Basement membrane extract potentiates the endochondral ossification phenotype of bone marrow-derived mesenchymal stem cell-based cartilage organoids.

Endochondral ossification is a developmental process in the skeletal system and bone marrow of vertebrates. During endochondral ossification, primitive cartilaginous anlages derived from mesenchymal stem cells (MSCs) undergo vascular invasion and ossification. In vitro regeneration of endochondral ossification is beneficial for research on the skeletal system and bone marrow development as well as their clinical aspects. However, to achieve the regeneration of endochondral ossification, a stem cell-based artificial cartilage (cartilage organoid, Cart-Org) that possesses an endochondral ossification phenotype is required. Here, we modified a conventional 3D culture method to create stem cell-based Cart-Org by mixing it with a basement membrane extract (BME) and further characterized its chondrogenic and ossification properties. BME enlarged and matured the bone marrow MSC-based Cart-Orgs without any shape abnormalities. Histological analysis using Alcian blue staining showed that the production of cartilaginous extracellular matrices was enhanced in Cart-Org treated with BME. Transcriptome analysis using RNA sequencing revealed that BME altered the gene expression pattern of Cart-Org to a dominant chondrogenic state. BME triggered the activation of the SMAD pathway and inhibition of the NK-κB pathway, which resulted in the upregulation of SOX9, COL2A1, and ACAN in Cart-Org. BME also facilitated the upregulation of genes associated with hypertrophic chondrocytes (IHH, PTH1R, and COL10A1) and ossification (SP7, ALPL, and MMP13). Our findings indicate that BME promotes cartilaginous maturation and further ossification of bone marrow MSC-based Cart-Org, suggesting that Cart-Org treated with BME possesses the phenotype of endochondral ossification.

Outcomes of transplant-eligible patients with myelodysplastic syndrome with excess blasts registered in an observational study: The JALSG-CS11-MDS-SCT.

Allogeneic hematopoietic stem cell transplantation (allo-SCT) is the sole curative therapy for myelodysplastic syndrome (MDS). However, whether bridging therapy (BRT) including azacitidine (AZA) and combination chemotherapy (CCT) prior to allo-SCT should be performed is unclear. We analyzed BRT and the outcomes of patients with myelodysplastic syndrome with excess blasts (MDS-EB) who were ≤ 70 years old at the time of registration for a prospective observational study to clarify the optimal allo-SCT strategy for high-risk MDS. A total of 371 patients were included in this study. Among 188 patients (50.7%) who were considered for allo-SCT, 141 underwent allo-SCT. Among the patients who underwent allo-SCT, 64 received AZA, 29 received CCT, and 26 underwent allo-SCT without BRT as the initial treatment. Multivariate analysis identified BRT as an independent factor influencing overall survival (AZA vs. without BRT, hazard ratio [HR] 3.33, P = 0.005; CCT vs. without BRT, HR 3.82, P = 0.003). In multivariate analysis, BRT was independently associated with progression-free survival (AZA vs. without BRT: HR, 2.23; P = 0.041; CCT vs. without BRT: HR, 2.94; P = 0.010). Transplant-eligible patients with MDS-EB should undergo allo-SCT when clinically acceptable, and upfront allo-SCT without BRT may be superior to AZA or CCT.

Periosteum-derived podoplanin-expressing stromal cells regulate nascent vascularization during epiphyseal marrow development.

Bone marrow development and endochondral bone formation occur simultaneously. During endochondral ossification, periosteal vasculatures and stromal progenitors invade the primary avascular cartilaginous anlage, which induces primitive marrow development. We previously determined that bone marrow podoplanin (PDPN)-expressing stromal cells exist in the perivascular microenvironment and promote megakaryopoiesis and erythropoiesis. In this study, we aimed to examine the involvement of PDPN-expressing stromal cells in postnatal bone marrow generation. Using histological analysis, we observed that periosteum-derived PDPN-expressing stromal cells infiltrated the cartilaginous anlage of the postnatal epiphysis and populated on the primitive vasculature of secondary ossification center. Furthermore, immunophenotyping and cellular characteristic analyses indicated that the PDPN-expressing stromal cells constituted a subpopulation of the skeletal stem cell lineage. In vitro xenovascular model cocultured with human umbilical vein endothelial cells and PDPN-expressing skeletal stem cell progenies showed that PDPN-expressing stromal cells maintained vascular integrity via the release of angiogenic factors and vascular basement membrane-related extracellular matrices. We show that in this process, Notch signal activation committed the PDPN-expressing stromal cells into a dominant state with basement membrane-related extracellular matrices, especially type IV collagens. Our findings suggest that the PDPN-expressing stromal cells regulate the integrity of the primitive vasculatures in the epiphyseal nascent marrow. To the best of our knowledge, this is the first study to comprehensively examine how PDPN-expressing stromal cells contribute to marrow development and homeostasis.

