Nonsense-codon mutations of the ornithine aminotransferase gene with decreased levels of mutant mRNA in gyrate atrophy.

A generalized deficiency of the mitochondrial matrix enzyme ornithine aminotransferase (OAT) is the inborn error in gyrate atrophy (GA), an autosomal recessive degenerative disease of the retina and choroid of the eye. Mutations in the OAT gene show a high degree of molecular heterogeneity in GA, reflecting the genetic heterogeneity in this disease. Using the combined techniques of PCR, denaturing gradient gel electrophoresis, and direct sequencing, we have identified three nonsense-codon mutations and one nonsense codon-generating mutation of the OAT gene in GA pedigrees. Three of them are single-base substitutions, and one is a 2-bp deletion resulting in a reading frameshift. A nonsense codon created at position 79 (TGA) by a frameshift and nonsense mutations at codons 209 (TAT----TAA) and 299 (TAC----TAG) result in abnormally low levels of OAT mRNA in the patient's skin fibroblasts. A nonsense mutation at codon 426 (CGA----TGA) in the last exon, however, has little effect on the mRNA level. Thus, the mRNA level can be reduced by nonsense-codon mutations, but the position of the mutation may be important, with earlier premature-translation termination having a greater effect than a later mutation.     

Splicing defect at the ornithine aminotransferase (OAT) locus in gyrate atrophy.

Gyrate atrophy (GA), a recessive eye disease involving progressive vision loss due to chorioretinal degeneration, is associated with the deficiency of the mitochondrial enzyme ornithine aminotransferase (OAT), with consequent hyperornithinemia. We and others have reported a number of missense mutations at the OAT locus which result in GA. Here we report a GA patient of Danish/Swedish ancestry in whom one OAT allele produces an mRNA that is missing a single 96-bp exon relative to the normal mRNA. Polymerase-chain-reaction amplification and sequencing revealed a 9-bp deletion covering the splice acceptor region of exon 5, resulting in the absence of exon 5 sequences from the mRNA with no disruption to the reading frame. This mutation, which was not present in 15 other independent GA patients, adds to the array of allelic heterogeneity observed in GA and represents the first example of a splicing mutation associated with this disorder.     

The ornithine aminotransferase (OAT) locus: analysis of RFLPs in gyrate atrophy.

A cDNA probe (HOAT1) for ornithine aminotransferase (OAT) has recently been used to map (1) the structural gene for this enzyme to chromosome 10 and (2) several related DNA sequences to the X chromosome. We have defined six RFLPs for OAT, to explore its possible role in gyrate atrophy (GA) of the choroid and retina, an autosomal recessive genetic disorder associated with a deficiency of OAT activity. The RFLPs, which are detected by noncoding single-copy probes from the OAT gene and by subclones of the HOAT1 cDNA, all map on human chromosome 10, producing an overall level of heterozygosity for the OAT locus of 83%. Using the RFLPs, we have determined that the OAT locus segregates concordantly with GA in one available pedigree. Furthermore, the RFLPs display significant disequilibrium with GA, providing genetic evidence implicating a defect in the OAT structural gene as the cause of this disorder. The RFLPs for OAT are potentially applicable to prenatal diagnosis and carrier detection in families with a previous history of GA. They will also allow identification of specific haplotypes associated with GA chromosomes, as a guide for more detailed molecular-genetic investigations of the mutations underlying the disorder.     

A single-base change at a splice acceptor site in the ornithine aminotransferase gene causes abnormal RNA splicing in gyrate atrophy

Gyrate atrophy (GA) is an autosomal recessive eye disease involving a progressive loss of vision due to chorioretinal degeneration in which the mitochondrial matrix enzyme ornithine aminotransferase (OAT) is defective. Two sisters with GA are described in this study in whom an A-to-G substitution at the 3 splice acceptor site of intron 4 in one allele of the OAT gene results in a truncated OAT mRNA devoid of exon 5 sequence. The mutation in the other allele was identified to be a missense mutation at codon 318 by denaturing gradient gel electrophoresis and direct sequencing of the polymerase chain reaction (PCR)-amplified DNA. Thus, these GA patients are compound heterozygotes with respect to mutations in the OAT gene that result in inactivation of OAT.     

