Nobuhisa Mizuki

HLA class II molecules and autoimmune hepatitis susceptibility in Japanese patients

To investigate the association between autoimmune hepatitis and HLA alleles in Japanese patients, serological typing and class II genotyping were performed using the polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLP) method. Serological typing showed that HLA-B54, -DR4, -DR53, and -DQ4 were significantly more frequent in patients with autoimmune hepatitis than in controls. HLA-DR4 was most frequently associated with autoimmune hepatitis (88.7%). In PCR-RFLP typing, the frequency of DRB1*0405 was significantly higher in autoimmune hepatitis than in controls. However, there was no significant difference in the frequency of Dw between the patients and the controls who were DR4-positive. The significant increase observed in DQA1*0301 and DQB1*0401 was explained by a linkage disequilibrium with DR4. Six DR4-negative patients had DR2, but there was no significant difference in the frequency of the DR2-associated Dw-alleles compared with the DR2-positive controls. No DPB1 allele was significantly associated with autoimmune hepatitis. These findings suggest that the basic amino acid at position 13, which is present only on the DR2 and DR4 B1 molecules (Arg on DR2 and His on DR4), contributes to the susceptibility to autoimmune hepatitis among the Japanese.

Allelic repertoire of the humanMHC class IMICA gene

The hallmark of the classical major histocompatibility complex (MHC) class I molecules is their astonishing level of polymorphism, a characteristic not shared by the nonclassicalMHC class I genes. A distinct family ofMHC class I genes has been recently identified within the humanMHC class I region. TheMICA (MHC classI chain-relatedA) gene in this family is a highly divergent member of theMHC class I family and has a unique pattern of tissue expression. We have sequenced exons encoding the extracellular α1, α2, and α3 domains of theMICA gene from twentyHLA homozygous typing cell lines and four unrelated individuals. We report the identification of eleven new alleles defined by a total of twenty-two amino acid substitutions. Thus, the total number ofMICA alleles is sixteen. Interestingly, a tentative superimposition ofMICA variable residues on theHLA-A2 structure reveals a unique pattern of distribution, concentrated primarily on the outer edge of the MICA putative antigen binding cleft, apparently bordering an invariant ligand binding site.

NUCLEOTIDE SEQUENCE OF THE HUMAN MHC CLASS I MICA GENE

In addition to the closely related genes encoding the conventional class I peptide-presenting molecules, the major histocompatibility complex (MHC) of humans and most mammals contains one or several highly diverged class I-like genes representing a second class I gene family (Bahram et al. 1994). In humans, two members of this gene family, MICA and MICB, are encoded near HLA-B and encode mRNA transcripts with intact long open reading frames (Bahram et al. 1994; Bahram and Spies 1996). Additional similar sequences, MICC, MICD, and MICE are widely spaced throughout the 2 megabase class I region and are pseudogenes because of several point mutations and/or gross deletions (S. Bahram and T. Spies, unpublished). Here we report the complete nucleotide sequence of the MICA gene comprising 11 722 basepairs (bp) of DNA 40 kilobases centromeric of HLA-B. The sequence was obtained from single (M13)and double (pUC19)-stranded templates of mapped or randomly shot-gun subcloned DNA fragments that were derived from the cosmids M32A (Spies et al. 1989) and pM67 (N. Mizuki and H. Inoko, unpublished). The first exon encoding the leader peptide is followed by an intron of 6840 bp, which is unusually large for a class I gene. The remainder of the MICA gene shows an organization quite similar to that of conventional class I genes, except for the presence of a relatively long intron following the transmembrane exon and the fusion of the cytoplasmic tail and 39 untranslated sequence in a single last exon. The translated amino acid sequence (not shown) corresponds to the previously identified MICA4 allele. Together with the recently documented sequence of an MICB cDNA (Bahram and Spies 1996), these data complete the primary genetic characterization of the highly diverged MHC class I MICA and MICB genes.

The Critical Region for Behcet Disease in the Human Major Histocompatibility Complex Is Reduced to a 46-kb Segment Centromeric of HLA-B, by Association Analysis Using Refined Microsatellite Mapping

The HLA-B51 allele is known to be associated with Behçet disease. Recently, we found a higher risk for Behçet disease in the MICA gene, 46 kb centromeric of HLA-B, by investigation of GCT repetitive polymorphism within exon 5 of MICA. The pathogenic gene causing Behçet disease, however, has remained uncertain. Here, eight polymorphic microsatellite markers, distributed over a 900-kb region surrounding the HLA-B locus, were subjected to association analysis for Behçet disease. Statistical studies of associated alleles detected on each microsatellite locus showed that the pathogenic gene for Behçet disease is most likely found within a 46-kb segment between the MICA and HLA-B genes. The results of this mapping study, and the results of an earlier study of ours, suggest that MICA is a strong candidate gene for the development of Behçet disease.

Immunology and functional genomics of Behçet's disease

Behçets disease (BD) is a multisystemic inflammatory disorder. Although the cause and pathogenesis of BD are still unclear, there is evidence for genetic, immunologic and infectious factors at the onset or in the course of BD. This review focusses on the functional genomics and immunology of BD. HLA-B51 is the major disease susceptibility gene locus in BD. An increased number of γδ T cells in the peripheral blood and in the involved tissues have been reported. However, the T cells at the sites of inflammation appear to be a phenotypically distinct subset. There is also a significant γδ T cell proliferative response to mycobacterial 65-kDa heat shock protein peptides. Homologous peptides derived from the human 60-kDa heat shock protein were observed in BD patients. There is evidence that natural killer T cells may also play a role in BD.

