Kazuo Ozawa

Mapping of a gene for May-Hegglin anomaly to chromosome 22q.

Abstract. May-Hegglin anomaly (MHA) is a rare autosomal dominant platelet disorder characterized by the triad of giant platelets, thrombocytopenia and leukocyte inclusions. Both the molecular and the genetic defects responsible for this disorder remain unknown. In order to map the gene responsible for MHA, we performed a genome-wide linkage study using highly polymorphic short tandem repeat markers in a single Japanese MHA family. Significant linkage was obtained for the markers on the long arm of chromosome 22 (22q12.3–q13.2), with a maximum two-point lod score of 4.52 at a recombination fraction of 0.00 for the markers D22S1142 and D22S277. Haplotype analysis mapped a critical region for the disease locus to a 13.6-centimorgan region, between D22S280 and D22S272. The relative proximity of the platelet GPIbβ gene (22q11.2) to this region, as well as its involvement in an isolated giant platelet disorder, suggested a possible involvement of GPIbβ mutations in MHA. However, DNA-sequencing analysis in two patients revealed no abnormality in the sequence of the GPIbβ gene. This is the first report of linkage for MHA, and further analysis of this locus may lead to the identification of a gene the product of which regulates platelet and leukocyte morphology.

Presence of Propionibacterium acnes in blood components

BACKGROUND: Sterility testing, as part of the QC of blood components at the Japanese Red Cross Aichi Blood Center between April 1998 and March 2000, showed that 10 of 5568 tested blood components (0.18%), all of which were RBC concentrates, were contaminated with bacteria. Nine isolates were Propionibacterium acnes and one was Staphylococcus capitis.

Cys97!Tyr mutation in the glycoprotein IX gene associated with Bernard-Soulier syndrome

Bernard‐Soulier syndrome (BSS) is an autosomal recessive bleeding disorder due to quantitative or qualitative abnormalities in the glycoprotein (GP) Ib/IX/V complex, the platelet receptor for von Willebrand factor. We describe here the genetic basis of the disorder in a patient with BSS. Flow cytometric analysis of the patients platelets showed a greatly reduced GPIbα and completely absent GPIX surface expression. Immunoblot analysis disclosed greatly reduced GPIbα and residual amounts of GPIbβ and GPIX in the platelets. DNA sequencing analysis revealed the patient to be homozygous for a novel missense mutation in the GPIX gene that converts Cys (TGT) to Tyr (TAT) at residue 97. Transient transfection studies confirmed that mutant GPIX was not expressed on the transfected cells, showing that the mutation was responsible for the BSS phenotype observed in the patient.

Cytomegalovirus Glycoprotein B Sequence Variation among Japanese Bone Marrow Transplant Recipients

Glycoprotein B (gB) of human cytomegalovirus (CMV) is the major target protein of neutralizing antibodies, and four variant types are known. A previously reported polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) method was modified by eliminating the cell culture step and amplifying a CMV fragment (corresponding to a.a. 441-450 of gB) by nested PCR. DNA was extracted from the sera of 27 pediatric bone marrow transplant recipients and subjected to nested PCR. Of the samples, 20 yielded PCR products, and the gB type was determined by RFLP. Of the 20 patients, 4 (20%) had gB type 1 (Towne type), 15 patients (75%) had gB type 2 (AD 169 type), and 1 patient (5%) had gB type 3. Previous studies showed that gB type was most common among Caucasians [Chou and Dennison: J Infect Dis 1991;163:1229-1234; Fries et al: J Infect Dis 1994;169:769-774]. Thus, gB genotypes seem to be distributed differently in Caucasians and Japanese. Further, nucleotide sequence analysis of the amplified region revealed that all the type 2 viruses had the same amino acid sequences. The type 3 sample had a novel amino acid substitution at position 498. Of the type 1 samples, 3 had amino acid substitutions in various positions: 1 sample at position 493, 1 sample at position 447 and 1 sample at position 452, 493 and 498.

Clinical evaluation of repeat apheresis donors in Japan.

