Improved sensitivity and specificity for citrin deficiency using selected amino acids and acylcarnitines in the newborn screening.

Citrin deficiency is an autosomal recessive disorder caused by a defect of citrin resulting from mutations in the SLC25A13 gene. Intrahepatic cholestasis and various metabolic abnormalities, including hypoglycemia, galactosemia, citrullinemia, and hyperammonemia may be present in neonates or infants in the "neonatal intrahepatic cholestasis caused by citrin deficiency" (NICCD) form of the disease. Because at present, newborn screening (NBS) for citrin deficiency using citrulline levels in dried blood spots (DBS) can only detect some of the patients, we tried to develop a new evaluation system to more reliably detect newborns with citrin deficiency utilizing parameters already in place in present NBS methods. To achieve this goal, we re-analyzed NBS profiles of amino acids and acylcarnitines in 96 NICCD patients, who were diagnosed through selective screening or positive family history. Hereby, we identified the combined evaluation of arginine (Arg), citrulline (Cit), isoleucine+leucine (Ile + Leu), tyrosine (Tyr), free carnitine (C0) / glutarylcarnitine (C5-DC) ratio in DBS as potentially sensitive to diagnose citrin deficiency in pre-symptomatic newborns. In particular, a scoring system using threshold levels for Arg (≥9 µmol/L), Cit (≥ 39 µmol/L), Ile + Leu (≥ 99 µmol/L), Tyr (≥ 96 µmol/L) and C0/C5-DC ratio (≥327) was significantly effective to detect newborns who later developed NICCD, and could thus be implemented in existing NBS programs at no extra analytical costs whenever citrin deficiency is considered to become a novel target disease.

Hexagonal Boron Nitride Seed Layer-Assisted van der Waals Growth of BaSnO3 Perovskite Films.

We have succeeded in obtaining BaSnO3 perovskite thin films with remarkable near-infrared luminescence by van der Waals growth. The films were grown on quartz glass substrates by pulsed laser deposition using hexagonal boron nitride as the seed layer, and their crystallinity was confirmed by X-ray diffraction and cross-sectional transmission electron microscopy. The near-infrared emission of the grown film exhibited a broad emission peak centered at 920 nm. The transparency of the BaSnO3 film (thickness = 1000 nm)/ hexagonal boron nitride /double-sided optically polished quartz glass substrate was approximately 90% at approximately 500 nm with or without the BaSnO3 film. Films showing remarkable near-infrared emission and high transparency obtained by van der Waals-type growth could be used in practical wavelength conversion devices that improve the efficiency of Si single-crystal solar cells. The hexagonal boron nitride seed layer supporting the van der Waals growth is an effective method for high-quality crystal growth of films. It can be used for perovskite-type oxides with many functionalities.

Low-Temperature Direct Synthesis of Multilayered h-BN without Catalysts by Inductively Coupled Plasma-Enhanced Chemical Vapor Deposition.

Low-temperature direct synthesis of thick multilayered hexagonal-boron nitride (h-BN) on semiconducting and insulating substrates is required to produce high-performance electronic devices based on two-dimensional (2D) materials. In this study, multilayered h-BN with a thickness exceeding 5 nm was directly synthesized on quartz and Si at low temperatures, between 400 and 500 °C, by inductively coupled plasma-enhanced chemical vapor deposition using borazine as the precursor material. The quality and thickness of the h-BN crystals were investigated with respect to synthesis parameters, namely, temperature, radio frequency power, N2 flow rate, and H2 flow rate. Introducing N2 and H2 carrier gases critically affected the deposition rate, and increasing the carrier gas flow rate enhanced the h-BN crystal quality. The typical optical band gap of synthesized h-BN was approximately 5.8 eV, consistent with that of previous studies. The full width at half-maximum of the h-BN Raman peak was 32-33 cm-1, comparable to that of commercially available multilayered h-BN on Cu foil. These results are expected to facilitate the development of 2D materials for electronics applications.

