Keisuke Otsubo

Identification of FOXP3-negative regulatory T-like (CD4+CD25+CD127low) cells in patients with immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome

Immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome is an autoimmune disorder caused by mutations in the FOXP3 gene, which plays a key role in the generation of CD4(+)CD25(+)regulatory T (Treg) cells. We selected CD127 as the surface marker of Treg cells to illustrate the development and function of Treg cells in IPEX syndrome. CD4(+)CD25(+)FOXP3(+) T cells, the putative Treg cells, were almost completely absent in all patients. Importantly, a substantial number of CD4(+)CD25(+)CD127(low) T cells were observed in 3 IPEX patients with hypomorphic mutations in the FOXP3 gene. We demonstrated that CD4(+)CD25(+)CD127(low) T cells isolated from these 3 patients exhibited an appreciable suppressive activity on effector T cell proliferation, although less than that displayed by Treg cells from healthy controls. These results suggest that genetically altered FOXP3 can drive the generation of functionally immature Treg cells, but that intact FOXP3 is necessary for the complete function of Treg cells.

ETV6–ARNT fusion in a patient with childhood T lymphoblastic leukemia

The ETS variant gene 6 (ETV6) gene is located at 12p13, and is frequently involved in translocations in various human neoplasms, resulting in the expression of fusion proteins consisting of the amino-terminal part of ETV6 and unrelated transcription factors or protein tyrosine kinases. Leukemia with t(1;12)(q21;p13) was previously described in a 5-year-old boy with acute myeloblastic leukemia (AML-M2) who exhibited a novel ETV6-aryl hydrocarbon receptor nuclear translocator (ARNT) fusion protein. We herein report the case of a 2-year-old boy with T-cell lymphoblastic leukemia (T-ALL) harboring t(1;12)(q21;p13). Fluorescence in situ hybridization (FISH) with a ETV6 dual-color DNA probe revealed that the split signals of the ETV6 gene in 96.7% of bone marrow cells, indicating rearrangement of the ETV6 gene. Therefore, we performed a FISH analysis with bacterial artificial chromosome (BAC) probes containing the ARNT, BCL9, and MLLT11 genes located at 1q21, and these results indicated that the ARNT gene might be involved in the t(1;12)(q21;p13). Reverse transcriptase-polymerase chain reaction analysis disclosed the existence of a ETV6-ARNT fusion gene. To our knowledge, the current report is novel in its report of the ETV6-ARNT fusion in childhood T-ALL. The ETV6-ARNT fusion is associated not only with AML but also with T-ALL.

Autoimmune lymphoproliferative syndrome mimicking chronic active Epstein–Barr virus infection

Chronic active Epstein–Barr virus infection (CAEBV) is defined as a systemic EBV-associated lymphoproliferative disease characterized by fever, lymphadenopathy, and splenomegaly in apparently immunocompetent persons. Recent studies have revealed that EBV infects T or natural killer cells in most patients with CAEBV; the etiology of CAEBV, however, remains unknown. Autoimmune lymphoproliferative disorder (ALPS) is an inherited disorder associated with defects in apoptosis, and clinically characterized by lymphadenopathy, splenomegaly, hypergammaglobulinemia, and autoimmune disease. ALPS is most often associated with mutations in the FAS gene, which is an apoptosis-signaling receptor important for homeostasis of the immune system. Based on the clinical similarity between ALPS and CAEBV with respect to lymphoproliferation, we have examined the possibility of the co-occurrence of ALPS in patients with a diagnosis of CAEBV. In this study, we have identified FAS gene mutations in three Japanese patients with lymphadenopathy, hepatosplenomegaly, and unusual EBV infection, who were diagnosed with CAEBV. These observations, which indicate that the clinical development of ALPS may be associated with EBV infection, alert us to a potential diagnostic pitfall of CAEBV.

Clinical and genetic analyses of presumed shwachman-diamond syndrome in Japan

Shwachman-Diamond syndrome (SDS) is a rare autosomal recessive disorder characterized by exocrine pancreatic insufficiency, bone marrow dysfunction, and skeletal abnormalities. SBDS was identified as a causative gene for SDS in 2003, and genetic analyses of SDS have been performed.We performed genetic analysis of 13 Japanese patients with presumed SDS and found that 10 of them had SBDS mutations. Most patients had recurrent mutations (181-184TA→CT and 258+2T→C); however, 2 patients had unique mutations (259-1G→A and 428C→G). Although genetic analysis is useful for definitive diagnosis and for genetic counseling of SDS patients and families, SDS appears to be a genetically heterogeneous disorder. In addition, presumed SDS patients without SBDS mutations may be included in other disorders.

