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Cancer treatment with a fluoropyrimidine (FP) is often accompanied by severe toxicity that may be dependent on the activity of catalytic enzymes encoded by the DPYD, DPYS, and UPB1 genes. Genotype‐guided dose individualization of FP therapy has been proposed in western countries, but our knowledge of the relevant genetic variants in East Asian populations is presently limited. To investigate the association between these genetic variations and FP‐related high toxicity in a Japanese population, we obtained blood samples from 301 patients who received this chemotherapy and sequenced the coding exons and flanking intron regions of their DPYD, DPYS, and UPB1 genes. In total, 24 single nucleotide variants (15 in DPYD, 7 in DPYS and 2 in UPB1) were identified including 3 novel variants in DPYD and 1 novel variant in DPYS. We did not find a significant association between FP‐related high toxicity and each of these individual variants, although a certain trend toward significance was observed for p.Arg181Trp and p.Gln334Arg in DPYS (P = .0813 and .087). When we focused on 7 DPYD rare variants (p.Ser199Asn, p.IIe245Phe, p.Thr305Lys, p.Glu386Ter, p.Ser556Arg, p.Ala571Asp, p.Trp621Cys) which have an allele frequency of less than 0.01% in the Japanese population and are predicted to be loss‐of‐function mutations by in silico analysis, the group of patients who were heterozygous carriers of at least one these rare variants showed a strong association with FP‐related high toxicity (P = .003). Although the availability of screening of these rare loss‐of‐function variants is still unknown, our data provide useful information that may help to alleviate FP‐related toxicity in Japanese patients with cancer., This is the first report to assess the clinical relevance of DPYD, DPYS, and UPB1 variants as predictors of severe FP‐associated toxicity in East Asians. This study shows rare DPYD variants which cause a loss‐of‐function in silico and a DPYS p.Gly334Arg polymorphism may be associated with severe FP‐related toxicity in Japanese patients with cancer. However, the common UPB1 pathogenic variant p.Arg326Gln in the Japanese population does not show a clear association with toxicity in heterozygous individuals.

Schwannomas occurring in the hepatoduodenal ligament are extremely rare, with only four cases reported. Here, we describe a case of a 30-mm schwannoma that originated in the hepatoduodenal ligament of a 38-year-old female found during a periodic medical check-up. Magnetic resonance imaging demonstrated a tumor in the hepatoduodenal ligament. Following an ultrasound-guided microbiopsy, histological examination showed solitary fibrous tumor or schwannomas in the liver or originating from the hepαtoduodenal ligament. The relationship between the tumor and associated organs was confirmed intraoperatively, and the tumor was removed safely in its entirety using indocyanine green. The postoperative histopathological examination revealed the presence of a schwannoma with typical characteristics. To our knowledge, this is the first case of hepatoduodenal ligament schwannoma treated by laparoscopic surgery using indocyanine green fluorescence imaging.

Arginase 1 (ARG1) deficiency is a rare urea cycle disorder (UCD), with an estimated frequency of 1 per 2,200,000 births in Japan. Patients with ARG1 deficiency develop symptoms in late infancy or pre-school age with progressive neurological manifestations and sometimes present with severe hepatic disease. We previously investigated the status of UCDs in Japan; however, only one patient was identified as having ARG1 deficiency. Therefore, we aimed to investigate the current status of patients with ARG1 deficiency in 2018–2021 because almost 10 years have passed since the previous study. We present the disease history, clinical outcome, and treatment of five surviving patients with ARG1 deficiency and discuss the features of ARG1 deficiency in Japan. We found that clinicians often face difficulty in diagnosing ARG1 deficiency at the early stage of onset because of interpatient variability in onset time and clinical manifestations. Blood L-arginine and guanidino compounds were considered to be the major factors causing adverse neurodevelopmental outcomes. Therefore, early detection and intervention of ARG1 deficiency is essential for improved neurodevelopmental outcomes. Liver transplantation has been considered an effective treatment option that can dramatically improve the quality of life of patients, prior to the neurological manifestation of symptoms caused by ARG1 deficiency.

