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Abstract Although killer Ig-like receptor ligands (KIR-L) mismatch has been associated with alloreactive natural killer cell activity and potent graft-versus-leukemia (GVL) effect among adults with acute myeloid leukemia (AML), its role among children with AML receiving cord blood transplantation (CBT) has not been determined. We conducted a retrospective study using a nationwide registry of the Japanese Society for Transplantation and Cellular Therapy. Patients who were diagnosed with de novo non-M3 AML and who underwent their first CBT in remission between 2000 and 2021 at under 16 years old were included. A total of 299 patients were included; 238 patients were in the KIR-L match group, and 61 patients were in the KIR-L mismatch group. The cumulative incidence rates of neutrophil recovery, platelet engraftment, and acute/chronic graft-versus-host disease did not differ significantly between the groups. The 5-year event-free survival (EFS) rate was 69.8% in the KIR-L match group and 74.0% in the KIR-L mismatch group (p = 0.490). Stratification by CD34 + cell dose into four groups revealed a significant correlation between CD34 + cell dose and EFS in the KIR-L mismatch group (p = 0.006) but not in the KIR-L match group (p = 0.325). According to our multivariate analysis, KIR-L mismatch with a high CD34 + cell dose (≥ median dose) was identified as an independent favorable prognostic factor for EFS (hazard ratio = 0.19, p = 0.029) and for the cumulative incidence of relapse (hazard ratio = 0.09, p = 0.021). Our results suggested that higher CD34 + cell doses are crucial for achieving a potent GVL effect in the context of KIR-L-mismatched CBT.

Abstract HOIL-1L deficiency was recently reported to be one of the causes of myopathy and dilated cardiomyopathy (DCM). However, the mechanisms by which myopathy and DCM develop have not been clearly elucidated. Here, we sought to elucidate these mechanisms using the murine myoblast cell line C2C12 and disease-specific human induced pluripotent stem cells (hiPSCs). Myotubes differentiated from HOIL-1L-KO C2C12 cells exhibited deteriorated differentiation and mitotic cell accumulation. CMs differentiated from patient-derived hiPSCs had an abnormal morphology with a larger size and were excessively multinucleated compared with CMs differentiated from control hiPSCs. Further analysis of hiPSC-derived CMs showed that HOIL-1L deficiency caused cell cycle alteration and mitotic cell accumulation. These results demonstrate that abnormal cell maturation possibly contribute to the development of myopathy and DCM. In conclusion, HOIL-1L is an important intrinsic regulator of cell cycle-related myotube and CM maturation and cell proliferation.

Abstract Hepatoblastomas (HB) display heterogeneous cellular phenotypes that influence the clinical outcome, but the underlying mechanisms are poorly understood. Here, we use a single-cell multiomic strategy to unravel the molecular determinants of this plasticity. We identify a continuum of HB cell states between hepatocytic (scH), liver progenitor (scLP) and mesenchymal (scM) differentiation poles, with an intermediate scH/LP population bordering scLP and scH areas in spatial transcriptomics. Chromatin accessibility landscapes reveal the gene regulatory networks of each differentiation pole, and the sequence of transcription factor activations underlying cell state transitions. Single-cell mapping of somatic alterations reveals the clonal architecture of each tumor, showing that each genetic subclone displays its own range of cellular plasticity across differentiation states. The most scLP subclones, overexpressing stem cell and DNA repair genes, proliferate faster after neo-adjuvant chemotherapy. These results highlight how the interplay of clonal evolution and epigenetic plasticity shapes the potential of HB subclones to respond to chemotherapy.

