Yoko Mizoguchi

Simple diagnosis of STAT1 gain-of-function alleles in patients with chronic mucocutaneous candidiasis

CMCD is a rare congenital disorder characterized by persistent or recurrent skin, nail, and mucosal membrane infections caused by Candida albicans. Heterozygous GOF STAT1 mutations have been shown to confer AD CMCD as a result of impaired dephosphorylation of STAT1. We aimed to identify and characterize STAT1 mutations in CMCD patients and to develop a simple diagnostic assay of CMCD. Genetic analysis of STAT1 was performed in patients and their relatives. The mutations identified were characterized by immunoblot and reporter assay using transient gene expression experiments. Patientsˈ leukocytes are investigated by flow cytometry and immunoblot. Six GOF mutations were identified, three of which are reported for the first time, that affect the CCD and DBD of STAT1 in two sporadic and four multiplex cases in 10 CMCD patients from Japan. Two of the 10 patients presented with clinical symptoms atypical to CMCD, including other fungal and viral infections, and three patients developed bronchiectasis. Immunoblot analyses of patientsˈ leukocytes showed abnormally high levels of pSTAT1 following IFN‐γ stimulation. Based on this finding, we performed a flow cytometry‐based functional analysis of STAT1 GOF alleles using IFN‐γ stimulation and the tyrosine kinase inhibitor, staurosporine. The higher levels of pSTAT1 observed in primary CD14+ cells from patients compared with control cells persisted and were amplified by the presence of staurosporine. We developed a flow cytometry‐based STAT1 functional screening method that would greatly facilitate the diagnosis of CMCD patients with GOF STAT1 mutations.

Wnt3a stimulates maturation of impaired neutrophils developed from severe congenital neutropenia patient-derived pluripotent stem cells

The derivation of induced pluripotent stem (iPS) cells from individuals of genetic disorders offers new opportunities for basic research into these diseases and the development of therapeutic compounds. Severe congenital neutropenia (SCN) is a serious disorder characterized by severe neutropenia at birth. SCN is associated with heterozygous mutations in the neutrophil elastase [elastase, neutrophil-expressed (ELANE)] gene, but the mechanisms that disrupt neutrophil development have not yet been clarified because of the current lack of an appropriate disease model. Here, we generated iPS cells from an individual with SCN (SCN-iPS cells). Granulopoiesis from SCN-iPS cells revealed neutrophil maturation arrest and little sensitivity to granulocyte-colony stimulating factor, reflecting a disease status of SCN. Molecular analysis of the granulopoiesis from the SCN-iPS cells vs. control iPS cells showed reduced expression of genes related to the wingless-type mmtv integration site family, member 3a (Wnt3a)/β-catenin pathway [e.g., lymphoid enhancer-binding factor 1], whereas Wnt3a administration induced elevation lymphoid enhancer-binding factor 1-expression and the maturation of SCN-iPS cell-derived neutrophils. These results indicate that SCN-iPS cells provide a useful disease model for SCN, and the activation of the Wnt3a/β-catenin pathway may offer a novel therapy for SCN with ELANE mutation.

Steroid-Dependent ACTH-Produced Thymic Carcinoid: Regulation of POMC Gene Expression by Cortisol via Methylation of Its Promoter Region

Aims: Metyrapone causes a decrease in the serum cortisol level without affecting ACTH production in ectopic tumors. We report a case who presented with Cushing’s syndrome due to an ectopic ACTH-producing thymic carcinoid. In the present case, it was demonstrated that metyrapone administration resulted in a significant decrease in the plasma ACTH and serum cortisol levels. We hypothesized that the steroid hormone may promote proopiomelanocortin (POMC) gene expression in the carcinoid cells. Methods: An 11-year-old boy presented with Cushing’s syndrome. Prior to the detection of a thymic tumor, metyrapone was administered to ameliorate the symptoms of Cushing’s syndrome. Interestingly, plasma ACTH as well as serum cortisol levels immediately decreased after metyrapone administration. The levels of cortisol and ACTH were observed to be normal after complete surgical resection of the tumor. Biological characterization of the tumor cells was by in vitro analysis. Results: Thein vitro culture of the tumor cells showed an increased expression of POMC in the presence of cortisol. A CpG methylation assay showed that the demethylation of the POMC promoter was induced by a steroid hormone. Conclusion: These findings suggest that the ectopic ACTH-producing tumor may partly be regulated by the elevated levels of cortisol.