Identification of a novel MYH9 mutation in a patient with May-Hegglin anomaly.

To the Editor: May-Hegglin anomaly (MHA) is characterized by a triad of giant platelets, thrombocytopenia, and Döhle body-like cytoplasmic inclusions in granulocytes [1]. In addition to MHA, Sebastian, Fechtner, and Epstein syndromes are also included in the inherited macrothrombocytopenias, and they are caused by mutations in the MYH9 gene, which encodes the non-muscle myosin heavy chain-A (NMMHCA) [2–4]. All the disorders have been proposed to be renamed as MYH9 disorders, and MYH9 disorders have been increasingly reported [5,6]. Here we described a sporadic case of MHA with a novel MYH9 mutation. A 1-year-old male was referred to Toyama University hospital for evaluation of thrombocytopenia. He was born weighing 1,710 grams at 35 weeks’ gestation, and laboratory test revealed a platelet count of 20,000/ml. Although the patient had no bleeding tendency, he showed persistent thrombocytopenia (20,000–63,000/ml). His peripheral blood smears with May–Grünwald–Giemsa (MGG) staining, which disclosed giant platelets and leukocyte inclusion bodies (Fig. 1A). Although the patient had no family history of thrombocytopenia, the diagnosis of MHA was considered. Thus, an immunofluorescence analysis of neutrophil NMMHCA and mutational analysis of MYH9 gene were performed, as described previously [4,7]. Abnormal subcellular localization of NMMHCA was observed in every neutrophil from the patient (Fig. 1B). His family members demonstrated normal diffuse homogeneous distribution of NMMHCA throughout the cytoplasm (data not shown). Genomic DNA of the patient revealed a heterozygous singlebase substitution (C to G) at nucleotide 2116, indicated Gln (Q) to Glu (E) at residue 706 (Fig. 1C) creating a missense mutation. This substitution was not observed in normal individuals and other patients with MYH9 disorders. The diagnosis of MHA has been clinically made by the presence of macrothrombocytopenia and inclusion bodies in neutrophils [1]. It is thus important to carefully examine the peripheral blood smears using MGG staining. In addition, the faint appearance of inclusion bodies occasionally hampers proper recognition. MYH9 disorders show autosomal dominant traits, and family histories of thrombocytopenia are diagnostically helpful. However, approximately 20% of patients, as is the case with the present patient, are considered to be de novo. A few cases with MYH9 disorders are associated with macrothrombocytopenia without inclusion bodies. In the present study, we identified a novel MYH9 mutation in a sporadic MHA patient. The Q706E substitution affects codon 706, which is highly conserved among species, suggesting the presence of a strong functional or structural constraint for this residue. Adjacent to the Q706 residue, the R702C, R702H, and R705H mutations were described in other MYH9 disorders [6,8,9]. The R702 and R705 residues are located within or adjacent to the ‘‘SH1-SH2’’ helix of the globular-head domain. The SH1 and SH2 helices may play a pivotal role in the conformational changes that occur in the myosin head during force generation coupled to ATP hydrolysis. As well as R702C, R702H, and R705H mutations, Q706E mutation may cause an altered conformation of SH1 helix that affects its flexibilities and movement, thereby disrupting the function of NMMHCA. To confirm the diagnosis of MYH9 disorders, an immunofluorescence analysis of NMMHCA and mutational analysis of MYH9 gene should be performed even if the patients have no family histories of thrombocytopenia.

Acute promyelocytic leukemia following aleukemic leukemia cutis harboring NPM/RARA fusion gene.

