Akira Ohtake

Cloning and sequence analysis of a full length cDNA encoding human mitochodrial 3-oxoacyl-CoA thiolase

The cDNA sequence of human mitochondrial 3-oxoacyl-CoA thiolase was determined. The nucleotide sequence contains an open reading frame of 1191 base pairs and encodes an amino acid sequence of 397 residues which exhibits 86.6% homology with that of the rat enzyme. Northern blot analysis gave a single mRNA species of 1.6 kb in the human liver, fibroblasts and intercostal muscle.

Molecular analysis of holocarboxylase synthetase deficiency: a missense mutation and a single base deletion are predominant in Japanese patients

Holocarboxylase synthetase (HCS) deficiency is an inherited disease of biotin metabolism characterized by a unique pattern of organic aciduria, metabolic acidosis, and skin lesions. By analysis of five patients in four unrelated families, two mutations were identified: a transition from T to C which causes an amino-acid substitution of proline for leucine at position 237 (L237P) and a single deletion of guanine (delG1067) followed by premature termination. One patient was homozygous for the L237P mutation, three patients in two families were compound heterozygotes of the missense and deletion alleles, and the other patient was heterozygous for the L237P mutation. Inheritance was successfully demonstrated in all of the patients families by a modified PCR followed by restriction enzyme digestion. The two mutations accounted for seven of eight mutant alleles, while neither mutation was detected in 108 normal healthy Japanese children (216 alleles). Transient expression in cultured fibroblasts from a patient showed that the L237P mutation was responsible for decreased HCS activity. These results suggest that the L237P and delG1067 mutations are frequent disease-causing mutations in Japanese patients with HCS deficiency. This PCR-based technique may therefore be useful for detecting mutations among Japanese patients.

A female case of ornithine transcarbamylase deficiency with marked computed tomographic abnormalities of the brain

The patient, 2 years and 9 months of age, was referred to our hospital with complaints of frequent vomiting, left hemiconvulsion and deep coma. The serum ammonia level was 251 micrograms/dl. Urine had a high orotate level (3,900 mumol/g creatinine). There was 7% residual of ornithine transcarbamylase (OTC) activity in the liver. Activities of other enzymes of the urea cycle were within normal limits. CT scanning on admission showed diffuse low density of both frontal lobes and of the right temporo-parietal lobe, narrowing of the right lateral ventricle and a shift of the mid-line to the left. The diffuse low density area was not enhanced after contrast medium injection. Follow-up CT scanning showed progressive bilateral ventricular dilatation and cerebral and cerebellar atrophy.

Ornithine transcarbamylase deficiency in spf and spf-ash mice: Genes, mRNAs and mRNA precursors

Two ornithine transcarbamylase-deficient mice are available, the spf with a variant enzyme and spf-ash with a markedly decreased enzyme protein. Genomic DNA, mRNA and the nuclear precursors for the enzyme in these mutants were analyzed. Southern blot analysis of genomic DNA showed no abnormality in the mutant mice. Blot analysis of hepatic mRNA revealed a slight decrease (67% of control) in spf and a marked decrease (12% of control) in spf-ash; no difference in size was found among the control and the mutant mice (about 1.8 kb). Blot analysis of nuclear mRNA precursors (greater than 25, approximately 9.0 and 4.0 kb) showed no significant difference in size and amount among the control, spf and spf-ash. These results suggest that ornithine transcarbamylase deficiency in the spf-ash results from a mutation, which to some extent affects mRNA processing.

Molecular basis of ornithine transcarbamylase deficiency inspf andspf-ash mutant mice

Ornithine transcarbamylase (OTC; ornithine carbamoyltransferase; EC 2.1.3.3) is a mitochondrial matrix enzyme of the urea cycle and is expressed only in the liver and intestinal mucosa. Since the X chromosome carries a gene responsible for OTC expression, its inherited deficiency (McKusick 31125) in man often produces lethal ammonia intoxication in affected males. Female heterozygotes have varying amounts of residual enzyme activity depending on the relative proportions of functionally active X chromosomes bearing the mutant or normal gene. Two mouse OTC mutations, spf(sparse fur) and spf-ash (sparse fur with abnormal skin and hair) have been reported (Doolittle et al.~, 1974; De Mars et al., 1976). Hepatic OTC activity in spf mice is reduced to 10% of control at pH7.4, affinity for ornithine is decreased, the pH optimum is shifted to pH 9.5, and the amount of OTC-related immunoreactive protein is somewhat increased. On the other hand, OTC activity and the amount of immunoreactive protein in spf-ash mice are both 5-10% of control, and affinity for substrates is normal (Briand et al., 1982; Qureshi et al., 1982). Thus, the deficiency in spf mice is of qualitative type, whereas that in spf-ash is of quantitative type. The translatable mRNA in spf mice is reduced to 60% of control, and the increased amount of the enzyme protein is presumably attributable to a diminution of the enzyme degradation. The mRNA activity in spf-ash mice is reduced to the same level (5-10%) of control as the activity and amount of the enzyme, indicating that the decreased enzyme amount is due to decreased synthesis (Briand et aI., 1983; Rosenberg et al., 1983). We report here an analysis of mRNA, its precursors in nuclei, and genomic DNA from these mutant mice, using a cloned rat OTC cDNA as a probe.