There are few case reports of hepatocellular carcinoma (HCC) metastasis to the skeletal muscle. A 78-year-old man developed a mass in the right shoulder. Washout of contrast medium during contrast-enhanced ultrasonography (CEUS) in both the primary HCC and the metastatic site was detected. Several nodules were scattered throughout the liver on an autopsy. In addition, the moderately differentiated HCC had metastasized to the right teres major muscle. Rare muscular metastasis should be considered if a hepatic tumor is moderately or poorly differentiated HCC. Early washout during CEUS is consistent with a pathological diagnosis of moderately or poorly differentiated HCC.

Background Mitochondrial DNA depletion syndrome (MTDPS) is part of a group of mitochondrial diseases characterized by a reduction in mitochondrial DNA copy number. Most MTDPS is caused by mutations in genes that disrupt deoxyribonucleotide metabolism. Methods We performed the whole‐exome sequencing of a hepato‐encephalopathy patient with MTDPS and functional analyses to determine the clinical significance of the identified variant. Results Here, whole‐exome sequencing of a patient presenting with hepato‐encephalopathy and MTDPS identified a novel homozygous frameshift variant, c.13_29del (p.Trp6Profs*71) in MICOS13. MICOS13 (also known as QIL1, MIC13, or C19orf70) is a component of the MICOS complex, which plays crucial roles in the maintenance of cristae junctions at the mitochondrial inner membrane. We found loss of MICOS13 protein and fewer cristae structures in the mitochondria of fibroblasts derived from the patient. Stable expression of a wild‐type MICOS13 cDNA in the patients fibroblasts using a lentivirus system rescued mitochondrial respiratory chain complex deficiencies. Conclusion Our findings suggest that the novel c.13_29del (p.Trp6Profs*71) MICOS13 variant causes hepato‐encephalopathy with MTDPS. We propose that MICOS13 is classified as the cause of MTDPS., We performed the whole‐exome sequencing of the hepato‐encephalopathy patient with MTDPS. The functional analyses revealed the clinical significance of the identified variant.

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. Electronic supplementary material The online version of this article (10.1186/s12881-018-0733-3) contains supplementary material, which is available to authorized users.

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.

BackgroundCongenital 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.MethodsWe 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.ResultsWe 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

Unlike complete deficiency of hypoxanthine phosphoribosyltransferase (HPRT) (i.e., Lesch–Nyhan syndrome), partial HPRT deficiency causes HPRT-related hyperuricemia without neurological symptoms. Herein, we describe a 22-year-old man without neurological symptoms that presented gout, hyperuricemia (serum urate level, 12.2 mg/dL), multiple renal microcalculi, and a family history of juvenile gout that was exhibited by his brother and grandfather. Genetic testing revealed a novel missense mutation, c.103G>A (p.V35M), in the HPRT1 gene, and biochemical testing (conducted using the patient’s erythrocytes) showed that the patient retained only 12.4% HPRT enzymatic activity compared to that exhibited by a healthy control subject. We thus diagnosed the patient with HPRT-related hyperuricemia caused by partial HPRT deficiency. After his serum urate level was controlled via treatment with febuxostat, his gout did not recur. Thus, this study emphasizes that HPRT deficiency should be considered as a potential cause of familial juvenile gout, even in the absence of neurological symptoms. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s13730-020-00459-9) contains supplementary material, which is available to authorized users.

Background Hepatic epithelioid hemangioendothelioma (HEH) is rare; it is reported in Case presentation The present report describes the histologically proven three HEH cases evaluated using Sonazoid® CEUS. Case 1 was a 33-year-old female patient with no relevant past medical history, who experienced right upper quadrant pain. Conventional abdominal US revealed multiple low echoic liver nodules with vague borderlines. In CEUS, the vascularity of the nodules was similar to that seen in the neighboring normal liver. Later in the portal venous and late phases (PVLP) and post vascular phase, washout of Sonazoid® was detected in the nodules. Case 2 was a 93-year-old female patient with a previous medical history including operations for breast cancer and ovary cancer in her 50’s. Conventional abdominal US revealed multiple low echoic nodules, some of which contained cystic lesions. In the early vascular phase of CEUS, nodules excluding the central anechoic regions were enhanced from peripheral sites. Although the enhancement inside the nodules persisted in both the PVLP and post vascular phase, anechoic areas in the center of some nodules were not enhanced at all. Case 3 was a 39-year-old male patient presented with right upper-quadrant pain, without any relevant past medical history. Conventional abdominal US revealed multiple low echoic liver nodules. In the early vascular phase of CEUS, nodules were gradually enhanced from the peripheral sites as ringed enhancement. Sonazoid®was washed out from the nodules in the PVLP and post vascular phase. Conclusions The most important feature was peripheral enhancement in the early vascular phase. In case 2, the enhancement of the parenchyma of liver nodules persisted even in the PVLP; indicating the lower degree of malignant potential than others. Actually, the tumors did not extend without any treatment in case 2. Since case 2 is the first case report of HEH with cystic lesions, in patients with liver nodules including cystic lesions, HEH is a potential diagnosis.

