Ikuyo Ueda

Characteristic perforin gene mutations of haemophagocytic lymphohistiocytosis patients in Japan

Summary. Perforin gene (PRF1) mutations appear to occur in about 30% of patients with haemophagocytic lymphohistiocytosis (HLH). We tested perforin expression and gene mutations in 14 HLH patients and six patients with Epstein–Barr virus‐associated HLH (EBV‐HLH) in Japan. Five of the 14 HLH patients had perforin abnormalities. The presence of PRF1 genetic abnormality correlated well with the lack of perforin expression as determined by flow cytometry. Sequencing showed that four patients had a compound heterozygous mutation while the fifth patient had a homozygous mutation. Three of the mutations we detected were novel. In contrast, none of the six EBV‐HLH patients showed perforin abnormalities. Our data, combined with the PRF1 mutations in three previously reported Japanese patients, suggest that the 1090–1091delCT and 207delC mutations of the perforin gene are frequently present in Japanese HLH patients (62·5% and 37·5% respectively). Examination of the geographical origins of the ancestors in the perforin‐mutant HLH patients revealed that they mostly came from the Western part of Japan, suggesting that the present‐day cases may largely derive from a common ancestor.

Correlation between phenotypic heterogeneity and gene mutational characteristics in familial hemophagocytic lymphohistiocytosis (FHL)

Classification of familial hemophagocytic lymphohistiocytosis (FHL) into FHL2, FHL3, and other subtypes based on genetic abnormalities has recently become possible. We studied the phenotypic differences among these subtypes in Japan.

Identification of novel MUNC13-4 mutations in familial haemophagocytic lymphohistiocytosis and functional analysis of MUNC13-4-deficient cytotoxic T lymphocytes.

Background: Familial haemophagocytic lymphohistiocytosis (FHL) has an autosomal recessive mode of inheritance and consists of at least three subtypes. FHL2 subtype with perforin (PRF1) mutation accounts for 30% of all FHL cases, while FHL with MUNC13-4 mutation was recently identified and designated as FHL3 subtype. Objective: To examine MUNC13-4 mutations and the cytotoxic function of MUNC13-4 deficient T lymphocytes in Japanese FHL patients Methods: Mutations of MUNC13-4 and the cytotoxicity of MUNC13-4-deficient cytotoxic T lymphocytes (CTL) were analysed in 16 Japanese families with non-FHL2 subtype. Results: Five new mutations of the MUNC13-4 gene were identified in six families. The mutations were in the introns 4, 9, and 18, and exons 8 and 19. Two families had homozygous mutations, while the remaining four had compound heterozygous mutations. Cytotoxicity of MUNC13-4 deficient CTL was low compared with control CTL, but was still present. Clinically, the onset of disease tended to occur late; moreover, natural killer cell activity was not deficient in some FHL3 patients. Conclusions:MUNC13-4 mutations play a role in the development of FHL3 through a defective cytotoxic pathway.

Occurrence of haemophagocytic lymphohistiocytosis at less than 1 year of age: analysis of 96 patients

We analysed data of 96 infants (under 1 year of age) with haemophagocytic lymphohistiocytosis (HLH) from the registry of an HLH study conducted during 1986–2002 in Japan. The cases were classified into five groups. The diagnosis of familial HLH (FHL) as group 1 (n =27) was made with positive family history and/or recent molecular test for perforin and Munc13–4 mutations. Neonatal enterovirus- or herpes simplex virus-associated HLH as group 2a (n =7), Epstein-Barr virus–associated HLH (n =12) as group 2b, adenovirus- or cytomegalovirus-associated HLH as group 3 (n =9) were mostly diagnosed by viral isolation or by the detection of viral genome. Juvenile rheumatoid arthritis-associated macrophage activation syndrome was classified as group 4 (n =4) and the remaining without known triggers as group 5 (n =37). The peak onset age was 1–2 months for group 1, 1–2 weeks for group 2a, 12 months for group 2b, none for group 3, 9 months for group 4 and 2 months for group 5. Future novel diagnostic measures are required to define the precise nature of HLH in group 5. Conclusion:These data may provide useful information for neonatologists/ paediatricians in the differential diagnosis of haemophagocytic lymphohistiocytosis in early infancy.