The usefulness of tranexamic acid for bleeding symptoms of chronic consumptive coagulopathy complicated by aortic disease: a single-institute, retrospective study of 14 patients.

BACKGROUND: Tranexamic acid (TXA) is an antifibrinolytic drug that blocks lysine-binding sites on the profibrinolytic enzyme plasminogen. Aortic diseases with chronic consumption coagulopathy may lead to disseminated intravascular coagulation (DIC) and cause fatal bleeding. Although the use of antifibrinolytic agents in DIC is generally not recommended due to enhanced fibrin deposition risking thrombotic symptoms, the efficacy of TXA has been reported in several cases of DIC with aortic diseases. However, the efficacy and safety of TXA for bleeding symptoms of chronic consumption coagulopathy with aortic diseases have not been studied in detail. METHODS: We evaluated the efficacy of TXA in 14 patients with chronic consumptive coagulopathy due to aortic disease complicated by bleeding symptoms. Changes in coagulation and fibrinolysis parameters from baseline were analyzed with Wilcoxon matched-pairs signed-rank tests, excluding missing values. Kaplan-Meier curves were used to analyze overall survival. RESULTS: Median age was 78.5 years (range, 66-89 years) and median observation period was 448 days (range, 0-2282 days). Twelve patients had chronic renal failure and 1 patient had chronic liver failure. Before starting treatment, median Japanese Ministry of Health and Welfare DIC diagnostic criteria score was 8 (range, 4-11) and median platelet count was 64 × 109/L (range, 25-97 × 109/L). Twelve patients underwent evaluation of bleeding symptoms after introduction of TXA, and 10 of those 12 patients showed improved bleeding tendencies within 30 days (median, 5.0 days). One patient with chronic liver failure showed worsening of bleeding symptoms. Although only one patient was initiated TXA in combination with anticoagulants, no significant worsening of thrombotic events was observed within 30 days. CONCLUSIONS: TXA therapy appears effective against chronic consumptive coagulopathy with bleeding due to aortic disease, with few side effects.

Elevated red blood cell distribution width is associated with poor outcome in osteoporotic vertebral fracture.

INTRODUCTION: Red blood cell distribution width (RDW) has been evaluated as a potential screening marker for cancer and prognostic marker in heart failure and coronary heart disease. Recent studies have been suggested the association of RDW with mortality in patients with hip fracture and arthroplasty. Objective of this study was to investigate whether RDW as a prognostic marker is significant in patients with osteoporotic vertebral fracture (OVF). MATERIALS AND METHODS: Total of 460 patients with fresh OVF from January 2014 and September 2017 were assessed for a 1-year follow-up period. The cutoff value for RDW was set at 15%, and outcomes of conservative treatment of OVF were evaluated using the Japanese Orthopaedic Association (JOA) scores, Barthel index, and walking state. RESULTS: Of the total 460 patients, 125 patients (27.2%) had an elevated RDW. RDW value was not correlated with osteoporotic parameters. Both JOA score and Barthel index were significantly lower at 1 year after treatment in the elevated RDW group. In the elevated RDW group, 21 patients died within 1 year (mortality 16.8%) compared with 7 patients (mortality 2.1%) in the non-elevated RDW group; this was statistically significant. Multivariate statistical analysis showed elevated RDW, independent walk before OVF and skeletal muscle mass index (SMI) remained independent factors associated with abasia after OVF affected. CONCLUSION: Elevated RDW was associated with the poor clinical outcomes of conservative treatment of an OVF, independent of osteoporosis or severity of the OVF. RDW provides prognostic information for risk stratification as a senescence biomarker.