Oxidation Resistance Improvement of Graphene-Oxide-Semiconductor Planar-Type Electron Sources Using h-BN as an Oxygen-Resistant, Electron-Transmissive Coating.

Graphene-oxide-semiconductor (GOS) planar-type electron emission devices with a hexagonal boron nitride (h-BN) protective layer have demonstrated improved oxidation resistance while maintaining their emission performance. The devices with a monolayer or a multilayer (13 nm in thickness) h-BN protective layer can emit electrons even after oxygen plasma exposure (ashing). Remarkably, the device with a monolayer h-BN was able to emit electrons with a maximum efficiency of 11% after a 4-min ashing, showing that a thin h-BN protection layer can provide oxygen tolerance to GOS devices without a significant emission loss. The thicker multilayer h-BN imparted higher oxidation resistance to the device but with decreased emission efficiency compared with the device with monolayer h-BN. Thus, the use of h-BN necessitates a trade-off between the device's emission performance and its oxidation resistance. In addition, the etching rate of h-BN by the oxygen plasma treatment was found to increase by exposure to air after the first plasma treatment, which indicates that the adherence of H2O to the surface of h-BN is one probable cause of h-BN etching during the ashing process.

Clinical manifestation and long-term outcome of citrin deficiency: Report from a nationwide study in Japan.

Citrin deficiency is an autosomal recessive disorder caused by mutations in the SLC25A13 gene. The disease can present with age-dependent clinical manifestations: neonatal intrahepatic cholestasis by citrin deficiency (NICCD), failure to thrive, and dyslipidemia by citrin deficiency (FTTDCD), and adult-onset type II citrullinemia (CTLN2). As a nationwide study to investigate the clinical manifestations, medical therapy, and long-term outcome in Japanese patients with citrin deficiency, we collected clinical data of 222 patients diagnosed and/or treated at various different institutions between January 2000 and December 2019. In the entire cohort, 218 patients were alive while 4 patients (1 FTTDCD and 3 CTLN2) had died. All patients <20 years were alive. Patients with citrin deficiency had an increased risk for low weight and length at birth, and CTLN2 patients had an increased risk for growth impairment during adolescence. Liver transplantation has been performed in only 4 patients (1 NICCD, 3 CTLN2) with a good response thereafter. This study reports the diagnosis and clinical course in a large cohort of patients with citrin deficiency and suggests that early intervention including a low carbohydrate diet and MCT supplementation can be associated with improved clinical course and long-term outcome.

Different pharmacoresistance of focal epileptic spasms, generalized epileptic spasms, and generalized epileptic spasms combined with focal seizures.

OBJECTIVE: Among standard treatments for infantile spasms, adrenocorticotropic hormone (ACTH) is reported as the best treatment, but ACTH is ineffective in one-half of the patients. To establish precision medicine, we examined pharmacoresistance of focal epileptic spasms (ES), generalized ES, and generalized ES combined with focal seizures, diagnosed based on the revised seizure classification of ILAE in 2017. METHODS: We conducted a retrospective nationwide study in Japan on the long-term seizure outcome of ES. Long-term seizure outcome was evaluated by seizure-free rate, seizure-free period, and Kaplan-Meier curve. Seizure-free was defined as seizure control for longer than 2 months. RESULTS: From the medical history of 501 patients, 325 patients had generalized ES only (GES group) at the start of the first treatment, 125 patients had generalized ES after focal seizure onset (FS-GES group), seven patients had focal ES after focal seizure onset (FS-FES group), and 24 patients had generalized ES combined with focal seizures after focal seizure onset (FS-GES + FS group). Seizure-free period of ES (generalized ES and focal ES) [mean (95% confidence interval)] was 2.7 (0.0-5.4) months in GES group, 1.1 (0.1-2.2) months in FS-GES group, 1.0 (0.2-1.9) months in FS-GES + FS group, and 0.1 (-0.2-0.5) months in FS-FES group. Seizure-free rate, seizure-free period, and Kaplan-Meier curve of generalized ES were almost the same in GES group and FS-GES group, with characteristics of superior response to ACTH. Mean seizure-free period of generalized ES combined with focal seizures was significantly shorter in FS-GES + FS group than in GES group. Mean seizure-free period of focal ES in FS-FES group was extremely short with exceedingly early relapse. SIGNIFICANCE: Pharmacoresistance was different in generalized ES, focal ES, and generalized ES combined with focal seizures. ES with focal features or with focal seizures may have focal lesions, thus consider surgical options earlier in the course.

