Akio Yamane

New polymorphisms of human CD80 and CD86: lack of association with rheumatoid arthritis and systemic lupus erythematosus

The crucial role of costimulatory molecules, CD28, CTLA-4, CD80 and CD86, for T cell activation and inhibition has been established. In the previous study, we reported the results of a polymorphism screening of human CTLA-4 gene. In this study, we screened for polymorphisms of human CD28, CD80 and CD86 genes, and detected that polymorphisms were tested for the association with rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). Variations were identified in the coding regions of CD80 (452G/A, 614C/G and 864A/G) and CD86 (1057A/G), while no variation was observed in the coding region of CD28. The variations at CD80 position 452 and CD86 position 1057 were present in a substantial proportion of the Japanese population, and were considered to be single nucleotide polymorphisms within the coding sequence (cSNPs). CD80 864 (G→A) leads to the amino acid substitution N186D, and CD86 1057 (A→G) results in A304T substitution. Furthermore, in the analysis of CD80 5′-flanking region, six SNPs, −454C/A, −387T/C, −232G/A, −79G/C, −7T/C and +5C/A, and one insertion, −558ins (CATGA), were identified. The combination of these variations was found to constitute four promoter alleles of CD80. None of the observed variations was significantly associated with RA or SLE. Further studies will be of particular interest to examine the functional difference of the promoter alleles for the transcriptional activity of CD80, as well as the evolutionary pathway of the four alleles.

Determination of granulocyte-specific antigens on neutrophil FcA receptor IIIb by PCR-preferential homoduplex formation assay, and gene frequencies in the Japanese population.

Background and Objectives: Granulocyte‐specific antigens play an important role in provoking immune neutropenia and transfusion reactions. We developed a new DNA‐typing method, PCR‐preferential homoduplex formation assay (PHFA), to determine granulocyte‐specific antigens on the neutrophil Fcγ receptor IIIb (FcγRIIIb, CD 16b), namely, the NA1, NA2, and SH antigens and their gene frequencies in the Japanese population. Materials and Methods: Four hundred unrelated healthy Japanese blood donors were typed using PCR‐PHFA. To confirm the accuracy of the results of FcγRIIIB genotyping using PCR‐PHFA, PCR‐sequence‐specific primer (SSP) typing and PCR‐restriction fragment length polymorphism (RFLP) typing were carried out in another 20 samples for comparison. Results: The results of PCR‐PHFA typing agreed well with other methods. The frequencies of the FcγRIIIB alleles were 62.2, 37.8, 0 and 0% for NA1, NA2, SH, and ‘NA‐null’, respectively. Conclusion: The PCR‐PHFA method can be semi‐automated easily with computer‐based assignment and is suitable for typing both small and large numbers of samples. In the Japanese population, the frequency of NA1 is about double that in Caucasians (32.5%), and the SH allele is rare.

Nomenclature of Granulocyte Alloantigens

and the detection of new ones has made a new nomenclature for these antigens necessary. Neutrophil alloantibodies are known to cause neonatal alloimmune neutropenia, immune neutropenia after bone marrow transplantation, refractoriness to granulocyte transfusions, and severe transfusion-related acute lung injury. With respect to the increasing use of granulocyte transfusions this is of special importance, since alloimmunization to granulocyte antigens can decrease the recovery and half-life of transfused granulocytes, can prevent their migration to sites of infection and can cause severe pulmonary transfusion reactions. The Granulocyte Antigen Working Party of the ISBT agreed at their meeting in S’Agaró, Spain, in 1998, to establish a new nomenclature system for well-defined neutrophil alloantigens, which is based on the following rules: (1) Neutrophil antigens are to be called HNA for ‘human neutrophil antigen(s)’. (2) MemISBT Working Party

Large-scale DNA typing for human platelet alloantigens by PCR-PHFA (preferential homoduplex formation assay)

Alloimmunization against human platelet alloantigens (HPA) is known to be involved in disorders such as neonatal alloimmune thrombocytopenic purpura, post‐transfusion purpura, and refractoriness to platelet transfusion therapy. HPA typing is essential in diagnosis and management of patients. Therefore a reliable and speedy method is necessary for HPA typing. We have successfully applied a new DNA typing method, PCR‐preferential homoduplex formation assay (PHFA) method, to typing for the HPA‐1, ‐2, ‐3, ‐4, ‐5 and ‐6 systems. This method is based  on DNA strand competition during hybridization under a precisely controlled temperature gradient between a double‐labelled amplicon (standard DNA), prepared from biotin‐ and DNP‐labelled primers, and an unlabelled amplicon (sample DNA). The results obtained by PCR‐PHFA typing were in good agreement with the allotypes determined by serological typing and by other DNA typing methods. The PCR‐PHFA method can be easily automated, is suitable for typing both small and large numbers of samples, and thus is applicable to routine HPA typing.

