Hisataka Awata

A nonsense mutation in the 4-hydroxyphenylpyruvic acid dioxygenase gene (Hpd) causes skipping of the constitutive exon and hypertyrosinemia in mouse strain III

4-Hydroxyphenylpyruvic acid dioxygenase (HPD; EC 1.13.11.27) is an important enzyme in tyrosine catabolism in most organisms. Decreased activity of 4-hydroxyphenylpyruvic acid dioxygenase in the liver of mouse strain III is associated with tyrosinemia. We report a nucleotide substitution that generates a termination codon in exon 7 of the 4-hydroxyphenylpyruvic acid dioxygenase gene in III mice. This mutation is associated with partial exon skipping, and most of the mRNA lacks sequences corresponding to exon 7. The partial exon skipping apparently is the result of a nonsense mutation in the exon. Mouse strain III is a model for human tyrosinemia type 3 (McKusick 276710), and this strain together with recently established models for tyrosinemia type 1 will facilitate studies of hereditary tyrosinemias.

Complete rescue of lethal albino c(14CoS) mice by null mutation of 4- hydroxyphenylpyruvate dioxygenase and induction of apoptosis of hepatocytes in these mice by in vivo retrieval of the tyrosine catabolic pathway

Hereditary tyrosinemia 1 (HT1) is characterized by progressive liver damage, from infancy, and by a high risk for hepatocellular carcinoma. HT1 is due to mutations in the fumarylacetoacetate hydrolase gene Fah, encoding the last enzyme in the tyrosine catabolic pathway. Lethal albino deletionc 14CoS mice and mice with target-disruptedFah are models for HT1, but they die in the perinatal period, albeit with a different phenotype from that seen in HT1 in humans. We first asked whether homozygous null mutation of the 4-hydroxyphenylpyruvate dioxygenase gene Hpd could rescue the homozygous c 14CoS mice (c 14CoS /c 14CoS orFah −/−). The double mutantFah −/− Hpd −/− mice appeared normal, at least until age 18 months, and there was no evidence of liver disease, findings that facilitated examination of the effect of Fah −/− on mature and unmodified hepatocytes in vivo. The hepatocytes ofFah −/− undergo rapid apoptosis, and acute death follows. Essentially the same phenomena were observed whenFah −/− Hpd −/− mice were administered homogentisate intraperitoneally. These changes in liver pathology in Fah −/− Hpd −/− mice after the administration of homogentisate were associated with massive urinary excretion of succinylacetone. These results suggest that accumulation of fumarylacetoacetate, maleylacetoacetate, or succinylacetone seems to trigger the endogenous process of apoptosis in hepatocytes that lack fumarylacetoacetate hydrolase activity. This apoptosis may be related to the development of hepatocellular carcinomas seen in HT1 patients and pharmaceutically treated fumarylacetoacetate hydrolase-deficient mice.

Structural organization and analysis of the human fumarylacetoacetate hydrolase gene in tyrosinemia type I

Fumarylacetoacetate hydrolase (FAH) is a metabolic enzyme functioning at the last step of tyrosine catabolism. Deficiency in this enzyme activity is associated with tyrosinemia type I, characterized by hypertyrosinemia, liver dysfunction, renal tubular dysfunction, liver cirrhosis, and hepatic tumors. We isolated from a human gene library a chromosomal gene related to FAH. The human FAH gene is 30 kilobases long and is split into 14 exons. All of the splice donor and acceptor sites conform to the GT/AG rule. We also analyzed findings in a patient with tyrosinemia type I with respect to the mutation responsible for defects in the enzyme. A nucleotide change from T to G was found in the exon 2 of the gene and this change was accompanied by an amino acid substitution (Phe62Cys). Transfection and expression analysis of the cDNA in cultured BMT-10 cells with the nucleotide substitution demonstrated that the substitution was indeed responsible for the decreased activity of the enzyme in the patient. These results confirmed that the T to G mutation was one of the causes of tyrosinemia type I. Structure of the FAH gene and tests for expression of the mutant FAH will facilitate further understanding of various aspects of FAH.