Saccharopinuria accompanied by hyperammonemia and hypercitrullinemia presented with elderly-onset epilepsy, progressive cognitive decline, and gait ataxia.

We report a case of saccharopinuria with hyperammonemia and hypercitrullinemia in a Japanese woman who presented with elderly-onset epilepsy, progressive cognitive decline, and gait ataxia. Blood amino acid analysis revealed an increase in citrulline, cystine, and lysine levels, and urine amino acid analysis showed increased citrulline and cystine levels. Urine metabolomics revealed an increased saccharopine level, leading to the definitive diagnosis of saccharopinuria. In western blots of liver biopsy samples, normal citrin levels were observed, suggesting that adult-onset citrullinemia type 2 (CTLN2) was not present. In addition, decreased argininosuccinate synthetase (ASS) levels were observed, and ASS1 gene, a causative gene for citrullinemia type 1 (CTLN1), was analyzed, but no gene mutations were found. Because the causes of hypercitrullinemia were not clear, it might be secondary to saccharopinuria. Muscle biopsy findings of the biceps brachii revealed diminished cytochrome c oxidase (COX) activity, mitochondrial abnormalities on electron microscopy and p62- positive structures in immunohistochemical analyses. Saccharopinuria is generally considered a benign metabolic variant, but our case showed elevated lysine and saccharopine levels causing ornithine circuit damage, mitochondrial dysfunction, and autophagy disorders. This may lead to so far unknown neurological disorders.

Highly Monochromatic Electron Emission from Graphene/Hexagonal Boron Nitride/Si Heterostructure.

In this work, a planar electron emission device based on a graphene/hexagonal boron nitride (h-BN)/n-Si heterostructure is fabricated to realize highly monochromatic electron emission from a flat surface. The h-BN layer is used as an insulating layer to suppress electron inelastic scattering within the planar electron emission device. The energy spread of the emission device using the h-BN insulating layer is 0.28 eV based on the full-width at half-maximum (FWHM), which is comparable to a conventional tungsten field emitter. The characteristic spectral shape of the electron energy distributions reflected the electron distribution in the conduction band of the n-Si substrate. The results indicate that the inelastic scattering of electrons at the insulating layer is drastically suppressed by the h-BN layer. Furthermore, the maximum emission current density reached 2.4 A/cm2, which is comparable to that of a conventional thermal cathode. Thus, the graphene/h-BN heterostructure is a promising material for planar electron emission devices to obtain a highly monochromatic electron beam and a high electron emission current density.

Early Detection and Diagnosis of Neonatal Intrahepatic Cholestasis Caused by Citrin Deficiency Missed by Newborn Screening Using Tandem Mass Spectrometry.

Citrullinemia is the earliest identifiable biochemical abnormality in neonates with intrahepatic cholestasis due to a citrin deficiency (NICCD) and it has been included in newborn screening panels using tandem mass spectrometry. However, only one neonate was positive among 600,000 infants born in Sapporo city and Hokkaido, Japan between 2006 and 2017. We investigated 12 neonates with NICCD who were initially considered normal in newborn mass screening (NBS) by tandem mass spectrometry, but were later diagnosed with NICCD by DNA tests. Using their initial NBS data, we examined citrulline concentrations and ratios of citrulline to total amino acids. Although their citrulline values exceeded the mean of the normal neonates and 80% of them surpassed +3 SD (standard deviation), all were below the cutoff of 40 nmol/mL. The ratios of citrulline to total amino acids significantly elevated in patients with NICCD compared to the control. By evaluating two indicators simultaneously, we could select about 80% of patients with missed NICCD. Introducing an estimated index comprising citrulline values and citrulline to total amino acid ratios could assure NICCD detection by NBS.

