expression was assessed using two different endogenous controls (PPIA, DRA). We have found statistically significant differences in HLA‐DQA1 allele 02:01 expression (PPIA normalization, P_corr = 0·041; DRA normalization, P_corr = 0·052) between healthy controls and patients with T1D. The complete methylation profile of the HLA‐DQA1 promoter was gained with the most methylated allele DQA1*02:01 and the least methylated DQA1*05:01 in both studied groups. Methylation profile observed in patients with T1D and healthy controls was similar, and no correlation between HLA‐DQA1 allele expression and DNA methylation was found. Although we have not proved significant methylation differences between the two groups, detailed DNA methylation status and its correlation with expression of each HLA‐DQA1 allele in patients with T1D have been described for the first time.     

CTLA-4 gene polymorphism confers susceptibility to insulin-dependent diabetes mellitus (IDDM) independently from age and from other genetic or immune disease markers

Apart from genes in the HLA complex (IDDM1) and the variable number of tandem repeats in the 5′ region of the insulin gene (INS VNTR, IDDM2), several other loci have been proposed to contribute to IDDM susceptibility. Recently, linkage and association have been shown between the cytotoxic T lymphocyte-associated protein 4 (CTLA-4) gene on chromosome 2q and IDDM. In a registry-based group of 525 recent-onset IDDM patients < 40 years old we investigated the possible interactions of a CTLA-4 gene A-to-G transition polymorphism with age at clinical disease onset and with the presence or absence of established genetic (HLA-DQ, INS VNTR) and immune disease markers (autoantibodies against islet cell cytoplasm (ICA); insulin (IAA); glutamate decarboxylase (GAD65-Ab); IA-2 protein tyrosine phosphatase (IA-2-Ab)) determined within the first week of insulin treatment. In new-onset IDDM patients, G-allele-containing CTLA-4 genotypes (relative risk (RR) = 1.5; 95% confidence interval (CI) = 1.2–2.0; P< 0.005) were not preferentially associated with age at clinical presentation or with the presence of other genetic (HLA-DR3 or DR4 alleles; HLA-DQA1*0301-DQB1*0302 and/or DQA1*0501-DQB1*0201 risk haplotypes; INS VNTR I/I risk genotype) or immune (ICA, IAA, IA-2-Ab, GAD65-Ab) markers of diabetes. For 151 patients, thyrogastric autoantibodies (anti-thyroid peroxidase, anti-thyroid-stimulating hormone (TSH) receptor, anti-parietal cell, anti-intrinsic factor) were determined, but association between CTLA-4 risk genotypes and markers of polyendocrine autoimmunity could not be demonstrated before or after stratification for HLA- or INS-linked risk. In conclusion, the presence of a G-containing CTLA-4 genotype confers a moderate but significant RR for IDDM that is independent of age and genetic or immune disease markers.     

HLA-DP and susceptibility to insulin-dependent diabetes mellitus in Japanese

ABSTRACT: Human leukocyte antigen (HLA) genes are candidates for susceptibility genes in insulin-dependent diabetes mellitus (IDDM). Recently, the association of DR and DQ with IDDM has been reported, but the role of HLA-DP genes remains uncertain. To address the question, we analyzed the DPB1 gene of 20 Japanese IDDM patients and 30 control subjects using a combination of polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis (PCR-RFLP method). DPB1*0501 was the most frequent allele both in Japanese patients and control subjects. There was no appreciable association between IDDM and the DPB1 allele in Japanese. The absence of association between IDDM and DP, in spite of the known association between this disease and both DR and DQ, suggests that the HLA locus (loci) telomeric to DP encodes susceptibility to IDDM.     

Genetic predisposition and environmental factors leading to the development of insulin-dependent diabetes mellitus in Chilean children

This study was designed to examine the hypothesis that some environmental factors increase the risk for insulin-dependent diabetes mellitus. Data on dietary history was collected from 80 diabetic children from the Santiago de Chile Registry and from 85 nondiabetic control subjects who were comparable in terms of age, sex, and ethnic characteristics. Early exposure was defined as the ingestion of food sources other than maternal milk before 3 months of age. To define genetic susceptibility to insulin-dependent diabetes mellitus each subject was typed in terms of HLA DQA1 and DQB1, and the possible conformation of susceptible heterodimers was considered as a risk marker. Fewer children were exclusively breast fed in the diabetic group than in the control group (21.55±15.05 vs. 33.95±20.40 weeks, P<0.01). In addition, exposure to cow's milk and solid foods occurred earlier in the diabetic group than in the control group (15.90±10.95 vs. 21.15 13.65 and 16.85±10.25 vs. 21.20±12.35 weeks, P<0.05). Our data show that a short duration of breast-feeding and early exposure to cow's milk and solid foods may be important factors in the development of insulin-dependent diabetes mellitus. The high relative risk observed in individuals genetically predisposed indicates an interaction effect between genetic and environmental components.Abbreviations IDDM Insulin-dependent diabetes mellitus - RR Relative risk     

T cell receptor haplotypes in families of patients with insulin-dependent diabetes mellitus.