Background: Hypophosphatasia (HPP) is a rare inborn error of metabolism that results from a dysfunctional tissue non-specific alkaline phosphatase enzyme (TNSALP). Although genotype-phenotype correlations have been described in HPP patients, only sparse information is currently available on the genetics of mild type HPP. Methods: We investigated 5 Japanese patients from 3 families with mild HPP (patients 1 and 2 are siblings; patient 4 is a daughter of patient 5) who were referred to Fujita Health University due to the premature loss of deciduous teeth. Physical and dental examinations, and blood, urine and bone density tests were conducted. Genetic analysis of the ALPL gene was performed in all patients with their informed consent. Results: After a detailed interview and examination, we found characteristic symptoms of HPP in some of the study cases. Mobile teeth or the loss of permanent teeth were observed in 2 patients, and 3 out of 5 patients had a history of asthma. The serum ALP levels of all patients were 30% below the lower limit of the age equivalent normal range. ALPL gene analysis revealed compound heterozygous mutations, including Ile395Val and Leu520Argfs in family 1, Val95Met and Gly491Arg in family 2, and a dominant missense mutation (Gly456Arg) in family 3. The 3D-modeling of human TNSALP revealed three mutations (Val95Met, Ile395Val and Gly456Arg) at the homodimer interface. Severe collisions between the side chains were predicted for the Gly456Arg variant. Discussion: One of the characteristic findings of this present study was a high prevalence of coexisting asthma and a high level serum IgE level. These characteristics may account for the fragility of tracheal tissues and a predisposition to asthma in patients with mild HPP. The genotypes of the five mild HPP patients in our present study series included 1) compound heterozygous for severe and hypomorphic mutations, and 2) dominant-negative mutations. All of these mutations were at the homodimer interface, but only the dominant-negative mutation was predicted to cause a severe collision effect between the side chains. This may account for varying mechanisms leading to different effects on TNSALP function. Keywords: Hypophosphatasia, ALPL, Premature loss of deciduous teeth, Dominant-negative mutations

Several immunosuppressants have been used to treat children with steroid-dependent nephrotic syndrome (SDNS). Mizoribine (MZR) is an immunosuppressant used to maintain remission in children with SDNS, although its effectiveness for treating SDNS remains controversial. Therefore, in this study, we assessed the clinical factors associated with children having SDNS who were successfully treated with MZR. A total of 47 children with SDNS who underwent MZR treatment were retrospectively evaluated. Clinical features including pharmacokinetics after MZR administration were compared between MZR responders and non-responders. The comparison of the two groups revealed no significant differences in age, body weight (BW), daily dose of MZR per BW, serum concentration 2 h after administration (C2), peak serum concentration (Cmax), and area under the concentration curve 0–4 h after administration (AUC0–4). C2/(single dose/BW), Cmax/(single dose/BW), and AUC0–4/(single dose/BW) were significantly higher in the MZR responders than in the non-responders (all p

Mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase (HMGCS2) deficiency is a metabolic disorder caused by mutations in the HMGCS2 gene. The present study describes the identification of four cases of HMGCS2 deficiency in Japan. Hepatomegaly and severe metabolic acidosis were observed in all cases. Fatty liver was identified in three cases, which suggested the unavailability of fatty acids. All patients presented with a high C2/C0 ratio, suggesting that the fatty acid oxidation pathway was normal during metabolic crisis. Genetic analyses revealed five rare, novel variants (p.G219E, p.M235T, p.V253A, p.S392L and p.R500C) in HMGCS2. To confirm their pathogenicity, a eukaryotic expression system and a bacterial expression system was adopted that was successfully used to obtain affinity-purified HMGCS2 protein with measurable activity. Purified M235T, S392L and R500C proteins did not retain any residual activity, whilst the V253A variant showed some residual enzymatic activity. Judging from the transient expression experiment in 293T cells, the G219E variant appeared to be unstable. In conclusion, the present study identified five novel variants of HMGCS2 that were indicated to be pathogenic in four patients affected by HMGCS2 deficiency.