Truncated RUNX1 Generated by the Fusion of RUNX1 to Antisense GRIK2 via a Cryptic Chromosome Translocation Enhances Sensitivity to Granulocyte Colony-Stimulating Factor.

Fusions of the Runt-related transcription factor 1 (RUNX1) with different partner genes have been associated with various hematological disorders. Interestingly, the C-terminally truncated form of RUNX1 and RUNX1 fusion proteins are similarly considered important contributors to leukemogenesis. Here, we describe a 59-year-old male patient who was initially diagnosed with acute myeloid leukemia, inv(16)(p13;q22)/CBFB-MYH11 (FAB classification M4Eo). He achieved complete remission and negative CBFB-MYH11 status with daunorubicin/cytarabine combination chemotherapy but relapsed 3 years later. Cytogenetic analysis of relapsed leukemia cells revealed CBFB-MYH11 negativity and complex chromosomal abnormalities without inv(16)(p13;q22). RNA-seq identified the glutamate receptor, ionotropic, kinase 2 (GRIK2) gene on 6q16 as a novel fusion partner for RUNX1 in this case. Specifically, the fusion of RUNX1 to the GRIK2 antisense strand (RUNX1-GRIK2as) generated multiple missplicing transcripts. Because extremely low levels of wild-type GRIK2 were detected in leukemia cells, RUNX1-GRIK2as was thought to drive the pathogenesis associated with the RUNX1-GRIK2 fusion. The truncated RUNX1 generated from RUNX1-GRIK2as induced the expression of the granulocyte colony-stimulating factor (G-CSF) receptor on 32D myeloid leukemia cells and enhanced proliferation in response to G-CSF. In summary, the RUNX1-GRIK2as fusion emphasizes the importance of aberrantly truncated RUNX1 in leukemogenesis.

Differences in incidence and trends of haematological malignancies in Japan and the United States.

The incidence of a malignant disease reflects the genetic and cumulative exposure to the environment of a population. Therefore, evaluation of the incidence and trends of a disease in different populations may provide insights into its aetiology and pathogenesis. To evaluate the incidence of haematological malignancies according to specific subtypes, we used population-based registry data in Japan (N = 125 148) and the United States (US; N = 172 925) from 1993 to 2008. The age-adjusted incidence of haematological malignancies in Japan was approximately one-half that in the US but has been increasing significantly, whereas no significant change was seen in the US [annual percent change (95% C confidence interval): Japan, +2·4% (1·7, 3·1); US, +0·1% (-0·1, 0·2)]. Hodgkin lymphoma (HL) and non-Hodgkin lymphoma (NHL) showed the largest differences in incidence, with the most remarkable differences observed for chronic lymphocytic leukaemia, HL-nodular sclerosis, mycosis fungoides and cutaneous T-cell lymphoma. HL and NHL are increasing substantially in Japan but not in the US, suggesting that environmental exposures, such as Westernization of the life style may be causing this increase. Differences in the incidence and trends for specific subtypes also showed a marked contrast across subtypes, which, in turn, may provide significant new insights into disease aetiology in the future.

Rhof promotes murine marginal zone B cell development.

RhoF is a member of the Rho GTPase family that has been implicated in various cell functions including long filopodia formation, adhesion, and migration of cells. Although RhoF is expressed in lymphoid tissues, the roles of RhoF in B cell development remain largely unclear. On the other hand, other members of the Rho GTPase family, such as Cdc42, RhoA, and Rac, have been intensively studied and are known to be required for B cell development in the bone marrow and spleen. We hypothesized that RhoF is also involved in B cell development. To examine our hypothesis, we analyzed B cell development in RhoF knockout (KO) mice and found a significant reduction in marginal zone (MZ) B cells in the spleen, although T cell development in the thymus and spleen was not affected. Consistent with these results, the width of the MZ B cell region in the spleen was significantly reduced in the RhoF KO mice. However, the antigen-specific antibody titer of IgM and IgG3 after MZ B cell-specific antigen (T cell-independent antigen, type I) stimulation was not affected by RhoF deletion. Furthermore, we demonstrated that RhoF was dispensable for stromal cell-derived factor-1α- and B lymphocyte chemoattractant-induced B cell migration. These results suggest that RhoF promotes MZ B cell development in the spleen.