Abstract Acute myeloid leukemia (AML) is a malignancy characterized by differentiation arrest of hematopoietic precursor cells. Differentiation therapy is effective for patients with acute promyelocytic leukemia; however, only a few effective differentiation therapies have been established for patients with other AML subtypes. In this study, seven benzimidazole anthelmintics were examined to determine the effects of differentiation on AML cells. The expression of monocyte markers (CD11b and CD14) was elevated after treatment with most benzimidazole anthelmintics. Among these drugs, parbendazole (PBZ) induced AML cell differentiation at low concentration. PBZ induced the monocyte marker expression, KLF4/DPYSL2A gene expression, and apoptosis for 21 AML cell lines with various subtypes and a primary AML sample. Finally, an in vivo analysis using an AML patient-derived xenograft mouse model showed a significant decrease in the chimerism level and prolonged survival in PBZ-treated mice. These findings could lead to a more effective differentiation therapy for AML.

BackgroundDuchenne muscular dystrophy (DMD), a severe degenerative skeletal and cardiac muscle disease, has a poor prognosis, and no curative treatments are available. Because decreased autophagy has been reported to contribute to skeletal muscle degeneration, therapies targeting autophagy are expected to improve skeletal muscle hypofunction. However, the role of this regulatory mechanism has not been evaluated clearly in DMD cardiomyocytes.MethodsIn this present study, we evaluated myocardial fibrosis and its mechanism in mdx mice, a model of DMD, and also evaluated changes in cardiac function.ResultsAs assessed by LC3 immunohistochemistry, a small number of autophagosomes were detected in cardiomyocytes of both mdx mice and control wild-type (WT) mice. The number of autophagosomes was significantly enhanced by 4 weeks of isoproterenol-induced cardiac stress in cardiomyocytes of mdx but not WT mice. Simultaneously, isoproterenol increased cardiomyocyte fibrosis in mdx but not WT mice. Administration of chloroquine significantly decreased cardiomyocyte fibrosis in mdx mice, even after isoproterenol treatment. Left ventricle size and function were evaluated by echocardiography. Left ventricular contraction was decreased in mdx mice after isoproterenol treatment compared with control mice, which was alleviated by chloroquine administration.ConclusionsHeart failure in DMD patients is possibly treated with chloroquine, and the mechanism probably involves chloroquine's anti-inflammatory effects.

The clinical utility of measuring cerebrospinal fluid placental alkaline phosphatase (CSF-PLAP) was evaluated in three patients with germinoma. CSF-PLAP was elevated in two patients with suprasellar germinoma who were symptomatic before developing typical radiological findings. The third patient, who had been radiologically diagnosed with a pineal mature teratoma, was suspected of having a germinoma component due to elevated CSF-PLAP; this patient received adjuvant radiochemotherapy. The presence of a germinoma-specific tumor marker may aid in the early diagnosis of suspicious cases at initial clinical presentation and in the accurate subtype classification of intracranial germ cell tumors.

The molecular heterogeneity of KMT2A-rearranged infant acute lymphoblastic leukemia (ALL) remains poorly characterised. Here, the authors perform multi-omics analysis for 84 ALL patients and suggest 5 distinct subgroups for risk stratification and personalised treatment.’

PurposeUpregulation of type I interferon (IFN) signaling has been increasingly detected in inflammatory diseases. Recently, upregulation of the IFN signature has been suggested as a potential biomarker of IFN-driven inflammatory diseases. Yet, it remains unclear to what extent type I IFN is involved in the pathogenesis of undifferentiated inflammatory diseases. This study aimed to quantify the type I IFN signature in clinically undiagnosed patients and assess clinical characteristics in those with a high IFN signature.MethodsThe type I IFN signature was measured in patients’ whole blood cells. Clinical and biological data were collected retrospectively, and an intensive genetic analysis was performed in undiagnosed patients with a high IFN signature.ResultsA total of 117 samples from 94 patients with inflammatory diseases, including 37 undiagnosed cases, were analyzed. Increased IFN signaling was observed in 19 undiagnosed patients, with 10 exhibiting clinical features commonly found in type I interferonopathies. Skin manifestations, observed in eight patients, were macroscopically and histologically similar to those found in proteasome-associated autoinflammatory syndrome. Genetic analysis identified novel mutations in the PSMB8 gene of one patient, and rare variants of unknown significance in genes linked to type I IFN signaling in four patients. A JAK inhibitor effectively treated the patient with the PSMB8 mutations. Patients with clinically quiescent idiopathic pulmonary hemosiderosis and A20 haploinsufficiency showed enhanced IFN signaling.ConclusionsHalf of the patients examined in this study, with undifferentiated inflammatory diseases, clinically quiescent A20 haploinsufficiency, or idiopathic pulmonary hemosiderosis, had an elevated type I IFN signature.