The frequencies of Bgl 11 and BamH1 restriction fragment length polymorphisms (RFLP) of C beta, V beta 8, V beta 11 and V beta 7.2 have been defined in a healthy Australian population. Linkage disequilibrium between alleles of the T cell receptor (TCR) V beta 8 and V beta 11 gene segments has been confirmed. We have also confirmed the lack of linkage disequilibrium between either of these loci and alleles at C beta or V beta 7.2. Using RFLPs at V beta 11 and V beta 8 loci TCR beta haplotypes have been identified in five families in which the probands have insulin-dependent diabetes mellitus (IDDM). An extremely rare haplotype, marked by the higher molecular weight BamH1 allele (H, H) at each of V beta 11 and V beta 8, was found in the DR4+ DR3- probands of two families (P = 0.004). In three families in which the probands had DR3, the more common TCR haplotype LH (V beta 11, V beta 8) was found. Taken together, these data confirm that linkage disequilibrium does exist in the TCR beta locus, at least in some regions, and suggest that detailed analysis of the relationship between TCR V beta haplotypes and HLA is warranted since these RFLPs may be markers for important allelic V gene sequence variations.     

Autologous mixed lymphocyte reaction in man: XV. Cellular and molecular basis of deficient autologous mixed lymphocyte response in insulin-dependent diabetes mellitus

The autologous mixed lymphocyte response (AMLR) and the allogeneic mixed lymphocyte response were deficient in a subset of patients with newly diagnosed insulin-dependent diabetes mellitus (IDDM). Using a single set of HLA-identical twins, the cellular and molecular basis of deficient AMLR was investigated and appears to be due to a defect in both responder T cells and stimulator non-T cells. Interleukin-2 production was diminished in the patient but not in the healthy twin. Thein vitro addition of purified interleukin-2 enhanced the depressed AMLR in the diseased twin. This suggests that the deficient AMLR in IDDM may be in part due to a deficiency in the production of interleukin-2.     

The virus susceptibility of skin-derived fibroblasts from families with insulin-dependent diabetes mellitus

We have studied the growth of eight different viruses on skin fibroblasts from three families each having one or more diabetic members and appropriate controls. The haplotypes of all of the family members had been previously characterized. In addition, we have investigated the growth of mumps virus on the lymphoblast cultures from four families of the same type. Our results show no difference between the growth of these viruses in cells derived from juvenile diabetics and cells derived from nondiabetic siblings and parents even when the haplotypes were identical. However, we noted a striking resistance of human skin fibroblast cultures from both normal and diabetic individuals to Coxsackie B virus infection.     

A new predictive model for insulin-dependent diabetes mellitus susceptibility based on combinations of molecular HLA-DRB1 and HLA-DQB1 pockets

With a view to establishing an accurate evaluation of the genetic predisposition to insulin-dependent type I diabetes (IDDM), we have built a model based on the characteristics of the relevant pockets of HLA-DR and -DQ molecules. Three independent populations were investigated. Group I and group II were Caucasoids, while group III was Japanese, including a total of 1,166 IDDM patients and 2,391 healthy controls. We formulate the hypothesis that suceptibility to IDDM is not only explained by the absence of Aspartate 57 (negative charge) from pocket 9 of DQB1 (P9DQ), but also by the presence of an electric charge (+/- vs. neutral), generated by residues 70, 71 and 74 in pockets 4 of DRB1 (P4DR) and DQB1 (P4DQ) molecules. The respective weight of each pocket, was evaluated in a multivariate analysis based on the logistic regression method. The 4 components (2 loci and 2 pockets) were systematically analysed in the computer model. It was clearly shown that the structural characteristics of pockets P9DQ-P4DR and, to a lesser degree that of P4DQ, account for IDDM predisposition. On applying the model to the whole international series, it appears that the highest risk concerns individuals with P9DQ non-Asp 57 and both the charged P4 of DRB1 and P4 of DQB1, conferring a 80% prediction of susceptibility. Conversely, P9DQ Asp and neutral P4DR and P4DQ give the lowest risk with a predictive value of 5%. This model of risk susceptibility prediction fits remarkably well with the observed distribution in a worldwide study. It allows a better evaluation of the respective role of HLA-DR and -DQ molecules as a major component of susceptibility to IDDM.     

Analysis of three glucose transporter genes in a Caucasian population: no associations with non-insulin-dependent diabetes and obesity

The significance of variation within the genes coding for three glucose transporter proteins in the aetiology of non-insulin dependent diabetes mellitus was assessed by analysing restriction fragment length polymorphisms in an English Caucasian population. Two polymorphisms at the HepG2/erythrocyte glucose transporter (GLUT1) locus, four at the liver/pancreatic glucose transporter (GLUT2) locus and one at the muscle/adipocyte glucose transporter (GLUT4) were analysed in a sample of diabetic and non-diabetic subjects. No significant differences in the allelic, genotypic or haplotypic frequencies of the polymorphisms at these three loci were observed between the diabetic or non-diabetic populations. No significant linkage disequilibrium was observed between the two GLUT1 polymorphic sites, whereas the four polymorphic sites at the GLUT2 locus, one of which appears to be due to a 100-200 base pair DNA insertion/deletion, were found to be in significant linkage disequilibrium. In order to study the possible role of glucose transporter gene variants contributing to the development of obesity, the body mass indexes were compared in the different genotypic groups of diabetic and non-diabetic subjects. No differences in body mass index between genotype groups were found at the p < 0.005 level of significance.