A C-terminal amino acid substitution in the γ-chain caused by a novel heterozygous frameshift mutation (Fibrinogen Matsumoto VII) results in hypofibrinogenaemia

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Aggressive NK-Cell Leukemia

Aggressive NK cell leukemia (ANKL) is a rare malignant lymphoproliferative disorder of mature NK cells closely associated with Epstein-Barr virus (EBV) and more common in East Asia than in other areas. Significant variations exist in the morphology of ANKL tumor cells, from typical large granular lymphocyte morphology to highly atypical features with basophilic cytoplasm containing azurophilc granules. The main involved sites are hepatosplenic lesions, bone marrow and peripheral blood, and nasal or skin lesions are infrequent. A fever and liver dysfunction with an often rapidly progressive course are the main clinical symptoms, including hemophagocytic syndrome and disseminated intravascular coagulation. Although the outcome had been dismal for decades, with a median survival of less than three months, the introduction of combined chemotherapy including L-asparaginase and allogeneic hematopoietic cell transplantation has helped achieve a complete response and potential cure for some patients. With the advent of next-generation sequencing technologies, molecular alterations of ANKL have been elucidated, and dysfunctions in several signaling pathways, including the JAK/STAT pathway, have been identified. Novel target approaches to managing these abnormalities might help improve the prognosis of patients with ANKL

Quantitative monitoring of single nucleotide mutations by allele-specific quantitative PCR can be used for the assessment of minimal residual disease in patients with hematological malignancies throughout their clinical course

BackgroundMonitoring of minimal residual disease (MRD) in patients with hematological malignancies is important for evaluating the patients\u27 therapeutic response and risk of relapse. Single nucleotide mutations associated with leukemogenesis can be considered as applicable MRD markers.MethodsWe developed an allele-specific quantitative polymerase chain reaction (AS-qPCR) for FLT3 2503G > T, KIT 2446G > T, and KIT 2447A > T and compared the change in the expression levels of the FLT3 or KIT mutations assessed by AS-qPCR to those of the RUNX1–RUNX1T1 fusion gene and WT1 by conventional quantitative PCR.ResultsThe AS-qPCR using primers including template-mismatched nucleotide or template-mismatched nucleotide plus locked nucleic acid substituted nucleotide provided higher selectivity for mutant nucleotides. The change in the expression levels of the FLT3 or KIT mutations at the time of relapse and just after hematopoietic stem cell transplantation correlated well with that of the RUNX1–RUNX1T1 fusion gene and WT1. Moreover, during complete remission, only AS-qPCR could detect low-level expression of residual mutations.ConclusionsThe AS-qPCR for analyzing single nucleotide mutations contributes to the monitoring of MRD in patients without recurrent fusion gene throughout the clinical course and thus broadens the spectrum of patients in whom MRD can be monitored

Simple generation of hairless mice for in vivo imaging

The in vivo imaging of mice makes it possible to analyze disease progress non-invasively through reporter gene expression. As the removal of hair improves the accuracy of in vivo imaging, gene-modified mice with a reporter gene are often crossed with Hos:HR-1 mutant mice homozygous for the spontaneous Hrhr mutation that exhibit a hair loss phenotype. However, it is time consuming to produce mice carrying both the reporter gene and mutant Hrhr gene by mating. In addition, there is a risk that genetic background of the gene-modified mice would be altered by mating. To resolve these issues, we established a simple method to generate hairless mice maintaining the original genetic background by CRISPR technology. First, we constructed the pX330 vector, which targets exon 3 of Hr. This DNA vector (5 ng/µl) was microinjected into the pronuclei of C57BL/6J mice. Induced Hr gene mutations were found in many founders (76.1%) and these mutations were heritable. Next, we performed in vivo imaging using these gene-modified hairless mice. As expected, luminescent objects in their body were detected by in vivo imaging. This study clearly showed that hairless mice could be simply generated by the CRISPR/Cas9 system, and this method may be useful for in vivo imaging studies with various gene-modified mice

Dual-alkylator Conditioning Regimen with Busulfan and Melphalan for Bone Marrow Allogeneic Hematopoietic Stem Cell Transplantation in Patients with Myelofibrosis : The Results of a Retrospective Study at a Single Institution in Japan

Myelofibrosis (MF) is a myeloproliferative neoplasm associated with significant morbidity and mortality, and allogeneic hematopoietic stem cell transplantation (allo-HSCT) remains the only curative approach. While the optimal conditioning regimen before allo-HSCT for MF patients remains to be determined, recent studies have suggested that a thiotepa-busulfan-containing dual-alkylator regimen, FBT regimen, may be associated with favorable outcomes. In Japan, however, thiotepa is not indicated for MF. Here we describe the results of 6 cases of MF treated with melphalan-busulfan containing dual-alkylator regimen, FBM regimen, followed by their first allo-HSCT at a single institution. Neutrophil and platelet engraftment was achieved in all patients. And a full donor chimerism was confirmed in all patients at ＋30 days after allo-HSCT. The relatively small size and short observation period of our study make it difficult to draw a definitive conclusion ; however, our results suggest that a dualalkylator regimen of FBM may be a candidate for an conditioning for allo-HSCT for MF, which should be verified with a large cohort of patients.Article信州医学雑誌 71(6) : 393-402, (2023)journal articl

