Toshiyuki Niiya

Activation of mouse phosphodiesterase 3B gene promoter by adipocyte differentiation in 3T3-L1 cells.

Activation of phosphodiesterase (PDE) 3B reduces free fatty acid output from adipocytes. Induction of PDE3B gene expression by adipocyte differentiation could improve insulin resistance. To examine whether the PDE3B promoter is activated by this differentiation, the 5′ flanking sequence of the mouse PDE3B gene was isolated. The transcription initiation site was determined to be located 195 bp upstream of the translation start site. No putative binding site for peroxisome proliferator‐activated receptor γ was found within 2 kb upstream of the transcription initiation site. This region had promoter activity, which was further activated on adipocyte differentiation in 3T3‐L1 cells.

CLTC-ALK fusion as a primary event in congenital blastic plasmacytoid dendritic cell neoplasm.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a subtype of acute myeloid leukemia, affecting mainly the elderly. It is thought to be derived from plasmacytoid dendritic cell precursors, which frequently present as cutaneous lesions. We have made a detailed analysis of an infant with BPDCN, who manifested with hemophagocytic lymphohistiocytosis. The peripheral blood leukocytes revealed the t(2;17;8)(p23;q23;p23) translocation and a CLTC‐ALK fusion gene, which have never been reported in BPDCN or in any myeloid malignancies thus far. Neonatal blood spots on the patients Guthrie card were analyzed for the presence of the CLTC‐ALK fusion gene, identifying the in utero origin of the leukemic cell. Although the leukemic cells were positive for CD4, CD56, CD123, and CD303, indicating a plasmacytoid dendritic cell phenotype, detailed analysis of the lineage distribution of CLTC‐ALK revealed that part of monocytes, neutrophils, and T cells possessed the fusion gene and were involved in the leukemic clone. These results indicated that leukemic cells with CLTC‐ALK originated in a multipotent hematopoietic progenitor in utero. This is the first report of the CLTC‐ALK fusion gene being associated with a myeloid malignancy, which may give us an important clue to the origin of this rare neoplasm.

A complete deficiency of coagulation factor XIII A-subunit due to a novel compound heterozygote of Ser 413 Leu missense and an nt 389 (ins G) frameshift mutation

Coagulation factor XIII consists of two A‐ and two B‐subunits, and either gene mutation can cause a complete deficiency. In a newborn patient with persistent bleeding from the umbilical cord stump, the plasma A‐subunit protein was not detectable. Direct PCR sequencing revealed an nt 389 (ins G) frameshift mutation in exon 4 resulting in a new stop codon and a Ser 413 Leu missense mutation in exon 10 in either allele. His mother and father were heterozygous for the nt 389 (ins G) and the Ser 413 Leu, respectively, with about 50% reduction of the plasma A‐subunit proteins. In all family members examined only those with either mutation showed the reduced subunit A protein levels. Thus, this complete deficiency of factor XIII was due to a novel compound heterozygous mutation in the A‐subunit gene.

Activation of T-cell receptor signaling in peripheral T-cell lymphoma cells plays an important role in the development of lymphoma-associated hemophagocytosis.

Peripheral T-cell lymphoma (PTCL) is a biologically diverse lymphoid malignancy. The clinical aggressiveness associated with hemophagocytic syndrome (HS) is a characteristic of PTCL, being more distinctive in CD8+ PTCL. However, the underlying mechanism of PTCL-associated HS has not yet been fully investigated. We newly established a novel IL-2-dependent CD8+ PTCL lymphoma cell line (T8ML-1) from a patient with CD8+ PTCL who suffered recurrent HS accompanying disease flare-up. Focusing on the lymphoma cell T-cell receptor (TCR), we examined the lymphoma cell functions responsible for such clinical manifestations. First, T8ML-1.1 in which endogenous TCR-α/β chains were silenced by siRNAs, and T8ML-1.2 in which endogenous TCR-α/β chains were replaced with HLA-A*24:02-restricted and WT1235–243-specific TCR-α/β, were established. T8ML-1 exerted phytohemagglutinin (PHA)-dependent cytotoxicity via granular exocytosis. Additionally, soluble factors produced by PHA-stimulated T8ML-1, which included INF-γ and TNF-α, but not by simple-cultured T8ML-1, caused human monocytes to exhibit erythrophagocytosis and thrombophagocytosis in vitro. PHA binding induced phosphorylation of CD3ζ chain. Furthermore, both cytotoxicity and hemophagocytosis were completely inhibited by T8ML-1.1, but eventually restored by T8ML-1.2. These data suggest that exogenous activation of TCR signaling in PTCL cells might play an important role in the formation of PTCL-associated HS.

Identification of novel C253Y missense and Y864X nonsense mutations in the insulin receptor gene in type A insulin‐resistant patients

Mutations in the insulin receptor gene have been reported in cases of type A insulin resistance. We report herein two cases of type A insulin resistance, which involve some novel mutations. Case 1 is a heterozygote of the C253Y missense mutation and case 2 is a heterozygote of the Y864X nonsense mutation. In the C253Y missense mutation in exon 3, a cysteine residue is replaced with tyrosine in the cysteine‐rich domain of the α subunit. The Y864X in exon 13 results in a truncated receptor, which is devoid of most of the β subunit. This mutant receptor could not be expressed on a cell membrane since the transmembrane domain is missing. Other significant mutations were not found for the entire coding regions and splice/donor sites.

