CRISPR correction of the Finnish ornithine delta-aminotransferase mutation restores metabolic homeostasis in iPSC from patients with gyrate atrophy.
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ABSTRACT:
SUBMITTER: Maldonado R
PROVIDER: S-EPMC9248217 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Gyrate atrophy of the choroid and retina (GACR) secondary to deficiency of ornithine aminotransferase (OAT) is a rare autosomal recessive metabolic disorder usually diagnosed in childhood when patients develop myopia and a characteristic retinal degeneration accompanied by hyperornithinemia. Plasma ammonia is normal or sub-normal after the neonatal period. A few GACR patients present in early infancy with hyperammonemia, encephalopathy and a biochemical profile of low plasma ornithine, citrulline and arginine, with increased urinary excretion of homocitrulline and orotic acid, resembling a primary urea cycle disorder. In these patients, ornithine levels do not increase until late infancy or following arginine or citrulline supplementation. We describe a patient with OAT deficiency who presented in the first month of life with episodes of lethargy, vomiting, and hypothermia. He had two episodes of hyperammonemia associated with subnormal levels of plasma ornithine, citrulline and arginine as well as elevated urinary excretion of homocitrulline and orotic acid. Unlike previously reported cases, intermittent hyperornithinemia was observed prior to the first hyperammonemic episode and citrulline supplementation. The latter alleviated the symptoms, normalized ammonia level, and led to increased plasma ornithine concentration. Furthermore, despite a protein restricted diet and ammonia scavenger treatment, continued supplemental citrulline was necessary to prevent hyperammonemia. Molecular analysis confirmed OAT deficiency, differentiating it from proximal urea cycle disorders and deficiency of the mitochondrial ornithine transporter, ORC1, (Hyperammonemia-Hyperornithinemia-Homocitrullinuria syndrome).SynopsisHyperornithinemia alternating with hypoornithinemia and hyperammonemia in a neonatal-onset case of gyrate atrophy with ornithine aminotransferase deficiency.
| S-EPMC9248225 | biostudies-literature
Project description:The deficit of human ornithine aminotransferase (hOAT) is responsible for gyrate atrophy (GA), a rare recessive inherited disorder. Although more than 60 disease-associated mutations have been identified to date, the molecular mechanisms explaining how each mutation leads to the deficit of OAT are mostly unknown. To fill this gap, we considered six representative missense mutations present in homozygous patients concerning residues spread over the hOAT structure. E. coli expression, spectroscopic, kinetic and bioinformatic analyses, reveal that the R154L and G237D mutations induce a catalytic more than a folding defect, the Q90E and R271K mutations mainly impact folding efficiency, while the E318K and C394Y mutations give rise to both folding and catalytic defects. In a human cellular model of disease folding-defective variants, although at a different extent, display reduced protein levels and/or specific activity, due to increased aggregation and/or degradation propensity. The supplementation with Vitamin B6, to mimic a treatment strategy available for GA patients, does not significantly improve the expression/activity of folding-defective variants, in contrast with the clinical responsiveness of patients bearing the E318K mutation. Thus, we speculate that the action of vitamin B6 could be also independent of hOAT. Overall, these data represent a further effort toward a comprehensive analysis of GA pathogenesis at molecular and cellular level, with important relapses for the improvement of genotype/phenotype correlations and the development of novel treatments.
| S-EPMC8360850 | biostudies-literature
Project description:Deficit of human ornithine aminotransferase (hOAT), a mitochondrial tetrameric pyridoxal-5'-phosphate (PLP) enzyme, leads to gyrate atrophy of the choroid and retina (GA). Although 70 pathogenic mutations have been identified, only few enzymatic phenotypes are known. Here, we report biochemical and bioinformatic analyses of the G51D, G121D, R154L, Y158S, T181M, and P199Q pathogenic variants involving residues located at the monomer-monomer interface. All mutations cause a shift toward a dimeric structure, and changes in tertiary structure, thermal stability, and PLP microenvironment. The impact on these features is less pronounced for the mutations of Gly51 and Gly121 mapping to the N-terminal segment of the enzyme than those of Arg154, Tyr158, Thr181, and Pro199 belonging to the large domain. These data, together with the predicted ΔΔG values of monomer-monomer binding for the variants, suggest that the proper monomer-monomer interactions seem to be correlated with the thermal stability, the PLP binding site and the tetrameric structure of hOAT. The different impact of these mutations on the catalytic activity was also reported and discussed on the basis of the computational information. Together, these results allow the identification of the molecular defects of these variants, thus extending the knowledge of enzymatic phenotypes of GA patients.
