Hydrops fetalis due to loss of function of hNav1.4 channel via compound heterozygous variants.

Hydrops fetalis, characterized by abnormal fluid accumulation in fetuses, presents a significant risk of stillbirth and neonatal mortality. Although the etiology of nonimmune hydrops fetalis (NIHF) is multifaceted, recent studies have highlighted genetic factors as crucial determinants. This study focused on a family with three consecutive stillbirths, each with pronounced hydrops fetalis. Using whole-exome sequencing (WES), we identified compound heterozygous variants of the SCN4A gene encoding the voltage-gated sodium channel of the skeletal muscle (hNav1.4), c.2429T>A p.L810Q and c.4556T>C p.F1519S, in all three deceased infants. A functional analysis conducted using the whole-cell patch-clamp technique revealed loss-of-function defects in both variant channels, with F1519S exhibiting a complete loss of ionic current and L810Q showing a reduced channel opening. These findings support the pathogenicity of SCN4A variants in NIHF and underscore the significance of functional studies in elucidating genotype-phenotype correlations. Furthermore, our study emphasizes the diagnostic value of WES in cases of NIHF in where standard genetic testing fails to identify causative variants.

[Supporting career development of female physicians and researchers].

The proportion of female doctors among younger generations has increased in recent years, and support for reemployment after childbirth and childcare leave is important for maintaining stability of local healthcare. We conducted a questionnaire with doctors in the Department of Pediatrics at Ehime University School of Medicine and it's affiliated hospitals to identify issues in the career development of female doctors. Although many female physicians want to pursue career development by obtaining subspecialty qualifications and PhD degrees, a high percentage have not actually obtained them. This is not only due to interruptions in work caused by childbirth and childcare but also because they are busy with housework, childcare, and daily work, and lack sufficient information about career development. In this regard, it appears that beyond improving work-life balance, female doctors must always keep in mind their career design and future goals, as well as their social mission as a physician. For administrators of these departments, acceptance of diversity, providing adequate support for female physicians to return to work after maternity/childcare leave, and balancing childcare and work are important for expanding female doctors' opportunities and career development.

Machine learning for rhabdomyosarcoma histopathology.

Correctly diagnosing a rare childhood cancer such as sarcoma can be critical to assigning the correct treatment regimen. With a finite number of pathologists worldwide specializing in pediatric/young adult sarcoma histopathology, access to expert differential diagnosis early in case assessment is limited for many global regions. The lack of highly-trained sarcoma pathologists is especially pronounced in low to middle-income countries, where pathology expertise may be limited despite a similar rate of sarcoma incidence. To address this issue in part, we developed a deep learning convolutional neural network (CNN)-based differential diagnosis system to act as a pre-pathologist screening tool that quantifies diagnosis likelihood amongst trained soft-tissue sarcoma subtypes based on whole histopathology tissue slides. The CNN model is trained on a cohort of 424 centrally-reviewed histopathology tissue slides of alveolar rhabdomyosarcoma, embryonal rhabdomyosarcoma and clear-cell sarcoma tumors, all initially diagnosed at the originating institution and subsequently validated by central review. This CNN model was able to accurately classify the withheld testing cohort with resulting receiver operating characteristic (ROC) area under curve (AUC) values above 0.889 for all tested sarcoma subtypes. We subsequently used the CNN model to classify an externally-sourced cohort of human alveolar and embryonal rhabdomyosarcoma samples and a cohort of 318 histopathology tissue sections from genetically engineered mouse models of rhabdomyosarcoma. Finally, we investigated the overall robustness of the trained CNN model with respect to histopathological variations such as anaplasia, and classification outcomes on histopathology slides from untrained disease models. Overall positive results from our validation studies coupled with the limited worldwide availability of sarcoma pathology expertise suggests the potential of machine learning to assist local pathologists in quickly narrowing the differential diagnosis of sarcoma subtype in children, adolescents, and young adults.

Postchemotherapy immune status in infants with acute lymphoblastic leukemia: A report from the JPLSG MLL-10 trial.