Association of transforming growth factor-β1 gene polymorphism in the development of Epstein-Barr virus-related hematologic diseases

Background and Objectives Epstein-Barr virus (EBV) is etiologically associated with various hematologic disorders, including primary acute infectious mononucleosis (IM), hemophagocytic lymphohistiocytosis (EBV-HLH), chronic active EBV infection (CAEBV) and malignant lymphomas. Although cytokines play a central role in EBV-related immune responses, the exact mechanisms causing different clinical responses remain unclear. In this study, the pattern of cytokine gene polymorphisms was comparatively analyzed in EBV-related diseases. Design and Methods Eighty-nine patients with EBV-related disease were analyzed; 30 with IM, 28 with EBV-HLH and 31 with CAEBV. Eighty-one EBV-seropositive healthy adults were also used as controls. Associations with polymorphisms of various cytokines, including interleukin (IL)-1α and IL-1β were evaluated. The gene polymorphisms were typed by polymerase chain reaction with sequence-specific primers. Results A significant difference of polymorphisms was found for transforming growth factor (TGF)-β1; the frequency of TGF-β1 codon 10 C allele was significantly higher in patients with EBV-related diseases than in controls (p<0.001). The difference was significant in patients with IM or HLH (p<0.001), but not in those with CAEBV (p=0.127), compared with controls. As regards other cytokines, the frequency of the IL-1α –889 C allele was significantly lower in patients with IM than in controls (p<0.05). Interpretation and Conclusions Our results suggests that TGF-β1 codon 10 C allele plays a role in the development of EBV-related diseases and that the IL-1α –889 C allele may be involved in response failure and sequential progression into the development of HLH.

Mutations of syntaxin 11 and SNAP23 genes as causes of familial hemophagocytic lymphohistiocytosis were not found in Japanese people

AbstractAlthough mutations of perforin, MUNC13-4 and syntaxin 11 genes have been found in children with familial hemophagocytic lymphohistiocytosis (FHL), the incidence of each genetic subtype varies in different ethnic groups. We evaluated mutations of syntaxin 11 and SNAP23 genes in 30 Japanese FHL patients. The patients had no mutations and 10% had one polymorphism (146G>A) of syntaxin 11, while no mutation of SNAP23 was observed. Our results indicate that aberrations in the SNARE system may not cause FHL in Japanese families.

A novel missense mutation (1060G → C) in the phosphoglycerate kinase gene in a Japanese boy with chronic haemolytic anaemia, developmental delay and rhabdomyolysis

Summary. We report the case of a 3‐year‐old Japanese boy with phosphoglycerate kinase 1 (PGK1) deficiency (Online Mendelian Inheritance in Man entry 311800). The patient had anaemia and jaundice at birth, necessitating exchange transfusions for 2 d. After one red blood cell transfusion at age 2 months, his Hb level was 8–9 g/dl, his reticulocyte counts were 300–500 × 109/l, and his total bilirubin level was 25·65–42·75 µmol/l. The patient suffered two episodes of respiratory infection‐associated haemolytic crisis and rhabdomyolysis during early infancy. At age 3·0 years, his developmental milestones (developmental quotients measured using the Tsumori–Inage methods) score was 49% (normal 74–131%), and his height was below average by −2·0 standard deviations. The diagnosis of PGK1 deficiency was made based on his remarkably low (< 10% of normal) erythrocyte PGK enzyme activity level and the identification of a novel missense (1060G→C) PGK1 gene mutation. This mutation results in the Ala‐353Pro amino acid substitution, which has been designated PGK Kyoto. The patient developed the full clinical symptoms of PGK1 deficiency including haemolytic anaemia, myopathy, central nervous system disorder and growth retardation, which is unusual.