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. 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. 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. Biallelic GALM pathogenic variants cause galactosemia, suggesting the existence of type IV galactosemia.

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 B12 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 μmol/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.

Background Severe combined immune deficiency (SCID) is a potentially fatal primary immunodeficiency due to the absence of T and B lymphocyte function. Early intervention for patients with SCID results in a higher survival rate. From 2017, we launched the first optional newborn screening (NBS) for SCID in Japan based on the detection of T-cell receptor excision circles (TREC). However, NBS for severe B-cell lymphopenia, such as X-linked agammaglobulinemia (XLA), has not been a standard screening test because of a high false-positive rate of Kappa-deleting recombination excision circles (KREC), which reflects the replication of B cells. XLA is characterized by severe B-cell lymphopenia and marked reduction of all classes of serum immunoglobulins. Patients with XLA require early diagnosis and immunoglobulin replacement therapy to prevent the development of bronchiectasis caused by recurrent infections. This study aimed to analyze the results of NBS for SCID and elucidate the utility of NBS for SCID and XLA using the TREC/KREC assay. Patients and Methods We enrolled infants who received NBS for SCID (n = 29,447) between April 2017 and June 2018. Using the EnLiteTM TREC kit, we measured TRECA and β-actin, which are used as controls for monitoring sample amplification. Samples with less than 30 copies/µL and adequate β-actin were defined as positive TRECA. All infants with positive TRECA were followed up for at least 12 months. We measured TRECB and KREC using the EnLiteTM TREC/KREC kit in these infants. As positive controls, we used TRECB and KREC in patients with SCID and XLA, respectively. Furthermore, all infants with positive TRECA were evaluated using flow cytometric analysis and target capture-based next-generation sequencing (NGS) analysis covering 349 primary immunodeficiency- and bone marrow failure-related genes to evaluate CD4+CD45RA+ T-cell counts and identify diagnostic variants. This study was approved by the institutional review board of Nagoya University Graduate School of Medicine. Results Of the infants who underwent NBS for SCID, 43 (0.15%) infants showed positive TRECA. All 43 infants were followed up in Nagoya University for at least 12 months. Of these, we identified one case with DiGeorge syndrome showing severe lymphopenia but did not identify typical SCID. TRECB and KREC were measured in 43 infants with positive TRECA. To determine which kit is more useful to detect T-cell lymphopenia, we compared TRECA with TRECB in 1454 infants with normal TRECA and 43 with positive TRECA. All healthy infants with normal TRECA showed TRECB with more than 30 copies/µL but nine patients with SCID showed extremely low TRECB (median [range], 0 [0-3] copies/µL). Only 6 of 43 (14%) infants showed TRECB with less than 30 copies/µL. Moreover, we analyzed the correlation between CD4+CD45RA+ T-cell counts and TRECB. Compared with 37 infants with normal TRECB, 6 infants with positive TRECB demonstrated significantly lower CD4+CD45RA+ T-cell counts (P = 0.026). However, target capture-based NGS did not identify any diagnostic variants among them. This finding suggested that using this kit, false-positive rates might be decreased from 0.15% (43/29,447) to 0.02% (6/29,447). Using this kit, we assessed KREC in 1454 infants with normal TRECA and 43 with positive TRECA. Of these, we identified one case with less than 30 copies/µL KREC who was diagnosed with congenital asplenia. As positive controls, all six patients with XLA showed quite low KREC (0 [0-9] copies/µL). Compared with previously reported KREC assay, this kit may result in lower false-positive rates. Furthermore, to demonstrate whether KREC reflects the replication of B cells, we analyzed the correlation with CD19+ B-cell counts and KREC. Among 43 infants with positive TRECA, infants with less than 500/µL CD19+ B cells showed significantly lower KREC than those with more than 500/µL CD19+ B cells (P = 0.014). Conclusion We conducted the first large-scale study to evaluate the utility of the newly released EnLiteTM TREC/KREC kit. This kit may be more useful than the current TREC kit to identify infants with T-cell lymphopenia and to avoid unnecessary follow up. Compared with previous NBS for XLA, the false-positive rate of this assay was within an acceptable range. Furthermore, TRECB and KREC were assessed with almost the same screening cost and labor. Therefore, we are considering switching from the current TREC kit to this TREC/KREC kit. Disclosures No relevant conflicts of interest to declare.

