Toshihiro Ohura

Primary systemic carnitine deficiency is caused by mutations in a gene encoding sodium ion-dependent carnitine transporter

Primary systemic carnitine deficiency (SCD; OMIM 212140) is an autosomal recessive disorder characterized by progressive cardiomyopathy, skeletal myopathy, hypoglycaemia and hyperammonaemia. SCD has also been linked to sudden infant death syndrome. Membrane-physiological studies have suggested a defect of the carnitine transport system in the plasma membrane in SCD patients and in the mouse model, juvenile visceral steatosis (jvs; ref. 6). Although the responsible loci have been mapped in both human and mouse, the underlying gene has not yet been identified. Recently, we cloned and analysed the function of a novel transporter protein termed OCTN2 (ref. 9). Our observation that OCTN2 has the ability to transport carnitine in a sodium-dependent manner prompted us to search for mutations in the gene encoding OCTN2, SLC22A5. Initially, we analysed the mouse gene and found a missense mutation in Slc22a5 in jvs mice. Biochemical analysis revealed that this mutation abrogates carnitine transport. Subsequent analysis of the human gene identified four mutations in three SCD pedigrees. Affected individuals in one family were homozygous for the deletion of a 113-bp region containing the start codon. In the second pedigree, the affected individual was shown to be a compound heterozygote for two mutations that cause a frameshift and a premature stop codon, respectively. In an affected individual belonging to a third family, we found a homozygous splice-site mutation also resulting in a premature stop codon. These mutations provide the first evidence that loss of OCTN2 function causes SCD.

Tetrahydrobiopterin-responsive phenylalanine hydroxylase deficiency

Serum phenylalanine concentrations decreased in 4 patients with hyperphenylalaninemia after loading with tetrahydrobiopterin. There were no abnormalities in urinary pteridine excretion or in dihydropteridine reductase activity. However, mutations were detected in the phenylalanine hydroxylase gene, suggesting a novel subtype of phenylalanine hydroxylase deficiency that may respond to treatment with cofactor supplementation.

Cystathionine β‐synthase mutations in homocystinuria

The major cause of homocystinuria is mutation of the gene encoding the enzyme cystathionine β‐synthase (CBS). Deficiency of CBS activity results in elevated levels of homocysteine as well as methionine in plasma and urine and decreased levels of cystathionine and cysteine. Ninety‐two different disease‐associated mutations have been identified in the CBS gene in 310 examined homocystinuric alleles in more than a dozen laboratories around the world. Most of these mutations are missense, and the vast majority of these are private mutations. The two most frequently encountered of these mutations are the pyridoxine‐responsive I278T and the pyridoxine‐nonresponsive G307S. Mutations due to deaminations of methylcytosines represent 53% of all point substitutions in the coding region of the CBS gene. Hum Mutat 13:362–375, 1999.

Mutations in SLC6A19 , encoding B 0 AT1, cause Hartnup disorder

Hartnup disorder, an autosomal recessive defect named after an English family described in 1956 (ref. 1), results from impaired transport of neutral amino acids across epithelial cells in renal proximal tubules and intestinal mucosa. Symptoms include transient manifestations of pellagra (rashes), cerebellar ataxia and psychosis. Using homozygosity mapping in the original family in whom Hartnup disorder was discovered, we confirmed that the critical region for one causative gene was located on chromosome 5p15 (ref. 3). This region is homologous to the area of mouse chromosome 13 that encodes the sodium-dependent amino acid transporter B0AT1 (ref. 4). We isolated the human homolog of B0AT1, called SLC6A19, and determined its size and molecular organization. We then identified mutations in SLC6A19 in members of the original family in whom Hartnup disorder was discovered and of three Japanese families. The protein product of SLC6A19, the Hartnup transporter, is expressed primarily in intestine and renal proximal tubule and functions as a neutral amino acid transporter.