Abstract Patients with refractory graft‐versus‐host disease (GVHD) have a dismal prognosis. Therefore, novel therapeutic targets are still needed to be identified. Runt‐related transcriptional factor (RUNX) family transcription factors are essential transcription factors that mediate the essential roles in effector T cells. However, whether RUNX targeting can suppress, and GVHD is yet unknown. Here, we showed that RUNX family members have a redundant role in directly transactivating NFATC2 expression in T cells. We also found that our novel RUNX inhibitor, Chb‐M’, which is the inhibitor that switches off the entire RUNX family by alkylating agent–conjugated pyrrole‐imidazole (PI) polyamides, inhibited T‐cell receptor mediated T cell proliferation and allogenic T cell response. These were designed to specifically bind to consensus RUNX‐binding sequences (TGTGGT). Chb‐M’ also suppressed the expression of NFATC2 and pro‐inflammatory cytokine genes in vitro. Using xenogeneic GVHD model, mice injected by Chb‐M’ showed almost no sign of GVHD. Especially, the CD4 T cell was decreased and GVHD‐associated cytokines including tissue necrosis factor‐α and granulocyte‐macrophage colony‐stimulating factor were reduced in the peripheral blood of Chb‐M’ injected mice. Taken together, our data demonstrates that RUNX family transcriptionally upregulates NFATC2 in T cells, and RUNX‐NFATC2 axis can be a novel therapeutic target against GVHD.

SPI1 fusion genes in T-cell acute lymphoblastic leukemia (T-ALL) are commonly found with co-occurring NRAS mutations. Here, the authors show that the combination of these oncogenes is necessary to drive T-ALL in a murine model and that the oncogenic activity of the SPI1 fusion is dependent on β-catenin.

Abstract Aims/Introduction It was reported that fetuses secrete endogenous incretin; however, the stimulants of fetal incretin secretion are not fully understood. To investigate the association between the passage of amniotic fluid through the intestinal tract and fetal secretion of incretin, we analyzed umbilical cord incretin levels of infants with duodenum atresia. Materials and Methods Infants born from July 2017 to July 2019 (infants with duodenum atresia and normal term or preterm infants) were enrolled. We measured and compared the concentrations of glucagon‐like peptide‐1 (GLP‐1) and gastric inhibitory peptide/glucose‐dependent insulinotropic polypeptide (GIP) in the umbilical vein and preprandial blood samples after birth. Results A total of 98 infants (47 term, 46 preterm and 5 with duodenum atresia) were included. In patients with duodenum atresia, umbilical vein GLP‐1 and GIP levels were the same as those in normal infants. In postnatal samples, there were positive correlations between the amount of enteral feeding and preprandial serum concentrations of GLP‐1 (r = 0.47) or GIP (r = 0.49). Conclusions Our results show that enteral feeding is important for secretion of GLP‐1 and GIP in postnatal infants, whereas the passage of amniotic fluid is not important for fetal secretion of GLP‐1 and GIP. The effect of ingested material passing through the digestive tract on incretin secretion might change before and after birth. Other factors might stimulate secretion of GLP‐1 and GIP during the fetal period.

