No major monoclonal lymphocyte population in the thyroid of patients with Graves' disease: study of gene rearrangement by restriction fragment length polymorphism and polymerase chain reaction.

The present study aimed at determining the mono-, oligo-, or polyclonal nature of intrathyroid lymphocytes at the DNA level in patients with Graves' disease. Two techniques were used to seek monoclonal rearrangement in DNA derived from intrathyroidal lymphocytes obtained from six patients. The first was restriction fragment length polymorphism using two specific probes from the B-chain of T-cell receptor and the other from the heavy chain immunoglobulin gene; the second was polymerase chain reaction using a couple of specific primers from the variable and joining regions of heavy chain immunoglobulins. The results for the patients with Graves' disease were compared with those obtained for circulating T-and B-lymphocytes, granulocytes (negative controls), and T- and B-leukemic cells (positive controls). The results with restriction fragment length polymorphism favored a polyclonal origin for the lymphocytes in all cases, since no rearrangement was visualized. The results with polymerase chain reaction were analogous, and the technique was 10 times more sensitive in the detection of rearrangement.

Implication of the HLA-DRB3 gene in Graves' disease: predominance of allele Dw24.

Using RFLP, the present study sets off to determine the MHC class II gene polymorphism in Graves' disease, in order to define the HLA-related genetic susceptibility. Considering the preferential link between Graves' disease and the HLA-DR3 antigen, 42 HLA-DR3 Graves' disease patients were studied and compared with 42 HLA-DR-matched controls. Hybridization with a DQ alpha probe of DNAs digested by Taq I revealed a polymorphism of the DR3 haplotype with an overrepresentation of a 2.1 kb(U) fragment in patients, but this was merely a sign of the linkage disequilibrium between U and B8DR3. Hybridization with the DR beta probe of DNAs digested by Taq I yielded more facts. It revealed the overrepresentation of the Dw24 specificity (Taq I:9.8 kb) in DR3 Graves' disease patients. This study thus enabled us to determine precisely the susceptibility linked to the DR3 haplotype, implicating the DRB3 gene and its Dw24 allele, which appear to be the most reliable markers of the disease, providing a higher relative risk than B8DR3.

HLA class II gene polymorphism in Tunisians.

The polymorphism of HLA class II genes (HLA-DRB, DQB, DPB) was investigated in 101 Tunisians using polymerase chain reaction. (PCR) amplification and reverse dot blot (RDB) hybridization. Allele and haplotype frequencies, as well as DRB1-DQB1 linkage disequilibria, were calculated. A total of 26 DRB1 alleles were detected and the most prevalent variant was DRB1*0301 with an allelic frequency at 21.87%. In the DR1 group, DRB1*0102 was most frequent than DRB1*0101. In the DR4 group, DRB1*0403 was the most common allele and was associated with DQB1*0402. Interestingly this DRB1-DQB1 association has not been observed in other populations. With regard to the DR8 group, DRB1*0804 was the unique variant detected, whereas with the DR13 specificity, the most common variant was DRB1*1303 in Algerians also. Although the DQB1 polymorphism analysis showed an allelic distribution very close to that observed in caucasoids, many DRB1-DQB1 associations which have not been reported in studies of other populations, were described. Finally at the DPB1 locus DPB1*1701 and *1301 allele frequencies distinguish clearly this Tunisian sample from a French caucasoïd panel of 83 subjects. In conclusion, a specific distribution of HLA components in terms of gene and haplotype frequencies characterizises this Tunisian population. This specific pattern may reflect the great ethnic diversity of this community. All these informations may be helpful in the future for HLA and disease association studies.

Role of HLA-DR-DR and DR-DQ associations in the expression of extraarticular manifestations and rheumatoid factor in rheumatoid arthritis.

OBJECTIVE: To investigate the role of HLA-DRB1 genotypes and HLA-DRB1*0401-DQB1*03 haplotypes in the expression of extraarticular manifestations and rheumatoid factor (RF) in rheumatoid arthritis (RA). METHODS: 189 patients with RA were classified according to the presence of vasculitis and seropositivity for RF. IgM RF were determined by at least 2 of the following methods: standard latex agglutination, the Waaler-Rose test, and nephelometry. HLA genotyping was performed by reverse dot blot hybridization for DRB1 alleles and the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method for DQB1 alleles. RESULTS: The QKRAA susceptibility sequence, which characterizes the HLA-DRB1*0401 allele, was observed in 71.5% of the 21 patients with vasculitis and 57.6% of the 158 RF positive patients. The influence of a 2nd allele within the major histocompatibility complex was observed but the allele differed according to the clinical features examined. Higher risk for vasculitis was observed in patients who carried 2 DRB1 susceptibility alleles, one characterized by the QKRAA sequence and the other by the QRRAA sequence (OR = 3). Conversely, the higher risk for IgM RF positivity was observed in patients who carried the QKRAA sequence of the DRB1 alleles with the DQB1*0301 alleles of the DQ region (OR = 4.7). CONCLUSION: Our data suggest that a distinct immunogenetic association is involved in the extraarticular manifestations of RA and in the expression of RF.

