Heard, Robert Nigel Stewart; (1991) Studies on the molecular immunogenetics of class II histocompatibility antigens in multiple sclerosis. Doctoral thesis (M.D), UCL (University College London).

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Abstract

Serological studies of HLA antigens in a number of different populations over many years have clearly demonstrated that MS is associated with particular products of the Major Histocompatibility Complex Class II region, particularly DR2/Dw2 and DQw1. It is widely accepted that immune mechanisms are responsible for the demyelination seen in MS, and there is a good deal of circumstantial evidence in favour of an autoimmune pathogenesis. Furthermore, it is now well established that MHC gene products play a pivotal role in T cell activation, and this raises the possibility that a true disease susceptibility gene might be encoded in the MHC. It has also been suggested that the rate of disease progression might be influenced by the MHC in a similar way. Since the genes encoded by the MHC exhibit at the genetic level an extraordinary degree of polymorphism, much of which is not detectable using serological techniques, genetic methods have been employed to investigate further the association between MS and the Class II region. An extensive analysis of restriction fragment length polymorphism of the Class II region was carried out in an attempt to identify genetic markers more strongly associated with MS than the classical serological markers. Initial studies using pooled DNA samples from patients and controls from the Grampian region of Northeast Scotland enabled the screening of 14 restriction endonucleases with five HLA-D region probes (DPA, DPB, DQA, DQB and DRB). A small number of discriminatory polymorphisms were observed with the DQ probes, although none with DP or DR. Based on these data, further studies were carried out on individual samples of DNA from 33 MS patients and 48 controls from the Grampian region. By identifying a number of DQA and DQB fragments of known specificity, it could be demonstrated that almost all of the antigens that typed serologically as DR2 and DQw1 were encoded by the Dw2 DQw6 alleles of DR and DQ, and also that these alleles were represented equally in both patients and controls. However, following Msp1 digestion and hybridization to a DQA1 probe a cluster of fragments was observed significantly more frequently in the patients (p<0.001), and furthermore, this association was found to be independent of DR2. These findings were confirmed in an independently conducted collaborative study in Northern Ireland. The DQA restriction fragment duster was further characterized by means of established RFLP allogenotyping systems. The cluster was seen in all DRw8 (DQw4) homozygous cell lines included in the Tenth International Histocompatibility Workshop, and in some DR4 and DR7 lines, but not in caucasoid DR2 lines. In a panel of healthy British donors, the cluster was again seen most commonly in association with DQw4 or E>Qw8. The apparent allelism thus demonstrated by the cluster raised the possibility that disease susceptibility was associated with transcomplementation between the DR2-positive and DQA cluster-positive haplotypes. Since the putative allele marked by the DQA cluster possessed many of the features of DQw4, an allele which is thought to be uncommon in caucasoids, an attempt to demonstrate this directly using monoclonal serology was made. A number of lymphoblastoid cell lines were raised from Scottish MS patients and their families, and studied using the DQw4 β chain-specific antibody HU46. However, the relation between the cluster and HU46 reactivity, though close, was not exact. Although 5 out of 6 HU46-reactive cell lines were cluster-positive, 3 out of 8 cluster-positive lines were HU46 non-reactive. In order to demonstrate that the DQA cluster corresponded to an expressed DQ α polymorphism, attempts were made to raise alloreactive T cell clones against cluster-positive cell lines. Using cloning strategies designed to eliminate the possibility of generating clones against the 'cluster-negative' haplotype, a number of CD4-positive clones were raised and maintained in stable culture. However, analysis of the fine specificities of these clones failed to demonstrate reactivity corresponding exclusively to the DQA cluster. DQα1-encoding exons from 3 cluster-positive Scottish MS patients, 2 unaffected siblings from multiple case families and 2 cluster-positive homozygous cell lines were amplified enzymatically by the polymerase chain reaction, and probed with allele-specific oligonucleotides. The data obtained suggested that in each case, the cluster-positive haplotype contained the DQA allele predicted by the linked DR genotype, and thus that the DQA cluster did not correspond to an expressed member of the DQA allelic series. This conclusion was confirmed by analysing the nucleotide sequences of PCR-amplified DQA α1-encoding exons from selected cluster-positive haplotypes. Taken together, these data suggest that an MS-associated polymorphism, detectable by RFLP analysis, lies in noncoding regions adjacent to the DQA gene.

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