Familial Mediterranean fever (FMF) is a hereditary, autoinflammatory disease that causes recurrent fever, arthritis, and serositis. The diagnosis of FMF is based on the presentation of typical clinical symptoms and the Mediterranean fever gene (MEFV) test. However, the challenge lies in diagnosing atypical cases. In this report, we have described a pediatric patient with complex FMF whose diagnosis required trio-whole exome sequencing (WES) and functional validation of a rare MEFV variant. A 3-year-old boy presented with recurrent episodes of elevated liver enzymes and arthralgia. He was diagnosed with autoimmune hepatitis (AIH), and his liver enzymes improved rapidly with steroid treatment. However, he exhibited recurrent arthralgia and severe abdominal attacks. Trio-WES identified compound heterozygous mutations in MEFV (V726A and I692del). Ex vivo functional assays of the patient’s monocytes and macrophages, which had been pre-treated with Clostridium difficile toxin A (TcdA) and colchicine, were comparable to those of typical FMF patients, thereby confirming the diagnosis of FMF. Although he was intolerant to colchicine because of liver toxicity, subsequent administration of canakinumab successfully ameliorated his abdominal attacks. However, it was ineffective against liver injury, which recurred after steroid tapering. Therefore, in this case, the pathogenesis of AIH was probably interleukin-1β (IL-1β)-independent. In fact, AIH might have been a concurrent disease with FMF, rather than being one of its complications. Nevertheless, further studies are necessary to determine whether FMF-induced inflammasome activation contributes to AIH development. Moreover, we must consider the possibility of mixed phenotypes in such atypical patients who present distinct pathologies simultaneously.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The anti-CD19 chimeric antigen receptor (CAR) T cells showed excellent effect against acute lymphoblastic leukemia (ALL) in bone marrow (BM) in clinical trials. However, it remains to be elucidated whether the CD19 CAR T cell therapy is effective for ALL cells in central nervous system (CNS) because the patients with isolated or advanced CNS disease were excluded from clinical trials of systemic intravenous (i.v.) delivery of CAR T cells. Therefore, the preclinical evaluation for the efficacy of CAR T cell therapy against ALL cells in CNS is essential for clinical application. We evaluated the effect and adverse reaction of CD19 CAR T cells against ALL in CNS using a xenograft mouse model by i.v. or intra-cerebroventricular (i.c.v.) delivery of CAR T cells. Injection of piggyBac CD19 CAR T cells by i.v. had partial effects, whereas all CAR T i.c.v.-delivered mice had eliminated ALL in CNS. Although some CAR T i.c.v.-delivered mice showed transient changes of clinical symptoms during the first few days after treatment, none of CAR T i.c.v.-delivered mice displayed fatal adverse events. In this study, we demonstrated that direct delivery into CNS of CAR T cells is a possible therapeutic approach with the xenograft mouse model.

Abstract Background Clear cell sarcoma of the kidney (CCSK) is the second most common pediatric renal tumor. Case A 2‐year‐old boy was diagnosed with CCSK, which relapsed four times until he yielded to the disease at the age of 7 years. To characterize the longitudinal genetic alterations occurring in the present case, we performed targeted‐capture sequencing by pediatric solid tumors panel (381 genes) for longitudinally sampled tumors, including autopsy samples of metastasis. Internal tandem duplication of BCOR (BCOR‐ITD) was the only truncal mutation, confirming the previously reported role of BCOR‐ITD in CCSK. Conclusion Acquisition of additional mutations along tumor relapses and detection of metastasis‐specific mutations were reminiscent of the tumor progression and therapeutic resistance of this case, leading to clonal selection and a dismal fate.

Hepatic manifestations of Epstein-Barr virus (EBV) infection are relatively common, mild, and self-limiting. Although fulminant hepatic failure has been reported in a few cases, the contributing factors are unclear. This report discusses a pediatric case of EBV-associated acute liver failure that required urgent liver transplantation; however, liver damage continued to progress post-liver replacement. Monoclonal CD8+ T cells that preferentially infiltrated the native and transplanted liver were positive for EBV-encoded small RNA, suggesting a pathophysiology similar to that of EBV-associated hemophagocytic lymphohistiocytosis and chronic active EBV infection. Therefore, subsequent chemotherapy and hematopoietic cell transplantation was conducted, which led to cure. This is the first case of EBV-associated acute liver failure that relapsed post-liver transplant. As such, it sheds light on an under-recognized clinical entity: liver-restricted hyperinflammation caused by EBV-infected monoclonal CD8+ T cells. This phenomenon needs to be recognized and differentiated from hepatitis/hepatic failure caused by EBV-infected B cells, which has a relatively benign clinical course.