Recurrent hemophagocytic lymphohistiocytosis accompanied by Kikuchi's disease

Hemophagocytic lymphohistiocytosis (HLH) is characterized by fever, splenomegaly, cytopenia, hypertriglyceridemia and/or hypofibrinogenemia, hemophagocytosis, low or absent natural killer (NK)-cell activity, increased levels of serum ferritin, and increased levels of soluble interleukin (IL)-2 receptor in the serum and the cerebrospinal fluid [1]. It is proposed that the diagnosis of HLH can be established if five of these eight criteria are met [1]. Other less-common features include lymphadenopathy and skin rash. HLH can be classified as primary or secondary HLH. Primary HLHs include several types of familial HLHs (FHLs) [2 – 4] and the HLHs associated with primary immune deficiencies, and shows continuous or recurrent hemophagocytosis. Secondary HLHs include HLHs associated with infections, malignancies, or autoimmune diseases, and are not usually recurrent. Both primary and secondary HLH can be fatal. Kikuchi’s disease (KD; also known as Kikuchi – Fujimoto disease or subacute histiocytic necrotizing lymphadenitis) is characterized by fever, fatigue, and lymphadenitis. Affected lymph nodes show patchy necrotic areas with karyorrhexis surrounded by activated histiocytes. The cause of KD is still unknown. Although an infectious etiology is suspected, bacteria, fungi, toxoplasma, and Epstein-Barr virus (EBV) have been shown not to be associated with the disease. KD is benign and shows a self-limited clinical course [5,6]. Only 11 cases of concurrent HLH and KD have been reported, none of which was recurrent [7 – 16]. To our knowledge, this is the first report of a case of recurrent HLH accompanied by KD. The patient was a 13-year-old Japanese boy with atopic dermatitis and bronchial asthma, but who was otherwise healthy. Left inguinal tender lymphadenopathy appeared 2 weeks before hospitalization and ameliorated without any medication within 1 week. Fever and tender lymphadenopathy of the left axillary and right inguinal lymph nodes appeared 2 days before hospitalization. Hepatosplenomegaly was noticed upon hospitalization. Laboratory examinations showed marked leukopenia (white blood cell count 1.7610/l; absolute neutrophil count 0.37610/l), mild thrombocytopenia (13.5610/l), hypertriglyceridemia (306 mg/dl), low NK-cell activity (15%, normal range 18 – 40%), and an increased level of soluble IL-2 receptor (982 U/ml). The bone marrow (BM) was hypocellular, which was confirmed by both aspiration and biopsy, and revealed increased number of histiocytes (9.5%), most of which showed hemophagocytosis [Figure 1(a)]. These results showed that five of the eight diagnostic criteria for HLH were met [1]. The karyotype of BM was 46, XY [12/12]. Clonal rearrangements of the TCRG and IgH genes were not detected. PCR assays for cytomegalovirus (CMV), parvovirus B19, and hepatitis C virus (HCV), and serological assays for rubella, hepatitis B virus (HBV), and EBV did not indicate that the HLH was associated with any of these viruses. Results of cerebrospinal fluid assays and cranial magnetic resonance imaging showed no abnormalities. The patient did not have albinism,

Cytophagic histiocytic panniculitis with fatal haemophagocytic lymphohistiocytosis in a paediatric patient with perforin gene mutation

Cytophagic histiocytic panniculitis (CHP), or histiocytic cytophagic panniculitis, is a rare form of panniculitis associated with haemorrhagic diathesis and histiocytic lymphohistiocytosis (HLH), initially described in 1980 as a benign lymphoproliferative disease.1 In 1991 Gonzalez et al reported a unique entity of subcutaneous T-cell lymphoma with haemophagocytosis, later designated as subcutaneous panniculitis-like T-cell lymphoma (SPTL).2 Marzano et al suggested that CHP and SPTL might span a clinicopathological spectrum in which there is a natural progression from CHP to SPTL.3 HLH is a clinical syndrome of immune deregulation with hypercytokinaemia causing dysfunctions of various organs and a high mortality. Familial HLH (FHL) is associated with several hereditary defects. Stepp et al first showed that the mutation in perforin gene ( PRF1 ) at chromosome 10q21 was responsible for 20–40% of FHL patients.4 An 11-year-old girl with CHP and HLH presented with spiking fever and indurated skin nodules over the left thigh (fig 1A). She had psychomotor retardation and spastic type cerebral palsy at 2 years of age and epilepsy at age 7. Physical examination revealed indurated skin nodules and hepatosplenomegaly without lymphadenopathy. Laboratory tests showed anaemia, thrombocytopenia, impaired liver function, and raised triglyceride level, without coagulopathy. Blood and urine cultures were negative. Her fever and skin lesions responded dramatically to prednisolone and intravenous immunoglobulins. She became prednisolone-dependent despite attempts at tapering off by adding methotrexate and cyclosporine A. Several episodes of HLH and infections led to three additional admissions in three years. Six months later, she developed prominent facial bruising and swelling. Virological surveys revealed prior/remote Epstein-Barr virus …

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.