伊藤 哲哉

溶連菌感染後急性糸球体腎炎(PSAGN)は、A群β溶連菌感染症後に血尿、浮腫、高血圧を三主徴として発症する小児には比較的頻度の高い腎疾患であるが、時に乏尿によりはっきりした尿所見を認めずに浮腫や高血圧のみが前面になって発症する例があり、腎外症候性急性糸球体腎炎といわれる。今回、無熱性けいれんで発症した1例を経験したので報告する。症例は10歳男児。入院1週間前より腹痛、嘔吐、下痢といった胃腸炎症状を認めていた。入院前日より頭痛があり、入院当日に急激な意識レベルの低下、両側上肢の強直性けいれんを認め当院に搬送された。来院時の血液検査・髄液検査・頭部CTでは明らかな異常所見認めず、無熱性けいれんとして精査、加療目的に入院とした。第4病日頃より強い頭痛の訴えと血圧191/103mmHgと著明高値を認め、高血圧緊急症と診断した。同日に行った頭部MRI T2強調像、FLAIR像にて後頭葉に高信号域を認め可逆性後頭葉白質脳症(PRES)と診断した。血液検査では補体価の著減を認め、さらにASLO、ASKの著増、咽頭よりA群溶連菌迅速抗原陽性を認めたことより、PSAGNとこれに伴う二次性高血圧と診断した。高血圧症に対してニカルジピン塩酸塩持続静注等の緊急治療を行い諸症状は改善した。その後の全身状態は良好で第13病日に頭部MRI再検、PRESの所見は不明瞭化していることを確認し退院とした。PSAGNの中に、本例のように臨床的に高血圧のみが前面に出現する例があり、溶連菌感染症流行期に高血圧や浮腫を呈する症例の鑑別疾患として本症があることを考慮する必要があると考えられた。(著者抄録)

PURPOSE: Galactosemia is caused by metabolic disturbances at various stages of galactose metabolism, including deficiencies in enzymes involved in the Leloir pathway (GALT, GALK1, and GALE). Nevertheless, the etiology of galactosemia has not been identified in a subset of patients. This study aimed to explore the causes of unexplained galactosemia. METHODS: Trio-based exome sequencing and/or Sanger sequencing was performed in eight patients with unexplained congenital galactosemia. In vitro enzymatic assays and immunoblot assays were performed to confirm the pathogenicity of the variants. RESULTS: The highest blood galactose levels observed in each patient were 17.3-41.9 mg/dl. Bilateral cataracts were observed in two patients. In all eight patients, we identified biallelic variants (p.Arg82*, p.Ile99Leufs*46, p.Gly142Arg, p.Arg267Gly, and p.Trp311*) in the GALM encoding galactose mutarotase, which catalyzes epimerization between β- and α-D-galactose in the first step of the Leloir pathway. GALM enzyme activities were undetectable in lymphoblastoid cell lines established from two patients. Immunoblot analysis showed the absence of the GALM protein in the patients' peripheral blood mononuclear cells. In vitro GALM expression and protein stability assays revealed altered stabilities of the variant GALM proteins. CONCLUSION: Biallelic GALM pathogenic variants cause galactosemia, suggesting the existence of type IV galactosemia.

BACKGROUND: Ornithine transcarbamylase deficiency (OTCD) is an X-linked recessive disorder involving a defect in the urea cycle caused by OTC gene mutations. Although a total of 417 disease-causing mutations in OTC have been reported, structural abnormalities in this gene are rare. We here describe a female OTCD case caused by an exonic duplication of the OTC gene (exons 1-6). CASE PRESENTATION: A 23-year-old woman with late-onset OTCD diagnosed by biochemical testing was subjected to subsequent genetic testing. Sanger sequencing revealed no pathogenic mutation throughout the coding exons of the OTC gene, but multiplex ligation-dependent probe amplification (MLPA) revealed duplication of exons 1-6. Further genetic analyses revealed an inversion of duplicated exon 1 and a tandem duplication of exons 2-6. Each of the junctions of the inversion harbored a microhomology and non-templated microinsertion, respectively, suggesting a replication-based mechanism. The duplication was also of de novo origin but segregation analysis indicated that it took place in the paternal chromosome. CONCLUSION: We report the first OTCD case harboring an exonic duplication in the OTC gene. The functional defects caused by this anomaly were determined via structural analysis of its complex rearrangements.

Dihydropyrimidinase (DHP) is the second enzyme of the pyrimidine degradation pathway and catalyzes the ring opening of 5,6-dihydrouracil and 5,6-dihydrothymine. To date, only 31 genetically confirmed patients with a DHP deficiency have been reported and the clinical, biochemical and genetic spectrum of DHP deficient patients is, therefore, still largely unknown. Here, we show that 4 newly identified DHP deficient patients presented with strongly elevated levels of 5,6-dihydrouracil and 5,6-dihydrothymine in urine and a highly variable clinical presentation, ranging from asymptomatic to infantile spasm and reduced white matter and brain atrophy. Analysis of the DHP gene (DPYS) showed the presence of 8 variants including 4 novel/rare missense variants and one novel deletion. Functional analysis of recombinantly expressed DHP mutants carrying the p.M250I, p.H295R, p.Q334R, p.T418I and the p.R490H variant showed residual DHP activities of 2.0%, 9.8%, 9.7%, 64% and 0.3%, respectively. The crystal structure of human DHP indicated that all point mutations were likely to cause rearrangements of loops shaping the active site, primarily affecting substrate binding and stability of the enzyme. The observation that the identified mutations were more prevalent in East Asians and the Japanese population indicates that DHP deficiency may be more common than anticipated in these ethnic groups.