Immunochromatography test for rapid diagnosis of Mycoplasma pneumoniae infection.

The sensitivity and specificity of a new rapid Mycoplasma pneumoniae antigen immunochromatography (IC) test, DK-MP-001, were determined using particle agglutination (PA) antibody response and loop-mediated isothermal amplification (LAMP) gene detection as the gold standard. Of 165 patients, 59 were diagnosed with M. pneumoniae infection based on a ≥fourfold rise of serum PA antibody during the course of the illness. Of the first visit swabs, 60 were positive for M. pneumoniae on LAMP, and 49 were positive for M. pneumoniae antigen on IC test. Compared with PA antibody and LAMP, the sensitivity/specificity of the IC test were 81.4% (48/59) and 99.1% (105/106); and 81.7% (49/60) and 100% (105/105), respectively. IC test detected antigen in pharyngeal swabs more sensitively than in nasal swabs for the same subjects (P < 0.05). The IC test performs well enough to be used with pharyngeal swabs at the first examination.

A nationwide survey of common viral infections in childhood among patients with primary immunodeficiency diseases.

OBJECTIVES: Patients with primary immunodeficiency diseases (PID) are highly susceptible to various microorganisms. However, no population-based studies have been performed among common viral pathogens, such as respiratory syncytial virus (RSV), rotavirus (RV), varicella-zoster virus (VZV) and influenza virus (IV). The objective of this study was to reveal the clinical burden of these four infections among PID patients in Japan. METHODS: We conducted a nationwide survey by sending questionnaires to 898 hospitals with pediatric departments throughout Japan. RESULTS: Nine hundred ten PID patients from 621 hospitals were registered (response rate: 69.2%). Fifty-four of the patients were hospitalized due to these viral infections. The durations of hospitalization due to RSV and RV infections differed significantly in the PID patients with and without cellular immunodeficiency (12.0 vs 6.5 days, p = 0.041; and 14.0 vs 6.0 days, p = 0.031, respectively). There was no significant difference in the duration of hospitalization in PID patients with and without cellular immunodeficiency who were hospitalized with IV infections (7.3 vs 6.1 days, p = 0.53). CONCLUSIONS: Special attention should be paid to PID patients with compromised cellular immunity who present with RSV and RV infection due to their high risk for severe disease.

Spectrum of mutations associated with methionine adenosyltransferase I/III deficiency among individuals identified during newborn screening in Japan.

Methionine adenosyltransferase I/III deficiency (MAT I/III deficiency) is an inborn error of metabolism that results in isolated persistent hypermethioninemia. Definitive diagnosis is now possible by molecular analyses of the MAT1A gene. Based on newborn screening (NBS) data collected between 2001 and 2012 in Hokkaido, Japan, the estimated incidence of MAT I/III deficiency was 1 in 107,850. 24 patients (13 males, 11 females) from 11 prefectures in Japan were referred to our laboratory for genetic diagnosis of MAT I/III deficiency. They were all found between 1992 and 2012 by the NBS program in each region. In these 24 individuals, we identified 12 distinct mutations; 14 patients were heterozygous for an R264H mutation; R264H caused an autosomal dominant and clinically benign phenotype in each case. The mutations in the other 10 patients showed autosomal recessive inheritance and included eight novel MAT1A mutations. Putative amino acid substitutions at R356 were observed with six alleles (three R356P, two R356Q, and one R356L). MAT I/III deficiency is not always benign because three of our cases involved brain demyelination or neurological complications. DNA testing early in life is recommended to prevent potential detrimental neurological manifestations.

Fatigue and quality of life in citrin deficiency during adaptation and compensation stage.