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 5μmol/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.

INTRODUCTION Severe combined immunodeficiency disease (SCID) is the most severe form of primary immunodeficiency disorders (PIDs). Impaired cellular and humoral immunity renders the affected infants susceptible to various infections and results in death within the first 2 years of life. Affected infants are asymptomatic at birth, untreated disease leads to death, and prompt treatment (i.e., hematopoietic stem cell transplantation, gene therapy, or enzyme replacement therapy) is linked to significant improvement in outcome. Thus, SCID meets the disease criteria for newborn screening (NBS). The T-cell receptor excision circle (TREC) is an excellent marker of recently formed T cells, and quantitative PCR-based measurement of TREC is an excellent tool in population-based NBS for SCID. Recent progress in next-generation sequencing (NGS) has enabled the simultaneous sequencing of numerous nucleic acids, detecting single nucleotide changes as well as copy number variants. We launched a pilot newborn optional screening program for SCID, combining the measurement of TREC and NGS in Japan. PATIENTS AND METHODS We measured TREC copy number using the Enlite™ Neonatal TREC assay (Perkin Elmer, Turku, Finland), which utilizes the duplex amplification of TREC and beta-actin in the same reaction for each specimen. We used TREC negative cutoffs as follows: TREC copy number of RESULTS From April 2017 to March 2018, we screened a total of 22,865 newborns, covering 57% of the total number of births in the Aichi prefecture, Japan. We identified 48 (0.21%) newborns with TREC negative results. These newborns were referred to the Nagoya University Hospital or Fujita Health University Hospital and received thorough immunological examination, including target capture sequencing. Among them, 12 (25%) newborns had background diseases, including Down syndrome (n = 4), gastrointestinal defects (n = 3), congenital diaphragmatic hernia (n = 2), congenital chylothorax (n = 2), and severe congenital heart anomaly (n = 1). Immunological assessment identified 11 (23%) infants with lymphocytopenia ( CONCLUSION We successfully launched a pilot newborn optional screening program for SCID, combining the measurement of TREC and NGS in Japan. We did not identify typical SCID patients probably because of the relatively small sample size. However, this newborn screening program, incorporating an NGS assay as a second test, achieved early accurate diagnoses of patients with other PIDs with TREC negative results. Consequently, this program may facilitate patient management and optimize treatment outcomes. Disclosures No relevant conflicts of interest to declare.

【目 的】 食道癌・肺癌患者の放射線食道炎の状況と対応を調査・比較し, 症状緩和のための看護介入法を検討する. 【対象と方法】 放射線治療を行った食道癌・肺癌患者を対象に, 治療前日から終了日までの症状出現時期, 嚥下スコア・嚥下障害のGradeの変化, 投薬状況の項目についてデータを診療録から収集した. 【結 果】 食道癌患者の半数以上が治療前から症状があった. 両疾患を比較して治療終了時のスコアの上昇は同程度であったが, 食道癌患者では早期にスコア・Gradeが上昇する傾向であった. 食道癌患者には症状自覚時期とほぼ同時期に粘膜保護剤の投与を開始していたが, 肺癌患者は投薬が遅い傾向であった. 両疾患共に粘膜保護剤の予防投与はなく, 約25％の患者が鎮痛剤を使用していた. 【結 語】 放射線治療を受ける患者のQOLの維持には, 十分な症状観察と早期対応が重要であり, 適切な看護介入のための前向き研究が必要であることが示唆された.

