Genetic similarities between patients from the United States and South African (SA) Addison's Disease (AD) strengthen evidence for genetic association. SA-AD (n = 73), SA healthy controls (N = 78), and US-AD patients (N = 83) were genotyped for DQA1, DQB1, DRB1, and HLA-B alleles. Serum was tested for the quantity of 21OH-AA and IFNα-AA at the Barbara Davis Center. Although not as profound as in US-AD, in SA-AD 21OH-AA + subjects the predominantly associated risk haplotypes were DRB1*0301-DQB1*0201 (DR3), DRB1*04xx-DQB1*0302 (DR4), and the combined DR3/4 genotype. DQB1*0302 associated DRB1*04xx haplotypes conferred higher risk than those DRB1*04xx haplotypes associated with other DQB1 alleles. We found negative association in 21OH-AA + SA-AD for DQA1*0201-DQB1*0202 and DQA1*0101-DQB1*0501 vs SA controls, and positive association for DQA1*0401-DQB1*0402 vs US-AD. Apart from the class II DR3 haplotype, HLA-B8 did not have an independent effect; however together DR3 and HLA-B8 conferred the highest risk vs 21OH-AA negative SA-AD and SA-controls. HLA-B7 (often with DR4) conferred novel risk in 21OH-AA + SA-AD vs controls. This study represents the first comparison between South African and United States AD populations utilizing genotyping and serology performed at the same center. SA-AD and US-AD 21OH-AA + patients share common HLA risk haplotypes including DR4 (with HLA-B7) and DR3 (with HLA-B8), strengthening previously described HLA associations and implicating similar genetic etiology.

OBJECTIVE: People with the human leukocyte antigen (HLA) genotype DRB1 *0301-DQA1 *0501- DQB1 *0201/ DRB1 *04- DQA1 *0301- DQB1 *0302 (DR3/4-DQ8) are at the highest risk of developing type 1 diabetes. We sought to find an inexpensive, rapid test to identify DR3/4-DQ8 subjects using two single nucleotide polymorphisms (SNPs). RESEARCH DESIGN AND METHODS: SNPs rs2040410 and rs7454108 were associated with DR3- DQB1 *0201 and DR4- DQB1 *0302. We correlated these SNPs with HLA genotypes and with publically available data of 5019 subjects from the Type 1 Diabetes Genetic Consortium (T1DGC). Additionally, we analyzed these SNPs in samples from 143 HLA-typed children of the Diabetes Autoimmunity Study of the Young (DAISY) using Taqman® probes (rs7454108) and restriction digest analysis (rs2040410). RESULTS: With a simple combinatorial rule, the SNPs of interest identified the presence or absence of the DR3/4-DQ8 genotype. A wide variety of genotypes were tested for both SNPs. In T1DGC samples, the two SNPs were 98.5% (1173/1191) sensitive and 99.7% (3815/3828) specific for DR3/4-DQ8. In the DAISY population the test was 100% (69/69) sensitive and 100% (74/74) specific. Overall, the sensitivity for the test was 98.57% and was 99.67% specific. CONCLUSIONS: A two SNP screening test can identify the highest risk heterozygous genotype for type 1 diabetes, in a time- and cost-effective manner.

CONTEXTHuman leukocyte antigen (HLA) DQ haplotypes have the strongest genetic association with type 1 diabetes (T1DM) risk.OBJECTIVEThe objective of the study was to analyze whether HLA DQ alleles influence the development of antiislet autoantibodies, the progression to T1DM among autoantibody-positive relatives, or both.DESIGNThe Diabetes Prevention Trial-1 screened more than 90,000 nondiabetic relatives of patients for cytoplasmic islet-cell autoantibody (ICA) expression between 1994 and 2002.SETTINGThe study was conducted in the general community.PARTICIPANTSThe Diabetes Prevention Trial-1 found 2817 ICA-positive relatives who were tested for biochemical autoantibodies (GAD65, ICA512, and insulin) and HLA-DQ haplotypes, and 2796 of them were followed up for progression to diabetes for up to 8 yr (median, 3.6 yr).MAIN OUTCOME MEASUREProgression to T1DM was measured.RESULTSHigh-risk DQ haplotypes and genotypes were associated with a higher percentage of relatives expressing multiple biochemical autoantibodies and higher T1DM risk (e.g., respectively, 59 and 36% at 5 yr for carriers of the DQA1*0301-DQB1*0302/DQA1*0501-DQB1*0201 genotype). The number of autoantibodies expressed significantly increased T1DM risk and across different DQ genotypes, autoantibody positivity directly correlated with diabetes risk. However, multivariate analyses indicated that the influence of most genotypes on T1DM risk was not independent from autoantibody expression, with the possible exception of DQA1*0102-DQB1*0602. Specific genotypic combinations conferred 5-yr diabetes risks significantly lower (e.g. 7%-DQA1*0201-DQB1*0201/DQA1*0501-DQB1*0201 and 14%-DQA1*0301-DQB1*0301/DQA1*0501-DQB1*0201) than when those haplotypes were found in other combinations.CONCLUSIONHLA DQ alleles determine autoantibody expression, which is correlated with diabetes progression. Among autoantibody-positive relatives, most HLA DQ genotypes did not further influence T1DM risk.

