E. Keller

IDDM2/insulin VNTR modifies risk conferred by IDDM1/HLA for development of Type 1 diabetes and associated autoimmunity

Aim/hypothesisType 1 diabetes (T1D) is an autoimmune disease with multiple susceptibility genes. The aim of this study was to determine whether combining IDDM1/HLA and IDDM2/insulin (INS) 5′ variable number of tandem repeat locus (VNTR) genotypes improves T1D risk assessment.MethodsPatients with T1D (n=488), control subjects (n=846), and offspring of parents with T1D (n=1122) were IDDM1 and IDDM2 genotyped. Offspring were followed for islet autoantibodies and T1D from birth until the age of 2 to 12 years.ResultsCompared to the I/I INS VNTR genotype, the I/III and III/III genotypes reduced T1D risk conferred by IDDM1/HLA in all HLA genotype categories of the case-control cohort by 1.6-fold to three-fold. The highest T1D risk was associated with INS VNTR class I/I plus HLA DR3/DR4-DQ8 (20.4% in patients, 0.6% in control subjects) or HLA DR4-DQ8/DR4-DQ8 (6.3% in patients, 0.2% in control subjects). In the offspring, HLA DR3/DR4-DQ8 and DR4-DQ8/DR4-DQ8 conferred increased risk for early development of islet autoantibodies (14.6% and 12.9% by age 2 years). Offspring with these high risk IDDM1 genotypes plus the INS VNTR class I/I genotype (n=71; 6.3%) had the highest risk of developing islet autoantibodies (21.8% by age 2 years vs 8.9% in offspring with high risk IDDM1 plus INS VNTR class I/III or III/III genotypes, p<0.05) and T1D (8.5% by age 6 years vs 4.3%). Offspring who developed autoantibodies to multiple antigens had increased frequencies of both high risk IDDM1 and IDDM2 genotypes (p<0.0001), whereas offspring who developed autoantibodies to GAD only had increased frequencies of high risk IDDM1 and protective IDDM2 genotypes, suggesting that IDDM2 influences the autoimmune target specificity.Conclusion/InterpretationCombining IDDM1 and IDDM2 genotyping identifies a minority of children with an increased T1D risk.

Polymorphism of the tumor necrosis factor beta gene in systemic lupus erythematosus: TNFB-MHC haplotypes.

We investigated the Nco I restriction fragment length polymorphism (RFLP) of the tumor necrosis factor beta (TNFB) gene in 173 patients with systemic lupus erythematosus (SLE), 192 unrelated healthy controls, and eleven panel families, all of German origin. The phenotype frequency of the TNFB*1 allele was significantly increased in patients compared to controls (63.6% vs 47.1%, RR = 1.96, p <0.002). The results of a two-point haplotype statistical analysis between TNFB and HLA alleles show that there is linkage disequilibrium between TNFB*1 and HLA-A1, Cw7, B8, DR3, DQ2, and C4A DE. The frequency of TNFB*1 was compared in SLE patients and controls in the presence or absence of each of these alleles. TNFB*1 is increased in patients over controls only in the presence of the mentioned alleles. Therefore, the whole haplotype A1, Cw7, B8, TNFB*1, C4A DE, DR3, DQ2 is increased in patients and it cannot be determined which of the genes carried by this haplotype is responsible for the susceptibility to SLE. In addition, two-locus associations were analyzed in 192 unrelated healthy controls for TNFB and class I alleles typed by serology, and for TNFB and class II alleles typed by polymerase chain reaction/oligonucleotide probes. We found positive linkage disequilibrium between TNFB*1 and the following alleles: HLA-A24, HLA-B8, DRB1*0301, DRB1*1104, DRB1*1302, DQA1*0501, DQB1*0201, DQB1*0604, and DPB1*0101. TNFB*2 is associated with HLA-B7, DRB1*1501, and DQB1*0602.

Early expression and high prevalence of islet autoantibodies for DR3/4 heterozygous and DR4/4 homozygous offspring of parents with Type I diabetes: The German BABYDIAB study