Background: Transplanted organs from female donors are associated with less favorable prognoses and outcomes. This study aimed to determine whether donor gender affects levels of serum terminal complement component C5a and oxidative stress in pediatric living related liver transplantation (LRLTx) recipients. Material/Methods: The subjects were 43 patients (20 males and 23 females) who underwent LRLTx during childhood (age range 1.2 years to 14.4 years; mean age 5 years). Serum samples were taken during the patients' regular outpatient visits after LRLTx. Serum C5a was measured using the specific human C5a ELISA kit. Serum total hydroperoxide (TH) and biological antioxidative potential (BAP) were measured using the free radical analytic system, and the oxidative stress index (OSI) was calculated as the ratio of TH to BAP. Serum glutamic pyruvic transaminase (GPT), glutamic oxaloacetic transaminase (GOT), gamma-glutamyl transpeptidase (gamma GTP), and lactate dehydrogenase (LDH) were also measured as part of a typical outpatient examination for such patients. Results: C5a serum levels were higher in the 29 recipients who received their grafted livers from female donors than in the 14 recipients who received their grafted livers from male donors. Recipients who received their grafted livers from female donors had higher incidence of post-LTx (liver transplantation) complications. Female recipients from female donors showed the highest serum GPT and GOT levels, but this difference was only significant when compared to the female recipients from male donors (41.4 +/- 9.8 IU/L vs. 17.3 +/- 1.8 IU/L for GPT and 42.2 +/- 7.5 IU/L vs. 23.4 +/- 2.2 IU/L for GOT; P<0.05). Conclusions: Pediatric LRLTx patients who receive their grafts from female donors exhibit higher levels of serum C5a that probably plays a role in the immunological response against grafted livers from female donors in LTx.

BACKGROUND: Congenital anomalies of the kidney and urinary tract (CAKUT) are the most prevalent etiologies of pediatric chronic kidney disease (CKD). However, no robust mass screening methods have been developed to detect patients with CAKUT, making early intervention to prevent progressive renal failure challenging. METHODS: We applied tandem mass spectrometry (MS/MS) to measure the creatinine (Cr) value from dried blood spot (DBS) analysis, which has been used for newborn mass screening. Subsequently, we evaluated the correlation between DBS Cr measured by MS/MS and serum Cr measured by the conventional method in pediatric patients with CKD. Finally, DBS Cr was measured in 190 full-term, healthy newborns on days 4-6 after birth. RESULTS: We established a system of MS/MS-based measurement of Cr from DBS. Measured DBS Cr in the pediatric patients showed a strong association with serum Cr (r=0.86; P<0.01). The median DBS Cr value in newborns was 0.222 (interquartile range: 0.189, 0.269) mg/dl. No significant correlations were found between DBS Cr values and body weight, Apgar score, gestational age, and sex in newborns. CONCLUSION: We successfully established a method for MS/MS-based measurement of Cr for newborn screening and determined normal reference values for full-term newborns.

Propionic acidemia (PA) is an inherited metabolic disease caused by low activity of propionyl coenzyme A (CoA) carboxylase (PCC), which metabolizes propionyl-CoA into methylmalonyl-CoA. Although many patients with PA have been identified by tandem mass spectrometry since the test was first included in neonatal mass screening in the 1990s, the disease severity varies. Thus, determining the specific level of PCC activity is considered to be helpful to grasp the severity of PA. We developed a new PCC assay method by the determination of methylmalonyl-CoA, which is formed by an enzyme reaction using peripheral lymphocytes, based on ultra high-performance liquid chromatography tandem mass spectrometry (UPLC MS/MS). With methylmalonyl-CoA concentrations of 0.05, 0.5, and 5 p,mol/L, the intra-assay coefficients of variation (CVs) were 8.2%, 8.7%, and 5.1%, respectively, and the inter-assay CVs were 13.6%, 10.5%, and 5.9%, respectively. The PCC activities of 20 healthy individuals and 6 PA patients were investigated with this assay. Methylmalonyl-CoA was not detected in one PA patient with a severe form of the disease, but the remaining PA patients with mild disease showed residual activities (3.3-7.8%). These results demonstrate that determination of PCC activity with this assay would be useful to distinguish between mild and severe cases of PA to help choose an appropriate treatment plan. (C) 2017 Elsevier B.V. All rights reserved.

MSUD is an autosomal recessive condition characterized by a deficiency in the enzyme, BCKDH, which catalyzes the breakdown of BCAAs. If left untreated, MSUD can result in mental retardation, central nervous system disorders, and even death. Most patients with MSUD are treated with a restricted protein diet and milk from which BCAAs have been removed. LT has been shown effective in patients with MSUD. This report describes the case of a 15-month-old boy who received a liver graft from his mother. Transplantation was successful, and the patient was then able to ingest a normal diet. Despite episodes of acute rejection, chylous ascites, and high fever (40 degrees C), he has shown no evidence of MSUD recurrence. These findings indicate that patients with MSUD can be successfully treated by LDLT, even when the donor is a heterozygous carrier of a mutated BCKDH gene.

Newborns are routinely screened for organic acidemias by acylcarnitine analysis. We previously reported the partial catalytic methylesterification of dicarboxylic acylcarnitines by benzenesulfonic acid moiety in the solid extraction cartridge during extraction from serum. Since the diagnosis of organic acidemias by tandem mass spectrometry is affected by the higher molecular weight of these derivatized acylcarnitines, we investigated the methylesterification conditions. The kinetic constants for the methylesterification of carboxyl groups on the acyl and carnitine sides of carnitine were 2.5 and 0.24 h (1), respectively. The physical basis underlying this difference in methylesterification rates was clarified theoretically, illustrating that methylesterification during extraction proceeds easily and must be prevented. (C) 2015 Elsevier Ltd. All rights reserved.

Dihydropyrimidinase (DHP) deficiency is an autosomal recessive disease caused by mutations in the DPYS gene. Patients present with highly elevated levels of dihydrouracil and dihydrothymine in their urine, blood and cerebrospinal fluid. The analysis of the effect of mutations in DPYS on pre-mRNA splicing is hampered by the fact that DHP is primarily expressed in liver and kidney cells. The minigene approach can detect mRNA splicing aberrations using cells that do not express the endogenous mRNA. We have used a minigene-based approach to analyze the effects of a presumptive pre-mRNA splicing mutation in two newly identified Chinese pediatric patients with DHP deficiency. Mutation analysis of DPYS showed that both patients were compound heterozygous for a novel intronic mutation c.1443+5G>A in intron 8 and a previously described missense mutation c.1001A>G (p.Q334R) in exon 6. Wild-type and the mutated minigene constructs, containing exons 7, 8 and 9 of DPYS, yielded different splicing products after expression in HEK293 cells. The c.1443+5G>A mutation resulted in altered pre-mRNA splicing of the DPYS minigene construct with full skipping of exon 8. Analysis of the DHP crystal structure showed that the deletion of exon 8 severely affects folding, stability and homooligomerization of the enzyme as well as disruption of the catalytic site. Thus, the analysis suggests that the c.1443+5G> A mutation results in aberrant splicing of the pre-mRNA encoding DHP, underlying the DHP deficiency in two unrelated Chinese patients.