Objective: Certain human leukocyte antigen (HLA)-DR,DQ genotypes have been associated with type 1 diabetes mellitus (T1DM) risk, although it is unknown whether the association is due to alleles, haplotypes, genotypes, the formation of heterodimers, or all of the above. To characterize the role of the HLA-DR,DQ genotype and ethnicity on the onset age of T1DM, we analyzed these factors in patients with T1DM and the general population.Methods: One thousand three hundred twenty-two well-characterized patients with T1DM were compared with 3339 children from the general population of Denver, Colorado, USA. Because of the extensive available data across age and ethnic groups, this study population is unique.Results: The HLA-DR3/4,DQB1*0302, DRX/4,DQB1*0302 (where X = 1, 4, 8, and 9), and HLA-DR3/3 genotypes were associated with T1DM, supporting previous research. Additionally, the DR3/9 genotype showed a positive association with T1DM, which has not previously been described in Caucasian populations. The HLA-DR3/4*0302 genotype was most strongly associated with T1DM in diabetic individuals with the youngest onset age. Genotype frequencies were similar between Hispanics and non-Hispanic whites, except for the DR3/3 genotype, which was more likely to be found in non-Hispanic whites.Conclusions: These results indicate that there are multiple alleles and genotypes associated with T1DM and that the risk associated with different genetic markers depends on the age of disease onset, suggesting that some markers may be involved in more rapid disease progression.

Addison's disease (AD) is a rare autoimmune disorder affecting approximately 1/10,000 in the general population. It typically occurs sporadically with a single member of a family affected (simplex families). Rarely, multiple members of a family can be affected with AD (multiplex families). The purpose of this study is to characterize the HLA haplotypes and genotypes of a group of individuals with AD and evaluate the differences between simplex and multiplex cases. Individuals with AD were recruited for participation in this study through the National Adrenal Diseases Foundation and through screening individuals with type 1 diabetes (T1D) for AD. A total of 62 families and 239 individuals with at least one member with AD were identified. They were divided into four groups: multiplex AD (9 families, 82 members), simplex AD (34 families, 91 members), multiplex T1D (5 families, 32 members) and T1D with AD (14 families, 34 members). HLA haplotypes were remarkably restricted in this group of families with AD. Seventy-seven percent of unrelated probands had at least one DR3 and 47% had at least one DR4. Five of the seven unrelated probands who had neither DR3 nor DR4 were either DR2/2, DR2/5 or DR 5/5. The DR2 haplotype was concentrated in the simplex AD probands in which there was a 16% haplotype frequency of DR2 compared with 0% in the other groups (p=0.0015). Unrelated probands from multiplex AD families had an increase of the DRB1*0404, DQ8/ DR3 genotype (56%) compared with 10% of simplex AD probands (p≤0.01). Similarly, unrelated probands of multiplex T1D families had a higher rate of this genotype compared with simplex T1D probands (80% vs. 23%, p=0.047). T1D tended to occur at an increased frequency in the patients of multiplex AD families compared with the patients of the simplex AD families (24% vs. 3%, p=0.07). In conclusion, there is a restricted pattern of HLA haplotypes in these individuals with AD. DRB1*0404,DQ8/DR3 was highly associated with multiplex AD and T1D families. This may suggest that refined HLA analysis can identify groups of people with risk for AD approaching that found in the population with T1D, a group in which some centers are screening for the adrenal autoimmunity associated with AD.

