Chie Kobayashi

Identification of Severe Combined Immunodeficiency by T-Cell Receptor Excision Circles Quantification Using Neonatal Guthrie Cards

OBJECTIVE To assess the feasibility of T-cell receptor excision circles (TRECs) quantification for neonatal mass screening of severe combined immunodeficiency (SCID). STUDY DESIGN Real-time PCR based quantification of TRECs for 471 healthy control patients and 18 patients with SCID with various genetic abnormalities (IL2RG, JAK3, ADA, LIG4, RAG1) were performed, including patients with maternal T-cell engraftment (n = 4) and leaky T cells (n = 3). RESULTS TRECs were detectable in all normal neonatal Guthrie cards (n = 326) at the levels of 10(4) to 10(5) copies/microg DNA. In contrast, TRECs were extremely low in all neonatal Guthrie cards (n = 15) and peripheral blood (n = 14) from patients with SCID, including those with maternal T-cell engraftment or leaky T cells with hypomorphic RAG1 mutations or LIG4 deficiency. There were no false-positive or negative results in this study. CONCLUSION TRECs quantification can be used as a neonatal mass screening for patients with SCID.

Identification of the novel AML1 fusion partner gene, LAF4, a fusion partner of MLL, in childhood T-cell acute lymphoblastic leukemia with t(2;21)(q11;q22) by bubble PCR method for cDNA

The AML1 gene is frequently rearranged by chromosomal translocations in acute leukemia. We identified that the LAF4 gene on 2q11.2–12 was fused to the AML1 gene on 21q22 in a pediatric patient having T-cell acute lymphoblastic leukemia (T-ALL) with t(2;21)(q11;q22) using the bubble PCR method for cDNA. The genomic break points were within intron 7 of AML1 and of LAF4, resulting in the in-frame fusion of exon 7 of AML1 and exon 8 of LAF4. The LAF4 gene is a member of the AF4/FMR2 family and was previously identified as a fusion partner of MLL in B-precursor ALL with t(2;11)(q11;q23), although AML1-LAF4 was in T-ALL. LAF4 is the first gene fused with both AML1 and MLL in acute leukemia. Almost all AML1 translocations except for TEL-AML1 are associated with myeloid leukemia; however, AML1-LAF4 was associated with T-ALL as well as AML1-FGA7 in t(4;21)(q28;q22). These findings provide new insight into the common mechanism of AML1 and MLL fusion proteins in the pathogenesis of ALL. Furthermore, we successfully applied bubble PCR to clone the novel AML1-LAF4 fusion transcript. Bubble PCR is a powerful tool for detecting unknown fusion transcripts as well as genomic fusion points.

A conditioning regimen of busulfan, fludarabine, and melphalan for allogeneic stem cell transplantation in children with juvenile myelomonocytic leukemia

Abstract:  A pilot study was undertaken using a myeloablative conditioning with fludarabine, busulfan, and melphalan to improve the outcome of HSCT in 10 children, aged six months to six yr, with JMML. All patients were conditioned with oral busulfan (560 mg/m2), fludarabine (120 mg/m2), and melphalan (180–210 mg/m2) prior to HSCT, and received stem cells from bone marrow in seven cases, and from cord blood in three cases. Engraftment was documented in eight patients, whereas graft failure occurred in two, one of whom had received HLA‐mismatched cord blood and other had received bone marrow from HLA‐mismatched mother. Three patients, including two in who graft failure had occurred, relapsed. Five patients developed acute GVHD and two developed chronic GVHD. Seven patients are alive and in remission 27–69 months after transplantation. Thus, our study showed that HSCT following conditioning with fludarabine, busulfan, and melphalan was well tolerated and appeared to be effective for JMML.

Wiskott-Aldrich syndrome presenting with a clinical picture mimicking juvenile myelomonocytic leukaemia.

Wiskott–Aldrich syndrome (WAS) is a rare X‐linked immunodeficiency caused by defects of the WAS protein (WASP) gene. Patients with WAS typically demonstrate micro‐thrombocytopenia.