Acquired factor X deficiency developed four years after autologous transplantation in a patient with multiple myeloma associated with systemic AL amyloidosis.

We describe a case of acquired factor X deficiency after high-dose melphalan with autologous stem cell transplantation (HDM/ASCT) for multiple myeloma (MM) with systemic AL amyloidosis. A 68-year-old woman with renal amyloidosis was diagnosed as having MM in 2007. She achieved a partial response after VAD (vincristine, adriamycin, dexamethasone) therapy and HDM/ASCT. In December 2011, coagulation tests revealed a prolonged prothrombin time (PT) of 17.6 sec and she was administered vitamin K. In January 2012, she received low anterior resection with colostomy for rectal cancer. She received fresh frozen plasma (FFP) infusion but the perioperative bleeding tendency persisted. In February 2012, she was referred from surgery for colostomy closure. She showed no progression of MM and had prolonged PT, corrected by mixing with normal plasma. Factor X activity was markedly decreased. She was diagnosed as having an acquired factor X deficiency and was given FFP infusion for colostomy closure. Although acquired factor X deficiency after HDM/ASCT for MM with systemic AL amyloidosis is rare, we should be aware of the possibility of this disease in MM patients with a bleeding tendency.

Variability in combinations of APTT reagent and substrate plasma for a one-stage clotting assay to measure factor VIII products.

INTRODUCTION: An investigation of the suitability of reagents for measuring FVIII products in a one-stage clotting assay (OSA) showed variations in their FVIII activity (FVIII:C). Most studies have focused on the activated partial thromboplastin time (APTT) reagent rather than FVIII-deficient plasma (F8DP), even though the APTT-based OSA is comprised of APTT reagents and factor-deficient plasma. AIM: A single-centre study was conducted to clarify variations in measurements of FVIII products in an OSA using a total of 12 reagent combinations, including four APTT reagents and three types of F8DP. METHODS: FVIII:C in nine types of FVIII product-spiked plasma was measured using an OSA with four different APTT reagents and three types of F8DP. RESULTS: F8DP-dependent variations were found in addition to differences derived from APTT reagents. Variations in target recovery (TR) were observed for NovoEight®, Eloctate®, and Jivi®. Reduced TR for Jivi was found only for Pathromtin SL in combination with congenital F8DP (F8DP-3). This lower TR was not observed with alternative manufacturing lots of F8DP-3. The reduced TR for Jivi might be related to impaired contact activation due to lower factor XI activity in F8DP-3. CONCLUSION: In addition to APTT reagents, variations in F8DPs used for OSAs can also affect FVIII:C results. F8DPs as well as the APTT reagent used for OSA should be chosen with caution, and laboratories should evaluate reagents for F8DPs as they currently do for APTT reagents, especially when lot changes occur.

Real-world data of AML in Japan: results of JALSG clinical observational study-11 (JALSG-CS-11).

This report covers acute myeloid leukemia (AML) results from a multicenter, prospective observational study of AML, myelodysplastic syndromes, and chronic myelomonocytic leukemia in Japan. From August 2011 to January 2016, 3728 AML patients were registered. Among them, 42% were younger than 65, and the male-to-female ratio was 1.57:1. With a median follow-up time of 1807 days (95% confidence interval [CI]: 1732-1844 days), the estimated 5-year overall survival (OS) rate in AML patients (n = 3707) was 31.1% (95% CI: 29.5-32.8%). Trial-enrolled patients had a 1.7-fold higher OS rate than non-enrolled patients (5-year OS, 58.9% [95% CI: 54.5-63.1%] vs 35.5% [33.3-37.8%], p < 0.0001). Women had a higher OS rate than men (5-year OS, 34% [95% CI; 31.4-36.7%] vs 27.7% [25.7-29.7%], p < 0.0001). The OS rate was lower in patients aged 40 and older than those under 40, and even lower in those over 65 (5-year OS for ages < 40, 40-64, 65-74, ≥ 75: 74.5% [95% CI; 69.3-79.0%] vs 47.5% [44.4-50.6%] vs 19.3% [16.8-22.0%] vs 7.3% [5.5-9.4%], respectively). This is the first paper to present large-scale data on survival and clinical characteristics in Japanese AML patients.

