Differential expression of HLA-DQ alleles in peripheral blood mononuclear cells: alleles associated with susceptibility to and protection from autoimmune type 1 diabetes.

Differential expression of human leucocyte antigen (HLA) class II genes has been postulated to influence the risk of developing autoimmune disease. In this study, we investigated the relationship between the level of mRNA expression of DQA1 and DQB1 alleles in peripheral blood mononuclear cells and the influence of the alleles on susceptibility to type 1 diabetes (T1D). Transcripts from pairs of DQA1 and DQB1 alleles were quantified in 59 DQ-heterozygous individuals (29 patients with T1D and 30 healthy control subjects). Luciferase reporter gene assays were used to investigate the relative promoter activities of the alleles associated with high and low risk of disease. DQA1*0301 and the DQB1*06 group of alleles (*0601, *0602, *0603 and *0604) were generally overexpressed in comparison to other alleles. In contrast, mRNA for DQB1*0201/*0202 was generally less abundant than other DQB1 transcripts. These data correlated well with the relative promoter activities observed for the diabetes-associated alleles; the strongest promoters were those derived from DQA1*0301 and DQB1*0602, while a 700-bp fragment derived from the DQB1*0201 promoter showed the lowest activity of the DQB1 constructs. There was no simple correlation between the level of expression of specific DQ alleles and their influence on the risk of diabetes. The functional relevance of our findings and their implications for the pathogenesis of autoimmunity remain to be determined.

HLA-DQA1 and -DQB1 alleles associated with genetic susceptibility to IDDM in a black population.

Transracial analysis provides a method of distinguishing primary associations between insulin-dependent diabetes mellitus (IDDM) and HLA class II alleles from those secondary to linkage disequilibrium. Blacks show DR-DQ relationships that are different from other races and are a useful group in which to investigate HLA-D region associations with IDDM. In this study, the frequencies of HLA-DQA1 and -DQB1 alleles in Afro-Caribbean IDDM and control subjects were compared. Alleles were identified with sequence-specific oligonucleotide probing. The DQA1 allele A3 was positively associated with IDDM (relative risk [RR] = 25.3, corrected P [Pc] less than 7.0 x 10(-6). The DQB1 alleles DQw2 and DQw8 were also positively associated (RR = 4.7, Pc less than 6.5 x 10(-3) and RR = 12.3, Pc = 3.4 x 10(-3), respectively). The A1.2 and DQw6 alleles were negatively associated (RR = 0.16, Pc less than 3.5 x 10(-3) and RR = 0.15, Pc = 2.4 x 10(-2), respectively). These findings were compared to data from other races. The positive associations with A3 and DQw2 are consistent with all racial groups investigated. The negative association with DQw6 is present in all racial groups in which it is a common allele. These findings suggest that DQ alleles, and hence DQ molecules, may directly affect predisposition to IDDM.

Susceptibility to IDDM in a Chinese population. Role of HLA class II alleles.

MHC associations with IDDM in a Chinese population were studied to investigate genetic susceptibility to the disorder. The frequency of HLA-DR3 was significantly higher in the diabetic patients (19/49 [38.7%] vs. control subjects, 11/105 [10.5%], Pc less than 1.3 x 10(-3), RR = 5.3 [CI 2.3-12.1]), whereas DR4 was not (11/49 [22.4%] vs. 28/105 [26.7%], NS). The frequency of DR3/4 heterozygosity was higher in the diabetic patients (6/49 [12.2%] vs. control subjects, 0/105 [0%], P = 1.7 x 10(-3), RR = 31.5 [CI 3.8-263.6]). The frequency of DR3/9 heterozygosity also was higher in the diabetic patients (6/49 [12.2%] vs. control subjects, 2/105 [1.9%], P = 0.03, RR = 6.2 [CI 3.0-12.7]). No significant associations were noted between DQB1 alleles and IDDM. Among DR4-positive subjects, the frequency of DQB1 allele DQB1*0302 was higher in the diabetic patients (10/11 [90.0%] vs. control subjects, 12/24 [50%], Pc less than 0.05, RR = 7.0 [CI 1.3-38.0]), and the frequency of DQB1*0401 was significantly lower in the diabetic patients (2/11 [18.2%] vs. control subjects, 16/24 [66.7%], Pc = 0.04, RR = 0.1 [CI 0.02-0.46]). No DR4 subtype was associated significantly with IDDM. The frequency of DQA1*0501, a DQA1 allele, was higher in diabetic patients (22/41 [53.7%] vs. control subjects, 20/95 [21.1%], Pc less than 3 x 10(-3), RR = 4.3 [CI 2.0-9.3]). The frequency of DQA1*0301, which has been associated consistently with IDDM in other ethnic groups, was not significantly higher in the diabetic patients in this study (27/41 [65.9%] vs. control subjects, 53/95 [55.8%], NS).(ABSTRACT TRUNCATED AT 250 WORDS)