Clinical features and outcome of X-linked lymphoproliferative syndrome type 1 (SAP deficiency) in Japan identified by the combination of flow cytometric assay and genetic analysis

To cite this article: Kanegane H, Yang Xi, Zhao M, Yamato K, Inoue M, Hamamoto K, Kobayashi C, Hosono A, Ito Y, Nakazawa Y, Terui K, Kogawa K, Ishii E, Sumazaki R, Miyawaki T. Clinical features and outcome of X‐linked lymphoproliferative syndrome type 1 (SAP deficiency) in Japan identified by the combination of flow cytometric assay and genetic analysis. Pediatric Allergy Immunology 2012: 23: 488–493.

Six-year-old girl with primary cutaneous aggressive epidermotropic CD8+ T-cell lymphoma

Yukiko Kikuchi, Yoshifumi Kashii, Yuji Gunji, Akira Morimoto, Aki Masuzawa, Yuka Takatsuka, Etsuko Fujita, Mayumi Komine, Mamitaro Ohtsuki, Daisuke Matsubara, Chie Kobayashi, Ayako Sakurai, Kentaro Yanase, Keisuke Kato, Kazutoshi Koike, Masahiro Tsuchida and Mariko Y. Momoi Departments of Pediatrics, Dermatology and Pathology, Jichi Medical University School of Medicine, Shimotsuke, Tochigi and Ibaraki Children’s Hospital, Mito, Ibaraki, Japan

Early and rapid detection of X-linked lymphoproliferative syndrome with SH2D1A mutations by flow cytometry

X‐linked lymphoproliferative syndrome (XLP) is a rare immunodeficiency with extreme vulnerability to Epstein‐Barr virus (EBV) infection. It presents with fatal infectious mononucleosis, lymphoproliferative disorder, or dysgammaglobulinemia. The majority of affected males have mutations in the SH2D1A/SLAM‐associated protein (SAP) gene. We previously generated an antihuman SAP monoclonal antibody (KST‐3) for a flow cytometric assay and described the activation of T cells to be necessary for the flow cytometric assessment of the SAP expression using an FITC‐conjugated secondary antibody.

Genotyping NUDT15 can predict the dose reduction of 6-MP for children with acute lymphoblastic leukemia especially at a preschool age

The pharmacokinetics among children has been altered dynamically. The difference between children and adults is caused by immaturity in things such as metabolic enzymes and transport proteins. The periods when these alterations happen vary from a few days to some years after birth. We hypothesized that the effect of gene polymorphisms associated with the dose of medicine could be influenced by age. In this study, we analyzed 51 patients with childhood acute lymphoblastic leukemia (ALL) retrospectively. We examined the associations between the polymorphism in NUDT15 and clinical data, especially the dose of 6-mercaptopurine (6-MP). Ten of the patients were heterozygous for the variant allele in NUDT15. In patients under 7 years old with NUDT15 variant allele, the average administered dose of 6-MP was lower than that for the patients homozygous for the wild-type allele (P=0.04). Genotyping of NUDT15 could be a beneficial to estimate the tolerated dose of 6-MP for patients with childhood ALL, especially at a preschool age in Japan. Furthermore, the analysis with stratification by age might be useful in pharmacogenomics among children.

Autoimmunity and persistent RAS-mutated clones long after the spontaneous regression of JMML

Autoimmunity and persistent RAS-mutated clones long after the spontaneous regression of JMML

FATAL SIBLING CASES OF FAMILIAL HEMOPHAGOCYTIC LYMPHOHISTIOCYTOSIS (FHL) WITH MUNC13–4 MUTATIONS: Case Reports

The authors report here sibling cases of familial hemophagocytic lymphohistiocytosis (FHL) type 3 that took fatal courses despite intensive treatment. The older brother achieved remission by immunochemotherapy, but a central nervous system lesion occurred before the introduction of allogeneic hematopoietic stem cell transplantation (allo-HSCT). The patient died on day +1 of allo-HSCT due to progression of the disease. The younger brother developed symptoms of hemophagocytic lymphohistiocytosis mimicking neonatal hemochromatosis at birth. He died without a chance to receive allo-HSCT. Both siblings showed low natural killer cell (NK) activity and the compound heterozygous Munc13–4 gene mutations 1596+1 and 1723insA were identified postmortem in the younger brother. With recent progress in the molecular diagnosis of FHL, prompt and most appropriate therapeutic measures should be introduced to improve the prognosis of FHL patients.