Real-world data of MDS and CMML in Japan: results of JALSG clinical observational study-11 (JALSG-CS-11).

We conducted a multicenter, prospective observational study of acute myeloid leukemia (AML), myelodysplastic syndromes (MDS), and chronic myelomonocytic leukemia (CMML) in Japan. From August 2011 to January 2016, we enrolled 6568 patients. Herein, we report the results for MDS (n = 2747) and CMML (n = 182). The percentage of patients aged 65 years or older was 79.5% for MDS and 79.7% for CMML. The estimated overall survival (OS) rate and cumulative incidence of AML evolution at 5 years were 32.3% (95% confidence interval: 30.2-34.5%) and 25.7% (23.9-27.6%) for MDS, and 15.0% (8.9-22.7%) and 39.4% (31.1-47.6%) for CMML. Both diseases were more common in men. The most common treatment for MDS was azacitidine, which was used in 45.4% of higher-risk and 12.7% of lower-risk MDS patients. The 5-year OS rate after treatment with azacitidine was 12.1% (9.5-15.1%) for of higher-risk MDS patients and 33.9% (25.6-42.4%) for lower-risk patients. The second most common treatment was erythropoiesis-stimulating agents, given to just 20% of lower-risk patients. This is the first paper presenting large-scale, Japanese data on survival and clinical characteristics in patients with MDS and CMML.

Prolonged COVID-19 Infection in a Patient with Complete Remission from Follicular Lymphoma with Hyperosmolar Hyperglycemic Syndrome.

An 81-year-old woman undergoing B-cell depletion therapy developed COVID-19 and a hyperglycemic hyperosmotic state. She had a history of multiple vaccinations against coronaviruses but had persistent antigen positivity. Strategies to prevent the development of COVID-19 in immunosuppressed patients have not been established. Moreover, there is no standard treatment for prolonged antigen positivity. In this case, we were able to follow IgG antibodies during the course of treatment. The absence of N-IgG antibody titer elevation despite an effective immune response triggered by the vaccine is of great interest. The impaired humoral response observed in patients with lymphoma after anti-CD20 treatment implies the need for a justified different vaccination strategy for these patients. Furthermore, negative N-IgG titers in the immunosuppressed state may serve as an indicator of resistance to therapy.

Y654 of ß-catenin is essential for FLT3/ITD-related tyrosine phosphorylation and nuclear localization of ß-catenin.

ß-Catenin plays a dual role as a key effecter in the regulation of adherens junctions as well as a transcriptional co-activator. Tyrosine phosphorylation of ß-catenin affects the cell adhesion, migration, and gene transcription in many types of human cancer cells, including acute myeloid leukemia cells with FLT3 internal tandem duplication (FLT3/ITD-AML). Here, we investigated the relationship between three tyrosine residues (Y86, Y142, and Y654) in ß-catenin and oncogenic FLT3/ITD kinase. In the experiments using COS-7 cells expressing FLT3/ITD and Wt or mutant ß-catenin, FLT3/ITD phosphorylated Y654, and this residue was essential for ß-catenin's nuclear localization by FLT3/ITD. Promoter-reporter assays demonstrated that Y654 phosphorylation of ß-catenin was closely related to TCF transcriptional activity. In vitro kinase assays, using recombinant FLT3 and biotinylated ß-catenin peptide including Y654 showed that FLT3 directly phosphorylated Y654 of ß-catenin. These results explain how FLT3/ITD affects the tyrosine phosphorylation, nuclear localization, and transcriptional activity of ß-catenin. Targeting Y654 phosphorylation may lead to the development of novel approaches to therapy for FLT3/ITD-AML.

