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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Myh9 R702C is associated with erythroid abnormality with splenomegaly in mice.

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.

Essential role of a carboxyl-terminal α-helix motif in the secretion of coagulation factor XI.

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.

Impact of variation in reagent combinations for one-stage clotting assay on assay discrepancy in nonsevere haemophilia A.

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.

Aberrant X chromosomal rearrangement through multi-step template switching during sister chromatid formation in a patient with severe hemophilia A.

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.

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.