The influence of HLA-DR and -DQ alleles on progression to multiple sclerosis following a clinically isolated syndrome.

A 5-year follow-up study was performed on 70 Caucasian patients presenting with isolated neurological syndromes of the optic nerve, brain stem, or spinal cord to assess the risk of progression to MS. The influence on patient prognosis of HLA-DR and -DQ alleles and presentation with disseminated brain lesions, demonstrated by MRI scanning, was determined. Clinical progression to MS was observed in 61% of optic neuritis patients, 50% of patients with a brain-stem syndrome, and 35% of patients with a spinal cord disturbance. MS and the isolated clinical syndromes were positively associated with DRB1*1501, DQA1*0102, and DQB1*0602; the frequency of these alleles in the latter group was intermediate between that seen in MS patients and healthy controls. Conversion to MS was positively associated with the DRB1*1501.DQA1*0102.DQB1*0602 haplotype, but the influence of HLA was only significant in patients with disseminated brain lesions at presentation (MRI positive); MS developed in 86% of MRI-positive, DRB1*1501-positive patients compared with 55% of MRI-positive, DRB1*1501-negative patients (p < 0.025). The data suggest that these HLA alleles are involved in susceptibility to initial demyelinating lesion formation and are important in the subsequent development of MS in MRI-positive patients.

An investigation of the association between HLA-DQ heterodimers and type 1 (insulin-dependent) diabetes mellitus in five racial groups.

The association between HLA-DQ alpha Arg52-HLA-DQ beta non-Asp57 heterodimers and type 1 (insulin-dependent) diabetes was compared in Japanese, Chinese, Caucasian, North Indian Asian, and Afro-Caribbean patients to determine their importance in disease susceptibility. The potential to encode four Arg52-non-Asp57 DQ heterodimers, two in cis and two in trans, was significantly associated with increased risk of type 1 diabetes in all races except the Japanese. The possibility of encoding two Arg52-non-Asp57 heterodimers was also significantly associated with increased risk of the disease in all races except the Japanese. The possibility of encoding one heterodimer was not significantly associated with type 1 diabetes in any of the races studied. Heterogeneity testing revealed significant differences in RR values for four, two, and one heterodimers in all races except the Japanese and significant differences in RR for four and two heterodimers when compared across the races. This, together with the lack of an association between Arg52-non-Asp57 heterodimers and type 1 diabetes in the Japanese, suggests that, assuming the same genetic basis for disease in all races, the heterodimer is unlikely to be of primary importance in susceptibility to the disease.

HLA alleles and IDDM in children in Hungary: a comparison with Finland.

It has been postulated that variation in the distribution of human leukocyte antigen (HLA)-encoded susceptibility alleles for insulin-dependent diabetes mellitus (IDDM) is the genetic basis for variation in the incidence of the disease between populations. The aim of this study was to characterize HLA-encoded susceptibility to IDDM in Hungary and to identify whether HLA-DRB1/DQ-encoded susceptibility could account for the five times lower incidence of disease in Hungary compared with Finland. The haplotypes DRB1*03-DQA1*05-DQB1*02 (DRB1*03-DQ2) and DRB1*04-DQA1*0301-DQB1*0302 (DRB1*04-DQ8) were significantly associated with disease in both populations. Three genotypes incorporating either or both of these haplotypes accounted for over 70% of the diabetic population in both races. The combined background frequency and the degree of risk as measured by odds ratios of these HLA-DRB1-DQ genotypes were not significantly different in the two countries. Comparison of the DRB1*0401-DQ8 haplotype between the two races suggested a role for HLA-B alleles in susceptibility. These data indicate that the susceptibility associated with high risk DRB1-DQ genotypes alone is insufficient to account for the fivefold variation in incidence of IDDM between Hungary and Finland. Other genetic and/or environmental influences must be involved.