Long-Term Treatment and Diagnosis of Tetrahydrobiopterin-Responsive Hyperphenylalaninemia with a Mutant Phenylalanine Hydroxylase Gene

A novel therapeutic strategy for phenylketonuria (PKU) has been initiated in Japan. A total of 12 patients who met the criteria for tetrahydrobiopterin (BH4)-responsive hyperphenylalaninemia (HPA) with a mutant phenylalanine hydroxylase (PAH) (EC 1.14.16.1) gene were recruited at 12 medical centers in Japan between June 1995 and July 2001. Therapeutic efficacy of BH4 was evaluated in single-dose, four-dose, and 1-wk BH4 loading tests followed by long-term BH4 treatment, and also examined in relation to the PAH gene mutations. The endpoints were determined as the percentage decline in serum phenylalanine from initial values after single-dose (>20%), four-dose (>30%), and 1-wk BH4 (>50%) loading tests. Patients with mild PKU exhibiting decreases in blood phenylalanine concentrations of >20% in the single-dose test also demonstrated decreases of >30% in the four-dose test. The 1-wk test elicited BH4 responsiveness even in patients with poor responses in the shorter tests. Patients with mild HPA, many of whom carry the R241C allele, responded to BH4 administration. No clear correlation was noted between the degree of decrease in serum phenylalanine concentrations in the single- or four-dose tests and specific PAH mutations. The 1-wk test (20 mg/kg of BH4 per day) is the most sensitive test for the diagnosis of BH4-responsive PAH deficiency. Responsiveness apparently depends on mutations in the PAH gene causing mild PKU, such as R241C. BH4 proved to be an effective therapy that may be able to replace or liberalize the phenylalanine-restricted diets for a considerable number of patients with mild PKU.

Clinical pictures of 75 patients with neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD)

SummaryWe clarified the clinical features of NICCD (neonatal intrahepatic cholestasis caused by citrin deficiency) by retrospective review of symptoms, management and long-term outcome of 75 patients. The data were generated from questionnaires to paediatricians in charge of the patients. Thirty of the patients were referred to hospitals before 1 month of age because of positive results in newborn screening (hypergalactosaemia, hypermethioninaemia, and hyperphenylalaninaemia). The other 45, the screen-negative patients, were referred to hospitals with suspected neonatal hepatitis or biliary atresia because of jaundice or discoloured stool. Most of the screen-negative patients presented before 4 months of age, and 11 had failure to thrive. Laboratory data showed elevated serum bile acid concentrations, hypoproteinaemia, low levels of vitamin K-dependent coagulation factors and hypergalactosaemia. Hypoglycaemia was detected in 18 patients. Serum amino acid analyses showed significant elevation of citrulline and methionine concentrations. Most of the patients were given a lactose-free and/or medium-chain triglyceride-enriched formula and fat-soluble vitamins. Symptoms resolved in all but two of the patients by 12 months of age. The two patients with unresolved symptoms suffered from progressive liver failure and underwent liver transplantation before their first birthday. Another patient developed citrullinaemia type II (CTLN2) at age 16 years. It is important to recognize that NICCD is not always a benign condition.

Overview of mutations in the PCCA and PCCB genes causing propionic acidemia.

Propionic acidemia is an inborn error of metabolism caused by a deficiency of propionyl‐CoA carboxylase, a heteropolymeric mitochondrial enzyme involved in the catabolism of branched chain amino acids, odd‐numbered chain length fatty acids, cholesterol, and other metabolites. The enzyme is composed of α and β subunits which are encoded by the PCCA and PCCB genes, respectively. Mutations in both genes can cause propionic acidemia. The identification of the responsible gene, previous to mutation analysis, can be performed by complementation assay or, in some instances, can be deduced from peculiarities relevant to either gene, including obtaining normal enzyme activity in the parents of many patients with PCCB mutations, observing combined absence of α and β subunits by Western blot of many PCCA patients, as well as conventional mRNA‐minus result of Northern blots for either gene or β subunit deficiency in PCCB patients. Mutations in both the PCCA and PCCB genes have been identified by sequencing either RT‐PCR products or amplified exonic fragments, the latter specifically for the PCCB gene for which the genomic structure is available. To date, 24 mutations in the PCCA gene and 29 in the PCCB gene have been reported, most of them single base substitutions causing amino acid replacements and a variety of splicing defects. A greater heterogeneity is observed in the PCCA gene—no mutation is predominant in the populations studied—while for the PCCB gene, a limited number of mutations is responsible for the majority of the alleles characterized in both Caucasian and Oriental populations. These two populations show a different spectrum of mutations, only sharing some involving CpG dinucleotides, probably as recurrent mutational events. Future analysis of the mutations identified, of their functional effect and their clinical relevance, will reveal potential genotype–phenotype correlations for this clinically heterogeneous disorder. Hum Mutat 14:275–282, 1999.

