Motohiro Hamaguchi

Mutation of the beta1-tubulin gene associated with congenital macrothrombocytopenia affecting microtubule assembly.

Congenital macrothrombocytopenia is a genetically heterogeneous group of rare disorders. We identified the first TUBB1 mutation, R318W, in a patient with congenital macrothrombocytopenia. The patient was heterozygous for Q43P, but this single-nucleotide polymorphism (SNP) did not relate to macrothrombocytopenia. Although no abnormal platelet beta1-tubulin localization/marginal band organization was observed, the level of beta1-tubulin was decreased by approximately 50% compared with healthy controls. Large and irregular bleb protrusions observed in megakaryocytes derived from the patients peripheral blood CD34(+) cells suggested impaired megakaryocyte fragmentation and release of large platelets. In vitro transfection experiments in Chinese hamster ovary (CHO) cells demonstrated no incorporation of mutant beta1-tubulin into microtubules, but the formation of punctuated insoluble aggregates. These results suggested that mutant protein is prone to aggregation but is unstable within megakaryocytes/platelets. Alternatively, mutant beta1-tubulin may not be transported from the megakaryocytes into platelets. W318 beta1-tubulin may interfere with normal platelet production, resulting in macrothrombocytopenia.

Successful ribavirin therapy for severe adenovirus hemorrhagic cystitis after allogeneic marrow transplant from close HLA donors rather than distant donors.

Intravenous ribavirin was given to nine patients who had developed severe adenovirus-induced hemorrhagic cystitis (AD-HC) which was resistant to conventional therapy or where there was involvement of other organs after allogeneic BMT. Three patients recovered completely from AD-HC, two of whom had been resistant to vidarabine. All three had received sibling BMTs (2 HLA matched, 1 HLA mismatched). Five patients who received BMTs from related (2 HLA mismatched) or unrelated (1 HLA matched, 2 HLA mismatched) showed an improvement in symptoms but had recurrent AD-HC after discontinuation of ribavirin. Improvement in clinical symptoms and termination of virus excretion were well correlated. The last patient who received a mismatched unrelated BMT died during ribavirin therapy. Ribavirin was notably more effective among patients receiving BMTs from siblings in contrast to patients receiving BMTs from alternative donors (<0.05). One patient experienced severe pancytopenia during the second treatment with ribavirin after hc recurrence and recovered after ceasing ribavirin. Thus, ribavirin seems to be very effective for severe ad-hc for some recipients who receive transplants from a genetically close donor. Bone Marrow Transplantation (2000) 25, 545–548.

Intestinal thrombotic microangiopathy after allogeneic bone marrow transplantation: a clinical imitator of acute enteric graft-versus-host disease

Summary:Thrombotic microangiopathy after bone marrow transplantation (post-BMT TMA) is a serious transplant-related complication. We identified 16 patients with TMA after allogeneic BMT who showed histopathological evidence of intestinal TMA in their gut specimens (six autopsies, 10 biopsies). In all, 14 patients had grade II–IV acute graft-versus-host disease (GVHD). The first seven patients were retrospectively diagnosed with TMA. Since six of them were diagnosed with progressive GVHD at that time because there was no awareness of the existence of intestinal TMA, they received more intensive treatment for GVHD, but all died between days +49 and +253. In contrast, the remaining nine patients were recently diagnosed with intestinal TMA on the basis of colonoscopic biopsies. For eight of these patients, the immunosuppressants were reduced, and the patients’ intestinal symptoms improved gradually. Six of the nine patients were still alive 12 months after the diagnosis of TMA. Our findings suggest that the gut may be a site involved in post-BMT TMA, presenting as ischemic enterocolitis. Differentiating intestinal TMA from acute GVHD is important in patients suffering from severe and refractory diarrhea after BMT.