Genetic susceptibility to multiple sclerosis in a Shanghai Chinese population. The role of the HLA class II genes.

The association of MS with the HLA class II loci DR and DQ was investigated in subjects of Shanghai Chinese and British Caucasian origin. Our aim was to determine whether common alleles predispose to the disease in both races. In the Caucasian population MS was significantly positively associated with the putative haplotype DRB1*1501, DQA1*0102, DQB1*0602. In contrast, HLA class II alleles were not found to predispose to the disease in the Shanghai Chinese, suggesting that this haplotype is unlikely to be a universal susceptibility determinant. The absence of a disease association with the HLA-DR and -DQ genes in the Chinese population has a number of possible explanations. Our study suggests that other genetic and/or environmental components may be more important in determining susceptibility to MS in this race.

Analysis of a Chinese population suggests that the TNFB gene is not a susceptibility gene for Graves' disease.

Graves' disease is associated with different HLA genes in different races. The TNFB gene lies between the class I and class II HLA genes and has two alleles, TNFB*1 and TNFB*2. Studies in Caucasians have suggested that the TNFB gene might be a susceptibility gene for Graves' disease. To investigate further the role of TNFB in predisposition to Graves' disease, we determined whether the TNFB disease associations in the Chinese were similar to those in Caucasians. A total of 57 patients with Graves' disease (32 male) were studied. A TNFB gene fragment was amplified from genomic DNA by using the polymerase chain reaction and digested with Nco I to distinguish the TNFB alleles (TNFB*1 and TNFB*2). Genotype frequencies were compared with those in a racially matched group of 92 controls. TNFB*1 homozygosity occurred in 15 (26%) Graves' and 22 (24%) control subjects. TNFB*1/TNFB*2 heterozygosity occurred in 29 (51%) and 48 (52%) and TNFB*2 homozygosity in 13 (23%) and 22 (24%), respectively (NS). There were gender differences in the frequencies of TNFB*1 homozygosity (13 male [41%], 2 female [8%]). TNFB*1/TNFB*2 heterozygosity (13 male [41%], 16 female [64%]) (chi 2 = 7.3, p = 0.02), and TNFB*2 frequency (19 male [59%], 23 female [92%]; pc = 0.04) in Graves' patients. We conclude that the TNFB associations with Graves' disease in the Hong Kong Chinese differ between the genders and from those described in Caucasians. The TNFB gene is not a susceptibility gene for Graves' disease.

Genetics of type 1 diabetes.

Type 1 diabetes mellitus is a T-cell-mediated autoimmune disease characterized by the selective destruction of pancreatic beta cells. Susceptibility to the disease is determined by a combination of genetic and environmental factors. The genetic factors are termed 'susceptibility genes' as they modify the risk of diabetes but are neither necessary nor sufficient for disease to develop. A large number of chromosomal regions have been identified as containing potential diabetes susceptibility genes. The IDDM1 locus, which encompasses the major histocompatibility complex on chromosome 6, is the major genetic risk factor. The HLA-DQ genes are the primary susceptibility genes within this region, although other genes may also contribute. The IDDM2 locus maps to a variable number of tandem repeats in the insulin gene region on chromosome 11. Further research is necessary to determine the precise location and identity of other diabetes susceptibility genes.

Sequencing of the second exon of the MHC class II DQ6 alleles in patients with type 1 diabetes.

The MHC class II molecule DQ6 is strongly associated with protection from type 1 diabetes. A small number of diabetic subjects have been typed as positive for DQ6, but it has been suggested that these individuals may possess a mutant form of the molecule, which is structurally altered in such a way as to abrogate its protective effect. In order to test this hypothesis, eight diabetic individuals positive for DQ6 were investigated. The second exons of the alleles encoding DQ6, DQA1*0102 and DQB1*0602, were sequenced using fluorescently labelled dideoxynucleotides. No mutations were found. This suggests that all the subjects possessed the "wild-type" second exons of the DQ6 alleles and, hence, that the protective effect associated with the antigen-binding domain of DQ6 is not absolute.

