HHH Syndrome Research Articles

The hyperornithinemia-hyperammonemia-homocitrullinuria syndrome.

BackgroundHyperornithinemia-hyperammonemia-homocitrullinuria (HHH) syndrome is a rare autosomal recessive disorder of the urea cycle. HHH has a panethnic distribution, with a major prevalence in Canada, Italy and Japan. Acute clinical signs include intermittent episodes of vomiting, confusion or coma and hepatitis-like attacks. Alternatively, patients show a chronic course with aversion for protein rich foods, developmental delay/intellectual disability, myoclonic seizures, ataxia and pyramidal dysfunction. HHH syndrome is caused by impaired ornithine transport across the inner mitochondrial membrane due to mutations in SLC25A15 gene, which encodes for the mitochondrial ornithine carrier ORC1. The diagnosis relies on clinical signs and the peculiar metabolic triad of hyperammonemia, hyperornithinemia, and urinary excretion of homocitrulline. HHH syndrome enters in the differential diagnosis with other inherited or acquired conditions presenting with hyperammonemia.MethodsA systematic review of publications reporting patients with HHH syndrome was performed.ResultsWe retrospectively evaluated the clinical, biochemical and genetic profile of 111 HHH syndrome patients, 109 reported in 61 published articles, and two unpublished cases. Lethargy and coma are frequent at disease onset, whereas pyramidal dysfunction and cognitive/behavioural abnormalities represent the most common clinical features in late-onset cases or during the disease course. Two common mutations, F188del and R179* account respectively for about 30% and 15% of patients with the HHH syndrome. Interestingly, the majority of mutations are located in residues that have side chains protruding into the internal pore of ORC1, suggesting their possible interference with substrate translocation. Acute and chronic management consists in the control of hyperammonemia with protein-restricted diet supplemented with citrulline/arginine and ammonia scavengers. Prognosis of HHH syndrome is variable, ranging from a severe course with disabling manifestations to milder variants compatible with an almost normal life.ConclusionsThis paper provides detailed information on the clinical, metabolic and genetic profiles of all HHH syndrome patients published to date. The clinical phenotype is extremely variable and its severity does not correlate with the genotype or with recorded ammonium/ornithine plasma levels. Early intervention allows almost normal life span but the prognosis is variable, suggesting the need for a better understanding of the still unsolved pathophysiology of the disease.Electronic supplementary materialThe online version of this article (doi:10.1186/s13023-015-0242-9) contains supplementary material, which is available to authorized users.

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

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.

Mitochondrial transporters of the SLC25 family and associated diseases: a review

To date, 14 inherited diseases (including phenotypes) associated to mitochondrial transporters of the SLC25 family have been well characterized biochemically and genetically. They are rare metabolic disorders caused by mutations in the SLC25 nuclear genes that encode mitochondrial carriers, a superfamily of 53 proteins in humans that shuttle a variety of solutes across the mitochondrial membrane. Mitochondrial carriers vary considerably in the nature and size of the substrates they transport, the modes of transport and driving forces. However, their substrate translocation mechanism at the molecular level is thought to be basically the same. Herein, the main structural and functional properties of the SLC25 mitochondrial carriers and the known carrier-related diseases are presented. Two of these disorders, ADP/ATP carrier deficiency and phosphate carrier deficiency, are caused by defects of the two mitochondrial carriers that provide mitochondria with ADP and phosphate, the substrates of oxidative phosphorylation; these disorders therefore are characterized by defective energy production by mitochondria. The mutations of SLC25 carrier genes involved in other cellular functions cause carnitine/acylcarnitine carrier deficiency, HHH syndrome, aspartate/glutamate isoform 1 and 2 deficiencies, congenital Amish microcephaly, neuropathy with bilateral striatal necrosis, congenital sideroblastic anemia, neonatal epileptic encephalopathy, and citrate carrier deficiency; these disorders are characterized by specific metabolic dysfunctions depending on the role of the defective carrier in intermediary metabolism.