Fructose-1,6-bisphosphatase (FBPase), an enzyme involved in gluconeogenesis, catalyzes the hydrolysis of fructose-1,6-bisphosphate to fructose-6-phosphate and inorganic phosphate. FBPase deficiency is an autosonnal recessive inherited disorder, characterized by episodic attacks of hypoglycemia, ketosis, and lactic acidosis during fasting. In general, urinary organic acid analysis using gas chromatography-mass spectrometry (GC/MS) is very useful for the diagnosis of FBPase deficiency, because the appearance of glycerol or glycerol-3-phosphate in the urine is characteristic of this disease. Here, we report a case of FBPase deficiency in a girl with a history of several severe lactic acidosis events, both as a neonate and after the age of 12 months. The patient was identified as a compound heterozygote with two mutations in the FBPase 1 gene: c.841G>A (p.G1u281Lys) and c.960_961insG (p.Ser321fs). The c.841G>A is a newly identified pathogenic mutation. An abnormal level of glycerol-3-phosphate was not detected in the conventional urinary organic acid analysis using GC/MS after solvent extraction. This method, which is a widely used diagnostic standard, could not detect increased levels of glycerol or glycerol-3-phosphate in the patient's urine, which was sampled during the episode. However, glycerol and glycerol-3-phosphate were detected in the same sample, when it was analyzed using GC/MS with the urease pretreatment non-extraction method. Patients with FBPase deficiency have good glycemic control after correct treatment. Therefore, accurate and early diagnosis is essential for a good prognosis. Accordingly, when a patient presents with hypoglycemia and lactic acidosis, it is important to select the appropriate method of urinalysis for organic acids by GC/MS.

Methylmalonic acidemia (MMA) is an inherited metabolic disease. In this condition, metabolism from methylmalonyl coenzyme A (CoA) to succinyl-CoA is inhibited because of either low methylmalonyl-CoA mutase (MCM) activity or adenosylcobalamin deficiency owing to altered vitamin B-12 metabolism. A high-precision assay for detecting MCM activity would facilitate not only MMA diagnosis but also the ability to determine the severity of MMA. We developed an MCM assay method based on ultrahigh-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) that involves the determination of succinyl-CoA, which is formed in an enzyme reaction, using peripheral lymphocytes. Using 0.05, 0.5, and 5 mu mol/L succinyl-CoA, the intra-assay coefficient of variation (CV) was less than 5.2 % and the inter-assay CV was less than 8.7 %. The MCM activities of five healthy individuals and four patients were investigated with this assay. The MCM activities of the patients were very low in relation to those of healthy individuals. Together, these results show that the UPLC-MS/MS method is useful for a detailed MCM activity assay.

Herein we report on three siblings with Leigh syndrome (LS) harboring a homoplasmic m. 3697G > A mutation (G131S) in the MT-ND1 gene. The siblings' phenotypically normal mother had the same, albeit heteroplasmic, mutation. Complex I deficiency (8% of average control values) was demonstrated in a biceps brachii muscle from one of the patients. Heteroplasmic m. 3697G > A has been reported in patients with Leber's hereditary optic neuropathy, mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes and Stuve-Wiedemann syndrome. Because all three patients in this series carried m. 3697G > A in a homoplasmic manner and had LS, we suggest that homoplasmy of m. 3697G > A may cause the LS phenotype.

Glycogen storage disease type Ib (GSDIb) is caused by a deficiency in the glucose-6-phosphate transporter (G6PT), which leads to neutrophil dysfunction. However, the underlying causes of these dysfunctions and their relationship with glucose homeostasis are unclear. Induced pluripotent stem cells (iPSCs) hold a great promise for advances in developmental biology, cell-based therapy and modeling of human disease. Here, we examined the use of iPSCs as a model for GSDIb. In this study, one 2-year-old patient was genetically screened and diagnosed with GSDIb. We established iPSCs and differentiated these cells into hepatocytes and neutrophils, which comprise the main pathological components of GSDIb. Cells that differentiated into hepatocytes exhibited characteristic albumin secretion and indocyanine green uptake. Moreover, iPSC-derived cells generated from patients with GSDIb metabolic abnormalities recapitulated key pathological features of the diseases affecting the patients from whom they were derived, such as glycogen, lactate, pyruvate and lipid accumulation. Cells that were differentiated into neutrophils also showed the GSDIb pathology. In addition to the expression of neutrophil markers, we showed increased superoxide anion production, increased annexin V binding and activation of caspase-3 and caspase-9, consistent with the GSDIb patient's neutrophils. These results indicate valuable tools for the analysis of this pathology and the development of future treatments.

To describe the case of a patient who had been receiving abatacept, a T-cell costimulatory molecule blocker for rheumatoid arthritis, and developed an acute encephalomyelitis associated with reactivation of the varicella zoster virus (VZV), Epstein-Barr virus (EBV) and cytomegalovirus (CMV). A 61-year-old woman receiving abatacept therapy for rheumatoid arthritis developed a disturbance of consciousness. MRI indicated multifocal parenchymal lesions in the brainstem, supratentorial areas and cervical spinal cord. Although steroid therapy significantly improved the neurological symptoms and MRI findings, the patient died of sepsis aggravated by coinfection with a fungal infection. Retrospectively, a PCR assay revealed continued systemic reactivation of VZV, EBV and CMV. Acute encephalomyelitis may be associated with VZV EBV and CMV reactivation during abatacept therapy. Clinicians must be aware of the possibility of acute encephalomyelitis associated with herpes virus reactivation during abatacept therapy for rheumatoid arthritis. Copyright 2013 BMJ Publishing Group. All rights reserved.

beta-Ureidopropionase is the third enzyme of the pyrimidine degradation pathway and catalyzes the conversion of N-carbamyl-beta-alanine and N-carbamyl-beta-aminoisobutyric acid to beta-alanine and beta-aminoisobutyric acid, ammonia and CO2. To date, only five genetically confirmed patients with a complete beta-ureidopropionase deficiency have been reported. Here, we report on the clinical, biochemical and molecular findings of 11 newly identified beta-ureidopropionase deficient patients as well as the analysis of the mutations in a three-dimensional framework. Patients presented mainly with neurological abnormalities (intellectual disabilities, seizures, abnormal tonus regulation, microcephaly, and malformations on neuro-imaging) and markedly elevated levels of N-carbamyl-beta-alanine and N-carbamyl-beta-aminoisobutyric acid in urine and plasma. Analysis of UPB1, encoding beta-ureidopropionase, showed 6 novel missense mutations and one novel splice-site mutation. Heterologous expression of the 6 mutant enzymes in Escherichia coli showed that all mutations yielded mutant beta-ureidopropionase proteins with significantly decreased activity. Analysis of a homology model of human beta-ureidopropionase generated using the crystal structure of the enzyme from Drosophila melanogaster indicated that the point mutations p.G235R, p.R236W and p.S264R lead to amino acid exchanges in the active site and therefore affect substrate binding and catalysis. The mutations L13S, R326Q and T359M resulted most likely in folding defects and oligomer assembly impairment. Two mutations were identified in several unrelated beta-ureidopropionase patients, indicating that beta-ureidopropionase deficiency may be more common than anticipated. (c) 2012 Elsevier B.V. All rights reserved.