The MLL-10 trial (UMIN000004801) modified a Children's Oncology Group (COG) AALL0631 therapy for infants with KMT2A-rearranged acute lymphoblastic leukemia (ALL). In 2016, one registered case developed secondary immunodeficiency during maintenance therapy and eventually died due to cytomegalovirus infection. Around the same time, fatal secondary immunodeficiencies were reported in five infants with ALL in North America who had received COG-based chemotherapy between 1996 and 2015. Given these cases, we decided to conduct a retrospective study on the postchemotherapy immune status of infants with ALL. A questionnaire collected data on posttreatment immune function, frequency of infections, and supportive care for the 34 infants in the MLL-10 trial. Patients receiving allogeneic hematopoietic stem cell transplantation in first remission were excluded. Responses to the survey were obtained in 28 cases (85%). Most patients were immunocompetent after the completion of chemotherapy (median follow-up duration from the day of chemotherapy completion was 431 days), except for the aforementioned case. There were seven patients with nonsevere viral infection, all of whom recovered. In conclusion, severe chemotherapy-induced immunodeficiency in infants with ALL appears to be rare, but prospective data collection of immune function is necessary to clarify this finding.

Metaplastic carcinoma of the breast and BRCA1 germline mutation: a case report and review.

BACKGROUND: Metaplastic carcinoma of the breast consists of both invasive ductal carcinoma and metaplastic carcinoma. This rare subtype of cancer has a poor prognosis. The development of metaplastic breast cancer and relationship with BRCA1 are not well known. Here, we report a rare case of germline BRCA1 mutation-positive breast cancer with chondroid metaplasia. CASE PRESENTATION: A 39-year-old Japanese woman with a family history of breast cancer in her mother and ovarian cancer in her maternal grandmother consulted at our hospital with a left breast mass. Needle biopsy for the mass was performed, leading to a diagnosis of invasive breast cancer with chondroid metaplasia. We performed left mastectomy + sentinel lymph node biopsy + tissue expander insertion and replaced with a silicone implant later. Pathological examination revealed that the patient had triple-negative breast cancer. Four courses of doxorubicin+ cyclophosphamide therapy were performed as adjuvant therapy after surgery. We performed genetic counseling and genetic testing, and the results suggested the germline BRCA1 mutation 307 T> A (L63*). She has currently lived without a relapse for 2 years post-surgery. CONCLUSIONS: There have been only 6 cases of metaplastic breast carcinoma with germline BRCA1 mutations including our case. Patients with BRCA1 mutations may develop basal-like subtypes or M type of triple-negative breast cancer besides metaplastic breast cancers.

[Leukemogenic pathway of infant leukemia with MLL fusion].

Acute lymphoblastic leukemia (ALL) in infants remains an intractable and difficult-to-treat leukemia as compared to other pediatric ALLs, for which considerable progress has been achieved in terms of treatment outcomes in recent years. The leukemic cells in infants with ALL frequently carry chromosome translocations involving 11q23, resulting in the rearrangement and fusion of the MLL (KMT2A) gene. Among many MLL fusion genes, MLL-AF4 (KMT2A-AFF1) fusion is characteristically observed in infants with ALL, representing a hallmark of poor prognosis. In MLL-AF4-positive infants with ALL, first leukemic cells with MLL-AF4 were generated in utero. Analysis of several murine and human leukemia models revealed that the target cells for tumorigenesis by MLL-AF4 were not the hematopoietic progenitor cells of the bone marrow, but the early hematopoietic progenitor cells present in the fetal liver during the embryonic period and possibly the undifferentiated cells prior to the commitment to hematopoietic cells in the fetus. Elucidation of the leukemogenic process of infant ALL with MLL-AF4 may lead to early, pre-symptomatic diagnosis of leukemia, resulting in the improvement of prognosis and prevention of the onset of ALL in infants.

[Acute leukemia of infants and neonates].