| S-EPMC9967328 | biostudies-literature
Project description:PurposeIn humans, deficiency of ornithine-δ-aminotransferase (OAT) results in progressive degeneration of the neural retina (gyrate atrophy) with blindness in the fourth decade. In this study, we used the Xenopus embryonic developmental model to study functions of the OAT gene on embryonic development.MethodsWe cloned and sequenced full-length OAT cDNA from Xenopus oocytes (X-OAT) and determined X-OAT expression in various developmental stages of Xenopus embryos and in a variety of adult tissues. The phenotype, gene expression of neural developmental markers, and enzymatic activity were detected by gain-of-function and loss-of-function manipulations.ResultsWe showed that X-OAT is essential for Xenopus embryonic development, and overexpression of X-OAT produces a ventralized phenotype characterized by a small head, lack of axial structure, and defective expression of neural developmental markers. Using X-OAT mutants based on mutations identified in humans, we found that substitution of both Arg 180 and Leu 402 abrogated both X-OAT enzymatic activity and ability to modulate the developmental phenotype. Neurogenesis is inhibited by X-OAT during Xenopus embryonic development.ConclusionsNeurogenesis is inhibited by X-OAT during Xenopus embryonic development, but it is essential for Xenopus embryonic development. The Arg 180 and Leu 402 are crucial for these effects of the OAT molecule in development.
| S-EPMC4554259 | biostudies-literature
Project description:This review deals with biochemical and physiological aspects of plant ornithine d-aminotransferase (OAT, EC 2.6.1.13). OAT is a mitochondrial enzyme containing pyridoxal-5'-phosphate as a cofactor, which catalyzes the conversion of L-ornithine to L-glutamate gamma-semialdehyde using 2-oxoglutarate as a terminal amino group acceptor. It has been described in humans, animals, insects, plants and microorganisms. Based on the crystal structure of human OAT, both substrate binding and reaction mechanism of the enzyme are well understood. OAT shows a large structural and mechanistic similarity to other enzymes from the subgroup III of aminotransferases, which transfer an amino group from a carbon atom that does not carry a carboxyl function. In plants, the enzyme has been implicated in proline biosynthesis and accumulation (via pyrroline-5-carboxylate), which represents a way to regulate cellular osmolarity in response to osmotic stress. However, the exact metabolic pathway involving OAT remains a subject of controversy.
| S-EPMC2633738 | biostudies-literature
Project description:Human ornithine δ-aminotransferase (hOAT) (EC 2.6.1.13) is a mitochondrial pyridoxal 5'-phosphate (PLP)-dependent aminotransferase whose deficit is associated with gyrate atrophy, a rare autosomal recessive disorder causing progressive blindness and chorioretinal degeneration. Here, both the apo- and holo-form of recombinant hOAT were characterized by means of spectroscopic, kinetic, chromatographic and computational techniques. The results indicate that apo and holo-hOAT (a) show a similar tertiary structure, even if apo displays a more pronounced exposure of hydrophobic patches, (b) exhibit a tetrameric structure with a tetramer-dimer equilibrium dissociation constant about fivefold higher for the apoform with respect to the holoform, and (c) have apparent Tm values of 46 and 67 °C, respectively. Moreover, unlike holo-hOAT, apo-hOAT is prone to unfolding and aggregation under physiological conditions. We also identified Arg217 as an important hot-spot at the dimer-dimer interface of hOAT and demonstrated that the artificial dimeric variant R217A exhibits spectroscopic properties, Tm values and catalytic features similar to those of the tetrameric species. This finding indicates that the catalytic unit of hOAT is the dimer. However, under physiological conditions the apo-tetramer is slightly less prone to unfolding and aggregation than the apo-dimer. The possible implications of the data for the intracellular stability and regulation of hOAT are discussed.
| S-EPMC5432616 | biostudies-literature
Project description:BackgroundLike many other plant species, Arabidopsis uses arginine (Arg) as a storage and transport form of nitrogen, and proline (Pro) as a compatible solute in the defence against abiotic stresses causing water deprivation. Arg catabolism produces ornithine (Orn) inside mitochondria, which was discussed controversially as a precursor for Pro biosynthesis, alternative to glutamate (Glu).ResultsWe show here that ornithine-delta-aminotransferase (deltaOAT, At5g46180), the enzyme converting Orn to pyrroline-5-carboxylate (P5C), is localised in mitochondria and is essential for Arg catabolism. Wildtype plants could readily catabolise supplied Arg and Orn and were able to use these amino acids as the only nitrogen source. Deletion mutants of deltaOAT, however, accumulated urea cycle intermediates when fed with Arg or Orn and were not able to utilize nitrogen provided as Arg or Orn. Utilisation of urea and stress induced Pro accumulation were not affected in T-DNA insertion mutants with a complete loss of deltaOAT expression.ConclusionOur findings indicate that deltaOAT feeds P5C exclusively into the catabolic branch of Pro metabolism, which yields Glu as an end product. Conversion of Orn to Glu is an essential route for recovery of nitrogen stored or transported as Arg. Pro biosynthesis occurs predominantly or exclusively via the Glu pathway in Arabidopsis and does not depend on Glu produced by Arg and Orn catabolism.
