Kenji Ihara

Three-Dimensional Structure of a DNA Repair Enzyme, 3-Methyladenine DNA Glycosylase II, from Escherichia coli

The three-dimensional structure of Escherichia coli 3-methyladenine DNA glycosylase II, which removes numerous alkylated bases from DNA, was solved at 2.3 A resolution. The enzyme consists of three domains: one alpha + beta fold domain with a similarity to one-half of the eukaryotic TATA box-binding protein, and two all alpha-helical domains similar to those of Escherichia coli endonuclease III with combined N-glycosylase/abasic lyase activity. Mutagenesis and model-building studies suggest that the active site is located in a cleft between the two helical domains and that the enzyme flips the target base out of the DNA duplex into the active-site cleft. The structure of the active site implies broad substrate specificity and simple N-glycosylase activity.

Association of vascular endothelial growth factor (VEGF) and VEGF receptor gene polymorphisms with coronary artery lesions of Kawasaki disease.

We analyzed the genetic polymorphisms of vascular endothelial growth factor (VEGF) and its receptors [Fms-related tyrosine kinase-1, kinase insert domain receptor (KDR)] in Japanese patients with Kawasaki disease (KD) and normal control subjects to examine whether these genes would contribute to the KD occurrence and/or the development of coronary artery lesion (CAL) in KD. We found that the frequency of G allele of VEGF g.−634 G>C single-nucleotide polymorphism in the promoter region was significantly higher in KD patients with CAL than in those without CAL (p = 0.012) or control subjects (p = 0.021) because of a significantly higher frequency of the GG genotype in KD patients with CAL. In addition, the frequency of the A1 allele with 11 AC repeats of KDR g.+4422(AC)11–14 dinucleotide repeat polymorphism in intron 2 was significantly higher in KD patients with CAL than in those without CAL (p = 0.013) or control subjects (p = 0.040) as a result of a significantly higher frequency of the A1A1 genotype in KD with CAL patients. The multivariate analysis of clinical features and genotypes of the two polymorphisms showed that the A1A1 genotype of KDR g.+4422(AC)11–14 polymorphism was an independent risk factor for the development of CAL with the highest odds ratio among several clinical parameters (odds ratio 6.76; 95% confidence interval 1.05–43.48). Dual luciferase assay demonstrated that the A1 allele with KDR g.+4422(AC)11 repeats showed a weaker silencer function than the A2 allele with 12 AC repeats. These findings suggested that VEGF and its receptor, KDR, genes contributed to the development of CAL in KD patients.

Oversecretion of IL‐18 in haemophagocytic lymphohistiocytosis: a novel marker of disease activity

We investigated the significance of interleukin (IL)‐18 levels in the pathophysiology of haemophagocytic lymphohistiocytosis (HLH). IL‐18 levels were significantly elevated in all nine patients with active HLH compared with those of healthy controls. Serial determination of IL‐18 levels in three cases, showed a gradual decrease compared with those of IL‐12, interferon (IFN)‐γ or soluble Fas ligand (sFasL) in the course of clinical improvement, and seemed to be elevated until complete disappearance of disease activity. IL‐18 and IFN‐γ (CC 0.711, P = 0.018), and IFN‐γ and sFasL (CC 0.849, P = 0.0049) levels were significantly correlated. On the other hand, correlation between IL‐12 and IFN‐γ, IL‐18 and sFasL, or IL‐18 and IL‐12 was not observed. IL‐18, IFN‐γ and sFasL levels significantly correlated with disease activity such as fever and alanine transaminase (ALT) levels. IL‐18 mRNA expression was enhanced in spleen, but not in peripheral blood mononuclear cells (MNC), bone marrow MNC, liver from patients of active HLH, or the tumour from a patient with lymphoma‐associated haemophagocytic syndrome (LAHS). These results suggest that IL‐18 may play important roles in the pathogenesis of HLH, particularly through induction of Th1 cells. IL‐18 measurement may be useful for the diagnosis and for the detection of smouldering disease activity.