Carbamyl phosphate synthetase I deficiency with no detectable mRNA activity

In the autopsied liver of a neonate with carbamyl phosphate synthetase (CPS)-I deficiency, the activity of CPS-I was about 9% of the normal neonatal control. The enzyme protein of CPS-I was hardly detectable by sodium dodecyl sulphate/polyacrylamide gel electrophoresis (SDS/PAGE) and an immunoblotting method using anti-rat liver CPS-I. The level of translatable mRNA for CPS-I was markedly decreased in a cell-free protein synthesis system consisting of rabbit reticulocyte lysate and total RNA extracted from the autopsied liver of the patient. These observations indicate that the enzyme deficiency in this case is probably mainly due to a diminished level of translatable mRNA, which would lead to a decrease in the synthesis of the CPS-I precursor.

NIEMANN-PICK DISEASE ASSOCIATED WITH LIVER DISORDERS

We report a case of Niemann‐Pick disease (NPD) with accumulation of sphingomyelin in reticuloendothelial system (RES), hepatocellular giant cell transformation (GCT), cirrhosis, and multiple hepatocellular adenomata in a 19‐month‐old girl. GCT, but no NP‐cells, were seen at age 3 months by biopsy. Cirrhosis and hepatocellular adenomata were demonstrated in the liver at 19 months of age. Cytoplasmic, probably locally synthesized, globules of alpha‐1‐antitrypsin (A‐l‐AT) were accumulated in the hepatocellular adenomata. A‐l‐AT and alpha‐fetoprotein (AFP) were present in the serum.

A Carbamyl Phosphate Synthetase I Deficiency with No Detectable Messenger RNA Activity

Carbamyl phosphate synthetase I (CPS) (EC 6.3.4.16.) is located in the mitochondria1 matrix of hepatocytes and catalyzes the first step of urea synthesis. Inherited deficiency of CPS is a rare cause of hyperammonemia and protein intolerance. Although data on several patients with this disorder have been reported [ 1-31 , there is no report which refers to the molecular mechanism responsible for CPS deficiency, except for one on the different forms of the deficiency (with and without enzyme protein [ 41 ). We now report our findings in two female siblings with complete CPS deficiency with no detectable immunoreactive enzyme and no messenger RNA (mRNA) activity.

A Case With the Infantile Type of Glycerol Kinase Deficiency

A male infant with the infantile type of glycerol kinase deficiency is described. At six years of age, he showed proximal dominant muscle atrophy and weakness, addisonian pigmentation and mental retardation. Laboratory investigations revealed muscular dystrophy, adrenal insufficiency and glycerol kinase deficiency. He has a small deletion in a band (Xp21) of the X chromosome. The clinical, biochemical and genetic findings in this patient are reported.

Ornithine transcarbamylase deficiency: a case with a truncated enzyme precursor and a case with undetectable mRNA activity

The cell-free translation of ornithine transcarbamylase (OTC) mRNA from the livers of two heterozygous patients (from different families) with OTC deficiency was performed. The enzyme activities and the immunoreactive proteins in both patients were about 5% of those in controls. Immunoblotting assay of liver extracts from both patients showed decreased amounts of the OTC protein. The mRNA from the liver of patient 1 directed the synthesis of a very small amount of OTC precursor of normal subunit size (40 000 Da), whereas that from patient 2 directed the synthesis of small amounts of two distinctin vitro products; one was 40 000 Da and the other was about 30 000 Da. Thein vitro product of normal precursor synthesized with mRNA from patient 2 was converted to mature-sized OTC by isolated rat liver mitochondria, whereas the smaller product was degraded during the incubation with the mitochondria. These results indicate that in both patients the translatable level of mRNA for active OTC from liver cells was much lower than that in the controls. The results also suggest that in patient 2, the smaller product presumably derived from an abnormal gene could not be transferred to the mitochondria.