Abstract t(17;19)(q21‐q22;p13), responsible for TCF3‐HLF fusion, is a rare translocation in childhood B‐cell precursor acute lymphoblastic leukemia(BCP‐ALL). t(1;19)(q23;p13), producing TCF3‐PBX1 fusion, is a common translocation in childhood BCP‐ALL. Prognosis of t(17;19)‐ALL is extremely poor, while that of t(1;19)‐ALL has recently improved dramatically in intensified chemotherapy. In this study, TCF3‐HLF mRNA was detectable at a high level during induction therapy in a newly diagnosed t(17;19)‐ALL case, while TCF3‐PBX1 mRNA was undetectable at the end of induction therapy in most newly diagnosed t(1;19)‐ALL cases. Using 4 t(17;19)‐ALL and 16 t(1;19)‐ALL cell lines, drug response profiling was analyzed. t(17;19)‐ALL cell lines were found to be significantly more resistant to vincristine (VCR), daunorubicin (DNR), and prednisolone (Pred) than t(1;19)‐ALL cell lines. Sensitivities to three (Pred, VCR, and l‐asparaginase [l‐Asp]), four (Pred, VCR, l‐Asp, and DNR) and five (Pred, VCR, l‐Asp, DNR, and cyclophosphamide) agents, widely used in induction therapy, were significantly poorer for t(17;19)‐ALL cell lines than for t(1;19)‐ALL cell lines. Consistent with poor responses to VCR and DNR, gene and protein expression levels of P‐glycoprotein (P‐gp) were higher in t(17;19)‐ALL cell lines than in t(1;19)‐ALL cell lines. Inhibitors for P‐gp sensitized P‐gp‐positive t(17;19)‐ALL cell lines to VCR and DNR. Knockout of P‐gp by CRISPRCas9 overcame resistance to VCR and DNR in the P‐gp‐positive t(17;19)‐ALL cell line. A combination of cyclosporine A with DNR prolonged survival of NSG mice inoculated with P‐gp‐positive t(17;19)‐ALL cell line. These findings indicate involvement of P‐gp in resistance to VCR and DNR in Pgp positive t(17;19)‐ALL cell lines. In all four t(17;19)‐ALL cell lines, RAS pathway mutation was detected. Furthermore, among 16 t(1;19)‐ALL cell lines, multiagent resistance was usually observed in the cell lines with RAS pathway mutation in comparison to those without it, suggesting at least a partial involvement of RAS pathway mutation in multiagent resistance of t(17;19)‐ALL.

Summary: For long QT syndrome (LQTS), recent progress in genome-sequencing technologies enabled the identification of rare genomic variants with diagnostic, prognostic, and therapeutic implications. However, pathogenic stratification of the identified variants remains challenging, especially in variants of uncertain significance. This study aimed to propose a phenotypic cell-based diagnostic assay for identifying LQTS to recognize pathogenic variants in a high-throughput manner suitable for screening. We investigated the response of LQT2-induced pluripotent stem cell (iPSC)-derived cardiomyocytes (iPSC-CMs) following IKr blockade using a multi-electrode array, finding that the response to IKr blockade was significantly smaller than in Control-iPSC-CMs. Furthermore, we found that LQT1-iPSC-CMs and LQT3-iPSC-CMs could be distinguished from Control-iPSC-CMs by IKs blockade and INa blockade, respectively. This strategy might be helpful in compensating for the shortcomings of genetic testing of LQTS patients. : The methods presented by the authors allowed recognition of long QT syndrome (LQTS) subtypes 1, 2, and 3 using specific ion-channel current blockade with a combination of patient-derived iPSCs and a multi-electrode array system. This strategy might potentially compensate for the shortcomings of genetic testing for LQTS, especially in patients who have variants of unknown significance or no identified mutations. Keywords: long QT syndrome, induced pluripotent stem cell, phenotype-based diagnosis, multi-electrode array, genome editing