Aims/hypothesis. Islet autoantibodies precede the clinical onset of Type I (insulin-dependent) diabetes mellitus. The cumulative development of such autoantibodies in infants followed from birth and in particular infants with high-risk HLA genotypes is poorly defined, but such information is essential to design trials to prevent islet autoimmunity. Methods. HLA genotypes were determined in offspring of parents with Type I diabetes who were followed from birth for at least 2 years (median follow-up: 3.1 years) and who were characterised for the expression of insulin, GAD65, IA-2 and islet cell autoantibodies at birth, 9 months, 2 and 5 years of age. Results. The HLA genotypes DRB1*03/04(DQB1 *57non-Asp) and DRB1*04/04(DQB1*57non-Asp) were present in 7.1 % and 5.0 % of offspring of parents with Type I diabetes. The frequency of both genotypes was increased in offspring who developed islet autoantibodies within the first 2 years of life (27.3 % vs 5.5 %, odds ratio 6.3 [p = 0.002] and 22.7 % vs 4.2 %, odds ratio 6.6 [p = 0.003]) and half of all offspring who developed antibodies had these genotypes. Other genotypes were not associated with an increase in risk. By life-table analysis, the cumulative risk of developing islet autoantibodies by the age of 2 years was 20 % (95 % CI 9.4,30.6) for offspring carrying either the DRB1*03/04(DQB1 *57non-Asp) or the DRB1*04/04(DQB1*57non-Asp) genotype compared with 2.7 % (95 % CI 1.2,4.2) for offspring without these genotypes (p < 0.0001). Conclusion/interpretation. These data show that early appearance of islet autoantibodies is remarkably frequent for DR3/4 heterozygous and DR4/4 homozygous offspring and indicate that primary prevention could be considered once available in an offspring cohort selected for these genotypes. [Diabetologia (1999) 42: 671–677]

HLA-DP/DR interaction in early onset pauciarticular juvenile chronic arthritis

We investigated the polymorphic second exon of the HLA-DPB1 and HLA-DRB1 genes, using in vitro DNA amplification by polymerase chain reaction (PCR) and oligonucleotide hybridization in 136 patients with early onset pauciarticular juvenile chronic arthritis (EOPA-JCA) and 199 healthy controls. The analysis of the HLA-DRB1 system revealed that most of the DRB1 alleles are not indifferent with respect to susceptibility to EOPA-JCA. There is a hierarchy of susceptible (DRB1*08, DR5), “permissive” (DRB1*01), moderately “protective” (DR2, DRB1*04), and “protective” (DRB1*07) alleles. In contrast, no hierarchy could be shown for the HLA-DPB1 system. DPB1*0201 was found to be susceptible. The relatively frequent alleles DPB1*0402 and DPB1*0401 seem to be indifferent. The associations with DPB1*0201, DR5, and DRB1*08 are independent of each other: that is to say they, are not brought about by linkage disequilibrium. The susceptible alleles DPB1*0201 and DR5 show evidence for interaction in the pathogenesis of EOPA-JCA. Interaction seems likely between DPB1*0201 and DRB1*08, DR5 and DRB1*08, or between DR6 and DRB1*08. The strongest interaction exists between DPB1*0201 and a common DQ factor associated with both DR5 and DRB1*08. Finally, we observed a hierarchy among the various marker combinations, where the risk of developing EOPA-JCA increases with the number of associated markers present in an individual.

A model for the role of HLA-DQ molecules in the pathogenesis of juvenile chronic arthritis

SummaryRestriction fragment length polymorphism (RFLP) typing of MHC-class II loci DRB, DQA1, DQB1, DQA2 and DPB1 was performed in 94 patients with seronegative juvenile chronic arthritis (JCA) and 184 random controls. Analysis of allele frequencies and MHC-class II 4-loci haplotypes indicate: (1) Susceptibility to JCA is more strongly associated with the HLA-DQ subregion than with the HLA-DR subregion, especially in early onset pauciarticular JCA (EOPA-JCA). (2) Haplotype and sequence analysis show two independent MHC-class II associations for susceptibility to EOPA-JCA, one located in DQA1, the other in DPB1. (3) Two RFLP defined patterns of the DQA1 locus, DQA1.5 (DQA1*0501) and DQA1.8 (DQA1*0401, *0601) are strongly associated with the disease. (4) Analysis of amino-acid (AA) sequences coded in exon 2 of DQA1 reveals an AA sequence of six AAs common to all three associated DQA1 alleles. This suggests a model that includes a functional role for HLA-DQ molecules in the pathogenesis of JCA.

Class I associations and frequencies of class II HLA-DRB alleles by RFLP analysis in children with rheumatoid-factor-negative juvenile chronic arthritis

SummaryA total of 94 patients with juvenile chronic arthritis (JCA) was tested for HLA class I by serology and for class II by RFLP typing. Early onset JCA (EOPA) is associated with HLA-A2, DR5 and DR8 in both males and females. The combination (joint occurrence) of these JCA associated alleles (A2, DR5, DR8) is frequently seen in patients with chronic iridocyclitis. Late onset pauciarticular disease has an increased frequency of HLA-B27, especially in males. Our data confirm that polyarticular JCA with early childhood onset (≤4 years) is associated with DR5 and DR8 and has a different immunogenetic background from polyarticular JCA with later childhood (>4 years) onset (associated with DR4).

