Toshiaki Oura

Molecular characterization of phenylketonuria in Japanese patients

We characterized phenylalanine hydroxylase (PAH) genotypes of Japanese patients with phenylketonuria (PKU) and hyperphenylalaninemia (HPA). PKU and HPA mutations in 41 Japanese patients were identified by denaturing gradient gel electrophoresis and direct sequencing, followed by restriction fragment length polymorphism analysis to find a large deletion involving exons 5 and 6. Of 82 mutant alleles, 76 (92%) were genotyped showing 21 mutations. The major mutations were R413P (30.5%), R243Q (7.3%), R241 C (7.3%), IVS4nt-1 (7.3%), T278I (7.3%), E6nt-96A→g (6.1%), Y356X (4.9%), R111X (3.7%), and 442–706delE5/6 (2.4%). Eight new mutations (L52 S, delS70, S70P, Y77X, IVS3nt-1, A132 V, W187 C, and C265Y) and a polymorphism of IVS10nt-14 were detected. In vitro PAH activities of mutant PAH cDNA constructs were determined by a COS cell expression system. Six mutations, viz., R408Q, L52 S, R241 C, S70P, V388 M, and R243Q, had 55%, 27%, 25%, 20%, 16% and 10% of the in vitro PAH activity of normal constructs, respectively. The mean pretreatment phenylalanine concentration (0.83±0.21 mmol/l) of patients carrying the R408Q, R241 C, or L52 S mutation and a null mutation was significantly lower (P<0.0005) than that (1.99±0.65 mmol/l) of patients with both alleles carrying mutations associated with a severe genotype. Simple linear regression analysis showed a correlation between pretreatment phenylalanine concentrations and predicted PAH activity in 29 Japanese PKU patients (y=31.9–1.03x, r=0.59, P<0.0001). Genotype determination is useful in the prediction of biochemical and clinical phenotypes in PKU and can be of particular help in managing patients with this disorder.

Treatment of phenylketonuria with a formula consisting of low-phenylalanine peptide

A method of preparation of a more palatable therapeutic formula for phenylketonuria (PKU), consisting of low-phenylalanine peptide (LPP), was reported. There were no adverse effects and, in fact, there was a reduced frequency of diarrhea in patients who received LPP formula for more than 6 months. The LPP formula can be used not only as a more palatable therapeutic milk for PKU, but also as an ingredient to make more palatable foods of low-phenylalanine content.

Seasonality of birth in sporadic cretinism

The seasonal distribution of birth dates of 31 patients with sporadic cretinism due to thyroid dysgenesis was analyzed in Osaka area for 14 years. The incidence was statistically high in the summer months. A hypothesis that some environmental factors such as viral infection may cause the disease is proposed.

Semiautomated Enzyme Immunoassay of Thyrotropin as a Mass Screening Test for Neonatal Hypothyroidism

ABSTRACT: A sensitive, simple, and rapid semiautomated sandwich enzyme immunoassay (EIA) was developed for measuring thyrotropin in dried blood samples on filter paper for use in screening for neonatal hypothyroidism. Good correlation was found between values for thyrotropin determined by this method and those determined by radioimmunoassay (RIA) (r = 0.94). In pilot tests on 17,160 newborn infants in the general population, five cases of primary hypothyroidism were detected by both EIA and RIA. The recall rate was slightly higher in EIA than in RIA.

Experimental Research on a New Treatment for Maternal Phenylketonuria(PKU)

More girls with phenylketonuria (PKU) enter childbearing ages, and most such women are mentally normal, having been born since newborn screening was initiated in the 1970s and treated from early infancy with a low phenylalanine (Phe) diet. Women with PKU not treated prior to conception can have a pregnancy that results in serious fetal damage1. Maternal PKU as a cause of mental retardation and birth defects is a new phenomenon. There will be an increased need for specific therapies in maternal PKU. Low Phe diet is essential for the treatment of maternal PKU. It should be started before pregnancy and it is necessary to maintain their plasma Phe levels around 5 mg/dl throughout their pregnancy2. However they are usually controlled around 10 mg/dl because of the difficulty of the diet therapy. We made an animal model of maternal PKU by the intravenous injection of Phe to pregnant guinea-pigs, and examined plasma, liver and brain Phe levels in their fetuses after an intravenous administration of 6R-5,6,7,8-tetrahydrobiopterin (R-BH4) to the mothers.

Zinc status of untreated histidinemic children

Summary: In order to study zinc status in histidinemia, serum and hair zinc cocentrations were measured in 40 untreated children with histidinemia (age 2 months-5 years). In 20 children (> 2 years of age) zinc content and carbonic anhydrase activity of erythrocytes and urinary excretion of zinc were also studied. The amount of zinc excreted was elevated in histidinemic children and showed a positive correlation with the urinary histidine concentration (γ = 0.57, p < 0.005). The means of serum zinc concentration, erythrocyte zinc concentration, and erythrocyte carbonic anhydrase activity were all similar in the histidinemic and the control children. Hair zinc concentration of histidinemic children was compared with that of controls of five different age groups: <5 months, 5–18 months, 18 months-2 years, 2–4 years, and 4–5 years. In all of these age groups, hair zinc content was similar. The incidence of low-hair-zinc level (<80 μg/g) in histidinemic children >5 months of age (9 of 34) was significantly higher than in controls (18 of 180, p < 0.05). The observation suggested the possibility that untreated histidinemia may cause chronic mild zinc deficiency in some histidinemic children.

Fetal heart malformations in experimental hyperphenylalaninemia in pregnant rats

Abstract Sprague‐Dawley rats were given L‐phenylalanine intraperitoneally from the 8th to 11th day of pregnancy. The hearts of the fetuses were examined on the 21st day of pregnancy. We found that the group given the higher doses of L‐phenylalanine had significantly more heart defects than the group given the lower dose and the controls. Ventricular septal defect was found in 80 % of the fetuses with congenital deformities of the heart. [14C] Leucine uptake by the embryos was significantly lower in the group loaded with L‐phenylalanine than in the controls.

Experimental Research on a Fetal Treatment for Tetrahydrobiopterin Deficiency

Tetrahydrobiopterin (BH4) synthase deficiency has a high incidence of low birth weight,1 and some of them had a mild mental retardation in spite of their early treatment2. In this study we performed an intravenous loading of 2,4-diamino-6-hydroxypyrimidine (DAHP) with a small amount of BHU, and successfully made a model of fetal BH4 deficiency. We investigated the possibility of the fetal therapy of BH4 deficiency in this model by measurements of phenylalanine(Phe), tyrosine(Tyr), BH4, dopamine and catecholamines.