A Case of Adolescent Primary Adrenal Natural Killer Cell Lymphoma

Primary adrenal lymphoma is uncommon, and the majority cases of this disorder are found in elderly individuals. We describe a 17-year-old boy with persistent fever, hemophagocytic lymphohistiocytosis, and a bilateral tumor of the adrenal glands. The disease was progressive and did not respond to treatment such as immunosuppression therapy or plasma exchange. Postmortem analysis revealed nasal-type natural killer cell lymphoma in association with Epstein-Barr virus infection. To our knowledge, this case is the first of primary adrenal lymphoma with the natural killer cell phenotype to be reported. The characterization of this unusual case should be included in the differential diagnosis of adrenal gland tumors.

Deficiency of regulatory T cells in children with autoimmune neutropenia.

CD4+ 25+ regulatory T cells (Tregs) play a role in controlling the development and progression of autoimmunity. The transcription factors Foxp3 and NFATC2 (NFAT1) play key roles in regulating the development and function of Tregs. The present study examined the involvement of Tregs in the pathophysiology of autoimmune neutropenia in children. Tregs were analysed by flow cytometry, based on the expressions of CD4, CD25, and intracellular Foxp3. The expressions of FOXP3 and NFATC2 mRNA in the CD4+ 25+ cells were determined by quantitative real‐time polymerase chain reaction. The percentage of CD4+ 25high Tregs in patients with autoimmune neutropenia was significantly lower than that in age‐matched healthy subjects. The intracellular expression of Foxp3 of CD4+ 25+ cells in patients similarly decreased in comparison to that in healthy subjects. The expression of FOXP3 and NFATC2 mRNA of CD4+ 25+ cells in patients also significantly decreased in comparison to that in healthy subjects. These results suggest that the deficiency of Tregs might thus play an important role in the immunopathophysiology of autoimmune neutropenia in children.

Decreased Expression in Nuclear Factor-κB Essential Modulator Due to a Novel Splice-Site Mutation Causes X-linked Ectodermal Dysplasia with Immunodeficiency

X-linked ectodermal dysplasia with immunodeficiency (XL-ED-ID) is caused by hypomorphic mutations in NEMO, which encodes nuclear factor-kappaB (NF-κB) essential modulator. We identified a novel mutation, 769−1 G>C, at the splicing acceptor site of exon 7 in NEMO in a Japanese patient with XL-ED-ID. Although various abnormally spliced NEMO messenger RNAs (mRNAs) were observed, a small amount of wild-type (WT) mRNA was also identified. Decreased NEMO protein expression was detected in various lineages of leukocytes. Although one abnormally spliced NEMO protein showed residual NF-κB transcription activity, it did not seem to exert a dominant-negative effect against WT-NEMO activity. CD4+ T cell proliferation was impaired in response to measles and mumps, but not rubella. These results were consistent with the clinical and laboratory findings of the patient, suggesting the functional importance of NEMO against specific viral infections. The 769−1 G>C mutation is responsible for decreased WT-NEMO protein expression, resulting in the development of XL-ED-ID.

A case of neonatal coxsackie B2 meningo-encephalitis in which serial magnetic resonance imaging findings reveal the development of lesions.