Long-term efficacy of hematopoietic stem cell transplantation on brain involvement in patients with mucopolysaccharidosis type II: a nationwide survey in Japan.

Hematopoietic stem cell transplantation (HSCT) has not been indicated for patients with mucopolysaccharidosis II (MPS II, Hunter syndrome), while it is indicated for mucopolysaccharidosis I (MPS I) patients <2 years of age and an intelligence quotient (IQ) of ≥ 70. Even after the approval of enzyme replacement therapy for both of MPS I and II, HSCT is still indicated for patients with MPS I severe form (Hurler syndrome). To evaluate the efficacy and benefit of HSCT in MPS II patients, we carried out a nationwide retrospective study in Japan. Activities of daily living (ADL), IQ, brain magnetic resonance image (MRI) lesions, cardiac valvular regurgitation, and urinary glycosaminoglycan (GAG) were analyzed at baseline and at the most recent visit. We also performed a questionnaire analysis about ADL for an HSCT-treated cohort and an untreated cohort (natural history). Records of 21 patients were collected from eight hospitals. The follow-up period in the retrospective study was 9.6 ± 3.5 years. ADL was maintained around baseline levels. Cribriform changes and ventricular dilatation on brain MRI were improved in 9/17 and 4/17 patients, respectively. Stabilization of brain atrophy was shown in 11/17 patients. Cardiac valvular regurgitation was diminished in 20/63 valves. Urinary GAG concentration was remarkably lower in HSCT-treated patients than age-matched untreated patients. In the questionnaire analysis, speech deterioration was observed in 12/19 patients in the untreated cohort and 1/7 patient in HSCT-treated cohort. HSCT showed effectiveness towards brain or heart involvement, when performed before signs of brain atrophy or valvular regurgitation appear. We consider HSCT is worthwhile in early stages of the disease for patients with MPS II.

Mucolipidosis II and III alpha/beta: mutation analysis of 40 Japanese patients showed genotype–phenotype correlation

Mucolipidosis (ML) II alpha/beta and III alpha/beta are autosomal recessive diseases caused by a deficiency of α and/or β subunits of the enzyme N-acetylglucosamine-1-phosphotransferase, which is encoded by the GNPTAB gene. We analyzed the GNPTAB gene in 25 ML II and 15 ML III Japanese patients. In most ML II patients, the clinical conditions ‘stand alone’, ‘walk without support’ and ‘speak single words’ were impaired; however, the frequency of ‘heart murmur’, ‘inguinal hernia’ and ‘hepatomegaly and/or splenomegaly’ did not differ between ML II and III patients. We detected mutations in GNPTAB in 73 of 80 alleles. Fourteen new mutations were c.914_915insA, c.2089_2090insC, c.2427delC, c.2544delA, c.2693delA, c.3310delG, c.3388_3389insC+c.3392C>T, c.3428_3429insA, c.3741_3744delAGAA, p.R334L, p.F374L, p.H956Y, p.N1153S and duplication of exon 2. Previously reported mutations were p.Q104X, p.W894X, p.R1189X and c.2715+1G>A causing skipping of exon 13. Homozygotes or compound heterozygotes of nonsense and frameshift mutations contributed to the severe phenotype. p.F374L, p.N1153S and splicing mutations contributed to the attenuated phenotype, although coupled with nonsense mutation. These results show the effective molecular diagnosis of ML II and III and also provide phenotypic prediction. This is the first and comprehensive report of molecular analysis for ML patients of Japanese origin.

Experimental evidence that phenylalanine is strongly associated to oxidative stress in adolescents and adults with phenylketonuria

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 μmol/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 μmol/l even in adult patients.