Satoshi Moriyama

Sequence-based association analysis of HLA class I and II alleles in Japanese supports conservation of common haplotypes

Abstract Alleles of HLA-A, B, C, DRB1, DQB1, and DPB1 loci were fully determined in 117 healthy Japanese. A*2402, A*3303, A*1101, A*0201, B*4403, B*5201, Cw*0102, Cw*1403, Cw*0304, Cw*0702, Cw*0801, and Cw*1202 showed frequencies of over 10%. Multi-locus haplotype frequencies were estimated by the maximum likelihood method. Strength of association between C and B loci was comparable with that between DRB1 and DQB1 loci. Alleles unidentified by a serological method and having very similar nucleotide sequences (A2: A*0201, A*0206, A*0207, B61: B*4002, B*4006) were carried by different haplotypes. Several frequent five-locus haplotypes were identified including A*3303-Cw*1403-B*4403-DRB1*1302-DQB1*0604, and A*2402-Cw*1202-B*5201-DRB1*1502-DQB1*0601. These sequence-based haplotypes corresponded to serology-based common haplotypes which have already been described in Japanese. These findings indicate that common HLA haplotypes consist of particular sets of HLA alleles and that these haplotypes have been conserved through recent human evolution.

MIC-A polymorphism in Japanese and a MIC-A-MIC-B null haplotype.

Abstract A polymorphic gene, MIC-A, is one of the MIC family of genes which is composed of a group of homologous genes interspersed in the class III and class I regions of the major histocompatibility complex. MIC-A is located 46 kilobases (kb) centromeric of HLA-B, and is preferentially expressed in the epithelial cells and intestinal mucosa. Recently, MIC-A and the closely related MIC-B were reported as the molecules that conferred specificity in the recognition by the Vδ1γδT cells. In the present study, polymorphic exons 2, 3, and 4 of the MIC-A gene were analyzed using the polymerase chain reaction-single-strand conformation polymorphism method. The number of patterns found in exons 2, 3, and 4 were 5, 6, and 4, respectively, in 114 healthy Japanese subjects. Eight MIC-A alleles were observed in Japanese individuals, among which one, tentatively named MIC-AMW, has not previously been reported. There was a strong linkage disequilibrium between MIC-A and HLA-B loci: each MIC-A allele showed strong association with a particular HLA-B group. In contrast, B*3901 showed association with multiple MIC-A alleles. Furthermore, the existence of a MIC-A-MIC-B null haplotype, which is associated with HLA-B*4801, was identified. In this haplotype, a large-scale deletion (of approximately 100 kb) including the entire MIC-A gene was indicated and the MIC-B gene possessed a stop codon.

Purification and Properties of an Extracellular Exoinulinase from Penicillium sp. Strain TN-88 and Sequence Analysis of the Encoding Gene

An exoinulinase, P-I, was purified from the culture filtrate of Penicillium sp. strain TN-88 grown on inulin. The enzyme was homogeneous as judged by SDS-polyacrylamide gel electrophoresis with an apparent Mr of 81 kDa. The purified enzyme had extremely high specific activity, 743 U/mg, toward inulin. Inulinase activity was optimal at pH 4.0 and 55°C. A genomic DNA and cDNAs encoding this protein were cloned and sequenced. The exoinulinase gene (inuD) was present as a single copy in the genome. An open reading frame of 2,106 bp was interrupted by a single intron of 56 bp, and encoded a 25-amino acid signal peptide and a 677-amino acid mature protein. The mature protein contained two Cys residues and eight potential N-linked glycosylation sites. The 5′-noncoding region had a putative CAAT box at position −239. Four distinct transcription start points were observed at positions −98 (A), −91 (A), −80 (A), and −76 (A) from the start codon. The exoinulinase gene inuD was located 860-bp upstream of the previously reported endoinulinase gene inuC in the opposite direction of transcription.