Measurement of blood holoceruloplasmin by EIA using a mouse monoclonal antibody directed to holoceruloplasmin. Implication for mass screening of Wilson disease

Wilson disease (McKusick 277900) is an autosomal recessive disorder in which the biliary excretion of copper and its incorporation into ceruloplasmin is impaired (Danks 1989). Copper accumulates in hepatocytes and hepatocellular damage ensues in early childhood. Recently the gene responsible for Wilson disease has been cloned and the primary amino acid sequence of the gene product has been reported (Bull et al 1993). The deduced protein encoded in the gene is homologous to the MNK transporter protein (Bull et al 1993; Tanzi et al 1993), suggesting that the metabolic defect in hepatocytes of Wilson disease patients is at the copper transport step. Hepatocellular damage in untreated patients with the disease is progressive and liver transplantation is required in advanced cases. Administration of chelating agents such as D-penicillamine and trientine tetramine is effective in preventing progression of the liver disease; however, early initiation of treatment is needed to minimize liver damage. The estimated frequency of the disease is approximately one in 3000040000 in Japan (Aoki et al, unpublished) and the disease is probably the most frequent cause of chronic liver disease in children. Owing to the high incidence of the disease and availability of effective treatment, screening procedures for Wilson disease are being discussed (Saito 1981; WHO 1968). The ceruloplasmin protein levels in blood from most patients with Wilson disease are low, although some patients with the disease do have normal levels of ceruloplasmin (Danks 1989). Earlier studies indicated that holoceruloplasmin levels in plasma from patients with the disease were reduced, but the amount of apoprotein was similar to that observed in normal individuals (Matsuda et al 1974). Measurement of holoceruloplasmin in blood seems to be useful for the diagnosis of the majority of patients with the disease. Assay of holoceruloplasmin in blood has been achieved by measuring oxidase activity. However, immunochemical detection of the holoceruloplasmin has not been described. We have developed a mouse monoclonal antibody

Characterization of a point mutation in the pyruvate dehydrogenase E1α gene from two boys with primary lactic acidaemia

SummaryWe report here a novel mutation in the codon for amino acid 263 resulting in the change from arginine to glutamine in the pyruvate dehydrogenase (PDH) E1α gene, in two boys with primary lactic acidaemia, from independent families. The mutation changes an amino acid located between the two serine residues which are the sites of phosphorylation of the subunit protein. In one family, the mutation wasde novo and in the other it was transmitted from mother to son. The amino acid substitution may affect function of the PDH complex via phosphorylation and dephosphorylation of the E1α subunit. Derangement in the regulation of activity of the PDH complex may explain the primary lactic acidaemia in the patients.

Abnormal mRNA and inactive polypeptide in a patient with prolidase deficiency

Prolidase deficiency (McKusick 170100) is an autosomal recessive disorder with a characteristic clinical syndrome that includes chronic dermatitis, mental retardation and recurrent infections (Phang and Scriver, 1989). These symptoms are caused by heterogeneous abnormalities of protidase gene products (Endo et at., 1990). We determined the nucleotide sequence and primary structure of the enzyme (Endo et al., 1989). The prolidase gene consists of 15 exons and 14 introns, the span being over t30 kb (Tanoue et al., 1990a). We have now examined the molecular events in a Japanese patient with potypeptide (cross-reacting material)-negative prolidase deficiency.

Tyrosinaemia type III: Immunochemical studies on 4-hydroxyphenylpyruvic acid dioxygenase and molecular cloning of cDNA for the enzyme

4-Hydroxyphenylpyruvic acid dioxygenase (EC 1.13.tl.24), an enzyme that participates in the catabolism of tyrosine in most organisms, is present in the liver and kidney of mammals. This enzyme catalyses the formation of homogentisic acid from 4-hydroxyphenylpyruvic acid (4HPA), and has been purified from pig liver (Roche et al., 1982), human liver (Lindblad et al., 1977) and avian liver (Wada et al., 1975). The enzyme activity is low or absent in type I tyrosinaemia (McKusick 27670) and type III tyrosinaemia (McKusick 27671). Type I disease is clinically characterized by liver dysfunction and renal tubular defects. Fumarylacetoacetase is defective (Berger et al., 1981) and 4HPA dioxygenase activity is markedly reduced. Type III disease has been attributed to a deficiency in 4HPA dioxygenase in the liver (Endo et al., 1983). While the biochemical and clinical features have been extensively studied in the type I disease, much less is known of these features in type III disease. We discovered a mouse model of hypertyrosinaemia. 4HPA dioxygenase activity in the liver from this strain is virtually absent (Endo et al., 1990). In addition, there is no immunologically detectable 4HPA dioxygenase enzyme protein in the liver from this mouse strain (Endo et al., 1991). Thus, the 4HPA dioxygenase gene is of interest with regard to type I tyrosinaemia and type III tyrosinaemia. To better comprehend structure-function relationships, gene organization and biosynthetic regulatory mechanisms, we attempted to isolate mammalian 4HPA dioxygenase cDNA.