Short-term methotrexate could reduce early immune reactions and improve outcomes in umbilical cord blood transplantation for adults

Post transplant immune disorders are problematic in cord blood transplantation (CBT) for adult patients, and optimal prophylaxis has not been established. We investigated whether intensive graft-versus-host disease (GVHD) prophylaxis using short-term methotrexate (MTX) has a prognostic impact on CBT. Post-CBT immune reactions were classified according to time course as pre-engraftment immune reaction (PIR), engraftment syndrome (ES) or acute GVHD. Between March 2001 and November 2005, a total of 77 patients underwent CBT at eight transplantation centers. Median age was 48 years (range, 18–69 years). Preparative regimens comprised myeloablative (n=31) or reduced-intensity (n=46). Acute GVHD prophylaxis included cyclosporine alone (n=23), tacrolimus alone (n=12), cyclosporine plus MTX (n=17), tacrolimus plus short-term MTX (n=23) or cyclosporine plus methylprednisolone (n=2). Cumulative incidences of PIR, ES and grade II–IV GVHD were 36, 12 and 23%, respectively. Short-term MTX exerted significant favorable effects on post-CBT immune reactions (hazard ratio, 0.55; 95% confidence interval (95% CI), 0.31–0.98; P=0.04) in multivariate analysis. Overall survival rates for patients with and without short-term MTX at day 180 were 59% (95% CI, 42–73%) and 16% (95% CI, 6.6–30%) (P=0.0001), respectively. Short-term MTX could offer one optimal regimen to reduce immune reactions and improve outcomes in CBT.

Differential expression of wild-type and mutant NMMHC-IIA polypeptides in blood cells suggests cell-specific regulation mechanisms in MYH9 disorders

MYH9 disorders such as May-Hegglin anomaly are characterized by macrothrombocytopenia and cytoplasmic granulocyte inclusion bodies that result from mutations in MYH9, the gene for nonmuscle myosin heavy chain-IIA (NMMHC-IIA). We examined the expression of mutant NMMHC-IIA polypeptide in peripheral blood cells from patients with MYH9 5770delG and 5818delG mutations. A specific antibody to mutant NMMHC-IIA (NT629) was raised against the abnormal carboxyl-terminal residues generated by 5818delG. NT629 reacted to recombinant 5818delG NMMHC-IIA but not to wild-type NMMHC-IIA, and did not recognize any cellular components of normal peripheral blood cells. Immunofluorescence and immunoblotting revealed that mutant NMMHC-IIA was present and sequestrated only in inclusion bodies within neutrophils, diffusely distributed throughout lymphocyte cytoplasm, sparsely localized on a diffuse cytoplasmic background in monocytes, and uniformly distributed at diminished levels only in large platelets. Mutant NMMHC-IIA did not translocate to lamellipodia in surface activated platelets. Wild-type NMMHC-IIA was homogeneously distributed among megakaryocytes derived from the peripheral blood CD34(+) cells of patients, but coarse mutant NMMHC-IIA was heterogeneously scattered without abnormal aggregates in the cytoplasm. We show the differential expression of mutant NMMHC-IIA and postulate that cell-specific regulation mechanisms function in MYH9 disorders.

Haematological characteristics of MYH9 disorders due to MYH9 R702 mutations

Objective: MYH9 disorders are characterised by giant platelets, thrombocytopenia, and Döhle body‐like cytoplasmic granulocyte inclusion bodies that result from mutations in MYH9, the gene for non‐muscle myosin heavy chain‐IIA (NMMHC‐IIA). MYH9 R702 mutations are highly associated with Alport manifestations and result in Epstein syndrome. The aim of our study was to determine the haematological characteristics of MYH9 disorders as a result of R702 mutations to aid in making a proper diagnosis. Patients and methods: Platelet size of patients with MYH9 disorders was determined as platelet diameter by microscopic observation of 200 platelets on stained peripheral blood smears. Double in situ hybridisation using a biotinylated oligo(dT) probe and immunofluorescence analysis of neutrophil NMMHC‐IIA was performed on peripheral blood smears. Results: Patients carrying R702 mutations had significantly larger platelets than those with other MYH9 mutations. Although granulocyte inclusion bodies were mostly invisible on stained blood smears, immunofluorescence analysis for NMMHC‐IIA showed an abnormal type II localisation in all neutrophils. We first showed that poly(A)+ RNA coincided with accumulated NMMHC‐IIA at inclusion bodies in patients with MYH9 disorders. However, no condensation of poly(A)+ RNA at inclusion bodies was observed in patients with R702 mutations. Conclusion: Our study shows that R702 mutations result in especially large platelets and inclusion bodies being faint and mostly invisible on conventionally stained blood smears. We further demonstrated that poly(A)+ RNA content but not NMMHC‐IIA accumulation is responsible for the morphological appearance/stainability of inclusion bodies on stained blood smears and the amount of poly(A)+ RNA is decreased in those with R702 mutations.