Few studies have looked at optimal or acceptable serum phenylalanine levels in later life in patients with phenylketonuria (PKU). This study examined the oxidative stress status of adolescents and adults with PKU. Forty PKU patients aged over fifteen years were enrolled, and were compared with thirty age-matched controls. Oxidative stress markers, anti-oxidant enzyme activities in erythrocytes, and blood anti-oxidant levels were examined. Nitric oxide (NO) production was also examined as a measure of oxidative stress. Plasma thiobarbituric acid reactive species and serum malondialdehyde-modified LDL levels were significantly higher in PKU patients than control subjects, and correlated significantly with serum phenylalanine level (P<0.01). Plasma total anti-oxidant reactivity levels were significantly lower in the patient group, and correlated negatively with phenylalanine level (P<0.001). Erythrocyte superoxide dismutase and catalase activities were higher and correlated significantly with phenylalanine level (P<0.01). Glutathione peroxidase activity was lower and correlated negatively with phenylalanine level (P<0.001). The oxidative stress score calculated from these six parameters was significantly higher in patients with serum phenylalanine of 700-800 mu mol/l. Plasma anti-oxidant substances, beta-carotene, and coenzyme Q(10) were also lower (P<0.001), although the decreases did not correlate significantly with the phenylalanine level. Serum nitrite/nitrate levels, as stable NO products, were higher together with low serum asymmetric dimethylarginine, as an endogenous NO inhibitor. Oxidative stress status is closely linked with serum phenylalanine levels. Phenylalanine level in should be maintained PKU below 700-800 mu mol/l even in adult patients. (C) 2011 Elsevier Inc. All rights reserved.

Some oral antibiotics contain a pivalate ester, because molecules with a pivalate entity show enhanced absorption in the intestine. Upon absorption, such a "prodrug" is broken down into the active form of a given antibiotic and a pivalate molecule, the latter of which is converted to pivaloylcarnitine through pivaloyl-CoA and is excreted in the urine. Long-term administration of drugs containing pivalate decreases blood carnitine level and causes defects in fatty acid oxidation. Here, we used liquid chromatography tandem mass spectrometry to measure carnitine and pivaloylcarnitine levels in two patients (Patient 1: 16-month-old boy and Patient 2: 18-month-old boy) with secondary carnitine deficiency and hypoglycemic convulsions caused by pivalate-containing antibiotics. Both patients were administered excessive doses of pivalate for the long-term treatment of recurrent infection, and consequently, the serum free carnitine levels were very low (Patient 1: 1.0 mu mol/L and Patient 2: 0.4 mu mol/L), compared to normal range of 33.3-43.0 mu mol/L, while the serum pivaloylcarnitine levels were elevated from normally undetectable level (Patient 1: 3.7 mu mol/L and Patient 2: 1.6 mu mol/L). Patient 1 recovered immediately after the glucose infusion, whereas Patient 2 remained symptomatic even after blood glucose level was normalized and fully recovered after carnitine supplementation. The urine pivaloylcarnitine level in Patient 2 was increased during carnitine supplementation (from 821.4 to 12,200 mu mol/g creatinine) even after discontinuing the antibiotics, indicating that a considerable amount of pivalate was accumulated in the tissues. In conclusion, long-term administration of pivalate-containing antibiotics should be avoided particularly in children.

Vigabatrin (VGB) is one of the most effective anti-epileptic drugs for tonic spasms, those accompanied with tuberous sclerosis complex (TSC), but is not available in Japan. We treated 7 patients with West syndrome (WS) and TSC with VGB. In these patients, VGB treatment was started at 5-65 months of age. Six patients (86%) had complete cessation of tonic spasms. Of these, 3 patients had complete cessation within 24 hours after VGB treatment. The mean initial dosage of VGB was 36.2 mg·kg-1·day-1, and the mean maintenance dosage was 38.4 mg·kg-1·day -1. At the beginning of VGB treatment, 3 patients had hypsarrhythmia, 2 had focal discharge with generahzation, and 2 had only focal discharge on electroencephalography. Hypsarrhythmia disappeared within 4-8 weeks after VGB treatment. Behavioral problems and sleep difficulty were observed in 6 patients. Visual field examination revealed no abnormalities in 3 patients. We hope that patients with WS and TSC can be treated with VGB as soon as possible in Japan.

Free radicals play an important role in the inflammatory process of sepsis. We hypothesized that edaravone, a novel free radical scavenger, can suppress pathophysiological events and prolong survival in a neonatal sepsis cecal ligation and perforation (CLP) model. Of 32 3-day-old anesthetized and mechanically ventilated piglets, 11 received CLP only, 10 received CLP and edaravone treatment starting 30 min after CLP, and 11 constituted a sham (control) group. Mean arterial pressure (MAP), heart rate, cardiac output, arterial blood gas, serum total hydroperoxide, nitrite and nitrate, TNF-alpha, and high-mobility group box 1 (HMGB1) were measured before CLP and at 1, 3, and 6 h after CLP. Compared with the CLP group, the edaravone group showed higher MAP at 6 h, lower heart rate at 1 and 3 h, lower total hydroperoxide at 1 h, lower nitrite and nitrate at 3 and 6 h, and higher (although not significantly so) mean cardiac output at 1, 3, and 6 h. TNF-alpha elevation was delayed from 1 h in the CLP group to 3 h in the edaravone group. In the edaravone group, HMGB1 did not change significantly at any time, whereas in the CLP group, it increased at 6 h. Survival times were longer in the edaravone group than in the CLP group (15.4 +/- 1.4 vs. 10.2 +/- 1 h; P < 0.005). In addition, each of the serial dilutions of edaravone had a higher biological antioxidant potential than tempol does. In conclusion, edaravone suppressed free radicals, delayed the TNF-alpha surge, and prevented HMGB1 elevation, thereby maintaining MAP and prolonging survival time in a neonatal sepsis CLP model.

