Yosuke Shigematsu

Newborn mass screening and selective screening using electrospray tandem mass spectrometry in Japan.

Electrospray tandem mass spectrometry was applied to detect a series of inherited metabolic disorders during a newborn-screening pilot study and a selective screening in Japan. In our mass screening of 102,200 newborns, five patients with propionic acidemia, two with methylmalonic acidemia, two with medium-chain acyl-CoA dehydrogenase deficiency, three with citrullinemia type II, and one with phenylketonuria were identified. In a selective screening of 164 patients with symptoms mainly related to hypoglycemia and/or hyperammonemia, 12 with fatty acid oxidation disorders and six with other disorders were found. The results indicated the importance of newborn screening using this technology in Japan.

Thermolabile phenotype of carnitine palmitoyltransferase II variations as a predisposing factor for influenza‐associated encephalopathy

To assess the etiology of influenza‐associated encephalopathy (IAE), a surveillance effort was conducted during 2000–2003 in South‐West Japan. All fatal and handicapped patients except one (4/34 patients) exhibited a disorder of mitochondrial β‐oxidation evoked by the inactivated carnitine palmitoyltransferase II (CPT II) with transiently elevated serum acylcarnitine ratios (C16:0 + C18:1)/C2 > 0.09 during high‐grade fever. Analyses of genotypes and allele compositions of CPT II revealed a thermolabile phenotype of compound heterozygotes for [1055T > G/F352C] and [1102G > A/V368I], which shows a higher frequency in IAE patients than healthy volunteers (P < 0.025). The thermolabile phenotype of CPT II variations may be a principal genetic background of IAE in Japanese.

Bone mineral status in ambulatory pediatric patients on long‐term anti‐epileptic drug therapy

Background : For ambulatory pediatric outpatients, reports of abnormalities of bone metabolism associated with anti‐epileptic drugs are inconsistent and may be difficult to interpret.

Prenatal diagnosis of organic acidemias based on amniotic fluid levels of acylcarnitines

Acylcarnitines in amniotic fluid samples were analyzed for the prenatal diagnosis of propionic acidemia, methylmalonic aciduria, isovaleric acidemia, and glutaric aciduria by electrospray tandem mass spectrometry. Although the levels of the specific acylcarnitine between affected and unaffected cases showed an overlap, the ratios of propionylcarnitine to 4-carbon acylcarnitine levels for propionic acidemia and methylmalonic aciduria, those of isovalerylcarnitine to propionylcarnitine for isovaleric acidemia, and those of glutarylcarnitine to propionylcarnitine for glutaric aciduria type I were shown to be reliable indicators in the prenatal diagnosis. In addition, it is suggested that the combination of the ratios of glutarylcarnitine, isovalerylcarnitine, and hexanoylcarnitine to propionylcarnitine may be useful for the prenatal diagnosis of glutaric aciduria type II.

Identification and Functional Analysis of Novel Human Growth Hormone Secretagogue Receptor (GHSR) Gene Mutations in Japanese Subjects with Short Stature

CONTEXT Short stature (SS) is a multifactorial developmental condition with a significant genetic component. Recent studies have revealed that rare deleterious mutations in the GH-secretagogue receptor type 1A (GHSR1A) gene could be a cause of familial SS or GH deficiency. OBJECTIVE The aim of this study was to evaluate the contribution of GHSR1A mutations to the molecular mechanism underlying SS in Japanese subjects. METHODS We performed mutational screening of the GHSR1A gene in 127 unrelated Japanese SS patients diagnosed with either isolated GH deficiency or idiopathic SS. Identified mutations were analyzed in 188 control subjects, and their functional properties were examined in a heterologous expression system. RESULTS Four novel heterozygous GHSR1A mutations were identified (ΔQ36, P108L, C173R, and D246A). Expression studies demonstrated that these mutations had varying functional consequences: 1) all mutations showed a loss-of-function effect on the constitutive signaling activity of GHSR1A, but the degree of loss varied widely; 2) C173R caused intracellular retention of the mutated protein, resulting in total loss of receptor function; 3) P108L resulted in a large decrease in binding affinity to ghrelin, without affecting its surface expression; 4) D246A uniquely impaired agonist- and inverse agonist-stimulated receptor signaling; and 5) ΔQ36 showed only a subtle reduction in constitutive activity. The cumulative frequency of these putative functional mutations was significantly higher in the patient group than in controls (4.72 vs. 0.53%; P = 0.019; odds ratio = 9.28; 95% confidence interval, 1.10-78.0). CONCLUSIONS Our results suggest that GHSR1A mutations contribute to the genetic etiology of SS in the Japanese population.

Fatal Propionic Acidemia in Mice Lacking Propionyl-CoA Carboxylase and Its Rescue by Postnatal, Liver-specific Supplementation via a Transgene

Propionic acidemia (PA) is an inborn error of metabolism caused by the genetic deficiency of propionyl-CoA carboxylase (PCC). By disrupting the α-subunit gene of PCC, we created a mouse model of PA (PCCA−/−), which died in 24–36 h after birth due to accelerated ketoacidosis. A postnatal, liver-specific PCC expression via a transgene in a far lower level than that in wild-type liver, allowed PCCA−/− mice to survive the newborn and early infant periods, preventing a lethal fit of ketoacidosis (SAP+PCCA−/− mice). Interestingly, SAP+PCCA−/− mice, in which the transgene expression increased after the late infant period, continued to grow normally while mice harboring a persistent low level of PCC died in the late infant period due to severe ketoacidosis, clearly suggesting the requirement of increased PCC supplementation in proportion to the animal growth. Based on these results, we propose a two-step strategy to achieve an efficient PA prevention in human patients: a partial PCC supplementation in the liver during the newborn and early infant periods, followed by a larger amount of supplementation in the late infant period.