Leukemias diagnosed in <1-year-old infants generally have an aggressive clinical nature and unique biological characteristics. Acute lymphoblastic leukemia (ALL) in infants is still intractable and difficult to treat as compared with other pediatric ALLs, for which considerable progress in treatment outcomes has been recently achieved. Infant leukemia cells frequently carry chromosome translocations involving the 11q23 locus, resulting in the rearrangement and fusion of the KMT2A (MLL) gene. Among several KMT2A fusion genes, KMT2A-AFF1 (MLL-AF4) fusion is characteristically observed in neonatal and infant ALL, representing a hallmark of poor prognosis. The cytogenetic/molecular abnormalities t (1;22)(p13.3;q13.1)/RBM15-MKL1 and t (8;16)(p11.2;p13.3)/KAT6A-CREBBP (MOZ-CBP) are also well-known in acute myeloblastic leukemia in this population. Although many neonatal leukemias occurring within the first 28 days of birth are refractory, spontaneous remissions are occasionally observed, especially in the case of t (8;16). Therefore, international collaborative studies are necessary to improve understanding and facilitate the development of better treatment for this rare disease. Thus, this study summarizes the recently reported clinical, cytogenetic, and molecular biology aspects of neonatal and infant leukemias.

Renal dysfunction can occur in advanced-stage Duchenne muscular dystrophy.

INTRODUCTION: With improved treatments, patients with Duchenne muscular dystrophy (DMD) can survive far beyond adolescence. However, advanced-stage DMD patients are at risk of developing renal dysfunction. In this study, long-term renal function outcomes and associated risk factors in advanced stage DMD were analyzed. METHODS: Fifty-one patients were classified into three different age groups (<20, 20-29, and ≥30 years of age), and cystatin C (CysC) levels were compared among groups. RESULTS: Median serum CysC levels were 0.74 mg/L, 0.63 mg/L, and 0.76 mg/L in the age groups of <20, 20-29, and ≥30 years, respectively (P = .003). Five of the nine patients in the ≥30 years age group showed elevated serum CysC and decreased cardiac function compared with the other four in the group (P = .014). DISCUSSION: Our results indicate an association between cardiac and renal dysfunction in patients with advanced-stage DMD.

Early detection of the PAX3-FOXO1 fusion gene in circulating tumor-derived DNA in a case of alveolar rhabdomyosarcoma.

Cell-free DNA (cfDNA), which are small DNA fragments in blood derived from dead cells including tumor cells, could serve as useful biomarkers and provide valuable genetic information about the tumors. cfDNA is now used for the genetic analysis of several types of cancers, as a surrogate for tumor biopsy, designated as "liquid biopsy." Rhabdomyosarcoma (RMS), the most frequent soft tissue tumor in childhood, can arise in any part of the body, and radiological imaging is the only available method for estimating the tumor burden, because no useful specific biological markers are present in the blood. Because tumor volume is one of the determinants of treatment response and outcome, early detection at diagnosis as well as relapse is essential for improving the treatment outcome. A 15-year-old male patient was diagnosed with alveolar RMS of prostate origin with bone marrow invasion. The PAX3-FOXO1 fusion was identified in the tumor cells in the bone marrow. After the diagnosis, cfDNA was serially collected to detect the PAX3-FOXO1 fusion sequence as a tumor marker. cfDNA could be an appropriate source for detecting the fusion gene; assays using cfDNA have proved to be useful for the early detection of tumor progression/recurrence. Additionally, the fusion gene dosage estimated by quantitative polymerase chain reaction reflected the tumor volume during the course of the treatment. We suggest that for fusion gene-positive RMSs, and other soft tissue tumors, the fusion sequence should be used for monitoring the tumor burden in the body to determine the diagnosis and treatment options for the patients.

Histological characterisation of visceral changes in a patient with type 2 Gaucher disease treated with enzyme replacement therapy.