DNA TYPING FOR HLA‐DPB1‐ALLELES IN GERMAN PATIENTS WITH SYSTEMIC LUPUS ERYTHEMATOSUS USING THE POLYMERASE CHAIN REACTION AND DIG‐ddUTP‐LABELLED OLIGONUCLEOTIDE PROBES

Genomic DNA of 178 German Caucasian patients with systemic lupus erythematosus are studied for HLA‐DP locus by using PCR and DIG‐ddUTP‐labelled oligonucleotide probes. A significant increase of DPB1*0101 is observed in SLE patients compared with healthy controls (χ < 2 < = 15.27, p.c. <0.004). DPB1*0501 and *0901 are also slightly increased (χ2= 5.85, P < 0.05, p.c. = NS; χ2= 5.64, P < 0.05, p.c. = NS). There is no significant difference in frequency of DP alleles between male and female patients. Since a linkage disequilibrium between HLA‐B, DR and DP loci is found in our SLE patients, an analysis is performed assessing the relative importance of these HLA‐markers to SLE. The results show that the increase of DPB1*0101 in SLE patients is associated with the HLA‐B8, DR3 haplotype and it suggests a more important role for HLA‐B8, DR3 or genes within this haplotype than for DPB1*0101 in the genetic predisposition for SLE.

DR2 haplotypes (DRB1, DQA1, DQB1) associated with systemic lupus erythematosus

Although the etiology of systemic lupus erythematosus (SLE) is still unknown, population and family studies have shown that genetic factors influence the onset and course of the disease (Arnett 1992). Various studies have revealed that one or more genes in the major histocompatibility complex (MHC) are associated with susceptibility to SLE (Hartung et al. 1992; Arnett 1992). The largest study to date is a joint investigation of 417 Central European patients with SLE (Hartung et al. 1989). The results of this study (Hartung et al. 1992) show that two haplotypes are increased in this group of patients: one carrying the alleles HLA-B8 and -DR3 and one carrying HLA-DR2. We investigated the DR2 subtypes at the DRB1 locus level and the alleles of the DQA1 and DQB1 loci in DR2-positive SLE patients from the multicenter study and in healthy controls. The aim of the study was to determine whether there is a characteristic DR2 (DRB1, DQA1, DQB1) haplotype responsible for the association of I-ILA-DR2 with susceptibility to SLE. The study encompassed 62 DR2-positive Caucasian SLE patients whose diagnosis fulfilled the revised American Rheumatism Association (ARA) criteria for SLE (Tan et al. 1982). The patients, part of the German multicenter SLE study, had been serologically typed for HLA-DR antigens (Hartung et al. 1992). One-hundredand-ninety-two unrelated healthy individuals from the panel of the Immunogenetics Laboratory in Munich were included as control group.

Pvu II polymorphism in the primate homologue of the mouse B144 (LST-1): A novel marker gene within the tumor necrosis factor region

A Pvu II RFLP was mapped within the LST-1 gene, the human homologue of the mouse B144 sequence, establishing LST-1 as a new marker gene within the TNF region. We investigated the distribution of this Pvu II RFLP in 274 unrelated individuals, 132 additional HLA-DR7-positive individuals, 86 homozygous lymphoblastoid cell lines, and in four families. Seventeen of 274 individuals (6.2%) were heterozygous for the Pvu II restriction site (ADB1 = lack and ADB2 = presence of the Pvu II restriction site). In our study population the polymorphism has a much wider distribution than that previously reported in an analysis of selected haplotypes. Besides a strong association of ADB1 with HLA-B14, -DR7, we found a further association with HLA-B35. These results were also validated by family segregation studies and analyses of homozygous cell lines. In addition, five of 17 individuals carrying the ADB1 allele had HLA types other than B14 or B35, emphasizing that the presence of ADB1 is not limited to the HLA-B14, DR7 haplotype. LST-1 and its polymorphism may be used as an additional marker of the TNF region, where genes responsible for autoimmune diseases are suspected to be localized.

UnusualHLA-DR/DQ haplotypes: two different breakpoints in two differentDR2-DQw3 haplotypes

Recently we had the opportunity to study a family from Bangkok in which unusual haplotypes, HLA-DRw15 (DR2) and DQw7 (DQw3), could be identified. The maternal haplotype is unusual in that it shows a combination of DRw15 and DQw7 (w3), which, to our knowledge, has thus far not been observed. To validate and extend the above findings, genomic DNA was prepared from peripheral blood of all family members and digested with the restriction enzymes Eco RI, Taq I, Pvu II, and Hind III