Shinji Kunishima

Genetic abnormalities of 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. BSS is characterized by giant platelets, thrombocytopenia, and prolonged bleeding time, and the hallmark of this disorder is the absence of ristocetininduced platelet agglutination. In the last 10 years, the molecular and genetic bases of many GPIb/IX/V defects have been elucidated, providing a better understanding of primary hemostasis and structure-function relations of the complex. Thus far, more than 30 mutations of the GPIbα, GPIbβ, or GPIX genes have been described in BSS. Recent studies also have shown that the phenotypes caused by mutations in the subunits of the GPIb/IX/V span a wide spectrum, from the normal phenotype, to isolated giant platelet disorders/macrothrombocytopenia, to full-blown BSS and platelet-type von Willebrand disease. Although recent progress in molecular biology has clarified the genotype-phenotype relationships of the GPIb/IX/V disorders, a close examination of platelet morphology on blood smears is still indispensable for a proper diagnosis. In this review, we summarize recent advances in the molecular basis of BSS with special emphasis on giant platelets and the genetic characteristics of Japanese BSS.Int J Hematol. 2002; 76: 319-327.

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.

In vitro characterization of missense mutations associated with quantitative protein S deficiency

Summary.u2002 Objective:u2002To elucidate the molecular consequences of hereditary protein S (PS) deficiency, we investigated the in vitro synthesis of the PS missense mutants in COS‐1 cells and their activated protein C (APC) cofactor activities. Patients:u2002Four patients with quantitative PS deficiency suffering from venous thrombosis were examined. Results: We identified three distinct novel missense mutations, R275C, P375Q and D455Y, and two previously reported missense mutations, C80Y and R314H. The P375Q and D455Y mutations were found in one patient and observed to be in linkage on the same allele. The R314H mutant showed the lowest level of expression (32.7%), and the C80Y, P375Qu2003+u2003D455Y, and R275C mutants exhibited a moderate impairment of expression, that is, 43.8%, 49.5%, and 72.3% of the wild type, respectively. Furthermore, pulse‐chase experiments demonstrated that all mutants showed impaired secretion and longer half‐lives in the cells than the wild type PS. In the APC cofactor assays, the C80Y mutant showed no cofactor activity, and the R275C mutant showed reduced activity, 62.3% of the wild type PS, whereas the R314H and P375Qu2003+u2003D455Y mutants exhibited normal cofactor activity. Conclusion:u2002These data indicate that the C80Y and R275C mutations affect the secretion and function of the PS molecule, and that the R314H and P375Qu2003+u2003D455Y mutations are responsible for only secretion defects, causing the phenotype of quantitative PS deficiency observed in the patients.

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