Gaucher disease is a lysosomal storage disease caused by deficiency of glucocerebrosidase and accumulation of glucocerebroside. Three major sub-types have been described, type 2 is an acute neurological form that exhibits serious general symptoms and poor prognosis, compared with the other types. This case was a girl diagnosed with type 2 Gaucher disease at 12months of age who presented with poor weight gain from infancy, stridor, hypertonia, hepatosplenomegaly, trismus and an eye movement disorder. Enzyme replacement therapy (ERT) was administered, but she had frequent myoclonus and developmental regression. She needed artificial ventilation because of respiratory failure. She died at 11years of age. An autopsy demonstrated infiltrating CD68-positive large cells containing abundant lipids in alveoli, while in the liver, kidney and bone marrow CD68-positive cells were small and round. In the bone marrow, myelodysplastic changes were present without Gaucher cells. The infiltration of Gaucher cells in alveoli was marked, suggesting that ERT was relatively ineffective in pulmonary involvement, particularly intra-alveolar. Additional treatments are necessary to improve the neurological and pulmonary prognosis of type 2Gaucher disease.

Manifestation of recessive combined D-2-, L-2-hydroxyglutaric aciduria in combination with 22q11.2 deletion syndrome.

22q11.2 deletion syndrome is one of the most common human microdeletion syndromes. The clinical phenotype of 22q11.2 deletion syndrome is variable, ranging from mild to life-threatening symptoms, depending mainly on the extent of the deleted region. Brain malformations described in association with 22q11.2 deletion syndrome include polymicrogyria, cerebellar hypoplasia, megacisterna magna, and agenesis of the corpus callosum (ACC), although these are rare. We report here for the first time a patient who manifested combined D-2- and L-2-hydroxyglutaric aciduria as a result of a hemizygous mutation in SLC25A1 in combination with 22q11.2 deletion. The girl was diagnosed to have ACC shortly after birth and a deletion of 22q11.2 was identified by genetic analysis. Although the patient showed cardiac anomalies, which is one of the typical symptoms of 22q11.2 deletion syndrome, her rather severe phenotype and atypical face prompted us to search for additional pathogenic mutations. Three genes present in the deleted 22q11.2 region, SLC25A1, TUBA8, and SNAP29, which have been reported to be associated with brain malformation, were analyzed for the presence of pathogenic mutations. A frameshift mutation, c.18_24dup (p.Ala9Profs*82), was identified in the first exon of the remaining SLC25A1 allele, resulting in the complete loss of normal SLC25A1 function in the patient's cells. Our results support the notion that the existence of another genetic abnormality involving the retained allele on 22q11.2 should be considered when atypical or rare phenotypes are observed.

Exon skipping in CYBB mRNA and skewed inactivation of X chromosome cause late-onset chronic granulomatous disease.

Chronic granulomatous disease (CGD) is a hereditary immunodeficiency syndrome caused by a defect in the NADPH oxidase complex, which is essential for bactericidal function of phagocytes. Approximately 70% of patients with CGD have a mutation in the CYBB gene on the X chromosome, resulting in defective expression of gp91phox, one of the membrane-bound subunits of NADPH oxidase. Although most patients with X-linked CGD are males, owing to transmission of this disease as an X-linked recessive trait, there are female patients with X-linked CGD. Here, we report the case of a teenage girl with X-linked CGD associated with a heterozygous mutation in exon 5 of the CYBB gene (c.389G > C; R130P), which causes skipping of exon 5, resulting in a premature stop codon in exon 6 of CYBB. Accurate pro-mRNA splicing for mature mRNA formation is regulated by several splicing mechanisms that are essential for appropriate recognition of exonic sequences. The c.389G > C mutation disrupts exonic-splicing regulator sequences, thereby resulting in the aberrant skipping of exon 5 in the CYBB transcript of the patient. The patient showed an extremely skewed (≥96%) X inactivation pattern of the HUMARA locus; this inactivation is thought to be responsible for the development of CGD not only in neutrophils but also in monocytic, T-cell, and B-cell lineages and in CD34-positive immature hematopoietic cells. Our case and other reports indicate that the onset of X-linked CGD in female patients tends to occur later in life, and that the symptoms tend to be milder as compared to male patients.

Usefulness of positron emission tomography-CT for diagnosis of primary bone marrow lymphoma in children.