A C-terminal amino acid substitution in the gamma-chain caused by a novel heterozygous frameshift mutation (Fibrinogen Matsumoto VII) results in hypofibrinogenemia

This article is not an exact copy of the original published article in THROMBOSIS AND HAEMOSTASIS. The definitive publisher-authenticated version of THROMBOSIS AND HAEMOSTASIS. 104(2):213-223 (2010) is available online at: https://doi.org/10.1160/TH09-08-0540 .We found a novel hypofibrinogenemia designated as Matsumoto VII (M-VII), which is caused by a heterozygous nucleotide deletion at position g.7651 in FGG and a subsequent frameshift mutation in codon 387 of the γ-chain. This frameshift results in 25 amino acid substitutions, late termination of translation with elongation by 15 amino acids, and the introduction of a canonical glycosylation site. Western blot analysis of the patient’s plasma fibrinogen visualized with anti-γ-chain antibody revealed the presence of two extra bands. To identify the extra bands and determine which of the above-mentioned alterations caused the assembly and/or secretion defects in the patient, 11 variant vectors that introduced mutations into the cDNA of the γ-chain orγ’-chain were transfected into CHO cells. In vitro expression of transfectants containingγΔ7651A and γΔ7651A/399T (γΔ7651A with an amino acid substitution of 399Asn by Thr and a variant lacking the canonical glycosylation site) demonstrated a reduction in secretion to approximately 20% of the level seen in the transfectants carrying the normal γ-chain. Furthermore, results from other transfectants demonstrated that 8 aberrant residues between 391 and 398 of the M-VII variant, rather than the 15 amino acid extension or the additional glycosylation, are responsible for the reduced levels of assembly and secretion of M-VII variant fibrinogen. Finally, the results of this study and our previous reports demonstrate that the fibrinogen γ-chain C-terminal tail (388-411) is not necessary for protein assembly or secretion, but the aberrant amino acid sequence observed in the M-VII variant (especially 391-398) disturbs these functions.ArticleTHROMBOSIS AND HAEMOSTASIS. 104(2):213-223 (2010)journal articl

Potential inhibitory effects of low-dose thoron inhalation and ascorbic acid administration on alcohol-induced hepatopathy in mice

Although thoron inhalation exerts antioxidative effects in several organs, there are no reports on whether it inhibits oxidative stress-induced damage. In this study, we examined the combined effects of thoron inhalation and ascorbic acid (AA) administration on alcohol-induced liver damage. Mice were subjected to thoron inhalation at 500 or 2000 Bq/m(3) and were administered 50% ethanol (alcohol) and 300 mg/kg AA. Results showed that although alcohol administration increased the levels of glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) in the serum, the combination of thoron inhalation (500 Bq/m(3)) and AA administration 24 h after alcohol administration effectively inhibited alcohol-induced liver damage. The combination of thoron inhalation (500 Bq/m(3)) and AA administration 24 h after alcohol administration increased catalase (CAT) activity. Alcohol administration significantly decreased glutathione (GSH) levels in the liver. The GSH content in the liver after 2000 Bq/m(3) thoron inhalation was lower than that after 500 Bq/m(3) thoron inhalation. These findings suggest that the combination of thoron inhalation at 500 Bq/m(3) and AA administration has positive effects on the recovery from alcohol-induced liver damage. The results also suggested that thoron inhalation at 500 Bq/m(3) was more effective than that at 2000 Bq/m(3), possibly because of the decrease in GSH content in the liver. In conclusion, the combination of thoron inhalation at 500 Bq/m(3) and AA administration promoted an early recovery from alcohol-induced liver damage

Relationship between respiratory muscle endurance and dyspnea during high-intensity exercise in trained distance runners

We hypothesized that the trained distance runners, who have a relatively high respiratory muscle endurance, but not high respiratory muscle strength, have lower dyspneic sensations during submaximal running. Twenty-one male collegiate distance runners participated. Incremental respiratory endurance tests (IRET) and maximal inspiratory mouth pressure (PImax) measurements were performed under resting conditions. A submaximal exercise test was also performed on a treadmill at two different speeds (16 and 18 km/h) for 4 min each, and the subjects reported the rate of dyspnea (range: 0–10). The time to endpoint during the IRET, an index of respiratory muscle endurance, ranged from 9.4 to 18.8 min, and PImax, as an index of inspiratory muscle strength, ranged from 74.1 to 137.0 cmH2O. The dyspnea rating during running at 16 and 18 km/h ranged from 1 to 6 and from 4 to 8, respectively. The relative exercise intensity was approximately 80 % of peak oxygen uptake (VO2peak) at 16 km/h and 90 %VO2peak at 18 km/h. The time to endpoint during the IRET was significantly negatively correlated with dyspnea during running at 18 km/h (r = -0.459, P = 0.040), but not at 16 km/h (r = -0.161, P = 0.470). There was no significant correlation between PImax and dyspnea during running at 16 km/h (r = -0.003, P = 0.989) or 18 km/h (r = 0.070, P = 0.755). These results suggest that dyspneic sensations during high-intensity running are related to respiratory muscle endurance, but not inspiratory muscle strength, in trained distance runners.journal articl