Inheritance of ornithine aminotransferase gene, mRNA, and enzyme defect in a family with gyrate atrophy of the choroid and retina.

We studied the human ornithine aminotransferase (OAT) gene, mRNA, and enzyme activity in fibroblasts from a family with gyrate atrophy (G.A.) of the choroid and retina, using a normal human OAT cDNA as a probe. The family consists of an affected patient, who is heterozygous for a partial deletion of the functional OAT gene and whose cells produce no mRNA, and of his father, mother, two sons, and a daughter. Southern blot analysis of the OAT gene showed the partial deletion in the patient and in his father and daughter and in one son. Northern blot analysis revealed no OAT mRNA in the patient and approximately 50% of the normal level of OAT mRNA in the father, mother, two sons, and daughter. Assay showed that the OAT activity in these individuals mirrored the OAT mRNA levels. The results indicate that an active allele of the OAT gene expresses 50% of the total normal OAT mRNA and activity and that both alleles of the gene are inactive in the patient in this pedigree, a situation resulting in a complete absence of the OAT mRNA, in accordance with the autosomal recessive mechanism of this disease; they also indicate a 50% decrease of OAT mRNA and enzyme activity in obligate heterozygous carriers carrying one defective allele and that these defects are stably inherited.     

The ornithine aminotransferase gene in gyrate,atrophy of the retina: Analysis of expression and gross structure of this gene in cultured fibroblasts

Summary Gyrate atrophy (GA), a degenerative disease of the human chorioretina, is associated with a deficiency of ornithine aminotransferase (OAT) activity, hyperornithinemia, and ornithinuria. We have characterized a cDNA clone for OAT (HLOAT) that was isolated from a cDNA library constructed from mRNA prepared from Hep G2, cells, a human hepatoma cell line. We have used HLOAT and a nearly full length OAT cDNA clone isolated from, a rat liver library (RLOAT) to examine in cultured fibroblasts from individuals with GA and control individuals, the expression of OAT mRNA and the gross structure of the OAT gene. Northern blot analyses of total cellular RNA indicated that 3 of 3 control cell lines and 5 of 6 GA cell lines are capable of expressing an OAT related mRNA of approximately 2100 bases, the size of OAT mRNA. To date, this is the only case of GA in which a complete lack of OAT mRNA has been observed. Southern blot analyses of DNA isolated from these cell lines indicated that the gross structure of the OAT gene is usually not detectably altered in individuals with GA. However, a unique pattern, of restriction fragments was observed upon digestion with Eco RI or Hind III of DNA from the GA cell line that does not express OAT mRNA. These unique Eco RI and Hind III fragments arise from the OAT structural gene and will serve as useful molecular markers that allow this particular defective OAT allele to be identified. When the cellular DNAs were digested with Hinf I and examined with a probe that corresponds to at least a portion of the active site of the enzyme, i. e., the pyridoxal phosphate binding site, identical patterns of fragments were detected in all samples. Therefore, it appears unlikely that the loss of OAT activity associated with these GA cases, 4 of which are pyridoxal phosphate responders, is the result of insertions or deletions in this region of the OAT gene. This study indicates that the lack of OAT enzyme activity associated with GA is the result of a variety of different molecular defects within the OAT gene. This project was initiated in the laboratory of H. C. P. and was supported by grants CA07175, CA22484, and 5 T32 CA09020 from the National Cancer Institute and Postdoctoral Fellowship PF-2414 from the American Cancer Society. The continuing work in the laboratory of J. D. S. was supported by grants CA36727 and HD24189 from the National, Institutes of Health, grants SIG-16, ACS-IN165A, and a Junior Faculty Research Award (JFRA-227) from the American Cancer Society, and by University of Nebraska Medical Center Seed Research Grant 88-10.     

The reaction of ornithine aminotransferase with ornithine.