DPB1 polymorphism in rheumatoid arthritis: evidence of an association with allele DPB1 0401.

HLA-DP polymorphism was examined in 71 rheumatoid arthritis patients and 148 controls, using dot-blot analysis with 14 synthetic oligonucleotide probes specific for the variable region of the DPB1 second exon. The DPB1 0401 allele was found to be significantly more frequent in RA patients than in controls (77.46% vs 55.40%, p less than 0.002, pc less than 0.03, relative risk value: 2.74). An association between DPB1 0401 and seropositivity for rhumatoid factors was also observed: 44 of the 55 seropositive RA patients were DPB1 0401 (p less than 0.001). Analyzing the HLA DPB1 alleles frequencies in 57 HLA-DR-typed RA patients did not show any linkage between the DPB1 0401 and the DR4 specificities. Furthermore, the DPB1 0401 homozygous frequency was increased in DR4-negative RA patients. Our findings suggest an independent role of the DPB1 0401 allele in the genetic susceptibility to RA.

HLA class I and class II allele and haplotype diversity in Martinicans.

The Martinican population is mainly the product of admixture between African people and French Caucasians. The aim of the present study is to investigate at the DNA level the polymorphism of HLA class I (HLA-A, HLA-B) and class II (HLA-DRB1, DQB1 and DPB1) genes in a population of 100 Martinicans. Allelic distributions and interlocus linkage disequilibria were compared to those observed in a French Caucasian population and in African or North American African populations. Our data revealed a higher degree of polymorphism in Martinicans than in Caucasians and showed a prominant contribution of African origin in the admixed genetic feature of this population. African characteristic alleles were significantly represented in Martinicans: A*30, *33 *34, *66, *74, *8001, B*1510, *35, *42, *53, DRB1*0302, *0804, *1202, *1304, *1503, DPB1*0101, *1701, *1801, *3901. Moreover a higher diversity of A*-B* and DRB1*-DQB1* associations was observed in Martinicans compared to Caucasians which also reflects the African genetic background of this population. In the whole, using PCR-based genotyping methods for HLA class I and class II loci, this study allows a preliminary description of HLA allele distribution in this Caribbean island and gives new elements which may be helpful in the anthropologic field as well as in HLA and disease association studies.

Definition of DRw10 specificity by restriction fragment length polymorphism.

The purpose of this study was the RFLP characterization of the DRw10 specificity. Twenty-two DRw10 cells were tested: the DNAs were digested by seven restriction enzymes and hybridized with DR beta, DQ beta and DQ alpha probes. Hybridization with DR beta revealed a pattern particular to the DRw10 specificity, with a specific TaqI 12.5Kb fragment. Hybridization with both DQ-specific probes showed that DRw10 is always associated with a special DQw1 subtype: DQw5. Furthermore, at DR and DQ levels, the 22 DRw10 cells behaved homogeneously.

Investigation of the association of major histocompatibility complex genes, including HLA class I, class II and TAP genes, with clinical forms of Crohn's disease.

The aim of this study was to determine immunogenetic markers of susceptibility in Crohn's disease (CD), taking the different features of the clinical course of the disease into account. HLA class I, HLA class II and TAP transporter gene polymorphisms were studied using DNA typing methods. Gene and antigen frequencies were analysed and compared in a group of 102 CD patients and 200 unrelated healthy controls from the same area. Analysis of the whole CD patient population revealed no definite association with either HLA or TAP gene alleles, with the exception of an association with DRB1*1302 (Pc < 0.05). However, when clinical subgroups of patients were considered, specific associations with some genetic markers were found. The most definitive results involved a genetic association in the group of patients who did not respond to glucocorticoid therapy. This group was characterized by a high frequency of HLA-DRB1*04 (P < 0.05). Conversely, a positive association with the TAP2-A allele was found in cortico-responder patients (Pc < 0.03). Furthermore, analysis of the distribution of HLA class II alleles in relation to the presence of extra-intestinal manifestations revealed an association with the DQB1*0501 or *0503 suballele of DQ5 (P < 0.05). Finally, patients with lesions in the small bowel were more frequently HLA DRB1*07 (P < 0.05). The present study supports the concept of clinical heterogeneity in Crohn's disease associated with a background of genetic heterogeneity.