VWF-Gly2752Ser, a novel non-cysteine substitution variant in the CK domain, exhibits severe secretory impairment by hampering C-terminal dimer formation.

BACKGROUND: Von Willebrand factor (VWF) is a multimeric glycoprotein that plays important roles in hemostasis and thrombosis. C-terminal interchain-disulfide bonds in the cystine knot (CK) domain are essential for VWF dimerization. Previous studies have reported that missense variants of cysteine in the CK domain disrupt the intrachain-disulfide bond and cause type 3 von Willebrand disease (VWD). However, type 3 VWD-associated noncysteine substitution variants in the CK domain have not been reported. OBJECTIVE: To investigate the molecular mechanism of a novel non-cysteine variant in the CK domain, VWF c.8254 G>A (p.Gly2752Ser), which was identified in a patient with type 3 VWD as homozygous. METHODS: Genetic analysis was performed by whole exome sequencing and Sanger sequencing. VWF multimer analysis was performed using SDS-agarose electrophoresis. VWF production and subcellular localization were analyzed using ex vivo endothelial colony forming cells (ECFCs) and an in vitro recombinant VWF (rVWF) expression system. RESULTS: The patient was homozygous for VWF-Gly2752Ser. Plasma VWF enzyme-linked immunosorbent assay showed that the VWF antigen level of the patient was 1.2% compared with healthy subjects. A tiny amount of VWF was identified in the patient's ECFC. Multimer analysis revealed that the circulating VWF-Gly2752Ser presented only low molecular weight multimers. Subcellular localization analysis of VWF-Gly2752Ser-transfected cell lines showed that rVWF-Gly2752Ser was severely impaired in its ER-to-Golgi trafficking. CONCLUSION: VWF-Gly2752Ser causes severe secretory impairment because of its dimerization failure. This is the first report of a VWF variant with a noncysteine substitution in the CK domain that causes type 3 VWD.

Development and validation of a novel qualitative test for plasma fibrinogen utilizing clot waveform analysis.

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.

Protein S-Leu17Pro disrupts the hydrophobicity of its signal peptide causing a proteasome-dependent degradation.

INTRODUCTION: Protein S is a vitamin K-dependent glycoprotein with important anticoagulant, fibrinolytic, anti-inflammatory, anti-apoptotic, and cytoprotective functions. Congenital protein S deficiency is an autosomal dominant thrombophilia due to protein S gene (PROS1) variations. Our group identified a variation in PROS1 that translates into protein S deficiency: c.50 T > C (p.Leu17Pro). Here, we investigated the mechanisms by which this variation results in protein S deficiency. MATERIALS AND METHODS: The effect of L17P substitution on protein S signal peptide was predicted by in silico (a computational prediction technique) analysis of hydrophobicity and signal peptide cleavage. Recombinant protein S was overexpressed in HEK293 and COS-7 cells. Intracellular kinetics and extracellular secretion of recombinant protein S-L17P were analyzed by western blotting and immunocytochemistry. RESULTS: In silico hydrophobicity analysis showed that protein S-L17P had disrupted hydrophobic status in the h-region of its signal peptide. Under normal culture conditions, recombinant protein S -L17P was not detected in either transfectant cell lysates or medium. Upon treatment with a proteasome inhibitor, recombinant protein S-L17P was clearly detected in the cell lysate, but not in the culture medium. Recombinant protein S-L17P did not undergo post-translational modification with N-glycosylation, suggesting that the nascent polypeptide of recombinant protein S-L17P is not transported to the endoplasmic reticulum lumen, but is mislocalized to the cytosol. CONCLUSION: PROS1-L17P variation translates into protein S deficiency. Protein S-L17P causes its cytosolic mislocalization resulting in its proteasome-dependent degradation.

F9 mRNA splicing aberration due to a deep Intronic structural variation in a patient with moderate hemophilia B.