Abstract Background Infliximab (IFX), a mouse-human chimeric monoclonal antibody against human tumor necrosis factor alpha, is used in refractory cases of Takayasu arteritis. Several factors influence the pharmacokinetics of therapeutic antibodies including IFX. Monitoring plasma levels of IFX could be a useful approach in optimizing treatment via individual dose adjustment. Case presentation Here, we report the case of a 4-year-old Takayasu arteritis girl who was resistant to standard therapy. IFX was started at 5 mg/kg (day 0). C-reactive protein (CRP) levels decreased from 8.7 (day 0) to 1.6 mg/dL (day 10). CRP levels were thereafter elevated again on day 23 (9.0 mg/dL), and body fluid leakage at the inflammation site in the legs was observed. Trough IFX levels decreased from 23.6 (day 10) to 2.5 μg/mL (day 23). Based on the trough levels, IFX was given biweekly at 8 mg/kg. Plasma IFX levels gradually increased, and CRP levels decreased to around 2 mg/dL. A similar pattern -initial decreases followed by increases- was observed between clinical course of IFX and IgG levels. It was speculated that IgG and IFX losses were due to fluid leakage from the patient’s necrotizing legs. Conclusions Monitoring of plasma IFX levels can be a potential tool to optimize the treatment in Takayasu arteritis patients.

Neuroblastoma, the most common extracranial solid malignancy among children, originates from undifferentiated neural crest cells (NCC). Despite recent intensified treatment, high-risk patients still have a high mortality rate. To explore a new therapeutic strategy, we performed an integrated genomic and transcriptomic analysis of 30 high-risk neuroblastoma cases. Based on the expression profiling of RNA sequencing, neuroblastoma was classified into Mesenchymal (MES; n = 5) and Noradrenergic (ADRN; n = 25) clusters, as previously reported in the super-enhancer landscape. The expression patterns in MES-cluster cases were similar to normal adrenal glands, with enrichment in secretion-related pathways, suggesting chromaffin cell-like features built from NCC-derived Schwann cell precursors (SCPs). In contrast, neuron-related pathways were enriched in the ADRN-cluster, indicating sympathoblast features reported to originate from NCC but not via SCPs. Thus, MES- and ADRN-clusters were assumed to be corresponding to differentiation pathways through SCP and sympathoblast, respectively. ADRN-cluster cases were further classified into MYCN- and ATRX-clusters, characterized by genetic alterations, MYCN amplifications and ATRX alterations, respectively. MYCN-cluster cases showed high expression of ALDH18A1, encoding P5CS related to proline production. As reported in other cancers, this might cause reprogramming of proline metabolism leading to tumor specific proline vulnerability candidate for a target therapy of metabolic pathway. In ATRX-cluster, SLC18A2 (VMAT2), an enzyme known to prevent cell toxicity due to the oxidation of dopamine, was highly expressed and VMAT2 inhibitor (GZ-793A) represented significant attenuation of cell growth in NB-69 cell line (high SLC18A2 expression, no MYCN amplification) but not in IMR-32 cell line (MYCN amplification). In addition, the correlation of VMAT2 expression with metaiodobenzylguanidine (MIBG) avidity suggested a combination of VMAT2 inhibitor and MIBG radiation for a novel potential therapeutic strategy in ATRX-cluster cases. Thus, targeting the characteristics of unique neuroblastomas may prospectively improve prognosis.

Preoperative computed tomography-guided marking is commonly performed for peripheral small lung lesions but rarely for abdominal tumors. This technique was useful for intraoperative identification of a tiny abdominal recurrence in a patient with ruptured pancreatoblastoma in remission. We report the case especially focusing on the technical aspect of the procedure.