Identification of genetic mutations in Japanese patients with fructose-1,6-bisphosphatase deficiency

Fructose-1,6-bisphosphatase (FBPase) deficiency is an autosomal recessive inherited disorder and may cause sudden unexpected infant death. We reported the first case of molecular diagnosis of FBPase deficiency, using cultured monocytes as a source for FBPase mRNA. In the present study, we confirmed the presence of the same genetic mutation in this patient by amplifying genomic DNA. Molecular analysis was also performed to diagnose another 12 Japanese patients with FBPase deficiency. Four mutations responsible for FBPase deficiency were identified in 10 patients from 8 unrelated families among a total of 13 patients from 11 unrelated families; no mutation was found in the remaining 3 patients from 3 unrelated families. The identified mutations included the mutation reported earlier, with an insertion of one G residue at base 961 in exon 7 (960/961insG) (10 alleles, including 2 alleles in the Japanese family from our previous report [46% of the 22 mutant alleles]), and three novel mutations--a G-->A transition at base 490 in exon 4 (G164S) (3 alleles [14%]), a C-->A transversion at base 530 in exon 4 (A177D) (1 allele [4%]), and a G-->T transversion at base 88 in exon 1 (E30X) (2 alleles [9%]). FBPase proteins with G164S or A177D mutations were enzymatically inactive when purified from E. coli. Another new mutation, a T-->C transition at base 974 in exon 7 (V325A), was found in the same allele with the G164S mutation in one family (one allele) but was not responsible for FBPase deficiency. Our results indicate that the insertion of one G residue at base 961 was associated with a preferential disease-causing alternation in 13 Japanese patients. Our results also indicate accurate carrier detection in eight families (73%) of 11 Japanese patients with FBPase deficiency, in whom mutations in both alleles were identified.

Unexpectedly high prevalence of the mild form of propionic acidemia in Japan: presence of a common mutation and possible clinical implications

Abstract. Propionic acidemia [MIM 606054] is a form of organic acidemia caused by genetic deficiency of propionyl-CoA carboxylase (PCC) and characterized by attacks of severe metabolic acidemia and hyperammonemia beginning in the neonatal period or in early infancy. There are, however, patients who have higher PCC activities and present later with unusual symptoms, such as mild mental retardation or extrapyramidal symptoms, sometimes even without metabolic acidosis. Through the neonatal screening of more than 130,000 Japanese newborns we detected a frequency of patients with propionic acidemia more than ten times higher than previously reported, most of them with milder phenotypes. The mutational spectrum was quite different from that of patients with the severe form and there was a common mutation (Y435C) in the β subunit of the PCC gene (PCCB). Since patients with the mild form could present with unusual symptoms and therefore could easily remain unrecognized, it is important to identify those patients and clarify their natural history. Molecularly, one of the mutations (A1288C) caused an unusual pattern of multiple exon skipping and another unidentified mutation lead to the absence of mRNA. Taking into consideration previous findings regarding PCCB mutations, it appears that this gene is particularly prone to posttranscriptional modifications such as missense mediated exon skipping, mRNA decay, or rapid product degradation.

Effects of Citrin Deficiency in the Perinatal Period: Feasibility of Newborn Mass Screening for Citrin Deficiency

Deficiency of citrin due to mutations of the SLC25A13 gene causes adult-onset type II citrullinemia (CTLN2) and one type of neonatal intrahepatic cholestasis (NICCD). About half of the NICCD patients are detected based on high galactose, phenylalanine, and/or methionine concentrations on newborn mass screening (NMS). To clarify the perinatal and neonatal effects and the inconsistent results on NMS, we examined aminograms, the levels of bile acids and galactose in dried blood spots for NMS from 20 patients with NICCD. Birth weight was low for gestational age (−1.4 ± 0.7 SD). Affected fetuses may have suffered intrauterine citrin deficiency. The first abnormality detected after birth was citrullinemia, and 19 of 20 patients had citrulline levels higher than +2 SD of controls. Tyrosine, phenylalanine, methionine, galactose, and bile acids were less affected than citrulline on d 5 after birth. Galactose and bile acids levels were increased at 1 mo in comparison with d 5 after birth due to impairment of the cytosolic NADH reducing–equivalent supply into mitochondria of hepatocytes. Patients with negative findings on NMS had low levels of total 20 amino acids. Citrulline/serine, citrulline /leucine plus isoleucine, and citrulline/total amino acids ratios, controlled for the confounding effect of low amount of total amino acids, were higher in all patients than +2 SD, +2 SD, and +3 SD of controls, respectively. NMS for citrin deficiency (frequency of homozygote with SLC25A13 mutation: 1/10,000–1/38,000 in East Asia) will be useful for clarification of the clinical course, treatment, and prevention of this disease.

Modifications in electrospray tandem mass spectrometry for a neonatal-screening pilot study in Japan.

In a neonatal-screening pilot study for inherited disorders in organic acid and amino acid metabolism, we analyzed butyrated acylcarnitines and amino acids in blood spots of more than 20,000 newborns by electrospray tandem mass spectrometry. In order to screen urea cycle disorders, we performed multiple scanning functions with additional stable isotope-labelled internal standards, since such reported functions as neutral loss of m/z 102 or 109 for butyrated amino acids were not sufficient. Arginine levels were measured with arginine-13C6. Hypocitrullinemia for the screening of some urea cycle disorders was detectable by measurement with synthesized citrulline-d6, although we did not find any such disorders. In the acylcarnitine analysis, we found a patient with propionic acidemia, who has been treated effectively. The increasing false positive rate due to the use of pivalic acid-containing antibiotics in the diagnosis of isovaleric acidemia was a problem in Japan.