研究者詳細 - 兼松　毅

Development and validation of a novel qualitative test for plasma fibrinogen utilizing clot waveform analysis 査読有り国際誌

Suzuki, A; Suzuki, N; Kanematsu, T; Shinohara, S; Kurono, H; Arai, N; Okamoto, S; Suzuki, N; Tamura, S; Kikuchi, R; Katsumi, A; Kojima, T; Matsushita, T

SCIENTIFIC REPORTS 12 巻 ( 1 ) 頁： 434 2022年1月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Scientific Reports

Plasma fibrinogen is commonly examined by Clauss fibrinogen assay, which cannot distinguish between quantitative and qualitative fibrinogen anomalies. However, our previously reported Clauss fibrinogen assay utilizing clot waveform analysis (Clauss-CWA) provides additional information that contributes to the classification of fibrinogen anomalies. In this study, we adopted the Clauss-CWA method for an autoanalyzer to automatically measure the antigenic estimate (eAg) of fibrinogen in addition to the functional amount (Ac), and to thus provide the Ac/eAg ratio as a qualitative indicator. Performance was validated by receiver operating characteristics (ROC) and precision recall (PR) curve analyses using a patient cohort, consisting of a training cohort (n = 519) and a validation cohort (n = 523), both of which contained cases of congenital (hypo)dysfibrinogenemia as qualitative defects. We obtained an optimal cutoff of 0.65 for Ac/eAg by ROC curve analysis of the training cohort, offering superior sensitivity (> 0.9661) and specificity (1.000). This cutoff was validated in the validation cohort, providing positive predictive value > 0.933 and negative predictive value > 0.998. PR curve analysis also showed that Clauss-CWA provided excellent performance for detecting qualitative fibrinogen anomalies. The Clauss-CWA method may represent a useful approach for detecting qualitative fibrinogen abnormalities in routine laboratory testing.

DOI： 10.1038/s41598-021-04464-5

その他リンク： https://www.nature.com/articles/s41598-021-04464-5

The influence of hepatitis C virus eradication on hepatocarcinogenesis in patients with hemophilia HCC after HCV eradication in hemophilia 査読有り国際誌

Inukai, Y; Imai, N; Yamamoto, K; Ito, T; Ishizu, Y; Honda, T; Okamoto, S; Kanematsu, T; Suzuki, N; Matsushita, T; Ishigami, M; Fujishiro, M

ANNALS OF HEPATOLOGY 27 巻 ( 1 ) 頁： 100545 - 100545 2022年1月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Annals of Hepatology

Introduction and objectives: Hepatitis C virus (HCV) infections in patients with hemophilia lead to the development of hepatocellular carcinoma (HCC) at a relatively younger age than that in patients without hemophilia. Although recent progress in direct-acting-antivirals has facilitated a high rate of sustained virological response (SVR), the clinical influence of HCV eradication in hemophilia patients remains unclear. This study aimed to compare the clinical outcomes of SVR against HCV in patients with and without hemophilia. Patients and methods: The study enrolled 699 patients who achieved SVR after HCV antiviral treatment. Patients were divided into two groups: 78 patients with hemophilia (H group) and 621 patients without hemophilia (NH group). We evaluated patient characteristics, clinical outcomes, and the cumulative incidence of HCC after SVR. Results: Compared with the NH group, patients in the H-group were significantly younger and had a lower hepatic fibrosis score. No difference was found in the incidence of liver-related disease or overall death between the two groups over a mean follow-up period of 7 years. Four patients in the H group and 36 patients in the NH group were diagnosed with HCC after SVR. Multivariate analysis showed that male sex, age, and cirrhosis were significant risk factors for HCC incidence. There was no significant difference in the cumulative incidence of HCC after propensity-score matching adjusting for the risk factors of HCC between the two groups. Conclusion: Hemophilia is not a significant risk factor for hepatocarcinogenesis after SVR against HCV.

DOI： 10.1016/j.aohep.2021.100545

21ヵ月の経過で自然寛解した高力価インヒビター保有自己免疫性第V因子欠乏症(AiF5D)の一例(Spontaneous remission of autoimmune factor V deficiency(AiF5D) with high titer inhibitor) 査読有り国際誌

兼松毅, 鈴木伸明, 岡本修一, 尾崎司, 惣宇利正善, 鈴木敦夫, 田村彰吾, 早川文彦, 小嶋哲人, 清井仁, 一瀬白帝, 松下正

日本血液学会学術集会 83回巻頁： PS - 6 2021年9月

Tolerability of Molecular-targeted Agents for Hepatocellular Carcinoma Treatment in Haemophiliacs 査読有り国際誌

Yamamoto, T; Imai, N; Yamamoto, K; Ito, T; Ishizu, Y; Honda, T; Okamoto, S; Kanematsu, T; Suzuki, N; Matsushita, T; Ishigami, M; Fujishiro, M