INTRODUCTION: Hemophilia B (HB) is a hereditary bleeding disorder caused by the genetic variation of the coagulation factor IX (FIX) gene (F9). Several F9 structural abnormalities, including large deletion and/or insertion, have been observed to cause HB development. However, there is limited information available on F9 deep intronic variations. In this study, we report about a novel large deletion/insertion observed in a deep region of F9 intron 1 that causes mRNA splicing abnormalities. PATIENT AND METHODS: The patient was a Japanese male diagnosed with moderate HB (FIX:C = 3.0 IU/dL). The genomic DNA of the patient was isolated from peripheral blood leukocytes. DNA sequences of F9 exons and splice donor/acceptor sites were analyzed via polymerase chain reaction and Sanger sequencing. Variant-affected F9 mRNA aberration and FIX protein production, secretion, and coagulant activity were analyzed by cell-based exon trap and splicing-competent FIX expression vector systems. RESULTS: A 28-bp deletion/476-bp insertion was identified in the F9 intron 1 of a patient with moderate HB. A DNA sequence identical to a part of the inverted HNRNPA1 exon 12 was inserted. Cell-based transcript analysis revealed that this large intronic deletion/insertion disrupted F9 mRNA splicing pattern, resulting in reduction of protein-coding F9 mRNA. CONCLUSION: A novel deep intronic F9 rearrangement was identified in a Japanese patient with moderate HB. Abnormal F9 mRNA splicing pattern due to this deep intronic structural variation resulted in a reduction of protein-coding F9 mRNA, which probably caused the moderate HB phenotype.

FLT3/ ITD regulates leukaemia cell adhesion through α4ß1 integrin and Pyk2 signalling.

Internal tandem duplication of FMS-like receptor tyrosine kinase 3 (FLT3/ITD) within its juxtamembrane domain is a frequent mutation in adult acute myeloid leukaemia (AML). This mutation causes constitutive activation of FLT3 and is associated with poor prognosis. The high relapse rate of FLT3/ITD-positive AML might be partly because of insufficient eradication of slow-cycling leukaemic stem cells in the bone marrow microenvironment. ß1 integrin mediates haematopoietic stem and progenitor cell homing along with their retention in the bone marrow and also inhibits haematopoietic proliferation and differentiation. Here, we demonstrate that inhibition of FLT3/ITD kinase activity by a FLT3 selective inhibitor named FI-700 decreases affinity of α4ß1 integrin to soluble VCAM-1. α4ß1 integrin deactivation by FI-700 is independent of Rap1, which is the critical regulator of integrin inside-out signalling. In addition, selective inhibition of FLT3/ITD induces Pyk2 dephosphorylation together with the inhibition of phosphatidylinositol-3-kinase (PI3K)/Akt pathway. Both wild-type and ITD-FLT3 proteins co-immunoprecipitated with ß1 integrin and Pyk2 indicating the signal crosstalk between FLT3, ß1 integrin and Pyk2. These results collectively indicated that the inhibition of FLT3 kinase might contribute not only to the induction of apoptosis, but also to the leukaemia cell detachment from the bone marrow microenvironment in the treatment of AML.

Anemia in older adults as a geriatric syndrome: A review.

Anemia, a frequently occurring condition in older patients, has no standard definition; however, in most studies, it is defined as hemoglobin level <12 and <13 g/dL in women and men, respectively. Approximately 10% of older adults living in the community have anemia. The prevalence of anemia is significantly correlated with advanced age and male sex. Anemia is associated with falls, frailty and other negative outcomes, including early mortality. However, there remains little consensus regarding whether anemia treatment favorably affects these adverse outcomes. Therefore, this article reviews the prevalence of anemia, and provides updates on its common causes and treatments in older adults. While excluding well-established hematopoietic diseases, the etiology of anemia in older adults has been grouped into four categories: (i) nutritional deficiency; (ii) inflammation; (iii) clonal hematopoiesis; and (iv) "unexplained anemia," when there is no clear mechanism to account for the anemia. Recently, clonal leukocytes were detected in a considerable number of older individuals. The number of somatic mutations in blood leukocytes increases with age; however, single mutations of DNMT3A, TET2 and ASXL1 are not correlated with the presence of unexplained anemia in older adults. With an increased understanding of anemia etiology and the availability of innovative anti-anemic drugs, future studies that evaluate the causes and benefits of treatment are required. Geriatr Gerontol Int 2021; 21: 549-554.