Rat liver ornithine aminotransferase is found to exchange the pro-S hydrogen on the delta-carbon atom of ornithine exclusively, thus showing that the mammalian enzyme transfers the delta-amino group and not the alpha-amino group as has been demonstrated with the plant enzyme [Mestichelli, Gupta & Spenser (1979) J. Biol. Chem. 254, 640-647]. The enzyme also transfers the alpha-amino group of glutamate and the kinetics of the half reactions between the enzyme and both amino acids are compared. Rate and dissociation constants for both reactions are determined.     

Deficient L-ornithine: 2-oxoacid aminotransferase activity in cultured fibroblasts from a patient with gyrate atrophy of the retina

Gyrate atrophy of the retina is an autosomal recessive condition characterized by a specific progressive retinal degeneration and increased urine and plasma ornithine. This study demonstrates a deficiency of L-ornithine: 2-oxoacid aminotransferase (EC 2.6.1.13) activity in cultured fibroblasts from a patient with this disease.     

Gyrate atrophy of the choroid and retina: assignment of the ornithine aminotransferase structural gene to human chromosome 10 and mouse chromosome 7.

Gyrate atrophy of the choroid and retina is an autosomal recessive, blinding human disease caused by a deficiency of the mitochondrial matrix enzyme ornithine aminotransferase (OAT). Since human OAT cDNA hybridizes to DNA sequences on both human chromosomes 10 and X, a locus coding for OAT enzyme activity may be present on one or both of these human chromosomes. We have used a series of mouse-human somatic cell hybrids, in combination with starch gel electrophoresis and a histochemical stain for OAT enzyme activity, to assign the structural gene for OAT to human chromosome 10. Our results suggest that the human X chromosome does not contain a locus coding for OAT enzyme activity. In addition, we have used a panel of Chinese hamster-mouse hybrids to assign the murine Oat structural gene to mouse chromosome 7. Our findings, combined with recent molecular studies, indicate that human OAT probes specific for chromosome 10 will be useful for the diagnosis and genetic counseling of individuals at risk for gyrate atrophy.     

Pyridoxine effects on ornithine ketoacid transaminase activity in fibroblasts from carriers of two forms of gyrate atrophy of the choroid and retina.

Gyrate atrophy of the choroid and retina that is due to ornithine ketoacid transaminase (OKT) deficiency is an autosomal recessive disorder. Fibroblasts from heterozygotes for the pyridoxine-responsive variant as well as those for the pyridoxine-nonresponsive variant contain intermediate levels of OKT activity. These two variants can be distinguished by the in vitro responsiveness of OKT activity to pyridoxal phosphate (PLP) stimulation. The ratios of OKT activity at 0.04 mM PLP compared with activity at 0 mM PLP were, respectively, lowest for controls (1.18 +/- 0.18; N = 12), intermediate for pyridoxine-nonresponsive heterozygotes (1.43 +/- 0.26; N = 5), and highest for pyridoxine-responsive heterozygotes (2.20 +/- 0.14; N = 3).     

Ornithine ketoacid transaminase deficiency in gyrate atrophy of the choroid and retina.

Gyrate atrophy of the choroid and retina is a chorioretinal degeneration associated with hyperornithinemia with an autosomal recessive mode of inheritance. Cultured skin fibroblasts from five affected patients showed a virtual absence of ornithine ketoacid transaminase (OKT) (L-ornithine:2-oxoacid aminotransferase E.C.2.6.1.13) activity. Fibroblasts from four carrier parents showed a 42%-65% reduction in OKT activity. Increasing the concentration of pyridoxal phosphate (vitamin B6 in the assay media resulted in partial restoration of OKT activity in fibroblasts from one out of five patients studied. We conclude that OKT deficiency is closely associated with the genetic defect in gyrate atrophy of the choroid and retina and that genetic heterogeneity exists in this disease.     

Ornithine delta-aminotransferase mutations in gyrate atrophy. Allelic heterogeneity and functional consequences.