Aims/hypothesis: Immunological and genetic markers can be used to assess risk of developing type 1 diabetes prior to the onset of clinical symptoms. Autoantibody-positive relatives of patients with type 1 diabetes are at increased risk for disease, while the presence of HLA DQA1* 0102/ DQB1* 0602 is thought to confer protection. Using the unique population identified by the Diabetes Prevention Trial - Type Diabetes (DPT-1), our aim was to determine if these individuals were protected from type 1 diabetes. Methods: We described metabolic and immunological characteristics of islet cell cytoplasmic autoantibodies-positive relatives with DQB1* 0602 identified as part of DPT-1. Results: We found that 32% of DQB1* 0602-positive relatives identified through the DPT1 had abnormalities of glucose tolerance despite the fact that only 19% had multiple type 1 diabetes-associated autoantibodies and only 13% had abnormal insulin secretion, markers typically associated with the disease. In addition, these markers were not associated with abnormal glucose tolerance. In contrast, the DQB1* 0602- positive relatives had elevated fasting insulin ( 117 +/- 10 pmol/l) and homeostasis model assessment of insulin resistance (HOMA-R) (4.90 +/- 0.5) values, which are more commonly associated with type 2 diabetes. The later marker of insulin resistance was associated with glucose tolerance status. Conclusions/ interpretation: Our data indicate that DQA1*0102/ DQB1* 0602 relatives identified through DPT-1 have a high frequency of abnormal glucose tolerance and a disease phenotype with characteristics of type 1 and type 2 diabetes. Thus, multiple pathways to abnormal glucose tolerance are present within families of these type 1 patients.

Abstract: The HLA genotype DRB1*03,DQB1*0201/DRB1*04,DQB1*0302 confers a 25‐fold increase in the risk of type 1 diabetes. In persons with this genotype, DRB1*0405, *0402, and *0401 subtypes have been reported to further increase risk, whereas the *0403 and *0406 alleles confer a relative protection. We compared the frequencies of the DRB1*04 alleles in 193 type 1 diabetic patients with the HLA‐DRB1*03,DQB1*0201/DRB1*04,DQB1*0302 genotype (140 non‐Hispanic white [NHW] and 53 Hispanic) and 205 nondiabetic controls (142 NHW and 63 Hispanic). In addition, 87 NHW first‐degree relatives of type 1 diabetes patients were studied: 33 positive and 54 negative for autoantibodies to insulin, GAD65, or IA‐2. The HLA‐DRB1 was typed using standard PCR SSOP methods. DRB1*0401 (OR, 2.19; 95% CI, 1.36‐3.54) in NHW and *0405 (OR, 3.78; 95% CI, 1.43‐10.0) in Hispanics were significantly associated with T1DM, whereas DRB1*0403 was protective (OR, 0.19; 95% CI, 0.04‐0.89 in NHWs; OR, 0.10; 95% CI, 0.01‐0.83 in Hispanics). Associations between the DRB1*04 alleles and prediabetic islet autoimmunity were generally in the same direction as those with diabetes. Among diabetic patients, the mean age of diagnosis appeared to be higher among those with the *0403 and *0407 allele compared with the others. In summary, on the DRB1*03,DQB1*0201/DRB1*04,DQB1*0302 genotypes, the *0403 allele confers relative protection from type 1 diabetes and development of islet autoantibodies in both Hispanics and NHWs and is associated with older age at diabetes diagnosis. Although the associations between diabetes and *0401 and *0405 appear to differ somewhat between Hispanics and NHWs, overall there is no significant difference between these two ethnic groups.

As insulin is a major autoantigen in autoimmune diabetes and because the insulin gene region locus in humans has been linked to diabetes risk, we have bred insulin gene knockouts onto the NOD mouse. Mice differ from humans in terms that they express two nonallelic genes of insulin. Insulin 2 is the murine homologue of the human insulin gene and is located on mouse chromosome 7. Insulin 1 is thought to have evolved by a gene duplication event, lacks the second intron of the insulin 2 gene, and is located on mouse chromosome 19. The differential thymic expression of the insulin gene may be important for central tolerance induction. Here, we present the initial establishment of congenic knockouts and characterization of the congenic intervals corresponding to insulin 1 and insulin 2 knockout genes on mouse chromosome 19 and 7, respectively.