Holocarboxylase synthetase (HCS) deficiency is an inborn error of biotin metabolism, leading to a Multiple carboxylases deficiency. As the affected fetus sometimes presents with enlargement of the cerebral ventricles and intrauterine growth retardation (IUGR), prenatal administration of biotin has been attempted in some pregnancies. We present herein the case of a Japanese neonate with HCS deficiency who received maternal administration of biotin (10 mg/day) from 33 weeks' gestation. After biotin administration, the fetal body weight increased and gestation was continued to full term. However, lactic acidemia and metabolic acidosis were observed after birth. To evaluate the effects of prenatal therapy, we collected serum samples and measured the acylcarnitine profiles using high-performance liquid chromatography electrospray ionization tandem mass spectrometry. At birth, levels of propionylcarnitine and 3-hydroxyisovalerylcarnitine had already increased. At 2 It after birth, these levels of acylcarnitines were further increased. At 3.5 h after the start of biotin, these chemical findings were slightly improved. In conclusion, we considered that prenatal biotin therapy at 10 mg/day may have been inadequate to avoid neonatal acidotic crisis in this case. (C) 2008 Elsevier B.V. All rights reserved.

Oxidative stress plays an important role in cystic periventricular leukomalacia (PVL). We performed a case-control study of preterm infants delivered at <35 weeks of gestation between January 2003 and December 2006. Patients were stratified into three groups, according to age at which cysts were initially identified: <= 10 days old (early cystic PVL; n = 10), >10 days old (late cystic PVL; n = 12); and no cystic PVL (controls; n = 22). Serum total hydroperoxide, biological antioxidant potential and oxidative stress index (calculated Lis total hydroperoxide/biological antioxidant potential) were measured within 3 h after birth. Frequencies of preterm rupture of membrane and chorioamnionitis were significant higher in early cystic PVL than in late cystic PVL or controls. Duration of oxygen treatment and mechanical ventilation and frequency of apnea were significantly higher in late cystic PVL than in controls or early cystic PVL. Serum total hydroperoxide levels and oxidative stress index were significantly higher in early cystic PVL than in late cystic PVL or controls (p < 0.05, respectively). Postnatal duration until cyst identification displayed a significant negative correlation with oxidative stress index and total hydroperoxide level (r = -0.497, p < 0.05; r = -0.50, p < 0.05, respectively). These findings suggest that early onset of cystic PVL might be due to either antenatal or intrapartum factors, but late onset might be due to postnatal factors. In the pathophysiology and therapy of cystic PVL, oxidative stress and onset timing appear crucial. This is the first study to reveal that neonates experiencing much more oxidative stress at birth show earlier onset of cystic PVL. Crown copyright (C) 2008 Published by Elsevier B.V. All rights reserved.

Recently, the number of reports of encephalitis/encephalopathy associated with influenza virus has increased, In addition, the use of a non-steroidal anti-inflammatory drug, diclofenac sodium (DCF), is associated with a significant increase in the mortality rate of influenza-associated encephalopathy. Activated astrocytes are a source of nitric oxide (NO), which is largely produced by inducible NO synthase (iNOS) in response to proinflammatory cytokines. Therefore, we investigated whether DCF enhances nitric oxide production in astrocytes stimulated with proinflammatory cytokines. We stimulated cultured rat astrocytes with three cytokines, interleukin-1 beta, tumor necrosis factor-alpha and interferon-gamma, and then treated the astrocytes with DCF or acetaminophen (N-acetyl-p-aminophenol: APAP). iNOS and NO production in astrocyte Cultures were induced by proinflammatory cytokines. The addition of DCF augmented NO production, but the addition of APAP did not. NF-kappa B inhibitors SN50 and MG132 inhibited iNOS gene expression in cytokine-stimulated astrocytes with or without DCF. Similarly, NF-kappa B p65 Stealth small interfering RNA suppressed iNOS gene expression in cytokine-stimulated astrocytes with or without DCF. LDH activity and DAPI staining showed that DCF induces cell damage in cytokine-stimulated astrocytes. An iNOS inhibitor, L-NMMA, inhibited the cytokine- and DCF-induced cell damage. In conclusion, this study demonstrates that iNOS and NO are induced in astrocyte cultures by proinflammatory cytokines. Addition of DCF further augments NO production. This effect is mediated via NF-kappa B signaling and leads to cell damage. The enhancement of DCF on NO production may explain the significant increase in the mortality rate of influenza-associated encephalopathy in patients treated with DCF. (c) 2009 Elsevier Inc. All rights reserved.

Congenital disorders of metabolism show a wide spectrum of symptoms as a consequence of impairment of a certain metabolic pathway by mutated enzymes resulting in abnormal accumulation of enzyme substrates, deficiency of expected products, and abnormal burden to collateral metabolic pathways, etc. However, in some occasions, depending on which pathway LIP to what degree of disturbance, it call be asymptomatic until a certain kind of burden is placed on to the patient. Enzyme deficiency involved in pyrimidine degradation, Such as Dihydropyrimidine dehydrogenase (DPD) and Dihydropyrimidinase (DHP), has been reported with convulsion or autism as symptoms, but many asymptomatic cases are also reported. However, when the patients are treated with 5-fluorouracil, a pyrimidine analogue anticancer drug, lethal side-effects call be seen even in asymptomatic patients. Some oral cephem antibiotics have pivalic acid side chain to increase absorption rate at intestine. These antibiotics degrade into active antibiotics and pivalic acid at the intestinal wall. This pivalic acid is carnitine-conjugated and excreted into Urine. Carnitine acts as a carrier of long chain fatty acid to mitochondria and to beta-oxidation, thus all important Molecule for energy production by beta-oxidation and maintenance of mitochondrial function. Because of this, long term administration Of Such antibiotics Could induce depletion of carnitine from the body and lead to low ketotic hypoglycemia, Convulsion and Consciousness disturbance. This paper reports some possible serious side effects closely linked to drug metabolism.

