Abstract

We recently characterized the mitochondrial ornithine transporter (ORNT1), the gene defective in the hyperornithinemia–hyperammonemia–homocitrullinuria (HHH) syndrome, a urea cycle disorder. Despite the apparent functional ablation of ORNT1 in 10 French-Canadian probands with the ORNT1-F188Δ allele, these patients are mildly affected when compared to patients with other urea cycle disorders such as deficiency of ornithine transcarbamylase. Given that the inner mitochondrial membrane is impermeable to solutes, we hypothesize that other unidentified carriers have some degree of functional redundancy with ORNT1. Using conserved sequences of mammalian and fungal mitochondrial ornithine transporters, we screened the Expressed Sequence Tag database for additional transporters belonging to the ORNT subfamily. Here we identify a new intronless gene, ORNT2, located on chromosome 5. The gene product of ORNT2 is 88% identical to ORNT1, targets to the mitochondria and is expressed in human liver, pancreas, kidney, and cultured fibroblasts from control and HHH patients. When ORNT2 is overexpressed transiently in cultured fibroblasts from HHH patients, it rescues the deficient ornithine metabolism in these cells. Our results suggest that ORNT2 may in part be responsible for the milder phenotype in HHH patients secondary to a gene redundancy effect. We believe ORNT2 arose from a retrotransposition event. To our knowledge, this is the first report of a functional retroposon (ORNT2) that can rescue the disease phenotype of the gene it arose from, ORNT1. As such, ORNT2 may eventually become a candidate for pharmacological-based approaches to correct a urea cycle disorder.

Author Keywords: Mitochondrial ornithine transporter; Urea cycle disorders; Mitochondrial carrier family of proteins

Article Outline

• Introduction

• Methods

• Human ORNT2 cDNA cloning, sequence analysis, and chromosome mapping

• RNA and DNA isolation and Northern blot analysis

• ORNT2 protein polymorphism and genomic PCR

• Tissue distribution of human ORNT1, ORNT2, and OTC by RT-PCR

• Expression pattern of human ORNT1 and ORNT2 in cultured human fibroblasts with the wild type, HHH-F188Δ and HHH-E180K ORNT1 alleles

• Expression constructs and site-directed mutagenesis

• Human cell lines, transfections, ornithine transport assay, and indirect immunoflourescence (IF)

• Results

• Identification of a second human mitochondrial ornithine transporter (HsORNT2)

• Comparison of human ORNT2 and ORNT1

• ORNT2 polymorphisms and allele frequencies

• Confirmation of the intronless nature of human ORNT2

• Tissue distribution of human ORNT2 and ORNT1

• Subcellular localization of human ORNT2 in cultured fibroblasts

• Expression of N-myc-HsORNT2 restores ornithine metabolism in cultured HHH-F188Δ and HHH-E180K fibroblasts

• Discussion

• Acknowledgements

• References

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