Cytomegalovirus antigenemia and outcome of patients treated with pre-emptive ganciclovir: Retrospective analysis of 241 consecutive patients undergoing allogeneic hematopoietic stem cell transplantation

Summary:CMV disease remains a major infectious complication after allogeneic hematopoietic stem cell transplantation (HSCT). To investigate the relationship between CMV antigenemia, treatment with ganciclovir (GCV), and outcome, we retrospectively analyzed 241 consecutive patients at risk for CMV infection who underwent allogeneic HSCT. Antigenemia-guided pre-emptive strategy with GCV was used for all patients. CMV antigenemia developed in 169 patients (70.1%), and CMV disease in 18 patients (7.5%). Multivariate analysis showed that acute GVHD (grades II–IV) was the only risk factor for developing antigenemia, and acute GVHD and advanced age for CMV disease. GCV use, as well as acute GVHD and advanced age, significantly increased the risk for bacterial and fungal infection after engraftment. Those who developed CMV antigenemia had a poorer outcome than those who did not (log-rank, P=0.0269), although the development of CMV disease worsened the outcome with only borderline significance (log-rank, P=0.0526). In conclusion, detection of antigenemia proved to be a poor prognostic factor for HSCT patients, which may be attributed to a combination of factors, including CMV disease itself, the effect of treatment, and a host status that allows for reactivation of CMV. Optimal pre-emptive strategy needs to be determined.

A case of purpura fulminans is caused by homozygous Δ8857 mutation (protein C‐Nagoya) and successfully treated with activated protein C concentrate

We report a Japanese patient who developed purpura fulminans and disseminated intravascular coagulation (DIC) shortly after birth. The patient was diagnosed to be homozygous for protein C deficiency and was treated with an activated protein C (APC) concentrate. Intravenous infusions of APC markedly improved the necrotic skin lesions and the anticoagulation by APC enabled successful DIC control. The identified mutation (Δ8857) results in impaired intracellular transport and protein maturation and would be the cause of the complete protein C deficiency. This is the seventh case of the mutation that has been exclusively reported in Japan, but is the first report of a homozygous case. Our findings propose new therapeutic and diagnostic tools for the management of this fatal thrombotic disease.

Novel heterozygous missense mutation in the second leucine rich repeat of GPIbalpha affects GPIb/IX/V expression and results in macrothrombocytopenia in a patient initially misdiagnosed with idiopathic thrombocytopenic purpura.

Abstract:  Recent studies have shown that heterozygous carriers of the bleeding disorder Bernard‐Soulier syndrome are occasionally identified as isolated case of giant platelet disorder/macrothrombocytopenia or misdiagnosed with idiopathic thrombocytopenic purpura (ITP). We describe here a patient with congenital macrothrombocytopenia who had been diagnosed with ITP. On peripheral blood smears, platelet diameter was ∼30% larger than normal controls. In the patients platelets, the expression level of the GPIbIX complex was slightly decreased (70–80% of normal control). Densitometric analysis of immunoblots showed GPIbα to be ∼52% of normal. DNA sequencing analysis revealed a novel heterozygous missense mutation in the GPIbα gene that converts Tyr to Asp at residue 54 (Y54D) in the second leucine‐rich repeat. Mutant GPIbα protein was not detected in the patients platelets. Transient transfection studies demonstrated that mutant GPIbα affects complex expression. These findings suggest that null expression of the mutant GPIbα causes decreased density of the complex and results in macrothrombocytopenia.

Risk and prognostic factors for Japanese patients with chronic graft-versus-host disease after bone marrow transplantation

Correction to: Bone Marrow Transplant (2006) 37: 289–296; advance online publication, 9 January 2006. doi:10.1038/sj.bmt.1705247 The authors have identified an error in Table 1 of the above paper; a corrected version of the table is given below. Eosinophilia (>4%) has been changed to Eosinophils (<4%) and the corresponding N has been changed to 57.