ANTICANCER RESEARCH 41 巻 ( 5 ) 頁： 2569 - 2573 2021年5月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Anticancer Research

Background: Hepatocellular carcinoma (HCC) is considered a leading cause of death in patients with haemophilia. Recent advances in the treatment of unresectable HCC with molecular-Targeted agents (MTAs) have led to better clinical outcomes. However, the tolerability of MTAs by haemophilic patients with HCC remains unclear. Aim: This study aimed to compare the tolerability of MTAs in such patients. Patients and Methods: From January 2011 to October 2020, five haemophilic patients with HCC were treated with MTAs. Adverse events were assessed in comparison with 265 non-haemophilic patients with HCC. Results: The prevalence of hand foot skin reaction was not higher in the haemophiliacs than in the non-haemophiliacs, whereas the rate of haemorrhagic events was higher in the haemophiliacs (6.0% versus 40.0%, p=0.037). Conclusion: Haemophiliacs tolerate long-Term MTA use, without the occurrence of life-Threatening complications. However, careful observation and prevention are needed for MTA-related gastrointestinal bleeding in haemophiliacs.

DOI： 10.21873/anticanres.15035

第VIII因子製剤の活性測定における試薬バリエーションを把握するための単施設研究査読有り国際誌

鈴木敦夫, 鈴木伸明, 兼松毅, 岡本修一, 田村彰吾, 小嶋哲人, 松下正

日本血栓止血学会誌 32 巻 ( 2 ) 頁： 198 - 198 2021年5月

F8 Int22h-1が関与するF8大規模欠失挿入変異による重症血友病A症例査読有り国際誌

大平晃也, 早川友梨, 田村彰吾, 鈴木伸明, 所真昼, 河島史華, 向出将人, 岡田健太郎, 桂木裕実, 藤井渉, 岡本修一, 兼松毅, 鈴木敦夫, 松下正, 小嶋哲人, 早川文彦

日本血栓止血学会誌 32 巻 ( 2 ) 頁： 228 - 228 2021年5月

周術期の止血に難渋した先天性第V因子欠乏症の1例査読有り国際誌

兼松毅, 鈴木伸明, 岡本修一, 鈴木敦夫, 田村彰吾, 早川文彦, 小嶋哲人, 清井仁, 松下正

日本血栓止血学会誌 32 巻 ( 2 ) 頁： 232 - 232 2021年5月

マウス血管結紮モデルによるアンチトロンビンレジスタンスの血栓形成解析査読有り国際誌

鈴木伸明, 鈴木敦夫, 田村彰吾, 向出将人, 岡本修一, 兼松毅, 小嶋哲人, 松下正

日本血栓止血学会誌 32 巻 ( 2 ) 頁： 202 - 202 2021年5月

プロテインS欠乏症に同定したシグナルペプチド内変異プロテインSの分子病態解析査読有り国際誌

岡田健太郎, 田村彰吾, 鈴木伸明, 大平晃也, 早川友梨, 向出将人, 藤井渉, 桂木裕実, 兼松毅, 岡本修一, 鈴木敦夫, 松下正, 小嶋哲人, 早川文彦

日本血栓止血学会誌 32 巻 ( 2 ) 頁： 206 - 206 2021年5月

フィブリノゲン強制発現系の最適化に向けた基礎検討査読有り国際誌

向出将人, 田村彰吾, 鈴木伸明, 大平晃也, 早川友梨, 岡田健太郎, 藤井渉, 桂木裕実, 鈴木敦夫, 兼松毅, 岡本修一, 松下正, 小嶋哲人, 早川文彦

日本血栓止血学会誌 32 巻 ( 2 ) 頁： 228 - 228 2021年5月

VWF G2752S変異体の分子病態解析査読有り国際誌

岡本修一, 大平晃也, 早川友梨, 河島史華, 田村彰吾, 鈴木伸明, 兼松毅, 鈴木敦夫, 早川文彦, 清井仁, 小嶋哲人, 松下正

日本血栓止血学会誌 32 巻 ( 2 ) 頁： 248 - 248 2021年5月

Essential role of a carboxyl-terminal α-helix motif in the secretion of coagulation factor XI 査読有り国際誌

Hayakawa, Y; Tamura, S; Suzuki, N; Odaira, K; Tokoro, M; Kawashima, F; Hayakawa, F; Takagi, A; Katsumi, A; Suzuki, A; Okamoto, S; Kanematsu, T; Matsushita, T; Kojima, T

JOURNAL OF THROMBOSIS AND HAEMOSTASIS 19 巻 ( 4 ) 頁： 920 - 930 2021年4月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Journal of Thrombosis and Haemostasis