Citrin-deficient children and adolescents between adult-onset type II citrullinemia and neonatal intrahepatic cholestasis by citrin deficiency do not have clear clinical features except for unusual diet of high-fat, high-protein, and low-carbohydrate food. The aims of the present study are to characterize fatigue and quality of life (QOL) in citrin-deficient patients during adaptation and compensation stage, and to define the relationship between fatigue and QOL. The study subjects were 55 citrin-deficient patients aged 1-22years (29 males) and 54 guardians. Fatigue was evaluated by self-reports and proxy-reports of the PedsQL Multidimensional Fatigue Scale. QOL was evaluated by the PedsQL Generic Core Scales. Both scale scores were significantly lower in child self-reports (p<0.01 and p<0.05, respectively) and parent proxy-reports (p<0.01 and p<0.01, respectively) than those of healthy children. Citrin-deficient patients with scores of 50 percentile or less of healthy children constituted 67.5% of the sample for the Fatigue Scale and 68.4% for the Generic Core Scales. The PedsQL Fatigue Scale correlated with the Generic Core Scales for both the patients (r=0.56) and parents reports (r=0.71). Assessments by the patients and their parents showed moderate agreement. Parents assessed the condition of children more favorably than their children. The study identified severe fatigue and impaired QOL in citrin-deficient patients during the silent period, and that such children perceive worse fatigue and poorer QOL than those estimated by their parents. The results stress the need for active involvement of parents and medical staff in the management of citrin-deficient patients during the silent period.

Improved neurologic prognosis for a patient with propionic acidemia who received early living donor liver transplantation.

Despite medical therapy, patients with propionic academia (PA) still display a tendency to develop epilepsy. Patients with neonatal-onset PA who have received early living donor liver transplantation (LDLT) are limited in number, and the effect on neurologic prognosis, including epilepsy, is not clear. We report a patient with PA whose EEG findings improved dramatically after undergoing LDLT at age 7 months. The patient's neurologic development and brain MRI findings were quite satisfactory at age 2 years and 3 months. LDLT is effective not only in preventing metabolic decompensation, but also in improving neurologic function to ensure better quality of life.

Methionine adenosyltransferase I/III deficiency: neurological manifestations and relevance of S-adenosylmethionine.

Methionine adenosyltransferase I/III (MAT I/III) deficiency, caused by mutations in the MAT1A gene, is an inherited metabolic disorder characterized by persistent hypermethioninemia, usually detected by newborn mass screening. There is a wide range of clinical manifestations, from completely asymptomatic to neurological problems associated with brain demyelination. Physiological role of S-adenosylmethionine (SAM), the metabolic product of methionine catalyzed by MAT, in the central nervous system has been investigated in vivo and in vitro, and case reports demonstrated an effectiveness of supplementary treatment of SAM in the improvement of neurological development and myelination. Methionine restriction can be an additional therapeutic strategy because hypermethioninemia alone may be neurotoxic; however, lowering methionine carries a risk to decrease the synthesis of SAM.

Living-donor liver transplantation for propionic acidemia.

Propionic acidemia is a rare autosomal recessive disorder affecting the catabolism of branched-chain amino acids because of a genetic defect in PCC. Despite the improvements in medical treatment with protein restriction, sufficient caloric intake, supplementation of l-carnitine, and metronidazole, patients with the severe form of propionic acidemia have life-threatening metabolic acidosis, hyperammonemia, and cardiomyopathy, which results in serious neurologic sequelae and sometimes death. This study retrospectively reviewed three children with neonatal-onset propionic acidemia who received LDLT. Between November 2005 and December 2010, 148 children underwent LDLT, with an overall patient survival of 90.5%, in our center. Three patients were indicated for transplantation because of propionic acidemia. All recipients achieved a resolution of metabolic derangement and better quality of life with protein restriction and medication, although urine methylcitrate and serum propionylcarnitine levels did not decrease markedly. LT can reduce the magnitude of progressive cardiac/neurologic disability as a result of poor metabolic control. Further evaluation is therefore required to determine the long-term suitability of this treatment modality.