Ornithine delta-aminotransferase is a nuclear-encoded mitochondrial matrix enzyme which catalyzes the reversible interconversion of ornithine and alpha-ketoglutarate to glutamate semialdehyde and glutamate. Inherited deficiency of ornithine delta-aminotransferase results in ornithine accumulation and a characteristic chorioretinal degeneration, gyrate atrophy of the choroid and retina. We have surveyed the ornithine delta-aminotransferase genes of gyrate atrophy patients for mutations. Using a variety of techniques, we discovered and molecularly characterized 21 newly recognized ornithine delta-aminotransferase alleles. We determined the consequences of these and three previously described mutations on ornithine delta-aminotransferase mRNA, antigen, and enzyme activity in cultured fibroblasts. The majority (20/24) of these alleles produce normal amounts of normally sized ornithine delta-aminotransferase mRNA. By contrast, only 2/24 had normal amounts of ornithine delta-aminotransferase antigen. Reproducing these mutations by site-directed mutagenesis and expressing the mutant ornithine delta-aminotransferase in Chinese hamster ovary cells confirms that several of these mutations inactivate ornithine delta-aminotransferase and cause gyrate atrophy in these patients.     

Localization of the ornithine aminotransferase gene and related sequences on two human chromosomes

Summary We have used a full length cDNA clone to determine the chromosomal location ofthegene encoding human ornithine aminotransferase (OAT), a mitochondrial matrix enzyme. Southern blot analysis of ScaI-digested DNA from 34 human-mouse somatic cell hybrids revealed 11 human fragments. Three fragments mapped to chromosome 10q23-10qter, confirming the previous provisional assignment of the functional gene to this autosome by analysis of OAT expression in somatic cell hybrids (O'Donnell et al. 1985). The remaining eight fragments were assigned to the X chromosome, and regionally assigned to Xp21-Xp11 by use of an X-chromosome mapping panel. These X chromosome sequences could represent pseudogenes, or related members of a multigene family. Two of the X chromosome fragments are alternate alleles of a restriction fragment length polymorphism (RFLP) making this OAT-related locus an excellent genetic marker. The RFLP may now be used to determine any possible relationship between this locus and several X-linked eye defects.     

Activities of ornithine aminotransferase and ornithine decarboxylase in chronically uremic rats

The activities of two enzymes mediating different pathways of ornithine catabolism were measured in liver and kidney of chronically uremic rats and their pair-fed controls. Two months following partial nephrectomy hepatic ornithine aminotransferase (OAT) activity tended to be lower in uremic rats and was correlated with urea clearance and with carbamoyl phosphate synthetase activity. Renal OAT activity in uremic rats was also correlated with urea clearance. When uremic rats were maintained for five months, OAT activity was significantly decreased in liver but not in kidney and the activity of ornithine decarboxylase (ODC), the enzyme regulating polyamine biosynthesis, was reduced in both liver and kidney. In cross-over experiments, evidence was obtained for a factor in uremic kidney cytosol which inhibited renal ODC activity.     

The regulation of ornithine aminotransferase synthesis by glucagon in the rat.

Ornithine aminotransferase (OAT) from rat liver mitochondria was purified to homogeneity. A monospecific antiserum against the enzyme protein was prepared in rabbits. Immunotitrations were performed on OAT present in crude mitochondrial extracts obtained from the livers and kidneys of rats in several hormonal and dietary states. No evidence was found for the existence of an immunologically reactive but enzymatically inactive form of OAT. The relative rate of enzyme synthesis in vivo was studied by pulselabeling rats with [4, 5-³H]leucine, isolating the enzyme protein by immunoprecipitation, and dissociating the immunoprecipitates on sodium dodecyl sulfate-acrylamide gels. Nine hours after a single subcutaneous injection of a glucagon oil emulsion, a 3-fold increase in OAT activity and a 12-fold increase in the synthetic rate of the enzyme were observed. Serine dehydratase activity increased on a time-course very similar to that of OAT following glucagon injection. These increases occurred only on low (0–12.5%) protein diets. At higher levels of dietary protein (30% and up), no further stimulation of OAT synthesis by glucagon was observed. Administration of actinomycin D within the first 2 h after glucagon injection resulted in an inhibition of OAT induction. When the administration of the antibiotic was delayed until 4 h after glucagon, no inhibition of OAT induction was observed. Glucose repression of the glucagon induction of the enzyme in hepatic mitochondria was demonstrated to be the result of a rapid inhibition of OAT synthesis.