A novel mutation in the SLC25A15 gene in a Turkish patient with HHH syndrome: Functional analysis of the mutant protein

The hyperornithinemia–hyperammonemia–homocitrullinuria syndrome is a rare autosomal recessive disorder caused by the functional deficiency of the mitochondrial ornithine transporter 1 (ORC1). ORC1 is encoded by the SLC25A15 gene and catalyzes the transport of cytosolic ornithine into mitochondria in exchange for citrulline. Although the age of onset and the severity of the symptoms vary widely, the disease usually manifests in early infancy. The typical clinical features include protein intolerance, lethargy, episodic confusion, cerebellar ataxia, seizures and mental retardation. In this study, we identified a novel p.Ala15Val (c.44C>T) mutation by genomic DNA sequencing in a Turkish child presenting severe tantrum, confusion, gait disturbances and loss of speech abilities in addition to hyperornithinemia, hyperammonemia and homocitrullinuria. One hundred Turkish control chromosomes did not possess this variant. The functional effect of the novel mutation was assessed by both complementation of the yeast ORT1 null mutant and transport assays. Our study demonstrates that the A15V mutation dramatically interferes with the transport properties of ORC1 since it was shown to inhibit ornithine transport nearly completely.

Disturbance of redox homeostasis by ornithine and homocitrulline in rat cerebellum: A possible mechanism of cerebellar dysfunction in HHH syndrome

AimsCerebellar ataxia is commonly observed in hyperornithinemia-hyperammonemia-homocitrullinuria (HHH) syndrome, an inherited metabolic disorder biochemically characterized by ornithine (Orn), homocitrulline (Hcit) and ammonia accumulation. Since the pathophysiology of cerebellum damage in this disorder is still unknown, we investigated the effects of Hcit and Orn on important parameters of redox and energy homeostasis in cerebellum of young rats. Material and methodsWe determined thiobarbituric acid-reactive substance (TBA-RS) levels, carbonyl content, nitrate and nitrite production, hydrogen peroxide production, GSH concentrations, sulfhydryl content, as well as activities of respiratory chain complexes I–IV, creatine kinase, Na+,K+-ATPase, aconitase and α-ketoglutarate dehydrogenase. Key findingsOrn and Hcit significantly increased TBA-RS levels (lipid oxidation), that was totally prevented by melatonin and reduced glutathione (GSH). We also found that nitrate and nitrite production was not altered by any of the metabolites, in contrast to hydrogen peroxide production which was significantly enhanced by Hcit. Furthermore, GSH concentrations were significantly reduced by Orn and Hcit and sulfhydryl content by Orn, implying an impairment of antioxidant defenses. As regards energy metabolism, Orn and Hcit provoked a significant reduction of aconitase activity, without altering the other parameters. Furthermore, Orn-elicited reduction of aconitase activity was totally prevented by GSH, indicating that the critical groups of this enzyme were susceptible to oxidation caused by this amino acid. SignificanceTaken together, our data indicate that redox homeostasis is disturbed by the major metabolites accumulating in HHH syndrome and that this mechanism may be implicated in the ataxia and cerebellar abnormalities observed in this disorder.

Determination of plasma dibasic amino acids following trimethylsilyl–trifluoroacyl derivatization using gas chromatography–mass spectrometry

A rapid analytical method was developed to quantify dibasic amino acids (ornithine, lysine and arginine) after two-step derivatization procedure with good sensitivity and specificity on human plasma. If early diagnosis has not been made, patients with inborn metabolic disorders such as HHH syndrome, Hyperornithinemia and dibasic aminoaciduria rapidly progress to sudden death, physical defect or mental retardation resulting in storage of the toxic material into the brain. Therefore, it is necessary to develop the analytical method for rapid screening and/or correct confirmation diagnosis. The formation of trimethylsilyl derivative of the carboxylic (COO-) functional group was performed by adding MSTFA. Five μL of methyl orange was added to the residue until the color changed into yellow. Consecutively, the trifluoroacyl derivative of the amino (-NH2) functional group was produced by adding MBTFA. Specific ions was chosen for quantification with following ions; m/z 166 and m/z 212 for ornithine, m/z 180 and m/z 395 for lysine, and m/z 292 and, m/z 519 for arginine. A calibration curve showed a linear relationship for the dibasic amino acids spiked to pooled normal plasma showing R(2) of 0.9955-0.9979 in the range of 0.1-600 ng investigated. The utility of the method for screening and diagnosis was demonstrated by recovery 80-125 % and reproducibility with RSD (9-17 %) at low, medium and high concentration fortified to pooled plasma. Collectively, the present study suggest that this method could be useful for diagnosis, screening, therapeutic monitoring of metabolic disorders on dietary therapy with excellent sensitivity and rapidity.