S-adenosylmethionine treatment in methionine adenosyltransferase deficiency, a case report.

Reported is a female patient with methionine adenosyltransferase I/III (MAT I/III) deficiency, who was found to have pronounced hypermethioninemia on newborn mass spectroscopy screening, and had two compound heterozygous missense mutations in the gene encoding human MAT1A protein. Hypermethioninemia persisted and her mental development was deficient. At 4 years and 8 months, we started with the supplementary treatment of S-adenosylmethionine, the metabolic product of methionine catalyzed by MAT, which was effective in her neurological development.

A novel proteolipid protein 1 gene mutation causing classical type Pelizaeus-Merzbacher disease.

Pelizaeus-Merzbacher disease (PMD) is a rare dysmyelinating disorder caused by mutations in the proteolipid protein 1 (PLP1) gene. PMD is generally classified according to its clinical or pathological features into classical or connatal forms. We describe here a 19-year-old male with classical form PMD who presented with stridor and nystagmus in early infancy and whose psychomotor development has been severely delayed. Brain magnetic resonance imaging revealed white matter abnormalities typical of PMD. Direct sequencing of the PLP1 gene identified two nucleotide substitutions. One was a C-to-T transition at -31 in the 5'-flanking region of exon 1; the other was a novel point mutation, T-to-C transition in exon 4, which led to substitution of cysteine for arginine at residue 184. Because Cys184 forms a disulphide bridge with Cys228, the Cys184Arg mutation probably removes the bridge and changes the tertiary structure of PLP protein. A defective disulfide bond in PLP protein could be important in the pathogenesis of PMD.

Progressive liver fibrosis in late-onset argininosuccinate lyase deficiency.

A 4-month-old boy, with late-onset argininosuccinate lyase (ASL) deficiency with hepatomegaly, was treated by protein restricted diet and arginine supplementation; he was followed for 3 years. Hepatomegaly and mild liver dysfunction persisted without significant hyperammonemia. He maintained normal psychomotor development to the age of 12 months, but, at 3 years of age, his developmental status is in the borderline normal range. Liver biopsy performed at 12 months of age demonstrated swollen and pale hepatocytes with abnormal glycogen deposition and mild periportal fibrosis. A subsequent liver biopsy at 3 years of age showed progressive liver fibrosis in the periportal and central areas, which extended into the liver lobule. These findings suggest that liver impairment in ASL deficiency may advance without significant hyperammonemia and underline the importance of repeated liver biopsy in this disorder, even when the plasma ammonia level is well controlled.

Application of mutation analysis for the previously uncertain cases of adult-onset type II citrullinemia (CTLN2) and their clinical profiles.

Type II citrullinemia (CTLN2) is characterized by a deficiency of argininosuccinate synthetase (ASS) in the liver. Mutation analysis of the SLC25A13 gene, which is responsible for CTLN2, provides a rapid and accurate diagnosis. We describe clinical, biochemical and histologic features of two patients, whose diagnosis was finally made by mutation analysis. They initially presented with symptoms related to hyperammonemia at 16 to 22 years of age. A patient had shown mental retardation and growth failure from early childhood. Laboratory findings including amino acids, were characteristic, such as elevated citrulline, arginine, and lysine concentrations, but definitive diagnosis had not been made. The patients died of liver cirrhosis and hepatoma at 31 and 34 years old, respectively. Fatty change in the hepatocytes was commonly observed in the autopsied specimens. ASS activity was decreased in the liver of both patients, and a concomitant decrease of arginase activity was found in one case. Investigation for the SLC25A13 mutation revealed that one patient was homozygous for IVS11 + 1G>A, and the other was compound heterozygote (851del4/S225X). Comparison of genetic, enzymatic and biochemical data among various cases of CTLN2 will be essential to understand the real nature of the disease.