Several fruit juices have been reported to cause food-drug interactions, mainly affecting cytochrome P450 activity; however, little is known about the effects of fruit juices on conjugation reactions. Among several fruit juices tested (apple, peach, orange, pineapple, grapefruit, and pomegranate), pomegranate juice potently inhibited the sulfoconjugation of 1-naphthol in Caco-2 cells. This inhibition was both dose- and culture time-dependent, with a 50% inhibitory concentration (IC(50)) value calculated at 2.7% (vol/vol). In contrast, no obvious inhibition of glucuronidation of 1-naphthol in Caco-2 cells was observed by any of the juices examined. Punicalagin, the most abundant antioxidant polyphenol in pomegranate juice, was also found to strongly inhibit sulfoconjugation in Caco-2 cells with an IC(50) of 45 microM, which is consistent with that of pomegranate juice. These data suggest that punicalagin is mainly responsible for the inhibition of sulfoconjugation by pomegranate juice. We additionally demonstrated that pomegranate juice and punicalagin both inhibit phenol sulfotransferase activity in Caco-2 cells in vitro, at concentrations that are almost equivalent to those used in the Caco-2 cells. Pomegranate juice, however, shows no effects on the expression of the sulfotransferase SULT1A family of genes (SULT1A1 and SULT1A3) in Caco-2 cells. These results indicate that the inhibition of sulfotransferase activity by punicalagin in Caco-2 cells is responsible for the reductions seen in 1-naphthyl sulfate accumulation. Our data also suggest that constituents of pomegranate juice, most probably punicalagin, impair the enteric functions of sulfoconjugation and that this might have effects upon the bioavailability of drugs and other compounds present in food and in the environment. These effects might be related to the anticarcinogenic properties of pomegranate juice.

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 0.16 micromol/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 1 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.

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 autosomal 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.Glu281Lys) 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.

Background Main indications for liver transplantation in the pediatric population include biliary atresia and inherited metabolic diseases. The present study evaluated whether there are differences between pediatric patients undergoing living-related liver transplantation due to the two diseases in terms of their oxidative and immunological status during their regular outpatient follow-up visits. Material and methods A clinical outpatient study measuring serum oxidative stress index (calculated as serum oxidant/antioxidant ratio, in the form of serum total hydroperoxide/serum biological antioxidative potential), serum terminal complement component 5a, as an indicator of complement activity and immunological status, and transforming growth factor-s1, as a marker of liver fibrosis, in 16 patients (6 males and 10 females, 2.5-15 years old) who received living-related liver transplantation due to inherited metabolic diseases (n=6; in the form of propionic acidemia [n=1], methylmalonic acidemia [n=1], arginase deficiency [n=1], tyrosinemia [n=2], and glycogen storage disease type 1b [n=1], with an age range of 2.4-14.6 years old) and due to biliary atresia ([n=10], with an age range of 2.9-14.5 years old). Results Serum oxidative stress index, complement component-5a, and transforming growth factor-s1 were significantly higher in the inherited metabolic diseases group than in the biliary atresia group. In all patients, serum oxidative stress index correlated positively with complement component-5a and transforming growth factor-s1. Conclusions Patients who receive living-related liver transplantation due to inherited metabolic diseases are prone to higher oxidative stress, complement activity, and serum transforming growth factor-s1.

We investigated the responses of experimentally produced hepatic metastases of colon carcinoma 26 tumor and subcutaneously (SC) implanted colon carcinoma 26 tumor in mice to 17 clinically-used and one under-development antitumor agents using same dose regimen. In intravenous administrations on days 7 and 14, there were no significant differences in their responses to most of the tested agents. However, there were big differences in the responses to some of the agents. Nimustine more effectively prolonged the lifespan of SC implanted tumor-bearing mice than of mice bearing hepatic metastases. Mitomycin C was, however, considerably more effective on hepatic metastases than on SC implanted tumor. ME2303, a new fluorinated anthracycline derivative, showed a similar effect to doxorubicin on both tumors. However, administrations of ME2303 on days 7, 11 and 15 showed more marked antitumor effect only on hepatic metastases than administrations on days 7 and 14. Doxorubicin was less active against both tumors for administrations on days 7, 11 and 15 than for those on days 7 and 14. These results suggest the importance of the site of tumor growth for the action of some drugs. ME2303 may be active against hepatic metastases if it is administered by multiple injections.

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.

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.

5-Fluorouracil (5FU) is rapidly metabolised in the liver by dihydrouracil dehydrogenase. Bromovinyluracil is formed in the liver from ( E )-5-(2-bromovinyl)-2′-deoxyuridine (BVDU) by pyrimidine nucleoside phosphorylase and is a potent inhibitor of dihydrouracil dehydrogenase. The co-administration of 5FU (intravenously) and BVDU (orally) was investigated in normal BDF 1 mice and in those bearing liver metastases of Lewis lung carcinoma. 5FU alone rapidly disappeared from plasma and liver within 60 min of dosing. Administered with BVDU, 5FU persisted in plasma and liver for 60–180 min. The combination also significantly enhanced the life-span of tumour-bearing mice. 5FU plus BVDU may have therapeutic potential in the treatment of primary and secondary liver tumours.