| S-EPMC2377265 | biostudies-literature
Project description:Gene-corrected patient-specific induced pluripotent stem (iPS) cells offer a novel approach to gene therapy. Yet it is unknown whether selective pressures during prolonged culture and multiple clonal events would introduce a mutational load incompatible with therapeutic use. Here we begin to assess whether the mutational load of gene-corrected iPS cells is compatible with use in the treatment of genetic causes of retinal degenerative disease. We isolated iPS cells free of transgene sequences from a patient with gyrate atrophy caused by a point mutation in the gene encoding ornithine-δ-aminotransferase (OAT) and used homologous recombination to correct the genetic defect. Cytogenetic analysis, array comparative genomic hybridization (aCGH), and exome sequencing were performed to assess the genomic integrity of an iPS cell line after three sequential clonal events: initial reprogramming, gene targeting, and subsequent removal of a selection cassette. No abnormalities were detected following standard G-band metaphase analysis. However, aCGH and exome sequencing identified two deletions, one amplification, and nine point mutations in protein-coding regions in the initial iPS cell clone. Except for the targeted correction of the single nucleotide in the OAT locus and a single synonymous base pair change, no additional mutations or copy number variation were identified in iPS cells following the two subsequent clonal events. These findings confirm that iPS cells themselves may carry a significant mutational load at initial isolation, but that the clonal events and prolonged cultured required for correction of a genetic defect can be accomplished without a substantial increase in mutational burden.
2011-03-01 | GSE26773 | GEO
Project description:Gene-corrected patient-specific induced pluripotent stem (iPS) cells offer a unique approach to gene therapy. Here, we begin to assess whether the mutational load acquired during gene correction of iPS cells is compatible with use in the treatment of genetic causes of retinal degenerative disease. We isolated iPS cells free of transgene sequences from a patient with gyrate atrophy caused by a point mutation in the gene encoding ornithine-?-aminotransferase (OAT) and used homologous recombination to correct the genetic defect. Cytogenetic analysis, array comparative genomic hybridization (aCGH), and exome sequencing were performed to assess the genomic integrity of an iPS cell line after three sequential clonal events: initial reprogramming, gene targeting, and subsequent removal of a selection cassette. No abnormalities were detected after standard G-band metaphase analysis. However, aCGH and exome sequencing identified two deletions, one amplification, and nine mutations in protein coding regions in the initial iPS cell clone. Except for the targeted correction of the single nucleotide in the OAT locus and a single synonymous base-pair change, no additional mutations or copy number variation were identified in iPS cells after the two subsequent clonal events. These findings confirm that iPS cells themselves may carry a significant mutational load at initial isolation, but that the clonal events and prolonged cultured required for correction of a genetic defect can be accomplished without a substantial increase in mutational burden.
| S-EPMC3080993 | biostudies-literature
Project description:Gene-corrected patient-specific induced pluripotent stem (iPS) cells offer a novel approach to gene therapy. Yet it is unknown whether selective pressures during prolonged culture and multiple clonal events would introduce a mutational load incompatible with therapeutic use. Here we begin to assess whether the mutational load of gene-corrected iPS cells is compatible with use in the treatment of genetic causes of retinal degenerative disease. We isolated iPS cells free of transgene sequences from a patient with gyrate atrophy caused by a point mutation in the gene encoding ornithine-δ-aminotransferase (OAT) and used homologous recombination to correct the genetic defect. Cytogenetic analysis, array comparative genomic hybridization (aCGH), and exome sequencing were performed to assess the genomic integrity of an iPS cell line after three sequential clonal events: initial reprogramming, gene targeting, and subsequent removal of a selection cassette. No abnormalities were detected following standard G-band metaphase analysis. However, aCGH and exome sequencing identified two deletions, one amplification, and nine point mutations in protein-coding regions in the initial iPS cell clone. Except for the targeted correction of the single nucleotide in the OAT locus and a single synonymous base pair change, no additional mutations or copy number variation were identified in iPS cells following the two subsequent clonal events. These findings confirm that iPS cells themselves may carry a significant mutational load at initial isolation, but that the clonal events and prolonged cultured required for correction of a genetic defect can be accomplished without a substantial increase in mutational burden. Array comparative genomic hybridization (aCGH) were performed to assess the genomic integrity of an iPS cell line after three sequential clonal events: initial reprogramming, gene targeting, and subsequent removal of a selection cassette. Human CGH 2.1M Whole-Genome Tiling v2.0D Array plus 4633 custom-designed probes which cover the gene targeting construct and the reprogramming plasmid vectors. Comparison of patient iPS cell with full skin fibroblast cells was performed on this design.
2011-03-01 | E-GEOD-26773 | biostudies-arrayexpress