Impairment of brain redox homeostasis caused by the major metabolites accumulating in hyperornithinemia–hyperammonemia–homocitrullinuria syndrome in vivo

Ornithine, ammonia and homocitrulline are the major metabolites accumulating in hyperornithinemia-hyperammonemia-homocitrullinuria syndrome, a genetic disorder characterized by neurological regression whose pathogenesis is still not understood. The present work investigated the in vivo effects of intracerebroventricular administration of ornithine and homocitrulline in the presence or absence of hyperammonemia induced by intraperitoneal urease treatment on a large spectrum of oxidative stress parameters in cerebral cortex from young rats in order to better understand the role of these metabolites on brain damage. Ornithine increased thiobarbituric acid-reactive substances (TBA-RS) levels and carbonyl formation and decreased total antioxidant status (TAS) levels. We also observed that the combination of hyperammonemia with ornithine resulted in significant decreases of sulfhydryl levels, reduced glutathione (GSH) concentrations and the activities of catalase (CAT) and glutathione peroxidase (GPx), highlighting a synergistic effect of ornithine and ammonia. Furthermore, homocitrulline caused increases of TBA-RS values and carbonyl formation, as well as decreases of GSH concentrations and GPx activity. Hcit with hyperammonemia (urease treatment) decreased TAS and CAT activity. We also showed that urease treatment per se was able to enhance TBA-RS levels. Finally, nitric oxide production was not altered by Orn and Hcit alone or in combination with hyperammonemia. Our data indicate that the major metabolites accumulating in hyperornithinemia-hyperammonemia-homocitrullinuria syndrome provoke lipid and protein oxidative damage and a reduction of the antioxidant defenses in the brain. Therefore, it is presumed that oxidative stress may represent a relevant pathomechanism involved in the brain damage found in patients affected by this disease.

Creatine metabolism in urea cycle defects

Creatine (Cr) and phosphocreatine play an essential role in energy storage and transmission. Maintenance of creatine pool is provided by the diet and by de novo synthesis, which utilizes arginine, glycine and s-adenosylmethionine as substrates. Three primary Cr deficiencies exists: arginine:glycine amidinotransferase deficiency, guanidinoacetate methyltransferase deficiency and the defect of Cr transporter SLC6A8. Secondary Cr deficiency is characteristic of ornithine-aminotransferase deficiency, whereas non-uniform Cr abnormalities have anecdotally been reported in patients with urea cycle defects (UCDs), a disease category related to arginine metabolism in which Cr must be acquired by de novo synthesis because of low dietary intake. To evaluate the relationships between ureagenesis and Cr synthesis, we systematically measured plasma Cr in a large series of UCD patients (i.e., OTC, ASS, ASL deficiencies, HHH syndrome and lysinuric protein intolerance). Plasma Cr concentrations in UCDs followed two different trends: patients with OTC and ASS deficiencies and HHH syndrome presented a significant Cr decrease, whereas in ASL deficiency and lysinuric protein intolerance Cr levels were significantly increased (23.5 vs. 82.6 μmol/L; p < 0.0001). This trend distribution appears to be regulated upon cellular arginine availability, highlighting its crucial role for both ureagenesis and Cr synthesis. Although decreased Cr contributes to the neurological symptoms in primary Cr deficiencies, still remains to be explored if an altered Cr metabolism may participate to CNS dysfunction also in patients with UCDs. Since arginine in most UCDs becomes a semi-essential aminoacid, measuring plasma Cr concentrations might be of help to optimize the dose of arginine substitution.

Insights into the Mutation-Induced HHH Syndrome from Modeling Human Mitochondrial Ornithine Transporter-1

Human mitochondrial ornithine transporter-1 is reported in coupling with the hyperornithinemia-hyperammonemia-homocitrullinuria (HHH) syndrome, which is a rare autosomal recessive disorder. For in-depth understanding of the molecular mechanism of the disease, it is crucially important to acquire the 3D structure of human mitochondrial ornithine transporter-1. Since no such structure is available in the current protein structure database, we have developed it via computational approaches based on the recent NMR structure of human mitochondrial uncoupling protein (Berardi MJ, Chou JJ, et al. Nature 2011, 476:109–113). Subsequently, we docked the ligand L-ornithine into the computational structure to search for the favorable binding mode. It was observed that the binding interaction for the most favorable binding mode is featured by six remarkable hydrogen bonds between the receptor and ligand, and that the most favorable binding mode shared the same ligand-binding site with most of the homologous mitochondrial carriers from different organisms, implying that the ligand-binding sites are quite conservative in the mitochondrial carriers family although their sequences similarity is very low with 20% or so. Moreover, according to our structural analysis, the relationship between the disease-causing mutations of human mitochondrial ornithine transporter-1 and the HHH syndrome can be classified into the following three categories: (i) the mutation occurs in the pseudo-repeat regions so as to change the region of the protein closer to the mitochondrial matrix; (ii) the mutation is directly affecting the substrate binding pocket so as to reduce the substrate binding affinity; (iii) the mutation is located in the structural region closer to the intermembrane space that can significantly break the salt bridge networks of the protein. These findings may provide useful insights for in-depth understanding of the molecular mechanism of the HHH syndrome and developing effective drugs against the disease.