Epstein-Barr Virus (EBV) Load and Cytokine Gene Expression in Activated T Cells of Chronic Active EBV Infection

To identify the role of T cells in chronic active Epstein-Barr virus (EBV) infection, EBV and cytokine gene expression was quantified by use of real-time polymerase chain reaction (PCR) among 6 patients who fulfilled the diagnostic criteria for chronic active EBV infection. Four of these patients showed clonal expansion of EBV-infected T cells. Quantitative PCR for EBV DNA in peripheral blood of patients with symptomatic chronic active EBV infection showed higher copy numbers of virus (mean, 1.45 x 10(5) copies/mL) than were seen in blood from patients with infectious mononucleosis (3.08 x 10(3) copies/mL) or with EBV-associated hemophagocytosis (2.95 x 10(4) copies/mL). Fractionated CD3(+) HLA-DR(+) cells from patients with chronic active EBV infection contained higher copy numbers than did CD3(+) HLA-DR(-) cells. Quantitative PCR for cytokines revealed that interferon-gamma, interleukin (IL)-2, IL-10, and transforming growth factor-beta genes were expressed at higher levels in HLA-DR(+) than in HLA-DR(-) T cells. These results suggest that activated T cells in chronic active EBV infection expressed high levels of EBV DNA and both Th1 and Th2 cytokines. EBV-infected T cells may contribute to the unbalanced cytokine profiles of chronic mononucleosis.

Perforin defects of primary haemophagocytic lymphohistiocytosis in Japan

Summary. The perforin gene was analysed in 15 Japanese patients with primary haemophagocytic lymphohistiocytosis (HLH). Perforin gene defects were found in two out of eight patients with familial HLH (FHL), and one out of seven without affected siblings. Four novel mutations were identified. Compound heterozygous mutations (one FHL and one sporadic HLH) and only one allele mutation (one FHL) were defined. Flow cytometry revealed no perforin expression in CD8+ or CD56+ cells from a surviving patient with a mutation. The frequency of mutation was at least 20% of FHL in Japan. Flow cytometry for intracellular perforin may be useful for the screening of FHL2.

MLL2 and KDM6A mutations in patients with Kabuki syndrome

Kabuki syndrome is a congenital anomaly syndrome characterized by developmental delay, intellectual disability, specific facial features including long palpebral fissures and ectropion of the lateral third of the lower eyelids, prominent digit pads, and skeletal and visceral abnormalities. Mutations in MLL2 and KDM6A cause Kabuki syndrome. We screened 81 individuals with Kabuki syndrome for mutations in these genes by conventional methods (n = 58) and/or targeted resequencing (n = 45) or whole exome sequencing (n = 5). We identified a mutation in MLL2 or KDM6A in 50 (61.7%) and 5 (6.2%) cases, respectively. Thirty‐five MLL2 mutations and two KDM6A mutations were novel. Non‐protein truncating‐type MLL2 mutations were mainly located around functional domains, while truncating‐type mutations were scattered through the entire coding region. The facial features of patients in the MLL2 truncating‐type mutation group were typical based on those of the 10 originally reported patients with Kabuki syndrome; those of the other groups were less typical. High arched eyebrows, short fifth finger, and hypotonia in infancy were more frequent in the MLL2 mutation group than in the KDM6A mutation group. Short stature and postnatal growth retardation were observed in all individuals with KDM6A mutations, but in only half of the group with MLL2 mutations.

Childhood Atopic Asthma: Positive Association with a Polymorphism of IL-4 Receptor α Gene but Not with That of IL-4 Promoter or Fc ε Receptor I β Gene

We examined the relative contributions of three representative candidate genes for atopy (Fc ε receptor I β, IL-4, and IL-4 receptor α) to the development of atopic asthma. Four polymorphisms of the three candidate genes including Ile50Val and Gln551Arg of IL-4 receptor α, -590C/T of IL-4 promoter and Glu237Gly of Fc ε receptor I β were studied in 100 patients with atopic asthma and 100 nonatopic controls in the northern Kyushu area in Japan. Among the four polymorphisms of the three candidate genes, the Ile50 allele of the IL-4 receptor α chain gene demonstrated an association with atopic asthma subjects (p = 0.044), especially in patients with onset at 2 years of age or earlier (p = 0.034) and in patients with moderate to severe atopic asthma (p = 0.031). Gln551Arg of IL-4 receptor α, -590C/T of IL-4 promoter and Glu237Gly of Fc ε receptor I β showed no association with atopic asthma. A slight linkage disequilibrium between Ile50Val and Gln551Arg polymorphisms of the IL-4 receptor α chain gene was observed in both patients and nonatopic controls. The identification of additional atopy genes in areas with a certain genetic background is essential for genetic diagnosis and to establish new therapeutic modalities for atopic asthma.

