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

Abstract

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 ...

Adult-Onset Presentation of a Hyperornithinemia-Hyperammonemia-Homocitrullinuria Patient Without Prior History of Neurological Complications

“…No orotic aciduria was observed nor increases in methionine (with normal plasma levels) in the urine amino acid profile, thus suggesting no urinary excretion of homocitrulline given that the peaks of these two amino acids overlap. This observation on our patient contrasts sharply with a group of five previously reported KaiserPermanente HHH patients, who consistently showed a urinary pattern suggestive of homocitrullinuria (Camacho et al 2006). The patient's plasma carnitine profile was normal.…”

“…Genomic DNA (blood) and total RNA (EBV-transformed lymphocytes) were used for mutation analysis utilizing previously published protocols (Torisu et al 2006;Camacho et al 2006). We identified two mutant ORNT1 alleles: c.658G>A (p.Gly220Arg) in Exon 6 and c.823C>T (p.Arg275X) in Exon 7.…”

“…Using a wild-type ORNT1-N-myc-tagged construct as a backbone, we generated the ORNT1 mutation p.Gly220Arg as previously reported (Camacho et al 2006). We performed electroporation (transient transfection), the ornithine transport assay, and immunofluorescence as previously described (Camacho and Rioseco-Camacho 2009).…”

“…We thought of further investigating the functional consequences of the Gly 220 to Arg 220 change given that, in the past, several reports have demonstrated that ORNT1 mutations that appeared to be nonfunctional have residual function (Camacho et al 2006). Overexpression studies shown in Fig.…”

Identification of novel mutations in theSLC25A15gene in hyperornithinemia-hyperammonemia-homocitrullinuria (HHH) syndrome: A clinical, molecular, and functional study

“…After the report of the first mutations in Québecois patients [Camacho et al, 1999], the array of allelic variants associated with the syndrome has expanded to Japan [Tsujino et al, 2000], Italy [Salvi et al, 2001a], and elsewhere [Camacho et al, 2003[Camacho et al, , 2006Korman et al, 2004], demonstrating that HHH has a panethnic distribution. With the exception of the French-Canadian founder effect, no common mutation has been detected in other populations, although the p.R179X variant is considered to be frequent in Japan [Miyamoto et al, 2001].…”