Behcet's Disease Associated With one of the HLA-B51 Subantigens, HLA-B* 5101

The strong association of Behçets disease with HLA-B51 in several ethnic groups is well known. Because the HLA-B51 antigen has been recently identified to comprise three alleles, HLA-B* 5101, HLA-B* 5102, and HLA-B* 5103, we sought to investigate whether there is any correlation of one particular allele among them with B51-positive patients with Behçets disease. Forty-six Japanese patients with Behçets disease and HLA-B51 were typed by using the alloantisera, which allowed the subdivision of B51 antigen by the microlymphocyte toxicity assay. All the patients were found to carry HLA-B* 5101. This result suggests that amino acid substitutions at residue 167 or 171 prevent the development of Behçets disease, because HLA-B* 5101 differs from HLA-B* 5102 and HLA-B* 5103 by single amino acid substitution at residues 171 and 167, respectively, or that another non-HLA gene tightly linked to the HLA-B* 5101-associated haplotype around the HLA class I gene region is responsible for the susceptibility to Bechçets disease. This study provides insight into the molecular mechanism underlying an HLA association with Behçets disease.

Pathogenic Gene Responsible for the Predisposition to Behçet's Disease

HLA-B51 is well known to be associated with Behçets disease (BD) in many different ethnic groups. The hypothesis may be presented that B51 molecules are primarily involved in BD development through specific antigen presentation. Furthermore, HLA-C genotyping by the polymerase chain reaction-sequence specific primers method suggests that the BD pathogenic gene is not the HLA-C gene itself but some other gene located near the HLA-B gene. Polymorphic analysis of the Tau-a microsatellite between the HLA-B and TNF genes indicates that the pathogenic gene of BD is not the HLA-B51 gene itself but other gene located around the HLA-B gene. Recent studies suggest that many novel genes exist in the region between the TNF and HLA-B or HLA-C genes such as MIC and PERB, etc. and furthermore, many unidentified new genes have been suggested to exist in this region. In this paper, the present situation of the investigations on the genetic predisposition responsible for BD was reviewed.

Microsatellite polymorphism between the tumor necrosis factor and HLA-B genes in Behçet's disease

Behçets disease is associated with the HLA-B51 antigen. However, it has not yet been clarified if the HLA-B51 gene itself is the susceptibility gene related to this disease or if it is some other non-HLA gene in linkage disequilibrium with HLA-B51. Therefore, we screened one of the HSP70 genes, HUM70t (HSP70-Hom), around the class III region and the microsatellite sequence located between the HLA-B and TNF genes for genetic polymorphism in BD. A comparison between patients with BD and healthy controls revealed no significant difference in the frequency of the HUM70t polymorphism. In the microsatellite sequence, Tau-a, in the region between the HLA-B and TNF genes, the frequency of 14 repetitions of GT was increased significantly and that of 11 repetitions was decreased significantly in the patient group. Further, the allelic distributions of the B51 antigen-associated microsatellite polymorphism differed significantly between patients and healthy controls, and in the B51 antigen-negative subjects, analysis of the microsatellite polymorphism also revealed a significant difference in the haplotype frequency between the patient and control groups. These results suggest that the HLA-B51 gene may not be the primary locus responsible for BD, and implicate some other gene(s) located between the TNF and HLA-B genes.

Analysis of genes within the HLA region affecting susceptibility to ulcerative colitis

To clarify the molecular relationship between HLA loci and ulcerative colitis (UC) in Japanese patients, we performed HLA-DP genotyping by the PCR-RFLP method and studied tumor necrosis factor beta-chain genetic polymorphism by Southern hybridization, in addition to conventional serologic typing. Significant increase was observed in Bw52, DPw9 (DPB1*0901), and DR2 (DRB1*1502) in Japanese patients with UC. Linkage analysis indicated that A24-Bw52-DR2-DPw9 alleles constitute a common haplotype in Japanese UC patients. Among the patients not carrying Bw52, B13 was significantly increased and B44 was relatively increased. These Bw52, B13, and B44 alleles share the unique amino acids, serine and aspartic acid at positions 67 and 77, respectively. These positions are in the second hypervariable region of the alpha 1-domain of the HLA-B13, B44, Bw52, and B49 antigens (B49 is quite rare in the Japanese population). The inflammatory region in UC patients was found to vary depending on their HLA-B alleles. These results suggest that the HLA-B locus itself plays an important role in the susceptibility to Japanese UC.

Allelic variants of the human MHC class I chain-related B gene (MICB).

Abstractu2003The human major histocompatibility complex (MHC) is located within a 4 megabase segment on chromosome 6p21.3. Recently, a highly divergent MHC class I chain-related gene family, MIC was identified within the class I region. The MICA and MICB genes in this family have unique patterns of tissue expression. The MICA gene is highly polymorphic, with more than 20 alleles identified to date. To elucidate the extent of MICB allelic variations, we sequenced exons 2 (α1), 3 (α2), 4 (α3), and 5 (transmembrane) as well as introns 2 and 4 of this gene in 46 HLA homozygous B-cell lines. We report the identification of eleven alleles based on seven non-synonymous, two synonymous, and four intronic nucleotide variations. Interestingly, one allele has a nonsense mutation resulting in a premature termination codon in the α2 domain. Thus, MICB appears to have fewer alleles than MICA, not unlike the allelic ratio between the HLA-C and -B loci. A preliminary linkage analysis of the MICB alleles with those of the closely located MICA and HLA-B genes revealed no conspicuous linkage disequilibrium between them, implying the presence of a potential recombination hotspot between the MICB and MICA genes.