Primary leiomyoma and leiomyosarcoma of the lung are rare, and in the majority of reported cases of leio myoma and leiomyosarcoma of the lung, neither bron choscopic examination nor sputum cytological examina tion was of diagnostic value. The definitive diagnosis was made after exploratory thoracotomy or at necropsy in most cases of leiomyosarcoma of the lung. The lei omyoma and leiomyosarcoma of the lung presented in this report were successfully diagnosed by bronchial brushing cytology and bronchial biopsy under the bro nchoscopic control.Imprint smear of the biopsied material of the leiom yoma showed apparently naked nucleus and was scant of cytoplasm. The nucleus was caterpillar-, spindle-, “B”-, or cigarshaped. Nuclear chromatin was fine and granular, and it was dispersed homogeneously. Small bodies of calcium deposits were seen in the cells.Cytological findings of the bronchial brushing of the leiomyosarcoma cells were pleomorphic.The tumor cells were essentially spindleshaped, but other varie ties were also observed.The nuclear chromatin was fine, and dispersed evenly in the nucleus.These two patients underwent lobectomies and both of them have been uneventful since surgery.Comparative cytological features of these tumors were interesting, and these suggest the usefulness of clinical cytology.

才の女性にみられた肺の硬化性血管腫を組織学的, 電顕的に検索した.腫瘤を形成する細胞は乳頭状あるいは充実性に増生し, その主体は形態学的特徴から肺胞上皮由来の細胞と考えられた.増生細胞には種々の分化段階が認められたことから, この腫瘤は過誤腫的性格をもつものと考えられた.この組織形成異常の基盤に “引き金” となる外因が加わって腫瘤形成細胞が増生した可能性が示唆され, 腫瘍組織発生上興味ある症例である.

When given orally in combination with l-cysteine, 5′-deoxy-5-fluorouridine (DFUR) brought about a significant reduction in the growth of adenocarcinoma 755 and a significant prolongation of life-span in mice bearing Lewis lung carcinoma without increased toxicity to the host as compared with DFUR alone, though l-cysteine alone did not show an appreciable antitnmor activity. Moreover, the combination of DFUR and l-cysteine resulted in a marked retardation of growth of human colon tumor LS174T transplanted into nude mice. Thus, the potency of DFUR was increased by l-cysteine. Pharmacokinetic studies revealed that after DFUR administration, plasma DFUR and 5-fluorouracil (5-FU) levels rapidly declined, but that, in the combination with l-cysteine, the plasma clearances of DFUR and 5-FU were slowed down considerably. In the tumor, DFUR and 5-FU levels were similar to those in the plasma. Such a prolongation of DFUR and 5-FU levels in plasma and tumor may produce the enhancement of antitumor effect seen with the combination of DFUR and l-cysteine.

The A or B-glycosyl transferases of hematological diseases showed low activities in the majority of malignant diseases, and their activities became to normal during recovery and remission of the disease. In the cases of leukemia accompanied with A or B-red blood cell type changes, the glycosyl transferases were also low. There was one exception having a loss of A-red blood cell antigen with normal level of A-transferase. These findings suggest that there are other factors which cause red blood type change. For example; H-transferase decrement, abnormal proliferation of erythrobasts in addition to A or B-transferase decrement.

We examined antibodies to adult T-cell leukemia (ATL) associated antigen (ATLA-Ab) by enzyme-immunoassay. In 811 normal subjects in Chiba prefecture, the results showed 4 positives (0.49%) with ATLA-Ab. This positive rate is compatible to other non-endemic areas of ATL in Japan. It was also noted that, all 6 patients were positives (100%) and, out of 14 family members of ATL, 6 persons (43%) were positive with ATLA-Ab.In 100 patients with fluorescent anti-nuclear antibody (FANA), there were 1 positive (1%) and 5 false-positives (5%) with ATLA-Ab by EIA method. But, in these false-positive samples, there was no ATLA-Ab, which could be inhibited by incubation with ATL virus. And these same false-positive samples were negative in the particle agglutination method and in Western blotting method.There was no significant correlation between the degrees of false-positive and the titers or types of FANA. These findings indicate that these false-positives were related to factors other than FANA.