The insulin gene region and susceptibility to insulin-dependent diabetes mellitus in four races; new insights from Afro-Caribbean race-specific haplotypes.

The IDDM2 component of the genetic susceptibility to insulin-dependent diabetes mellitus (IDDM) has been mapped to chromosome 11p15.5. The exact identity of IDDM2 remains uncertain. It has been suggested that IDDM2 maps within the 5' VNTR (variable number tandem repeat) polymorphism upstream of the insulin gene (INS). This has not been confirmed and a contribution from other INS gene region polymorphisms cannot be excluded. We present INS region genotype data from four racial groups: the Japanese, Hong Kong Chinese, North Indian Asians and Afro-Caribbeans (two groups; one born and resident in the UK, one in Jamaica). These races have not been previously studied with the range of INS region polymorphisms included here. No INS polymorphism was associated with IDDM across all races. These data from this study thus do not identify any INS polymorphism as IDDM2. The Afro-Caribbean race showed a very different distribution of INS genotypes from the other races and novel race-specific INS haplotypes were identified. Analysis of these excluded a contribution to susceptibility to IDDM from the- 23HphI INS polymorphism. An Afro-Caribbean INS haplotype which differed only at the VNTR from the very protective INS haplotype (VPH) identified in white Caucasians was detected. Population analysis of this haplotype will allow direct assessment of the role of the VNTR in susceptibility to IDDM. In conclusion, the diverse Afro-Caribbean TH/INS/IGF2 haplotypes identified in this study will be valuable in mapping IDDM2 more precisely.

Genetics of diabetes. Trans-racial gene mapping studies.

A major component of inherited susceptibility to IDDM is associated with one or more loci in the MHC. Identification of the primary susceptibility genes has been complicated by the low frequency of recombination, i.e. linkage disequilibrium, within the MHC. It is difficult to distinguish whether a detected genetic association with the disease is primary, or secondary due to linkage disequilibrium with an allele at another locus which is directly predisposing. During the evolution of different races, however, recombination within the MHC has occurred and population-specific MHC haplotypes exist. Primary susceptibility allels should be associated with disease in all racial groups, regardless of genetic background. It is unlikely that disease associations secondary to linkage disequilibrium will be consistent in these groups. This chapter reviews the known associations of candidate class II susceptibility alleles with IDDM in the five largest racial groups; white Caucasians, Asian Indians, Negroids, Japanese and Chinese. These trans-racial studies suggest that the DQ molecule has a primary role in predisposition to IDDM. There are consistent findings of a positive association with the DQA1*0301 allele and negative associations with the DQB1*0602 and DQB1*0603 alleles. These two alleles differ by a single codon and so the encoded DQ beta chains are likely to have similar functions. DR4-associated susceptibility is associated with the DQA1*0301 allele in all races tested so far but this allele cannot be the only susceptibility factor on this haplotype. The identity of the DR3-associated susceptibility factor remains unclear but the DQB1*0201 allele is a candidate. If DQB1*0201 is involved, the existence of a protective factor on the neutral DR7-DQB1*0201 haplotypes is indicated. Analysis of DR9 associated susceptibility implicates a non-DR/DQ predisposing factor.

The aetiology of Type I diabetes.

The aetiology of Type I diabetes involves both genetic and environmental factors. The genes implicated are 'susceptibility genes', which modify risk. Individual susceptibility genes may not be required and are not sufficient for disease development. The strongest genetic risk component is encoded within the major histocompatibility complex (MHC) and is designated IDDM I. The HLA-DQ genes contribute to the risk, but so may other MHC-encoded genes. The susceptibility encoded by IDDM2 refers to a variable number of tandem repeats in the insulin gene region. Many other genomic regions have been designated as susceptibility intervals potentially containing candidate genes. Environmental factors appear to be important in disease expression in either a causative or a protective role. Epidemiological data indicate that such factors operate from early in life. Viral infection(s) may have a disease-initiating and/ or accelerating effect. A potential diabetogenic role for cows' milk protein remains unconfirmed. Further research is necessary to elucidate fully the aetiological factors involved and how they interact.