A novel MYH9 mutation in a patient with MYH9 disorders and platelet size-specific effect of romiplostim on macrothrombocytopenia.

Dear Editor, M Y H 9 d i s o r d e r s a r e c h a r a c t e r i z e d b y macrothrombocytopenia, leukocyte inclusion bodies, and Alport manifestations including nephropathy, deafness, and cataract, all of which derive frommutations of theMYH9 gene encoding nonmuscle myosin heavy chain IIA (NMMHC-IIA) [1]. The severity of Alport manifestations observed in patients with MYH9 disorders generally depends on mutation sites of MYH9 gene and such a genotype-phenotype relationship has been constructed and extended by a series of case reports [2, 3]. We report here a patient with MYH9 disorders carrying a novelMYH9mutation. In addition, we report the platelet sizespecific effect of romiplostim on macrothrombocytopenia during the treatment for neurosurgery with romiplostim. A 42-year-old female had long-standing purpura and thrombocytopenia. There were many giant platelets in her peripheral blood, and the platelet count, as determined by phase contrast microscopy, was 25×10/L, indicating macrothrombocytopenia. This finding led us to sequence analysis of the MYH9 gene, which revealed a novel missense mutation of c.5507A>G (p.Q1836R). Immunofluorescence of neutrophil NMMHC-IIA showed fine aggregate formation, although leukocyte inclusion bodies were not visible in a May-Grunwald-Giemsa-stained blood smear. Audiograms showed high-frequency hearing loss. Neither proteinuria nor renal dysfunction was observed. No cataract was detected by ophthalmological evaluation. She had large cerebral aneurysm, and craniotomy was planned in order to prevent the rupture of aneurysm. We considered short-term romiplostim as a means of hemostatic management for neurosurgery [4–7]. After approval of the ethical committee of Ehime University Hospital, 1 μg/kg romiplostim was started 6 weeks before the scheduled day of surgery and gradually increased to 5 μg/kg. Romiplostim increased the platelet count, but there were little increase in giant platelets and predominant increase in intermediateand normal-sized platelets (Fig. 1). When her platelet count was 84×10/L, the surgical procedure was performed without any bleeding complications, and no neurological sequelae were left. Romiplostim was tapered and discontinued after 1 month. This report has two medical implications. First, we have added p.Q1836R to the list of MYH9 mutation. The Q1836R residue is located at a long alpha-helical coiled coil region of the tail domain, which connects two NMMHC-IIA molecules to form functional myosin filaments [8]. The mutations in this region cause Alport manifestations variably [2, 3]. The E1841K, a mutation close to Q1836R, is a hot spot of MYH9mutation, and the incidences of nephropathy, deafness, and cataract were 18, 43, and 23 %, respectively [3]. The mutations around residues 1836–1841 may predispose to deafness among Alport manifestations. Second, this study shows that romiplostim preferentially increased non-giant platelets. It remains to be determined whether this effect is a * Jun Yamanouchi yamanouc@m.ehime-u.ac.jp

Vasodilator-stimulated phosphoprotein (VASP) phosphorylation assay for platelet response to cilostazol.

Recent clinical trials have demonstrated that cilostazol, an antiplatelet drug competent to inhibit phosphodiesterase 3, is effective in secondary prevention of atherothrombosis including ischemic stroke, myocardial infarction, and peripheral arterial disease. However, there is no reliable assay for detection of the platelet response to cilostazol. We attempted to establish such an assay for clinical use. Phosphorylation of vasodilator-stimulated phosphoprotein (VASP) subsequent to the pharmacological action of cilostazol was measured by a platelet VASP assay kit that has been widely used for monitoring platelet response to ADP receptor antagonists in clinical settings. We modified the kit and found the optimal conditions for detection of cilostazol-dependent VASP phosphorylation. The assay could detect the in vitro and in vivo platelet responses to cilostazol effectively in the presence of 50 nM PGE1. ROC analysis showed that our assay had 97% sensitivity and 75% specificity when blood drawn between 3 and 9 h after cilostazol ingestion was subjected to the assay. The assay results were verified by immunoblotting specific for VASP phosphorylation. Standard light transmission platelet aggregometry could not detect significant inhibition of agonist-induced platelet aggregation by cilostazol except for the in vitro effect of high concentrations of cilostazol. These results demonstrate that the platelet VASP assay can detect the platelet response to cilostazol with high sensitivity and specificity.