Primary bone marrow lymphoma (PBML) is hard to diagnose in children, due to the difficult identification of malignant cells in bone marrow. The first case, a 5-year-old boy, showed knee swelling with an intermittent fever. The second case, a 12-year-old girl, showed fever of unknown origin without lymphadenopathy or hepatosplenomegaly. In both cases, the diagnosis was not confirmed despite the repeated bone marrow aspirations. Finally, bone marrow aspiration and biopsy at the positive site by positron emission tomography (PET)-CT contributed to definitive diagnosis of PBML. The PET-CT is useful for the accurate diagnosis of PBML in children with non-specific symptoms.

Prolonged adrenal insufficiency after high-dose glucocorticoid in infants with leukemia.

Although outcomes for infant leukemia have improved recently, transient adrenal insufficiency is commonly observed during treatment, especially after glucocorticoid administration. We identified three infants with acute leukemia who suffered from prolonged adrenal insufficiency requiring long-term (from 15 to 66 months) hydrocortisone replacement. All infants showed life-threatening symptoms associated with adrenal crisis after viral infections or other stress. Severe and prolonged damage of hypothalamo-pituitary-adrenal (HPA) axis is likely to occur in early infants with leukemia, therefore routine tolerance testing to evaluate HPA axis and hydrocortisone replacement therapy are recommended for infants with leukemia to avoid life-threatening complications caused by adrenal crisis.

Identification of CD34+ and CD34- leukemia-initiating cells in MLL-rearranged human acute lymphoblastic leukemia.

Translocation of the mixed-lineage leukemia (MLL) gene with AF4, AF9, or ENL results in acute leukemia with both lymphoid and myeloid involvement. We characterized leukemia-initiating cells (LICs) in primary infant MLL-rearranged leukemia using a xenotransplantation model. In MLL-AF4 patients, CD34(+)CD38(+)CD19(+) and CD34(-)CD19(+) cells initiated leukemia, and in MLL-AF9 patients, CD34(-)CD19(+) cells were LICs. In MLL-ENL patients, either CD34(+) or CD34(-) cells were LICs, depending on the pattern of CD34 expression. In contrast, in patients with these MLL translocations, CD34(+)CD38(-)CD19(-)CD33(-) cells were enriched for normal hematopoietic stem cells (HSCs) with in vivo long-term multilineage hematopoietic repopulation capacity. Although LICs developed leukemic cells with clonal immunoglobulin heavy-chain (IGH) rearrangement in vivo, CD34(+)CD38(-)CD19(-)CD33(-) cells repopulated recipient bone marrow and spleen with B cells, showing broad polyclonal IGH rearrangement and recipient thymus with CD4(+) single positive (SP), CD8(+) SP, and CD4(+)CD8(+) double-positive (DP) T cells. Global gene expression profiling revealed that CD9, CD32, and CD24 were over-represented in MLL-AF4, MLL-AF9, and MLL-ENL LICs compared with normal HSCs. In patient samples, these molecules were expressed in CD34(+)CD38(+) and CD34(-) LICs but not in CD34(+)CD38(-)CD19(-)CD33(-) HSCs. Identification of LICs and LIC-specific molecules in primary human MLL-rearranged acute lymphoblastic leukemia may lead to improved therapeutic strategies for MLL-rearranged leukemia.

Azacitidine successfully maintained the second remission in an infant with KMT2A-rearranged acute lymphoblastic leukemia who relapsed after unrelated cord blood transplantation.

The outcome for infants with KMT2A (MLL)-rearranged acute lymphoblastic leukemia (MLL-r ALL) is dismal despite intensive therapy, including hematopoietic stem cell transplantation (HSCT). Epigenetic dysregulation is considered a key driver of MLL-r leukemogenesis, which theoretically supports the use of epigenetic modifiers as a treatment option. We report an infant MLL-r ALL case with post-HSCT relapse. After achieving a second remission, which was maintained for 10 months using only the DNA methyltransferase inhibitor, azacitidine, the patient successfully received the second HSCT. This report describes the clinical effectiveness of azacitidine for the treatment of infant MLL-r ALL.