Molecular aspects of type 1 diabetes.

Type 1 diabetes is a T cell mediated autoimmune disease, characterised by the selective destruction of pancreatic beta cells, and susceptibility is determined by a combination of genetic and environmental factors. The environmental agents implicated include viruses and dietary factors, although none has yet been shown to be directly responsible for triggering beta cell autoimmunity. The genetic factors that influence disease risk have been subjected to more intensive study and two gene regions of major importance have been identified: the human leucocyte antigen locus and the insulin gene. This review will focus on the mechanisms by which these genes might influence the risk of developing type 1 diabetes.

Assessment of the non-HLA-DR-DQ contribution to IDDM1 in British Caucasian families: analysis of LMP7 polymorphisms.

AIMS: Whilst HLA-DRB1 and HLA-DQ alleles contribute to IDDM1, the major determinant of genetic susceptibility to Type 1 diabetes mellitus, other major histocompatibility complex (MHC)-encoded genes may also be involved. The LMP7 (large multifunctional proteasome 7) gene is a potential candidate. The aim of this study was to assess whether LMP7 confers susceptibility to Type 1 diabetes independently of linkage disequilibrium with HLA-DRB1 and HLA-DQ. METHODS: The diallelic LMP7 polymorphism (LMP7*A or *B) was determined in 142 multiplex families from the British Diabetic Association Warren Repository. At least one parent was heterozygous for LMP7 in 112 families and these were informative for calculation of the statistic Tsp. This gives a valid chi2 test of the null hypothesis of no association or no linkage. RESULTS: An excess of transmissions of LMP7*A was observed from parents to affected offspring and the Tsp statistic was significant for association in the presence of linkage. LMP7*A was in positive, and LMP7*B in negative, linkage disequilibrium with the HLA-DRB1*03-DQ2, DRB1*04-DQ8 (group of all DRB1*04 subtypes), DRB1*0401-DQ8 and DRB1*0404-DQ8 haplotypes, although the linkage disequilibrium coefficient (delta) value was not statistically significant for DRB1*0404-DQ8. Analysis of HLA-DR-DQ-LMP7 haplotypes and Tsp analysis of HLA-matched-homozygous parents showed no association between LMP7 alleles and Type I diabetes independent of linkage disequilibrium with HLA-DR-DQ haplotypes associated with increased risk of disease. A contribution of LMP7 alleles to susceptibility to Type 1 diabetes in subjects with low-risk HLA-DR-DQ haplotypes could not be excluded. CONCLUSIONS: LMP7 alleles do not contribute to genetic susceptibility to Type 1 diabetes in subjects with high-risk-associated HLA-DR-DQ haplotypes.

The nucleotide sequence of two new DP alleles, DPA1*02015 and DPB1*8401, identified in a Chinese subject.

The HLA-DP genes (HLA-DPA1 and -DPB1) are encoded by the major histocompatibility complex (MHC) on human chromosome 6. They are involved in the presentation of antigen to CD4+ T cells as part of the class II antigen-presentation pathway. During a small study of Oriental subjects (11 Chinese and 26 Japanese subjects), one Chinese subject was identified as having numerous heterozygous sites within the second exon of both DPA1 and DPB1. These were further analysed using novel codon-specific primers. Sequencing analysis using these primers determined the subject to have DPA1*0103/*02015 and DPB1*0501/*8401; these new alleles have been submitted to GenBank and assigned the accession numbers AF098794 and AF077015, respectively.

The nucleotide sequence of a new DMB allele, DMB*0106.

The HLA-DM genes (DMA and DMB) are encoded in the major histocompatibility complex (MHC) on human chromosome 6. They are involved in the loading of antigenic peptide onto class II molecules as part of the class II antigen-processing and -presentation pathway. During a study of Oriental subjects (11 Chinese and 26 Japanese subjects), one Chinese subject was identified as having a new DMB allele using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). This was confirmed with sequencing. The new allele was found to have Glu (GAG) and Thr (ACT) at positions 144 and 179 respectively. This was a previously unpublished combination of polymorphic sites, and was submitted to GenBank and assigned the accession number AF072680, and named HLA-DMB*0106.

Long-term expression of an HLA-DQ molecule in the EBV-transformed bare lymphocyte cell line, BLS-1, using a plasmid vector.