We report a patient with neonatal Coxsackie B2 meningo-encephalitis in whom magnetic resonance imaging (MRI) could be performed serially from the early stage of the disease. The patient was a 12-day-old girl born at a gestational age of 37 weeks. She was hospitalized due to poor suckling. During her hospital stay, she developed clonic seizures in the right upper and lower limbs. Coxsackie B2 virus was detected, and a diagnosis of viral encephalitis was made. The first diffusion-weighted images (DWI) showed an abnormal high-intensity area restricted to the corpus callosum and posterior limb of the internal capsule 8 h after onset of seizures. Repeated MRI revealed damage to the white matter, which finally changed into diffuse excessive necrosis followed by cystic leukomalacia. Early DWI is valuable for the early detection and diagnosis of neonatal meningo-encephalitis. This is the first report of the detailed neuroradiological course of neonatal Coxsackie B2 meningo-encephalitis.

Juvenile myelomonocytic leukemia with t(7;11)(p15;p15) and NUP98-HOXA11 fusion†

The t(7;11)(p15;p15) translocation has been reported as a rare and recurrent chromosomal abnormality in acute myeloid leukemia (AML) patients. The NUP98‐HOXA9 fusion gene with t(7;11)(p15;p15) was identified and revealed to be essential for leukemogenesis and myeloproliferative disease. To date, t(7;11)(p15;p15) with NUP98‐HOXA11 fusion has been reported only in one case of ph‐negative chronic myeloid leukemia (CML). Here, we report a case of a 3‐year‐old girl with juvenile myelomonocytic leukemia (JMML) carrying t(7;11)(p15;p15) abnormality with NUP98‐HOXA11 fusion. AML chemotherapy followed by bone marrow transplantation (BMT) was found to be effective in treating this disorder, and she remains in complete remission for 3 years after BMT. We suggest the possibility that AML chemotherapy might be effective for treating JMML with t(7;11)(p15;p15) abnormality and NUP98‐HOXA11 fusion. Am. J. Hematol. 2009.

Neutropenia (In Infancy and Childhood)

Neutropenia is defined as a decrease in the number of circulating neutrophils in the peripheral blood with absolute neutrophil count less than 1000–1500/μL. Chronic neutropenia in pediatric patients is divided into three groups. Extrinsic factors, such as antibodies, some drugs, and nutritional deficiencies, lead to excessive destruction of neutrophils. Autoimmune neutropenia is a benign form of neutropenia shown in infancy to early childhood. Spontaneous recovery of neutropenia usually occurs within a few months to a few years. Acquired disorders of myeloid and stem cells present hypoplasia of myeloid cells. Congenital neutropenia is intrinsic defects in granulocytes or their progenitors and includes a heterogenous group of disorders. More than ten responsible gene mutations have been identified in congenital neutropenia. Most common congenital neutropenia is due to the gene mutation of neutrophil elastase. The hallmark of profound neutropenia is increased susceptibility to bacterial infections, cutaneous cellulitis, deep tissue abscesses, pneumonia, and septicemia. Almost patients with congenital neutropenia have been responded to administration of G-CSF. However, long-term use of G-CSF has the risk of the development of MDS/AML, suggesting the necessity of the careful follow-up. Hematopoietic stem cell transplantation should be considered for the curable treatment in severe congenital neutropenia.

Management of advanced-stage neuroblastoma in a patient with 21-hydroxalase deficiency

A 2‐year‐old boy presented with a 21‐hydroxylase deficiency, associated with advanced‐stage neuroblastoma primarily occurring in the left adrenal gland. He required intensive chemotherapy with polypharmacy, followed by cord blood stem cell transplantation to treat the neuroblastoma. The precise adjustment of cortisol levels was crucial in this patient to prevent adrenal crisis. We administered hydrocortisone by continuous infusion while monitoring blood cortisol levels. As there are no published reports on the target cortisol levels for children, we used two control infants with advanced‐stage neuroblastoma, also undergoing chemotherapy and cord blood stem cell transplantation, to guide the continuous hydrocortisone therapy. The daily dose of hydrocortisone during chemotherapy required about threefold the normal treatment to avoid adrenal insufficiency. Continuous hydrocortisone therapy is feasible for preventing adrenal crisis and this report may provide an effective management for hydrocortisone replacement in 21‐hydroxylase‐deficient patients undergoing chemotherapy and hematopoietic stem cell transplantation.