Background/Aim: The R450H mutation of the TSH receptor (TSHR) gene has been frequently observed in Japanese patients with resistance to TSH. The purpose of this study was to clarify the phenotype of patients with a homozygous R450H mutation of the TSHR gene; the mutant receptor has previously demonstrated moderately impaired function in vitro. Methods: We performed a clinical investigation of 5 Japanese patients who had hyperthyrotropinemia as neonates, in whom a homozygous R450H mutation of the TSHR gene had been demonstrated by genetic sequencing analysis. Results: The thyroid hormone levels of the patients were normal in early infancy, although their serum levels of TSH were mildly elevated. After supplemental treatment with levothyroxine sodium (L-T4) was started, we had to increase the dose to maintain the level of TSH within the normal range in all patients. Thyroid dysfunction became obvious in one patient at reexamination during adolescence when L-T4 treatment was stopped for 1 month. Four patients were examined for intelligence quotient and their scores were normal. Conclusions: Thyroid hormone replacement therapy should be considered based on biological data in patients with hyperthyrotropinemia who have a homozygous R450H mutation of the TSHR gene even if they do not exhibit obvious hypothyroidism in infancy. Copyright (C) 2009 S. Karger AG, Basel

Periventricular leukomalacia is a major neuropathology in preterm infants associated with adverse motor and cognitive outcome. The cerebral blood flow volume of the internal carotid artery and the vertebral artery was measured by ultrasonography at the neck in 36 low-birth-weight infants with gestational age of 25-34 weeks in order to investigate the pathophysiology of cerebral white-matter injury: 30 infants, normal and 6 infants, diagnosed as PVL. The mean blood flow velocity and diameter of each vessel were measured at postnatal days from day 0 to day 70. The intravascular flow volume was determined by calculating the mean blood flow velocity and the cross-sectional area. The mean blood pressures were recorded and PaCO2 was determined. The total blood flow volume was significantly lower in infants with PVL than in normal infants on days 0, 1, 21, 28, 35, 42, and 63. The mean blood pressure was significantly lower in infants with PVL than in normal infants on days 7, 14, 21 28, and 42. We suggest that the total cerebral blood supply is decreased in cases of PVL in the few days after birth and front day 21 to day 42. The results of the present study suggest that a dip in the blood flow volume in the few days after birth might result in subsequent PVL. (D 2008 Elsevier B.V. All rights reserved.

Females with salt-wasting (SW) 21-hydroxylase deficiency (21OHD) may present with mild external genitalia virilization, despite complete or almost complete enzyme inactivation. We therefore analyzed genotype/phenotype correlation in 13 Japanese female patients with SW 21OHD. Criteria for classification into the SW phenotype included history of a salt-losing crisis with documented hyponatremia, hyperkalemia, and markedly elevated plasma renin activity. Urologists and pediatricians determined the Prader genital stage and classified the location of the vaginal entrance into the common urogenital sinus as low, moderate, or high. CYP21A2 gene, coding for 21-hydroxylase, was analyzed with Southern blotting and direct sequencing. Genotypes were categorized into four mutation Groups, based on the degree of enzymatic activity (N, complete enzyme inactivation; groups, A, < 2%, B, 3-7%, and C > 30%). Basal androgen levels were available from only six out of thirteen patients, so we could not relate androgen levels with the severity of external genitalia virilization. We compared the degree of external genitalia virilization with genotype. The severity of external genitalia virilization varied from Prader stage 1 to 4. One patient who presented with Prader 1 had a genotype consistent with Group B. In addition, discordance between Prader classification and the location of the vaginal entrance was noted, one patient classified as Prader 4 showed low vaginal entrance, while another patient classified as Prader 3 showed high vaginal entrance. The degree of the impairment of 21-hydroxylase activity does not correlate with the severity of virilization of the external genitalia in female patients with the SW type of 21OHD.

Due to its increased concentration in blood, 3-hydroxyisovalerylcarnitine (C5OH-I) is an important indicator for the diagnosis of organic acidemias in newborns. However, C5OH-I has not been used as a standard in tandem mass spectrometric (MS/MS) assays because its isolation is difficult. We developed a new synthesis of C5OH-I and investigated its behavior by MS/MS. A method using the multiple reaction monitoring (MRM) mode of MS/MS with HPLC was developed which provides high accuracy, precision and reproducibility. Acylcarnitine profiles in the serum and urine of a patient with multiple carboxylase deficiency (MCD) showed increased levels compared to a healthy patient. (C) 2007 Elsevier B.V. All rights reserved.

During neonatal respiratory support, maintaining optimal humidity minimizes the risk of airway occlusion and chronic lung disease. With neonatal respiratory support using a heated humidifier,condensation following decreases in temperature within the unheated part of the inspiratory circuit represents a serious problem, due to the resulting drop in absolute humidity. Several reports describing the temperature/humidity gradient in the unheated inspiratory limb have excluded the endotracheal tube (ETT). The present study investigated the extent to which the temperature gradient in the ETT affects breathing gas conditioning in premature infants, who display tiny minute volumes. By measuring temperature/dew point at various sites along the inspiratory circuit, including inside the ETT, we evaluated the effects of temperature change in the ETT using an in vitro model of a micropremie on mechanical respirator care in an incubator We confirmed significant moisture loss (absolute humidity loss; 7.5-10.1 mg/L) with decreasing gas temperature in the ETT external to the body, with subsequent drying of the gas (relative humidity drop, 10.7-22.3%) as temperature increased in the ETT inside the body. The present results suggest that temperature decreases in the ETT represent an important issue in the respiratory care of very premature infants.

Hypoxic ischemic brain can result in cerebral palsy, mental retardation, and learning disabilities in surviving children. The purpose of this study was to elucidate the cerebral blood flow volume in infants complicated with brain damage after the birth. Nine term infants with hypoxic ischemic encephalopathy and 41 normal term infants were studied. Four infants with HIE suffered from CP or mental retardation, and the other five infants exhibited normal neurodevelopment. The mean blood flow velocity and diameter of the internal carotid artery and the vertebral artery were measured for 28 days. The intravascular flow volume was determined by calculating the flow velocity and the cross-sectional area. The ejection fraction and cardiac output were obtained, and the mean blood pressures were recorded. The summed flow volumes in both the ICA and VA, and the total CBFV increased after the birth in both the normal infants and the infants diagnosed with HIE with no disability complications. The total blood flow volume was significantly lower in infants with HIE and CP than in normal infants on days 0, 2, 5, 7 10, 21, and 28, and significantly lower in infants with HIE and CP than in normal infants with HIE on days 2, 4, and 7. The ejection fraction was significantly lower in infants with HIE than in normal infants only on day 0. Our results suggest that the total cerebral blood supply is decreased in infants with HIE in those complicated with brain damage. (c) 2008 Published by Elsevier B.V.