A new four-way variant t(5;17;15;20)(q33;q12;q22;q11.2) in acute promyelocytic leukemia

Acute promyelocytic leukemia (APL) is characterized by t(15;17)(q22;q21), which results in the fusion of the promyelocytic leukemia (PML) gene at 15q22 with the retinoic acid alpha-receptor (RARA) at 17q21. We report a patient with APL carrying a new complex variant translocation (5;17;15;20). Spectral karyotyping analysis of bone marrow cells revealed t(5;17;15;20)(q33;q12;q22;q11.2). Fluorescence in situ hybridization with a PML/RARA dual-color DNA probe showed a single fusion signal, and RT-PCR analysis showed PML/RARA fusion transcripts. Complete remission was attained with a course of conventional chemotherapy with all-trans retinoic acid (ATRA). To our knowledge, this is the first report of a four-way translocation of 5q33 and 20q11 involvement in APL.

Severe immune thrombocytopenia secondary to Waldenström's macroglobulinemia with anti-GPIb/IX monoclonal IgM antibody

Dear Editor, Waldenströms macroglobulinemia (WM) is a lymphoproliferative disorder characterized by infiltration of small lymphocytes and plasmacytoid cells into bone marrow and by serum IgM monoclonal gammopathy [1, 2]. The paraprotein may include an autoantibody resulting in autoimmune complications in 5–16 % of patients with WM [3, 4]. A few reports have referred to the mechanism of immune thrombocytopenia (ITP) associated with WM, and platelet-associated IgM (PAIgM) and IgG (PA-IgG) have been shown to be a possible cause [4, 5]. However, whether monoclonal IgM in WM induces ITP is poorly understood. We report here a patient with WM and thrombocytopenia whose IgM recognized GPIb/IX on the surface membrane of platelets and inhibited GPIb-mediated platelet aggregation. A 68-year-old male was referred to our department because of extensive purpura and macrohematuria. His blood counts revealed 5.3 g/dL hemoglobin, 1.4×10/L white blood cells, and 6.0×10/L platelets. Platelet size was normal. Serum levels of IgM, IgG, and IgA were 1,980, 1,470, and 55 mg/ dL, respectively. Immunoelectrophoresis revealed that almost all IgM was monoclonal. Bone marrow-nucleated cells comprised 44.2 % lymphoid cells and 0.6 % plasmacytoid cells, which were positive for CD10, CD19, CD20, CD38, and SmIg-κ, but negative for CD5 and CD23. He was diagnosed as having WM and treated with cyclophosphamide. He was refractory to platelet transfusion and died of brain hemorrhage 1 week later. Because WM-induced ITP was suspected, we examined the effect of his IgMon platelets. Flow cytometry assay showed that his IgM bound to normal platelets whereas his IgG did not. To identify the platelet antigen recognized by IgM, we employed a PakAuto assay. This assay enables us to characterize the antigen of platelet-bound Ig. However, we failed to obtain Ig eluted from patients platelets because of very low platelet count. Then the patients serum was incubated with normal platelets and the platelet-bound Ig was eluted in order to concentrate plateletspecific Ig from the patients whole serum. Furthermore, we employed alkaline phosphatase-conjugated goat anti-human IgG and IgM (BioFX, Owings Mills, MD, USA) as secondary antibody to detect IgG and IgM separately. We confirmed that these antibodies produced efficient and specific signals when the positive and negative controls in the PakAuto assay kit were employed. Using this modified assay, we found that the eluted IgM bound to the immobilized platelet membrane GPIb/IX, but not to GPIIb/IIIa or GPIa/IIa (Fig. 1). The eluted IgG did not bind to any type of GP. Then we examined the effect of patients IgM on GPIb-mediated platelet aggregation. When the patients plasma was incubated with normal washed platelets, 1.2 mg/mL ristocetin-induced platelet aggregation was inhibited significantly although the expression of GPIb on the platelets was normal. These results suggest that the patients monoclonal IgM derived from WM has antiplatelet activity against GPIb/IX. This activity leads to ITP and platelet dysfunctions, both of J. Yamanouchi (*) : T. Azuma :M. Yasukawa Department of Hematology, Clinical Immunology and Infectious Diseases, Ehime University Graduate School of Medicine, 454 Shitsukawa, Toon, Ehime 791-0295, Japan e-mail: yamanouc@m.ehime-u.ac.jp

Development of exogenous FVIII‐specific inhibitor in a mild haemophilia patient with Glu272Lys mutation

bleeding which caused respiratory distress, patient was admitted to the emergency unit. Treatment with aPCC was started immediately at a maximum dose of 200 U kg 1 day 1 in PICU for 5 days. Then the patient was admitted to a paediatric haematology ward and treated for 10 days of 50 U kg 1 per dose every 8 h and complete treatment was achieved in 15 days. Thus, even a simple dental extraction operation can be life-threatening in haemophilia patients with inhibitors. Every bleeding should be examined carefully, and must be treated at the closest qualified centre. All major and minor surgery including dental extraction in haemophilia patients with inhibitors should be performed cautiously by surgeons together with haematologists under appropriate conditions in comprehensive haemophilia centres. Nevertheless, it should be taken into consideration that bleeding and other complications may be more serious in dental extraction in haemophiliac patients with inhibitors and should be monitored more carefully. We should keep in mind that the haemophilia patients with inhibitors can still experience the problem of bleeding despite appropriate bypass agent therapy, experienced dentistry and haematology.