To the Editor: Leukemia cutis is occasionally documented in patients with infant leukemia, and it is observed more frequently in acute myeloid leukemia with monocytic or myelomonocytic differentiation [1]. Leukemia cutis can occur before bone marrow involvement and systemic manifestations, and spontaneous resolution has been described [2]. We previously described 6-month-old male with aleukemic leukemia cutis harboringNPM/RARA fusion gene,whohad spontaneous regression of leukemia associated with cutaneous mastocytosis at the age of 19 months [3]. At 4 years and 4 months of age, the patient presented with fever and leg pain. Physical examination disclosed swelling of the right cheek and hepatosplenomegaly. Laboratory studies showed a hemoglobin level of 9.2 g/dl, a platelet count of 15.2 10/ml, elevated white blood cell count of 25.56 10/ml with 15.5% promyelocytes, 26.5% myelocytes, and 8.5% metamyelocytes, but no obvious blastic cells, and an elevated level of lactate dehydrogenase of 1,456 IU/L. Coagulopathy was associated with fibrinogen of 142mg/dl, FDP of 47.1 mg/ml (normal: <4.9), and D-Dimer of 21.5 mg/ml (normal: <1.0). Bone marrow specimens showed pathologic promyelocytic cells, which contained cytoplasmic azurophilic granules, but no Auer rods (Fig. 1A). The leukemic cells were strongly positive for myeloperoxidase staining (Fig. 1B). Karyotype analysis of the bone marrow cells showed 46,XY,t (5;17)(q35;q12),i(21)(q10) [16/20], as previously described [3]. Fluorescence in situ hybridization analysis with PML and RARA probes revealed two split RARA signals in 91.0% cells, but no fusion signal, and reverse transcriptase polymerase chain reaction showed a transcript of NPM/RARA fusion gene. The patient was finally diagnosed as having overt acute promyelocytic leukemia (APL) with NPM/RARA fusion gene as the relapse of aleukemic leukemia cutis. Therefore, he was treated with systemic chemotherapy followed by all-trans retinoic acid (ATRA) administration and achieved complete remission with the lesion in the head disappearing. Among eight patients with spontaneous resolution following aleukemic leukemia cutis, four patients were reported to have monocytic or myelomonocytic leukemia and one patient had acute myeloid leukemia (AML) M2, though AML subtypes of three patients were not described [2]. Cytogenetic and molecular studies suggested that APL clone was the same as that of the first presentation in our case.

Nocturnal enuresis as a first manifestation of acute lymphoblastic leukemia.

1 Ohlsson A, Walia R, Shah SS. Ibuprofen for the treatment of patent ductus arteriosus in preterm and/or low birth weight infants. Cochrane Database Syst. Rev. 2010; (4): CD003481. 2 Szymonowicz W, Yu VY. Periventricular haemorrhage: Association with patent ductus arteriosus and its treatment with indomethacin or surgery. Aust. Paediatr. J. 1987; 23 (1): 21–5. 3 Ment LR, Oh W, Ehrenkranz RA et al. Low-dose indomethacin therapy and extension of intraventricular hemorrhage: A multicenter randomized trial. J. Pediatr. 1994; 124: 951–5. 4 Bada HS, Green RS, Pourcyrous M et al. Indomethacin reduces the risks of severe intraventricular hemorrhage. J. Pediatr. 1989; 115: 631–7. 5 Maher P, Lane B, Ballard R, Piecuch R, Clyman RI. Does indomethacin cause extension of intracranial hemorrhages: A preliminary study. Pediatrics 1985; 75: 497–500.

Atypical lymphoproliferative disorder in a patient with X-linked thrombocytopenia