AbstractBackground and Objectives: To ascertain the safety of repeat apheresis donation, hematological and biochemical tests were performed on 511 donors with a donation rate of over 6 times per year for a period of 12–19 months. Materials and Methods: Repeat donors who had apheresis more than 6 times in the previous year were chosen. Data for the repeat donors at the start of the experiments were compared with those at the end of the end of the study. Blood samples were taken prior to donation. Serum protein, albumin, immunoglobulin G, A, and M, serum ferritin levels were determined by biochemical tests. Results: When compared to prospective donors of 400 ml, WBC, lymphocytes, and serum ferritin levels were lower in a roughly frequency‐dependent manner in female and male donor groups at the beginning of the study. All the data for the male group remained almost constant with increasing frequency of apheresis donation. However, in the female group, ferritin levels significantly decreased with over 21 donations. Conclusions: The present data showed that the serum ferritin level of the female donors decreased the most with increasing frequency of apheresis donation. The cumulative RBC left in the collecting chamber and for the laboratory test is discussed in relation to a possible cause of iron deficiency in frequent apheresis donors.

Application of 16S ribosomal RNA gene amplification to the rapid identification of bacteria from blood culture bottles.

In Japan, sterility testing as part of the QC of blood components is performed by sampling bags and culturing in anaerobic and aerobic culture bottles. If a culture bottle develops bacteria growth, biochemical identification is performed. Although culturing of blood components followed by confirmatory identification is standardized, this process usually requires as long as 1 month. Therefore, there is a need for a rapid and reliable alternative method. As a rapid diagnostic technique, PCR can potentially overcome the limitations of sensitivity and specificity.1 Although the detection of a single pathogenic species by the use of target-specific primers has proven to be highly sensitive, it cannot be a generic method in sterility testing.2 Gene amplification with 16S ribosomal RNA (16S rRNA) is an ideal alternative and has been employed for both identification and phylogenetic resolution of bacteria at the species level.3 We report here the utility of 16S rRNA gene amplification for the rapid detection and identification of bacteria isolated from blood culture bottles. Genomic DNA was extracted from culture bottles by using a tissue kit (QIAamp, QIAGEN GmbH, Hilden, Germany). Oligonucleotide primers used were 8F (AGAGT TTGAT CCTGG CTCAG) and 350R (CTGCT GCCTC CCGTA G); these primers target a highly conserved region of the bacterial 16S rRNA gene and amplify a segment of approximately 350 bp from all bacteria.3 PCR amplification was performed in a volume of 50 μL containing 1× PCR buffer, 1.5 mM of MgCl2, 1.25 U of AmpliTaq DNA polymerase LD (PE Applied Biosystems [PE ABI], Foster City, CA), 0.2 mM of dNTPs, 0.4 mM of primers, and approximately 100 ng of DNA template. Amplification proceeded in a thermal cycler (GeneAmp PCR System 9600, Perkin-Elmer Cetus, Norwalk, CT) for 30 cycles of 30 seconds at 95°C, 30 seconds at 55°C, and 45 seconds at 72°C. After amplification, samples were investigated for the presence of an approximately 350-bp fragment by electrophoresis on 2-percent agarose gels. Amplified DNA fragments were purified and subjected to direct cycle sequence analysis on a DNA sequencer (373A, PE ABI). In another experiment, a whole 16S rRNA gene was amplified with primers 8F and 1492R (GGTTA CCTTG TTACG ACTT) for 33 cycles of 45 seconds at 95°C, 45 seconds at 55°C, and 90 seconds at 72°C. A total of 2781 sampling inspections as QC were performed from April 1998 to March 1999 at our blood center. Three units (0.108%), all of which were RBC concentrates, were infected with Propionibacterium acnes. Amplification of the 16S rRNA gene followed by sequence homology analysis showed that the DNA sequences of all three isolates were completely identical to those from P. acnes. This procedure is accomplished within 2 days and offers greater speed and specificity than does biochemical identification. In another experiment, a total of 1486 bp of the 16S rRNA gene was amplified, sequenced, and aligned for each isolate with 8F/ 1492R primers. Sequences from the two isolates were identical. In contrast, in the remaining isolate, two bases did not match those of the two isolates (GenBank accession number AB041618 and AB041617). The existence of sequence variation revealed that these isolates were distinct strains. The major advantage of 16S rRNA gene amplification is the use of one set of universal primers that can detect any bacteria present, because it is not necessary to predict which bacteria may be present.3 Furthermore, one can perform a series of procedures without any knowledge or training in microbiology, which is a prerequisite for biochemical identification. Thus, it is reasonable to adopt this method as a rapid sterility test in regional blood centers where the incidence of bacterial contamination is low, and where keeping appropriate reagents and media for bacterial culture as well as skilled personnel is not practical. We anticipate that 16S rRNA gene analysis will be useful in future attempts to confirm the sterility of blood collection as well as in sterility testing itself. Shinji Kunishima, MT Chikako Inoue, MD Zenichiro Nishimoto, BS Tadashi Kamiya, MD Kazuo Ozawa, MD Japanese Red Cross Aichi Blood Center 539-3 Minamiyamaguchi Seto 489-8555, Japan e-mail: kunisima@fujita-hu.ac.jp