The incidence of type 1 diabetes in Korea is less than 1/10th of that in the United States, and it has been suggested that human leukocyte antigen (HLA) alleles of Asian patients associated with diabetes differ from those of Caucasians. In this study we analyzed the common susceptibility and transmission pattern of a series of HLA DRB1-DQB1 haplotypes to Korean and Caucasian patients with type 1 diabetes. We performed HLA DR and DQ typing of 158 type 1 diabetic patients in a case control study, 140 nondiabetic subjects from the same geographical area, 49 simplex families from Seoul, and 283 families from the Human Biological Data Interchange. Although the haplotype frequencies in the two populations are quite different, when identical haplotypes are compared, their odds ratios are nearly the same. For all parental haplotypes, the transmission to diabetic offspring was similar for Korean and Caucasian families (r = 0.8; P: < 10(-)(4)). Allowing for ethnic differences in allelic associations due to different frequencies of DRB1 and DQB1 haplotypes (linkage disequilibrium), these data show, not only by case-control comparison but also by transmission analyses of the haplotypes, that the susceptibility effects of DRB1-DQB1 haplotypes are consistent in Koreans and Caucasians. Thus, the influence of class II susceptibility and resistance alleles appears to transcend ethnic and geographic diversity of type 1 diabetes.

Type 1 diabetes and celiac disease are both immunologic disorders where specific HLA alleles are associated with disease risk. We have developed a radioassay for autoantibodies to tissue transglutaminase (tTG) following the report that this enzyme is ‘the’ endomysial autoantigen (EMA) of celiac disease. The radioassay for transglutaminase autoantibodies is similar to that utilized for detecting anti-islet autoantibodies. The ‘cut-off’ for the IgA autoantibody assay was established as 3×100th percentile of 184 healthy control subjects at an index of 0.05. Ninety-eight of 847 patients with type 1 diabetes (11.6%) had tissue transglutaminase autoantibodies (tTG). All EMA-positive patients were positive (49/49) for transglutaminase autoantibodies, as were 49/540 EMA-negative patients. Twenty transglutaminase-positive patients consented to intestinal biopsy and 15 biopsies were positive for celiac disease. All patients with a transglutaminase level greater than 0.70 (13/13) had a positive biopsy, while none (0/3) with a level <0.3 had a positive biopsy. The prevalence of transglutaminase autoantibodies was higher in diabetic patients with HLA DQ2 or DQ8. One third of DQ2 homozygous patients (22/68) expressed transglutaminase autoantibodies vs . less than 2% of patients lacking DQ2 or DQ8. A simple radioassay for IgA transglutaminase autoantibodies detects all endomysial antibody positive patients and detects transglutaminase autoantibodies in 5% of endomysial autoantibody negative patients. The prevalence of transglutaminase autoantibodies is associated with DQ2 and DQ8 and in particular DQ2 homozygosity. Autoimmunity to transglutaminase is remarkably prevalent amongst patients with type 1 diabetes expressing certain class II HLA alleles.

Wilson and coworkers (Wilson SB, Kent SC, Patton KT, Orban T, Jackson RA, Exley M, Porcelli S, Schatz DA, Atkinson MA, Balk SP, Strominger JL, Hafler DA: Extreme Th1 bias of invariant V alpha24J alpha Q T-cells in type 1 diabetes. Nature 391:177-181, 1998) have recently reported raised serum levels of interleukin-4 (IL-4) in anti-islet autoantibody-positive first-degree relatives of patients with type 1A diabetes who did not progress to diabetes. Protection from diabetes has been noted for several human lymphocyte antigen (HLA) alleles, such as HLA DR2-DQA1*0102-DQB1*0602. We, therefore, wanted to determine whether this cytokine phenotype was associated with HLA genes protective for type 1A diabetes. We used a two-site fluoroimmunoassay with the same monoclonal antibodies as those reported by Wilson et al. Using this assay, we have found evidence for human heterophile antibodies mimicking serum IL-4: all serum IL-4 reactivity was lost if mouse serum or mouse immunoglobulin were added to the assay; serum IL-4 activity was bound and then eluted by protein A/G chromatography; and levels of anti-mouse antibodies correlated with apparent serum IL-4. This pseudo-IL-4 activity was found in a subset of control subjects, patients with type 1A diabetes, and their relatives and was primarily associated with specific HLA alleles protective for type 1A diabetes (e.g., DQB1*0602). After adjustment for HLA, positive levels of heterophile antibodies were not associated with protection from diabetes. The confounding effect of protective HLA alleles associated with heterophile antibodies could explain the previously reported association between raised serum IL-4 and protection from type 1A diabetes. The mechanism by which specific DQ alleles protect from diabetes and are associated with increased heterophile antibodies is currently unknown.