Pleuropulmonary blastoma (PPB) is a rare malignant mesenchymal tumor. It is classified into 3 subgroups, and PPB type 1 is known to be a cystic lesion with good prognosis. Here, we report a case of PPB type 1 seen in a 1-year-old girl whose pulmonary cystic lesion had been followed-up as a congenital pulmonary airway malformation since neonate. The cyst had been diagnosed as congenital pulmonary airway malformation before surgery but the final diagnosis was type 1 PPB. The tumor had a somatic mutation in exon 25 of the DICER 1 gene whereas no germline mutation was found.

Infantile-onset Pompe disease (IOPD) is a life-threatening multi-organ disease caused by an inborn defect of lysosomal acid α-glucosidase (GAA), which can degrade glycogen into glucose. Lack of GAA causes abnormal accumulation of glycogen in the lysosomes, particularly in the skeletal muscle, liver, and heart. Enzyme replacement therapy (ERT) with recombinant human GAA (rhGAA) is the only available treatment; however, its effect varies by organ. Thus, to fully understand the pathomechanism of IOPD, organ-specific disease models are necessary. We previously generated induced pluripotent stem cells (iPSCs) from three unrelated patients with IOPD and establish a skeletal muscle model of IOPD. Here, we used the same iPSC lines as the previous study and differentiated them into hepatocytes. As a result, hepatocytes differentiated from iPSC of IOPD patients showed abnormal accumulation of lysosomal glycogen, the hallmark of Pompe disease. Using this model, we also demonstrated that glycogen accumulation was dose-dependently restored by rhGAA treatment. In conclusion, we have successfully established an in vitro liver model of IOPD using patient-specific iPSCs. This model can be a platform to elucidate the underlying disease mechanism or to be applied to drug-screening. Moreover, our study also suggest that an iPSC-based approach is suitable for modeling of diseases that affect multiple organs like Pompe disease.

Duchenne muscular dystrophy (DMD) is the most common and severe form of muscular dystrophy. The major symptoms of this condition are walking difficulties, dyspnea caused by progressive skeletal muscle weakness, and cardiomyopathy. Recent advances in ventilator support devices have dramatically decreased mortality caused by respiratory distress. Consequently, cardiomyopathy resulting in heart failure is currently the major cause of death among DMD patients. One mechanism by which skeletal muscle is damaged in DMD patients involves elevation of the intracellular Ca2+ concentration. By contrast, the mechanisms underlying the development of cardiomyopathy are unclear. To investigate this, we examined the intracellular Ca2+ concentration and calcium transients in cardiomyocytes differentiated from human induced pluripotent stem cells (hiPSCs). hiPSCs were derived from a DMD patient (DMD-hiPSCs), in whom exon 44 of the gene encoding dystrophin was deleted, and from his parents (control-hiPSCs), who did not carry this mutation. The intracellular Ca2+ concentration was measured using the fluorescent indicator indo-1. The fluorescence ratio (410/490 nm) of indo-1 at rest (R0), the peak of this ratio (Rmax), and the amplitude (Rmax-R0) were significantly higher in cardiomyocytes differentiated from DMD-hiPSCs than in those differentiated from control-hiPSCs. Moreover, mechanical stretching significantly increased the intracellular Ca2+ concentration in cardiomyocytes differentiated from DMD-hiPSCs, but not in those differentiated from control-hiPSCs. These findings indicate that elevation of the intracellular Ca2+ concentration can cause cardiac damage leading to cardiomyopathy in DMD patients.