Background: Coagulation factor XI (FXI) is a plasma serine protease zymogen that contributes to hemostasis. However, the mechanism of its secretion remains unclear. Objective: To determine the molecular mechanism of FXI secretion by characterizing a novel FXI mutant identified in a FXI-deficient Japanese patient. Patient/Methods: The FXI gene (F11) was analyzed by direct sequencing. Mutant recombinant FXI (rFXI) was overexpressed in HEK293 or COS-7 cells. Western blotting and enzyme-linked immunosorbent assay were performed to examine the FXI extracellular secretion profile. Immunofluorescence microscopy was used to investigate the subcellular localization of the rFXI mutant. Results: We identified a novel homozygous frameshift mutation in F11 [c.1788dupC (p.E597Rfs*65)], resulting in a unique and extended carboxyl-terminal (C-terminal) structure in FXI. Although rFXI-E597Rfs*65 was intracellularly synthesized, its extracellular secretion was markedly reduced. Subcellular localization analysis revealed that rFXI-E597Rfs*65 was abnormally retained in the endoplasmic reticulum (ER). We generated a series of C-terminal–truncated rFXI mutants to further investigate the role of the C-terminal region in FXI secretion. Serial rFXI experiments revealed that a threonine at position 622, the fourth residue from the C-terminus, was essential for secretion. Notably, Thr622 engages in the formation of an α-helix motif, indicating the importance of the C-terminal α-helix in FXI intracellular behavior and secretion. Conclusion: FXI E597Rfs*65 results in the pathogenesis of a severe secretory defect resulting from aberrant ER-to-Golgi trafficking caused by the lack of a C-terminal α-helix motif. This study demonstrates the impact of the C-terminal structure, especially the α-helix motif, on FXI secretion.

DOI： 10.1111/jth.15242

Impact of variation in reagent combinations for one-stage clotting assay on assay discrepancy in nonsevere haemophilia A 査読有り国際誌

Suzuki, A; Suzuki, N; Kanematsu, T; Okamoto, S; Tamura, S; Kikuchi, R; Katsumi, A; Kiyoi, H; Kojima, T; Matsushita, T

INTERNATIONAL JOURNAL OF LABORATORY HEMATOLOGY 43 巻 ( 1 ) 頁： 131 - 138 2021年2月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：International Journal of Laboratory Hematology

Introduction: Factor VIII activity (FVIII:C) is measured by one-stage clotting assay (OSA) or chromogenic substrate assay (CSA). Significant differences in FVIII:C between OSA (FVIII:C1st) and CSA (FVIII:CChr) are described as assay discrepancy in nonsevere haemophilia A (HA). A large number of reagent combinations (APTT reagent and FVIII-deficient plasma) are used for OSA, but the impact of variations in reagent combinations on assay discrepancy has not been fully characterized. Aim: To clarify the variations in FVIII:C1st/FVIII:CChr ratios according to OSA reagent combination in HA subjects with/without assay discrepancy. Methods: Thirty-nine patients previously diagnosed with nonsevere HA were enrolled, and their FVIII genes were investigated and FVIII:C levels were assessed by a single CSA reagent and 11 OSA reagent combinations. Receiver operating characteristic (ROC) curve analysis was used to predict possible cut-off values of the FVIII:C1st/FVIII:CChr ratio to define FVIII assay discrepancy for each reagent combination. Results: Patients were categorized into nondiscrepant (n = 25), discrepant (n = 5) and unclassified (n = 9) groups according to their genotypes and information in the database. The FVIII:C1st/FVIII:CChr ratio in nondiscrepant HA varied widely, depending on the APTT reagents and FVIII-deficient plasma used. The ratio in discrepant HA patients differed with respect to their genotype and the reagent combination used. ROC curve analyses revealed that cut-off values to distinguish the assay discrepancy differed depending on the reagents used, but revealed two novel genotype variants, p.Cys573Gly and p.Gly582Arg, associated with FVIII assay discrepancy. Conclusion: Our findings showed that the FVIII:C1st/FVIII:CChr ratio is dependent on the reagent combination used for OSA.

DOI： 10.1111/ijlh.13335

<i>Myh9</i> R702C is associated with erythroid abnormality with splenomegaly in mice 査読有り

Kanematsu, T; Suzuki, N; Tamura, S; Suzuki, A; Ishikawa, Y; Katsumi, A; Kiyoi, H; Saito, H; Kunishima, S; Kojima, T; Matsushita, T

NAGOYA JOURNAL OF MEDICAL SCIENCE 83 巻 ( 1 ) 頁： 75 - 86 2021年2月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Nagoya Journal of Medical Science