MICA allele typing of HLA-B27 positive Japanese patients with seronegative spondylarthropathies and healthy individuals : Differential linkage disequilibrium with HLA-B27 subtypes

OBJECTIVE To examine whether MICA (major histocompatibility complex [MHC] class I-related chain gene A) confers additional susceptibility for seronegative spondylarthropathies in HLA-B27 positive Japanese individuals. METHODS A polymerase chain reaction-single-strand conformation polymorphism method was developed, and the MICA alleles of 18 Japanese patients with ankylosing spondylitis, 1 patient with Reiters syndrome, and 17 healthy HLA-B27 positive Japanese subjects were determined. RESULTS Among 26 individuals with HLA-B*2704 (13 patients and 13 healthy subjects), all except 1 healthy individual were positive for MICA010, whereas all 9 HLA-B*2705 positive subjects (6 patients and 3 healthy subjects) possessed MICA007. One healthy individual with HLA-B*2711 also carried MICA010. CONCLUSION Strong linkage disequilibrium is present between HLA-B*2704 and MICA010, as well as between HLA-B*2705 and MICA007. Although HLA-B*2704 and B*2705 are highly homologous, each subtype participates in a different MHC haplotype. Direct involvement of MICA polymorphism in the pathogenesis seems to be unlikely; however, such information will provide a useful tool for elucidating the evolutional pathway of HLA-B27 subtypes as well as the contribution of other genes within the MHC region in the pathogenesis of these diseases.

MULTIPLEX ARMS‐PCR‐RFLP METHOD FOR HIGH‐RESOLUTION TYPING OF HLA‐DRB1

A reliable method for high‐resolution HLA‐DRB1 typing using the combination of group‐specific amplification and RFLP analysis is described. Group‐specific PCR amplification (multiplex ARMS‐PCR) was carried out under the same conditions for all groups using seven different primer pairs divided into four groups: (1) DR1 and DR10; (2) DR2, DR7 and DR9; (3) DR3, DR5, DR6 and DR8, and (4) DR4. The subsequent polyacrylamide gel electrophoresis was used to determine the group(s) contained in each sample. DR1, DR2/7, DR3/5/6/8, DR4, DRB1*0901 and DRB1 * 1001 could be distinguished easily using this system. Computer analysis of the various restriction enzyme cleavage sites was carried out on 105 DRB1 allele sequences. It was shown that all DRB1 alleles, except for five allele pairs and some alleles possessing silent mutations, could be distinguished with commonly available restriction endonucleases. Computer analyses on the discrimination of the heterozygous and homozygous combinations were also carried out. Although some heterozygous combinations could not be distinguished with single digestion, double digestion using two restriction enzymes could distinguish most of such heterozygotes. The results of the typing of 100 Japanese individuals using this method showed good agreement with those obtained by other methods.

EVALUATION OF QUALITIES ON A CLOSED-BAG SYSTEM WITH WHOLE BLOOD LEUKOCYTE FILTER (WBF2) FOR PRE-STORAGE LEUKOREDUCTION

In order to avoid a wide variety of side effects associated with blood transfusion such as nonhemolytic febrile transfusion reactions (NHFTR), HLA-sensitization and Cytomegalovirus infection. Leukocyte filters have come into wide use in bedside blood transfusion in Japan. During the storage of blood components, however, residual leukocytes disintegrate and release various biologically active substances, which constitute one of the causes of transfusion-related adverse events. Therefore, removal of leukocytes immediately or at an early stage after blood collection is expected to decrease the incidence of transfusion-related side effects by controlling the deterioration of blood cells or preventing the accumulation of cytokines.The WBF2 closed-bag system is a blood collection-component separation system designed for the removal of leukocytes from whole blood and the preparation of blood components by centrifugation. In the present study, we evaluated the usefulness of the system in the preparation of leukocyte-reduced RC-MAP and FFP using WBF2, and compared the quality of these preparations with those by conventional post-storage leukoreduction or non-leukoreduction. The WBF2 system yielded red cell components with superior red cell recovery and lower accumulation of some cytokines, demonstrating its suitability for use in a blood center program. It was also noted that there was almost no macroaggregation in red cell components prepared by prestorage leukoreduction.