Cloning and characterization of human ORNT2: a second mitochondrial ornithine transporter that can rescue a defective ORNT1 in patients with the hyperornithinemia–hyperammonemia–homocitrullinuria syndrome, a urea cycle disorder

Camacho, Jose A.; Rioseco-Camacho, Natalia; Andrade, Darío; Porter, John; Kong, Jin

doi:10.1016/s1096-7192(03)00105-7

Cited by 37 publications

(54 citation statements)

References 41 publications

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“…Primers were designed to amplify the exon-intron regions covering the ORNT1 open reading frame (ORF), exons 2 through 7, based on genomic clone AL161614 and previously published reports (4,6,7). Results of our scan uncovered a homozygous missense mutation, T32R, in exon 3.…”

Section: Methodsmentioning

confidence: 99%

“…To confirm that ORNT1-T32R was a mutation not a polymorphism, we screened exon 3 of the ORNT1 gene of 116 control individuals from different ethnic backgrounds (25 Mayan Indian, 28 Spanish, 35 French Canadian, 15 U.S. Hispanic, 8 Caucasian, and 5 African American). The primers and PCR conditions for amplification and sequencing of the human ORNT2 ORF have been previously published (6). Sequences of all new primers used in this study are presented in Table 2.…”

Section: Methodsmentioning

confidence: 99%

“…Total RNA from untransformed cultured control and HHH fibroblasts (HHH015) plus EBV-transformed lymphocytes (HHH013 and HHH015) was isolated as previously reported (4,6). We then isolated poly A ϩ RNA from total RNA utilizing the Oligotex mRNA mini kit (QIAGEN, Valencia, CA).…”

Section: Methodsmentioning

confidence: 99%

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Clinical and Functional Characterization of a Human ORNT1 Mutation (T32R) in the Hyperornithinemia-Hyperammonemia-Homocitrullinuria (HHH) Syndrome

et al. 2006

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ABSTRACT:We studied two related families (HHH013 and HHH015) with the hyperornithinemia-hyperammonemia-homocitrullinuria (HHH) syndrome, a disorder of the urea cycle and ornithine degradation pathway, who have the same novel ornithine transporter (ORNT1) genotype (T32R) but a variable phenotype. Both HHH015 patients are doing well in school and are clinically stable; conversely, the three affected HHH013 siblings had academic difficulties and one suffered recurrent episodes of hyperammonemia and ultimately died. Overexpression studies revealed that the product of the ORNT1-T32R allele has residual function. Ornithine transport studies in HHH015 fibroblasts, however, showed basal activity similar to fibroblasts carrying nonfunctional ORNT1 alleles. We also examined two potential modifying factors, the ORNT2 gene and the mitochondrial DNA lineage (haplogroup). Haplogroups, associated with specific diseases, are hypothesized to influence mitochondrial function. Results demonstrated that both HHH015 patients are heterozygous for an ORNT2 gain of function polymorphism and belong to haplogroup A whereas the HHH013 siblings carry the wild-type ORNT2 and are haplogroup H. These observations suggest that the ORNT1 genotype cannot predict the phenotype of HHH patients. The reason for the phenotypic variability is unknown, but factors such as redundant transporters and mitochondrial lineage may contribute to the neuropathophysiology of HHH patients. T he HHH syndrome (OMIM #238970) is an autosomal recessive disorder of the urea cycle and ornithine degradation pathway caused by the deficient transport of ornithine across the inner mitochondrial membrane (1,2). The gene defective in HHH syndrome is the mitochondrial ornithine transporter (ORNT1) that is localized in the q14.1 region of Ch13 and is a member of the MCF of proteins that includes the uncoupling protein, carnitine/acyl-carnitine translocase, and the ADP/ATP transporter (3,4). The human ORNT1 gene is expressed in the periportal hepatocytes, which contain the urea cycle pathway, and in the pericentral hepatocytes and skin fibroblasts that express the ornithine degradation pathway (1,2). Physiologically, ORNT1 allows ornithine to serve as a substrate for the ornithine transcarbamylase (OTC) and ornithine amino transferase (OAT) reactions that produce citrulline and the two amino acids, glutamate and proline. In vitro studies have demonstrated that ORNT1 transports ornithine, lysine, and arginine across the inner mitochondrial membrane in exchange for a hydrogen ion and citrulline (5).Biochemically, HHH syndrome is characterized by persistent elevation of plasma ornithine, episodic or postprandial hyperammonemia, and the urinary excretion of homocitrulline and orotic acid (1). The homocitrulline is believed to be the product of transcarbamoylation of lysine whereas the orotic aciduria occurs secondary to decreased OTC activity. Numerous studies have clearly demonstrated a link between hyperammonemia and CNS pathology in urea cycle disorders. However, little is known ...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Clinical and Functional Characterization of a Human ORNT1 Mutation (T32R) in the Hyperornithinemia-Hyperammonemia-Homocitrullinuria (HHH) Syndrome