MYH9 disorders are characterized by giant platelets, thrombocytopenia, and Döhle body-like cytoplasmic inclusion bodies in granulocytes. However, whether these disorders cause any changes in erythroid cells has yet to be determined. This study analyzed the influence of Myh9 R702C, as one of the most commonly detected MYH9 disorders, on erythroid cells in a mouse model. Knock-in mice expressing Myh9 R702C mutation either systemically or specific to hematological cells (R702C and R702C vav1 mice, respectively) were used in this study. Both displayed lower hemoglobin and higher erythropoietin levels than wild-type (WT) mice, along with significant splenomegaly. Flow cytometric analysis revealed erythroblasts present at a higher rate than WT mice in the spleen. However, no obvious abnormalities were seen in erythroid differentiation from megakaryocyte/erythroid progenitor to erythrocyte. Cell culture assay by fetal liver and colony assay also showed normal progression of erythroid differentiation from erythroid burst-forming unit to red blood cell. In conclusion, R702C and R702C vav1 mice displayed erythroid abnormality with splenomegaly. However, erythroid differentiation showed no obvious abnormality. Further research is required to elucidate the underlying mechanisms.

DOI： 10.18999/nagjms.83.1.75

Safety and efficacy of percutaneous radiofrequency ablation for hepatocellular carcinoma patients with haemophilia 査読有り国際誌

Yamamoto, T; Imai, N; Yamamoto, K; Ito, T; Ishizu, Y; Honda, T; Okamoto, S; Kanematsu, T; Suzuki, N; Matsushita, T; Ishigami, M; Fujishiro, M

HAEMOPHILIA 27 巻 ( 1 ) 頁： 100 - 107 2021年1月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Haemophilia

Haemophilia is an X-linked inherited bleeding disorder caused by coagulation factor deficiency. Hepatocellular carcinoma (HCC) is a major complication associated with the disease. No study thus far has investigated the safety and efficacy of percutaneous radiofrequency ablation (RFA) for HCC in patients with haemophilia. Aim: This study aimed to evaluate the safety and efficacy of RFA for HCC in haemophilia patients. Methods: From July 2008 to June 2019, 217 patients with HCC underwent 300 RFA sessions. Of these, 18 sessions were performed in ten haemophilia patients (H group) and 282 in 207 non-haemophilia patients (NH group). The patients' characteristics, incidence of haemorrhagic complications and rates of local tumour recurrence were compared between the groups. Results: A majority of the haemophilia patients received clotting factor concentrate replacement therapy before and after RFA treatment, with the aim of reaching a plasma clotting factor level of higher than 60%–80%. Twelve haemorrhagic complications were observed in the NH group (4.2%; 12/282). Major bleeding requiring control procedures was observed in two patients and minor bleeding with careful observation was noted in ten patients. No bleeding complications were observed in the H group (0/18). There were no significant differences in the 5-year local tumour recurrence rates after RFA treatment between the groups (35.0% in the H group and 32.1% in the NH group). Conclusion: RFA could be an effective and a safe method for HCC treatment in patients with haemophilia.

DOI： 10.1111/hae.14220

Aberrant X chromosomal rearrangement through multi-step template switching during sister chromatid formation in a patient with severe hemophilia A 査読有り国際誌

Tokoro, M; Tamura, S; Suzuki, N; Kakihara, M; Hattori, Y; Odaira, K; Suzuki, S; Takagi, A; Katsumi, A; Hayakawa, F; Okamoto, S; Suzuki, A; Kanematsu, T; Matsushita, T; Kojima, T

MOLECULAR GENETICS & GENOMIC MEDICINE 8 巻 ( 9 ) 頁： e1390 2020年9月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Molecular Genetics and Genomic Medicine

Background: Hemophilia A (HA) is an X-linked recessive bleeding disorder caused by pathogenic variants of the coagulation factor VIII gene (F8). Half of the patients with severe HA have a recurrent inversion in the X chromosome, that is, F8 intron 22 or intron 1 inversion. Here, we characterized an abnormal F8 due to atypical complex X chromosome rearrangements in a Japanese patient with severe HA. Methods: Recurrent F8 inversions were tested with inverse shifting-PCR. The genomic structure was investigated using PCR-based direct sequencing or quantitative PCR. Results: The proband's X chromosome had a 119.5 kb insertion, a reverse duplex of an extragenic sequence on the F8 telomere region into the F8 intron 1 with two breakpoints. The telomeric breakpoint was a joining from the F8 intron 1 to the inverted FUNDC2 via a two-base microhomology, and the centromeric breakpoint was a recombination between F8 intron 1 homologous sequences. The rearrangement mechanism was suggested as a multi-step rearrangement with template switching such as fork stalling and template switching (FoSTeS)/microhomology-mediated break-induced replication (MMBIR) and/or homologous sequence-associated recombination during a sister chromatid formation. Conclusion: We identified the aberrant X chromosome with a split F8 due to a multi-step rearrangement in a patient with severe HA.

DOI： 10.1002/mgg3.1390