To the Editor: X-linked thrombocytopenia (XLT), which is a milder variant of Wiskott–Aldrich syndrome (WAS), is characterized by congenital thrombocytopenia without immunodeficiency [1]. Mutations in theWASP gene are responsible for WAS and XLT [2]. There is a high prevalence of lymphoproliferative disorder (LPD), which includes reactive hyperplasia, atypical LPD and malignant lymphoma, in patientswithWAS [3]. LPD is sometimes a life-threatening consequence of WAS, but has seldom been observed in XLT. We herein describe a patient with XLT who developed atypical LPD. The patient was diagnosed to have XLT by a flow cytometric and genetic analysis at 5 years of age [4]. Thereafter, at 8 years of age, he was associated with IgA nephropathy and heparin-induced thrombocytopenia [5]. At 14 years of age, he presented with an urticaria and cervical lymph node swelling. He showed no hepatosplenomegaly; laboratory data demonstrated thrombocytopenia (75 10/ml) and an increased level of soluble interleukin 2 receptor (sIL-2R; 1,383 U/ml). Epstein–Barr virus (EBV) DNA was not detected in a peripheral blood. 18F-fluorodeoxyglucose positron emission tomography (FDG-PET) revealed increased accumulations on nasopharynx, cervical, supraclavicular, axillar, and inguinal lymph nodes (Fig. 1A). A biopsy of the nasopharynx and cervical lymph node was performed. In both specimens, the paracortex was found to be markedly expanded and contain lymphocytes, plasma cells, immunoblasts, eosinphils, and histiocytes (Fig. 1B,C). In situ hybridization for EBER-1 was negative. There were no rearrangements of immunoglobulin heavy chain and T cell receptor genes. The patient was diagnosedwith atypical LPD. Prednisolone (1 mg/kg/day) was administered and the cervical lymph node swelling thereafter improved. The sIL-2R level decreased to the normal range and the FDG-PET showed normalized. The patient has since been waiting for an unrelated bone marrow transplantation. InWAS, the risk of malignancy is estimated to be approximately 100 times greater than that of the age-matched control [3]. EBV infection is a major cofactor contributing to the increased risk of LPD, but our patient was not associated with an EBV infection. The clinical features of atypical LPD are an acute onset, generalized lymphadenopathy, hepatosplenomegaly, hypersensitivity, recurrent infections, dysgammaglobulinemia, and bone marrow plasmacytosis [6]. The present case showed the acute onset of generalized lymphadenopathy and urticaria, which may be compatible with atypical LPD. The present case improved after the administration of prednisolone, but another reported case with atypical LPD showed a spontaneous regression [7].WAShas been associatedwith autoimmune diseases and malignancies in 40% and 13% of the patients, respectively [8]. The incidence of malignancy in patients with the XLT phenotype is lower than that in patients with classic WAS [9]. Although the present case showed a regression of LPD, there is a chance that the patient still might develop a malignancy in the future. WAS-associated malignancies have a poor prognosis, and a bonemarrow transplantationwould be indicated in this patient if a malignancy were to develop at a later time. Keisuke Otsubo, MD Hirokazu Kanegane, MD, PhD* Keiko Nomura, MD, PhD Toshio Miyawaki, MD, PhD Department of Pediatrics Graduate School of Medicine University of Toyama, Toyama, Japan —————— *Correspondence to: Hirokazu Kanegane, Department of Pediatrics, Graduate School of Medicine, University of Toyama, 2630 Sugitani, Toyama, Toyama 930-0194, Japan. E-mail: kanegane@med.u-toyama.ac.jp

Acute lymphoblastic leukemia after living donor liver transplantation

Advances in liver transplantation have produced long survival and improved the quality of life of transplant recipients. 1 Despite these advances, liver transplantation is still confronted with clinically important problems. One of these is dealing with the donor shortage. In Japan, living donor liver transplantation has become increasingly popular because of the lack of cadaver donors. The other problems are complications related to prolonged immunosuppressive therapy. Many patients experience diabetes, hypercholesterolemia, progressive renal insufficiency, hypertension, and osteoporosis. Furthermore, the chronic use of immunosuppressive agents seems to increase the risk of development of de novo malignancies as well as major infections. De novo malignancies in liver transplant recipients include skin cancer, non-Hodgkin’s lymphoma, post-transplant lymphoproliferative disease (LPD), sarcoma, renal cell carcinoma, and hepatobiliary carcinomas. Nevertheless, acute leukemia is a rare complication after liver transplantation, and only seven patients who developed post-liver transplant leukemia have been reported. 2–6 Although liver injuries may occur as a side-effect of the chemotherapy for leukemia, liver dysfunction as an initial manifestation of leukemia is generally unusual. 7

Giardiasis in a patient undergoing chemotherapy for retinoblastoma and acute myelogenous leukemia

Giardiasis is a common cause of diarrhea in undeveloped countries, but is very rare in developed countries. A patient with acute myelogenous leukemia and retinoblastoma presented with a high fever and severe watery diarrhea during induction chemotherapy. On microscopy, cysts were seen in her stool, suggesting Giardia intestinalis, which was confirmed on polymerase chain reaction (PCR). G. intestinalis was also detected in the patients asymptomatic parents, who may have transmitted it to the patient. Giardiasis should be tested for in patients with severe and persistent diarrhea during chemotherapy, when other etiologies have been excluded. PCR used to amplify the DNA of G. intestinalis is rapid and sensitive.