et al. 2006

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“…Furthermore, the enzymatic activity of recombinant ORNT2 (i.e., ornithine shuttling across reconstituted liposomes) was at least three times less than that of ORNT1 (Fiermonte et al 2003). It is likely that ORNT2 could retain in vivo some residual ability to shuttle ornithine across the mitochondrial membrane, insufficient to rescue the viability of DArg11 yeast cells, but still able to improve ornithine metabolism in human fibroblasts carrying HHH mutations under overexpression conditions (Camacho et al 2003). While this work was under review, another group shows the same result (Marobbio et al 2015).…”

Section: Discussionmentioning

confidence: 64%

“…Most placental mammals possess also a second gene, SCL25A2 or ORNT2 (MIM #608157), an intronless gene probably derived from a retro-transposition event of ORNT1. The high level of identity (87%) between the ORNT1 and 2 proteins suggested that ORNT2 could act as a second mitochondrial ornithine transporter, possibly explaining the milder phenotype observed in HHH patients, compared to other urea cycle disorders (Camacho et al 2003). Nevertheless, the cellular role(s) of the human ORNT2 protein is not precisely established yet.…”

Section: Introductionmentioning

confidence: 99%

Heterologous Expression in Yeast of Human Ornithine Carriers ORNT1 and ORNT2 and of ORNT1 Alleles Implicated in HHH Syndrome in Humans

et al. 2015

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Hyperornithinemia-hyperammonemia-homocitrullinuria (HHH) syndrome is an autosomal recessive metabolic disorder usually presenting in the neonatal period with intermittent episodes of hyperammonemia, psychomotor delay, and progressive encephalopathy. Adult cases usually evolve into frank spastic paraparesis. The syndrome is caused by mutations in SLC25A15/ORNT1 encoding the mitochondrial ornithine transporter; a second ornithine transporter, ORNT2 of unknown function, is also present in most placental mammals. ORNT2 is believed to originate from an ancient retro-transposition event. In yeast Saccharomyces cerevisiae the major function of the transporter (encoded by Arg11) is to shuttle ornithine from the mitochondrial matrix to the cytosol. Its inactivation abolishes growth in the absence of arginine.In this work, we used functional complementation in S. cerevisiae to characterize the function of human ORNT2 and to test the pathogenicity of ORNT1 mutations found in HHH patients. Notably, we found that human ORNT1 but not ORNT2 complements the deletion of the yeast gene, despite their high level of homology. However, we identified some key residues in ORNT2, which may recover its functional competence when replaced with the corresponding residues of ORNT1, suggesting that roles of the two transporters are different. Moreover, we used this system to test a series of missense mutations of ORNT1 identified in patients with HHH syndrome. All mutations had a detrimental effect on the functionality of the human gene, without however clear genotype-phenotype correlations. Our data support yeast as a simple and effective model to validate missense mutations occurring in patients with HHH.

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Metabolism

Boden¹,

Scapa²,

Kanno³

et al. 2007

Textbook of Hepatology

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Cloning and characterization of human ORNT2: a second mitochondrial ornithine transporter that can rescue a defective ORNT1 in patients with the hyperornithinemia–hyperammonemia–homocitrullinuria syndrome, a urea cycle disorder

Cited by 37 publications

References 41 publications

Clinical and Functional Characterization of a Human ORNT1 Mutation (T32R) in the Hyperornithinemia-Hyperammonemia-Homocitrullinuria (HHH) Syndrome

Clinical and Functional Characterization of a Human ORNT1 Mutation (T32R) in the Hyperornithinemia-Hyperammonemia-Homocitrullinuria (HHH) Syndrome

Heterologous Expression in Yeast of Human Ornithine Carriers ORNT1 and ORNT2 and of ORNT1 Alleles Implicated in HHH Syndrome in Humans

Metabolism

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