The HLA class II molecule, DQ6, confers strong natural protection against the development of type 1 diabetes. The mechanism of disease protection is unknown, but is likely to be related to the function of the molecule in antigen presentation. In order to investigate this function, we have created an in vitro model which expresses DQ6 in isolation by introducing the relevant DQ alleles into an Epstein-Barr virus (EBV)-transformed, human leucocyte antigen (HLA) class II-deficient B cell line, bare lymphocyte syndrome (BLS)-1. A recent report suggested that the expression of transferred genes in human EBV-transformed B cells might be limited in duration. We present a plasmid-based transfection method that allows long-term stable expression of the DQ molecule. The DQA1*0102 and DQB1*0602 alleles were cloned into the pCIneo expression vector and the constructs were introduced into BLS-1 by electroporation. Stable transfectants were selected using magnetic sorting and cloned by limiting dilution. Two clones were shown to express functionally active DQ6 molecules even after 14 months of continuous culture. These clones will be used in functional studies to investigate the antigen binding and T-cell activation properties of the DQ6 molecule.

HLA-DQ and DRB1 polymorphism and susceptibility to type 1 diabetes in Jamaica.

Genetic susceptibility to type 1 diabetes is determined by a combination of HLA-DQ and DRB1 alleles. In the present study, HLA associations with type 1 diabetes were investigated in the Jamaican population. DRB1 and DQ genotyping was performed on 45 type 1 diabetic patients and 132 control subjects born and resident in Jamaica. The small number of patients available for study reflected the low prevalence of type 1 diabetes in Jamaica. The results were compared with those from other African heritage populations and white Caucasians. The highest relative risk was associated with the DRB1*03-DQ2/DRB1*04-DQ8 genotype. Both DRB1*0401-DQ8 and DRB1*0408-DQ8 were positively associated with disease. DRB1*0408-DQ8 is uncommon amongst white Caucasians, where DRB1*0401-DQ8 is the major predisposing haplotype. The DRB1*1503-DQ6 haplotype was associated with protection from diabetes in the Jamaican population. This haplotype is rare amongst white Caucasians, where DRB1*1501-DQ6 is the protective haplotype. Data from African heritage populations suggest that DRB1*1503-DQ6 might be less protective than DRB1*1501-DQ6. DRB1*03-DQA1*0401-DQB1*0402 was associated with protection from diabetes in the Jamaican population, whereas in white Caucasians DRB1*08-DQA1*0401-DQB1*0402 is predisposing. These data demonstrate that comparison of genetic associations with type 1 diabetes in races with population-specific DRB1-DQ haplotypes provides new information as to the exact determinants of disease susceptibility. Further support is provided for roles of the DQ genes and the DRB1 gene (or a gene in linkage disequilibrium with it) in determining susceptibility to type 1 diabetes.

Tumour necrosis factor-beta polymorphism is unlikely to determine susceptibility to type 1 (insulin-dependent) diabetes mellitus.

Tumour necrosis factor gene polymorphism has been proposed as a determinant of Type 1 (insulin-dependent) diabetes mellitus. Tumour necrosis factor-beta gene polymorphisms were analysed in 40 North Indian Asian Type 1 diabetic patients and 63 control subjects. A 5.5 kilobase gene fragment was significantly increased among the patients (82.5% vs 52%, pc less than 0.01). A 10.5 kilobase fragment was significantly reduced among the patients (70% vs 90.5%, pc less than 0.02). The 5.5 kilobase fragment was associated with DR3, and was not significantly increased among DR3-positive patients compared with DR3-positive control subjects. The 5.5 kilobase/5.5 kilobase genotype was increased among the diabetic subjects (30% vs 9.5%, pc less than 0.03). The 10.5 kilobase/10.5 kilobase genotype was reduced among the diabetic subjects (17.5% vs 47.5%, pc less than 0.02). The 5.5 kilobase/10.5 kilobase genotype was not significantly associated with disease. These findings contrast with those in a white Caucasian population, suggesting that tumour necrosis factor-beta polymorphisms do not predispose to Type 1 diabetes directly, but are in linkage disequilibrium with disease susceptibility alleles at other MHC loci.