Medium-chain acyl-CoA dehydrogenase deficiency (MCADD) is rare among Asian individuals, and the clinical course and biochemical findings remain unclear. We report herein a 3-year-old Japanese girl with MCADD. The diagnosis was suggested by acylcarnitine profiles and confirmed by enzyme activity and genetic analysis after clinical presentation. Our described method with high-performance liquid chromatography/tandem mass spectrometry allows quantification of levels of n-octanoylcarnitine (C8-N) and other isomers (e.g. valproylcarnitine). We examined the patient's acylcarnitine profiles in serum and urine samples during carnitine loading and 14-hr fasting tests with/without carnitine supplementation. Under hypocarnitinemia, serum level of C8-N was 0. 1 6,mu mol/l and C8-N/ decanoylcarnitine (C10) ratio was 1.8, which did not correspond to the diagnostic criteria for MCADD. However, intravenous carnitine loading test (100 mg/kg/day for 3 days and 50 mg/kg/day for I day) led to increased serum C8-N levels and urinary excretion was obvious, strongly suggesting MCADD. In the fasting test with carnitine supplementation, marked production of acylcarnitines (C8-N > C2 >> C6 > C10) was found, compared to the fasting test without carnitine supplementation. These results indicate that carnitine supplementation may be useful for detoxification of accumulated acylcarnitines even in an asymptomatic state. Moreover, the one-point examination for serum C8-N level and/or C8-N/C10 ratio may make the diagnosis of MCADD difficult, particularly in the presence of significant hypocarnitinemia. To avoid this pitfall, attention should be given to serum levels of free carnitine, and carnitine loading may be demanded in hypocarnitinemia.

The single-strand conformation polymorphism (SSCP) procedure has been applied in routine testing for hereditary diseases. Temperature, running buffer, gel composition, and fragment length can influence its sensitivity. Mutation detection in the clinical setting depends on the development of automated technology, especially for large genes, such as the dihydropyrimidine dehydrogenase (DPYD) gene, which codes the initial, rate-limiting enzyme in the catabolism of 5-fluorouracil (5FU). The authors have optimized the condition of SSCP with an automated system (GenePhor system, GE Healthcare UK Ltd.) to screen genetic polymorphisms in the DPYD gene. The efficiency of the method was evaluated using 21 positive controls (DNA samples with polymorphisms in the DPYD gene, previously characterized) and DNA samples from 35 Japanese. Results showed that the use of three different running buffers (pH 7.4, 8.3, and 9.0) in combination with other optimized conditions (10% polyacrylamide gel, 60-90 min at constant 900 V at 5 degrees C) resulted in a high polymorphism detection rate (95.3%), which was considered appropriate for routine screening. Therefore, this strategy could be useful for pharmacogenetic studies on 5FU.

An 18-month-old boy was treated with an antibiotic containing pivalic acid for 6 months for intractable otitis media and then developed repeated convulsions and loss of consciousness. Laboratory data showed hypoglycemia and hypocarnitinemia. Intravenous administration of glucose was ineffective against the seizures and loss of consciousness. However, the patient regained consciousness and recovered soon after intravenous infusion of carnitine. To our knowledge, intravenous carnitine administration that contributed to marked improvements in neurologic deficit caused by administration of an antibiotic containing pivalic acid has not been reported previously. These findings indicate that long- term use of such antibiotics should be avoided.

Newborn males are more sensitive to brain injury than newborn females are. The aim of the present study was to find an explanation for this. We used the neuron-specific enolase (NSE) levels in the cerebrospinal fluid (CSF) for the classification of 32 newborns (19 males and 13 females) on their fifth postnatal day. The NSE levels were higher than normal (8.4 +/- 1.6 ng/mL) in 10 newborn males and 6 females and were, respectively, considered asphyxiated male and female groups. The remaining newborns, 9 males and 7 females, had normal CSF levels of NSE and were considered normal newborn male and female groups. The CSF samples were measured for 12 cytokines, using a cytokine array kit, and for total hydroperoxide and biological antioxidant potentials (BAPs), using the free radical analytic system. Among the 12 cytokines measured, only interleukin 8 (IL-8) was properly detected. The CSF levels of IL-8 were higher in the asphyxiated newborn females than in the other three groups. The mean CSF levels of BAPs in the asphyxiated newborn females were higher compared with the other three groups, but significance was detected only in comparison with the BAP levels in the CSF samples of the normal newborn males. There were no differences in total hydroperoxide levels among the groups. There are sex-related differences in the CSF levels of IL-8 and antioxidants in asphyxiated newborns, with higher levels in newborn females; this might contribute in the sexual dimorphism regarding the fact that females have better protection from brain injury than the males.

Tandem mass spectrometry (MS/MS) has become a prominent method for screening newborns for diseases such as organic acidemia and fatty acid oxidation defects, although current methods cannot separate acylcarnitine isomers. Accurate determination of dicarboxylic acylcarnitines such as methylmalonylcarnitine and glutarylcarnitine has not been carried out, because obtaining standards of these acylcarnitines is difficult. We attempted the individual determinations of acylcarnitines with isomers and dicarboxylic acylcarnitines by applying high-performance liquid chromatography (HPLC). Chromatographic separation was performed by gradient elution using a mixture of 0.08% aqueous ion-pairing agent and acetonitrile as the mobile phase. Mass transitions of m/z 161.8 -> 84.8 for carnitine and m/z 164.8 -> 84.8 for deuterated carnitine were monitored in positive ion electrospray ionization mode. One carnitine and 16 acylcarnitines were quantified. The limit of quantitation (LOQ) was 0.1 mu mol/L for methylmalonylcarnitine and 0.05 mu mol/L for the other acylcarnitines. Intra-day and inter-day coefficients of variance (CVs) were < 8.3% and < 8.8%, respectively, for all acylcarnitines in serum, and both were < 9.2% in urine. Mean recoveries were > 90% for all acylcarnitines. Human samples were quantified by this method. After addition of deuterated acylcarnitines as internal standards, acylcarnitines in serum or urine were extracted using a solid-phase extraction cartridge. In healthy adult individuals, isobutyryl-, 2-methylbutyryl- and isovalerylcarnitine were detected in serum and urine. Dicarboxylic acylcarnitines were detected in urine. High concentrations of methylmalonylcarnitine and propionylcarnitine were found in both the serum and the urine of a patient with methylmalonic acidemia. The described HPLC/MS/MS method could separate most acylcarnitine isomers and quantify them, potentially allowing detailed diagnoses and follow-up treatment for those diseases. Copyright (c) 2007 John Wiley & Sons, Ltd.