An identification of the HLA-F null allele in Japanese.

The class I major histocompatibility complex ( MHC) genes consist of class Ia and class Ib. Class Ia genes ( HLA-A, -B, and -C) encode highly polymorphic polypeptides which present antigens to cytotoxic T lymphocytes. In contrast, class Ib genes ( HLA-E, -F, and -G) encode significantly fewer polymorphic polypeptides. In the present study, we identified anHLA-F allele which had a non-sense mutation in the exon 3. Single-strand conformational polymorphism (SSCP) analysis was carried out for exon 3 of the HLA-F gene. The polymerase chain reaction (PCR) primers were 5’AGGGGGGCGGGGCTAGCTGGGCGGGG-3’ (nucleotide 1512 to 1536) for the forward primer and 5’-ACAGGAGATAGGGAAGGGCGCCCATG-3’ (nucleotide 1845 to 1869) for the reverse primer. These primers are located in the intron regions encompassing exon 3 of the HLA-F gene (Geraghty et al. 1990). The PCR reaction consisted of 30 cycles of 1 min denaturation at 94 °C, 1 min annealing at 70 °C, and 1 min extension at 72 °C. A total of 50 samples from unrelated individuals were examined. One sample obtained from individual C.U. had a different banding pattern (Fig. 1). The amplified region was cloned into a plasmid pCRII (Invitrogen, San Diego, CA). Ten independent clones were chosen for two samples, one with a distinct banding pattern and the other with the same banding pattern as the rest of the samples. The DNA sequencing analysis showed that the latter sample had an sequence identical to the published sequence. The former sample had two sequences, one which was identical to the published sequence and the other which had a nonsense mutation at amino acid residue 28 (Fig. 2). These sequenced plasmids were used as PCR templates and confirmed that the

Epitope analysis of antibodies in Japanese to human cytomegalovirus phosphoprotein 150 with synthetic peptides.

Serological detection of antibodies specific to human cytomegalovirus (HCMV) is not reliable because the assay uses the whole HCMV protein fraction. Antigenic materials composed of well-characterized viral proteins are being tried for serodiagnosis in Europe. Epitopes of antibodies to HCMV phosphoprotein 150 (pp150) encoded by UL32 in Japanese individuals were investigated for comparison with the results in Europe. The major epitopes on amino acid residues 496 to 652 of HCMV pp150 were identified and the detection of antibodies with an enzyme-linked immunosorbent assay (ELISA) of synthetic peptides against the main epitopes was established. Fifteen seropositive and five seronegative serum samples for the epitope mapping and 131 seropositive and 50 seronegative samples for ELISA were investigated. Overlapping 15-mer peptides moving by two amino acids through V496-H652 were synthesized. The main epitope regions were V508-D530, L526-Q544, S536-D554, T616-G634, S624-P642, and L632-H652. When each peptide was conjugated with bovine serum albumin for ELISA, 80.9% of the seropositive samples were judged to be positive. The results of this study are the same as those for European sera, so the antigenic materials developed in Europe might be used to replace the whole HCMV protein fraction in Japanese.