A Colorimetric DNA Diagnostic Method for Falciparum Malaria and Vivax Malaria: A Field Trial in the Solomon Islands

Abstract We have developed a colorimetric assay, “microtiter plate-hybridization”, for the detection of malaria parasites Plasmodium falciparum and P. vivax in human blood, in which the target DNA sequences (18s small subunit ribosomal RNA gene) amplified by polymerase chain reaction (PCR) are hybridized with the species-specific probes immobilized on a microtiter well. This assay system was tested in Guadalcanal, Solomon Islands, where malaria is highly endemic. We obtained blood samples by finger puncture from 130 asymptomatic donors. Among the 130 samples, 30 (23 %) were P. falciparum positive, 28 (22 %) were P. vivax positive, and 8 (6 %) were mixed infections. The results of our DNA diagnostic method showed good correlation with those of acridine orange microscopy. §This paper is dedicated to Dr. Yoshihisa Mizuno, Emeritus Professor of Hokkaido University, on the celebration of the 75th birthday.

Cloning and mapping of the CYS4 gene of Saccharomyces cerevisiae

SummaryA DNA fragment containing the CYS4 gene of Saccharomyces cerevisiae was isolated from a genomic library. The cloned fragment hybridized to the transverse-alternating-field-electrophoresis band corresponding to chromosomes VII and XV. According to the 2 μm DNA chromosome-loss procedure, the cys2 and cys4 mutations, which are linked together and co-operatively confer cysteine dependence, were assigned to chromosome VII. By further mapping involving tetrad analysis, the cys2-cys4 pair was localized between SUP77 (SUP166) and ade3 on the right arm of chromosome VII.

Detection of DNA mutations by fluorescence resonance energy transfer-based preferential homoduplex formation assay

Molecularly targeted agents for cancer therapy are recognized as being effective and are gaining in popularity. However, the efficacy of the agents depends on the status of the targeted molecule such as the number of molecules expressed, activity, and mutation. Therefore, the use of companion diagnostics for investigating the status of the targeted molecule prior to therapy is highly important. We developed a simple and cost-effective somatic mutation detection method called the fluorescence resonance energy transfer-based preferential homoduplex formation assay (FRET-PHFA). By using double-stranded labeled DNA and fluorescence measurement with thermal control, this method provides higher reproducibility, easier handling, less risk for contamination, shorter assay time (only ∼15min), and less cost compared with conventional PHFA. Here we report the evaluation of FRET-PHFA on the detection of multiallelic KRAS mutations in codons 12 and 13 compared with the TheraScreen clinical diagnostics kit. We found that FRET-PHFA detected KRAS mutations (1.25-50%) from all cell line DNA titration samples.

Relationship between chromosomal α-specific suppressors of sir4-11 and polymorphism of the HMRa-bearing fragment

In the course of studying sir4-11 which is responsible for the non-mating phenotype of asd-homothallism of Saccharomyces cerevisiae, we detected chromosomal α-specific suppressors of it. By examining the mating-type cassette constitution of two strains and the spore-clones derived from a diploid culture formed by a mass mating between these strains, we obtained the following results; (1) the HMRa-bearing HindIII-BglII restriction fragment was dimorphic, as judged by size, within each tetrad, (2) while one form was common to all tetrads, the other varied among tetrads, (3) spore-clones with the α-specific suppressor of sir4-11 had the variant forms while those without had the common form, and (4) while the two parents had the common form, each independent diploid clone had the common form plus a variant form. From these results, we conclude that the mating of cells in certain combinations induces a change of DNA structure at or near HMRa in a mating-pair specific manner and that the change makes HMRa non-derepressible or non-functional when derepressed.

Genotypic Identification of Staphylococcal Enterotoxins and Toxic Shock Syndrome Toxin 1 by the Enzymatic Detection of Polymerase Chain Reaction-Amplified DNA

The enzymatic detection of a polymerase chain reaction product (ED-PCR), a new detection method of PCR-amplified DNA, was evaluated for the identification of staphylococcal enterotoxin (SE) and toxic shock syndrome toxin 1 (TSST-1) genes. A total of 61Staphylococcus aureus strains, including reference strains and strains isolated from clinical specimens and food poisoning outbreaks, were examined by ED-PCR and by reverse passive latex agglutination (RPLA) phenotypic identification. There was 100% agreement between the genotypic and phenotypic identification of SEA, SEB, SEC, SEE strains and TSST-1. In the case of SED, however, 4 strains were positive by ED-PCR and negative by RPLA. ED-PCR offers an accurate alternative to traditional immunoassays or conventional PCR using electrophoresis for the detection of SE and TSST-1 production yielding results that are more precise than with older techniques.