Background: Respiratory syncytial virus (RSV) infection causes asthma-like symptoms in infants and young children. Although an increase in several mediators in the airway during RSV infection has been reported, the mechanisms involved in airway inflammation are not fully understood. The aim of this study was to investigate the immunological deviation associated with airway inflammation by measuring cytokine and chemokine levels in the airway during RSV infection. Methods: One hundred and ten children under 3 years of age with respiratory symptoms were enrolled in this study from November 2004 through January 2005. Nasopharyngeal secretions (NPAs) were gently aspirated and analyzed with RSV antigen, thereafter the concentrations of IL-4, IL-10, IFN-γ, and RANTES were measured using an ELISA kit. We also investigated the prognosis of each child after 1 year by reference to clinical records or by interviews and re-evaluated the cytokine and chemokine levels. Results: Of the subjects, 70 children were RSV positive and 40 were negative. Only 4 children were given a diagnosis of asthma by the pediatrician when NPAs were collected. The levels of IL-4, IL-10, and RANTES were significantly higher in the RSV-positive patients than RSV-negative patients with P values at 0.0362, 0.0007, and 0.0047, respectively. In contrast, there was no significant difference in the levels of IFN-γ. Furthermore, there was a significant positive correlation between IL-10 and RANTES. Conclusions: The increased production of IL-4, IL-10, and RANTES in the airway may play an important role in the pathophysiological mechanisms of RSV infection. ©2007 Japanese Society of Allergology.

Oxidant/antioxidant imbalance plays an important role in septic shock. The present study examined changes in circulating oxidative components in a neonatal sepsis model. Subjects were 14 newborn mixed-strain piglets randomly divided into two groups: a cecal ligation and perforation (CLP) model (n = 7) and sham (n = 7). Blood samples for total hydroperoxide (TH), biological antioxidant potential (BAP), tumor necrosis factor (TNF) alpha, interleukin (IL)-6, and IL-10 were collected pre-CLP and at 1, 3, and 6 h post-CLP. TH and BAP levels at 1 h post-CLP were significantly higher in the CLP group than in the sham group. In the CLP group, TH decreased gradually and reached baseline levels by 6 h post-CLP, while BAP remained elevated. Linear correlations were identified between serum TH and BAP at 1 h post-CLP, serum TH and TNF-alpha at 1 h post-CLP, and BAP and IL-6 at 6 h post-CLP. Changes in and correlations between circulating oxidative and inflammatory state components in a neonatal sepsis model were clarified. This is the first study to reveal that the presence of oxidant/antioxidant imbalance in sepsis and septic shock changes during the disease course.

CYP2C19 is a clinically important enzyme involved in the metabolism of therapeutic drugs such as (S)-mephenytoin, omeprazole, proguanil, and diazepam. Individuals can be characterized as either extensive metabolizers (EM) or poor metabolizers (PM) on the basis of CYP2C19 enzyme activity. The PM phenotype occurs in 2-5% of Caucasian populations, but at higher frequencies (18-23%) in Asians. CYP2C19*2 and CYP2C19*3, which are single-nucleotide polymorphisms of CYP2C19, are the main cause of PM phenotyping in homozygotes or compound heterozygotes. We report two novel mutations in the CYP2C19 gene identified by direct sequencing and subcloning procedures. One of these mutations was considered to be CYP2C19*3 by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP). This result suggests that mutations classed as CYP2C19*3 might include other mutations. Further studies are needed to clarify the relationship between these novel mutations and enzyme activity.

Adenosine is a physiologically active molecule produced locally in many sites of the body to regulate various cell functions. Measurement of levels of the factor in organs and biological fluids provides clues to its role and we reported an accurate quantitative high-performance liquid chromatography method for urinary adenosine requiring no preliminary sample preparation, other than filtration. Analyses were performed isocratically with a reversed-phase and a molecular exclusion columns connected by a column switch. Each sample was analyzed automatically in 35 min. Linearity could be verified up to 1,000 mu mol/L (r = 0.999) and recovery of adenosine was 94.6 - 98.0%. The coefficients of variation (CV) were established to be 0.56 - 1.32%, intra-assay, and 1.61 - 4.67%, inter-assay. Based on analyses of healthy individuals at different ages, we are here able to provide age-related values, infants (1.51 +/- 0.71 mu mol/mmol creatinine) and children (1.06 +/- 0.36 and 0.83 +/- 0.27 mu mol/mmol creatinine; aged I - 5 and 6 - 10 years), excreting significantly higher amounts of adenosine than adults (0.44 +/- 0.08 mu mol/mmol creatinine). We also measured urinary adenosine from patients suffering from metabolic disease or severe respiratory failure and found that unfavorable pathophysiologic conditions are associated with appreciable elevation of adenosine.

Inosine triphosphate pyrophosphohydrolase (ITPase) is an enzyme that catalyzes the conversion of inosine triphosphate (ITP) to inosine monophosphate and pyrophosphate. In Caucasian populations it is reported that the frequency of cases showing decreased ITPase activity is 5%. The structure of ITPA gene along with five single nucleotide polymorphisms has been reported in Caucasians. We examined ITPase activity and frequency of two polymorphisms (94C > A and IVS2 + 21A > C) in 100 Japanese individuals. Among these individuals, we observed that three cases with zero activity were homozygote for 94C > A, and were accompanied by abnormal accumulation of ITP in erythrocytes. The cases included in the low ITPase activity group were heterozygote for 94C > A polymorphism. The activity of the heterozygote cases was approximately 27% of the mean value of the wild type. The allele frequency of the 94C > A polymorphism was 0.155, which was 2.6 times higher than that of the Caucasians (0.06). The IVS2 + 21A > C was not detected in Japanese cases, although it occurred with a frequency of 0.130 in Caucasians. Furthermore, we identified a novel mutation IVS2 + 68T > G in intron 2 in the case with the lowest enzyme activity in the 94C > A wild type. Since the frequency of ITPA 94C > A polymorphism is higher in the Japanese population than that in Caucasians, it is more important to examine ITPA 94C > A polymorphism in the Japanese population to prevent thiopurine drug toxicity. Pretherapeutic screening of individuals for ITPA polymorphisms should be considered for safer and more tolerable treatment with thiopurine drugs. (c) 2005 Elsevier Inc. All rights reserved.