Fusion genes involving ZNF384 have recently been identified in B-cell precursor acute lymphoblastic leukemia, and 7 fusion partners have been reported. We further characterized this type of fusion gene by whole transcriptome sequencing and/or polymerase chain reaction. In addition to previously reported genes, we identified BMP2K as a novel fusion partner for ZNF384. Including the EP300-ZNF384 that we reported recently, the total frequency of ZNF384-related fusion genes was 4.1% in 291 B-cell precursor acute lymphoblastic leukemia patients enrolled in a single clinical trial, and TCF3-ZNF384 was the most recurrent, with a frequency of 2.4%. The characteristic immunophenotype of weak CD10 and aberrant CD13 and/or CD33 expression was revealed to be a common feature of the leukemic cells harboring ZNF384-related fusion genes. The signature gene expression profile in TCF3-ZNF384-positive patients was enriched in hematopoietic stem cell features and related to that of EP300-ZNF384-positive patients, but was significantly distinct from that of TCF3-PBX1-positive and ZNF384-fusion-negative patients. However, clinical features of TCF3-ZNF384-positive patients are markedly different from those of EP300-ZNF384-positive patients, exhibiting higher cell counts and a younger age at presentation. TCF3-ZNF384-positive patients revealed a significantly poorer steroid response and a higher frequency of relapse, and the additional activating mutations in RAS signaling pathway genes were detected by whole exome analysis in some of the cases. Our observations indicate that ZNF384-related fusion genes consist of a distinct subgroup of B-cell precursor acute lymphoblastic leukemia with a characteristic immunophenotype, while the clinical features depend on the functional properties of individual fusion partners.

Recently, loss of heterozygosity (LOH) studies suggest that more than two tumor suppressor genes lie on the short arm of chromosome 1 (1p) in neuroblastoma (NB). To identify candidate tumor suppressor genes in NB, we searched for homozygous deletions in 20 NB cell lines using a high-density STS map spanning chromosome 1 p36, a common LOH region in NB. We found that the 45-kDa subunit of the DNA fragmentation factor (DFF45) gene was homozygously deleted in an NB cell line, NB-1. DFF45 is the chaperon of DFF40, and both molecules are necessary for caspase 3 to induce apoptosis. DFF35, a splicing variant of DFF45, is an inhibitor of DFF40. We examined 20 NB cell lines for expression and mutation of DFF45 gene by reverse transcription (RT)-polymerase chain reaction (PCR) and RT-PCR-single-strand conformation polymorphism. Some novel variant transcripts of the DFF45 gene were found in NB cell lines, but not in normal adrenal gland and peripheral blood. These variants may not serve as chaperons of DFF40, but as inhibitors like DFF35, thus disrupting the balance between DFF45 and DFF40. No mutations of the DFF45 gene were found in any NB cell line, suggesting that the DFF45 is not a tumor suppressor gene for NB. However, homozygous deletion of the DFF45 gene in the NB-1 cell line may imply the presence of unknown tumor suppressor genes in this region.

Background SINE- VNTR- Alu (SVA) retrotransposons move from one genomic location to another in a 'copyand- paste' manner. They continue to move actively and cause monogenic diseases through various mechanisms. Currently, disease- causing SVA retrotransposons are classified into human- specific young SVA_E or SVA_F subfamilies. In this study, we identified an evolutionarily old SVA_D retrotransposon as a novel cause of occipital horn syndrome (OHS). OHS is an X- linked, copper metabolism disorder caused by dysfunction of the copper transporter, ATP7A. Methods We investigated a 16- year- old boy with OHS whose pathogenic variant could not be detected via routine molecular genetic analyses. Results A 2.8 kb insertion was detected deep within the intron of the patient's ATP7A gene. This insertion caused aberrant mRNA splicing activated by a new donor splice site located within it. Long- read circular consensus sequencing enabled us to accurately read the entire insertion sequence, which contained highly repetitive and GC- rich segments. Consequently, the insertion was identified as an SVA_D retrotransposon. Antisense oligonucleotides (AOs) targeting the new splice site restored the expression of normal transcripts and functional ATP7A proteins. AO treatment alleviated excessive accumulation of copper in patient fibroblasts in a dose- dependent manner. Pedigree analysis revealed that the retrotransposon had moved into the OHScausing position two generations ago. Conclusion This is the first report of a human monogenic disease caused by the SVA_D retrotransposon. The fact that the evolutionarily old SVA_D is still actively transposed, leading to increased copy numbers may make a notable impact on rare genetic disease research. [ABSTRACT FROM AUTHOR]