Th1 and Th1-inducing cytokines in Salmonella infection

Thl and Thl‐inducing cytokines and T cell responses were investigated in human salmonellosis. Serum IFN‐γ, IL‐12 and IL‐18 levels were increased significantly in patients with salmonellosis. The increase in serum IL‐15 and IL‐18 levels was more significant and prolonged in patients with the systemic form of salmonellosis than in those with the gastroenteric form. The serum IFN‐γ level was correlated significantly with IL‐12 and IL18 levels, and the IL‐15 level was correlated significantly with IL‐18. Upon stimulation with Salmonella in vitro, mononuclear cells from salmonellosis patients produced significantly higher amounts of IFN‐γ and IL‐12 compared with those from healthy controls. Anti‐IL‐12 moAb or anti‐IL18 MoAb significantly inhibited Salmonella‐induced IFN‐γ production in vitro. γδ T cells expressed significantly higher levels of IFN‐γ mRNA in salmonellosis patients than in healthy controls. The results suggest that Th1‐inducing cytokines appear to be involved in the in vivo response against Salmonella infection, promoting IFN‐γ production by αβ and γδ T cells which plays a protective role against Salmonella.

Newborn screening for Fabry disease in Japan: prevalence and genotypes of Fabry disease in a pilot study

Fabry disease (FD) is an X-linked lysosomal storage disorder caused by a deficiency of α-galactosidase A (GLA) activity. Enzyme replacement therapy (ERT) for FD is available, and newborn mass screening for FD is being implemented. Here, we undertook a pilot study of newborn mass screening for FD in Japan. GLA activity in dried blood spots was measured using a fluorescence assay and confirmed by measurement of GLA activity in white blood cells (WBCs) in infants with abnormally low GLA activity. This was followed up by genetic testing. A total of 21 170 neonates were enrolled in the study. Of these, seven (five boys, two girls) had low GLA activities, which were verified by the WBC GLA activity assay. Thus, the initial fluorescence assay was suitable for newborn mass screening for FD. Pathogenic mutations of the GLA gene, that is, V199M and IVS4+919G>A, were found in two boys and one boy, respectively. Functional mutations, E66Q and c.−10C>T: g.1170C>T, were found in two boys and one girl, respectively. The prevalence of test-positive newborns was 1/3024, while that of those with a pathogenic mutation was 1/7057. The numbers are higher than those previously anticipated. Standardized management for FD found during newborn mass screening, including an ERT regimen, remains to be established.

Physical Growth and Retinopathy in Preterm Infants: Involvement of IGF-I and GH

GH and IGF-I are important for physical growth. We measured serum levels of these factors in preterm infants. The study population (n = 81) was divided into three groups according to the gestational age. We evaluated differences in serum GH and IGF-I levels among groups with regard to physical growth and development of retinopathy of prematurity. Serum GH levels in extremely preterm infants born at <28 wk of gestational age were significantly higher than levels in those born between 28 and 34 wk at 1 and 2 mo of age. In contrast, serum IGF-I levels in extremely preterm infants remained low, whereas those in the other two groups gradually increased. Evaluation of the effects of GH and IGF-I on physical growth in very low birth weight infants (<1500 g) showed that IGF-I concentrations were positively related to physical growth for several months after birth, whereas no relationship was observed between GH and physical growth. Multivariate analysis demonstrated that high GH concentration at 1 mo of age was significantly associated with development of severe retinopathy of prematurity. In conclusion, persistent low serum IGF-I levels may explain the slow physical growth during neonatal life, and exposure of high GH may cause, at least in part, severe retinopathy of prematurity in preterm infants.