Dual mechanism of brain damage induced in vivo by the major metabolites accumulating in hyperornithinemia–hyperammonemia–homocitrullinuria syndrome

Hyperornithinemia–hyperammonemia–homocitrullinuria (HHH) syndrome is an autosomal recessive disorder caused by a defect in the mitochondrial ornithine transporter, leading to accumulation of ornithine (Orn), homocitrulline (Hcit) and ammonia. Progressive neurological regression whose pathogenesis is not well established is common in this disease. The present work investigated the in vivo effects of intracerebroventricular administration of Orn and Hcit on important parameters of oxidative stress and energy metabolism in cerebral cortex from young rats. Orn and Hcit significantly increased thiobarbituric acid-reactive substances values and carbonyl formation, indicators of lipid and protein oxidative damage, respectively. Furthermore, N-acetylcysteine and the combination of the free radical scavengers ascorbic acid plus α-tocopherol attenuated the lipid oxidation and totally prevented the protein oxidative damage provoked by Orn and Hcit, suggesting that reactive species were involved in these effects. Hcit, but not Orn administration, also decreased glutathione concentrations, as well as the activity of catalase and glutathione peroxidase, indicating that Hcit provokes a reduction of brain antioxidant defenses. As regards to the parameters of energy metabolism, we verified that Orn and Hcit significantly inhibited the citric acid cycle function (inhibition of CO2 synthesis from [1-14C] acetate), the aerobic glycolytic pathway (reduced CO2 production from [U-14C] glucose) and complex I–III activity of the respiratory chain. Hcit also inhibited the activity of aconitase, an enzyme very susceptible to free radical attack. Taken together, our data indicate that mitochondrial homeostasis is disturbed by Orn and especially by Hcit. It is presumed that the impairment of brain bioenergetics and the oxidative damage induced by these metabolites may possibly contribute to the brain deterioration and neurological symptoms affecting patients with HHH syndrome.

Evidence that the major metabolites accumulating in hyperornithinemia–hyperammonemia–homocitrullinuria syndrome induce oxidative stress in brain of young rats

Ornithine and homocitrulline are the major metabolites accumulating in hyperornithinemia–hyperammonemia–homocitrullinuria syndrome, a genetic disorder characterized by neurological regression whose pathogenesis is still not understood. The present work investigated the in vitro effects of ornithine and homocitrulline on important parameters of oxidative stress in cerebral cortex from young rats. Ornithine significantly increased chemiluminescence and thiobarbituric acid-reactive substances levels, indicators of lipid peroxidation, while homocitrulline only augmented chemiluminescence values. Furthermore, ornithine-induced increase of thiobarbituric acid-reactive substances levels was attenuated (melatonin and reduced glutathione) or totally prevented (α-tocopherol) by free radical scavengers, suggesting that reactive species were involved in the lipid oxidative damage. We also observed that ornithine and homocitrulline significantly decreased the tissue antioxidant defenses, determined by reduced glutathione concentrations, the major non-enzymatic antioxidant defense found in the brain. Homocitrulline reduction of glutathione levels was completely prevented by melatonin and α-tocopherol, whereas ornithine-induced decrease of glutathione levels was only attenuated by these free radical scavengers. Ornithine and homocitrulline also induced protein oxidative damage, increasing carbonyl formation and sulfhydryl oxidation. In contrast, these amino acids did not affect nitric oxide production, indicating that nitrogen reactive species were not implicated in the lipid and oxidative damage provoked by ornithine and homocitrulline. Therefore, it is presumed that the major metabolites accumulating in hyperornithinemia–hyperammonemia–homocitrullinuria syndrome elicit oxidative stress and that this pathomechanism may possibly be involved in the brain damage found in patients affected by this disorder.

Experimental evidence that ornithine and homocitrulline disrupt energy metabolism in brain of young rats

Tissue accumulation of ornithine (Orn), homocitrulline (Hcit), ammonia and orotic acid (Oro) is the biochemical hallmark of patients affected by hyperornithinemia–hyperammonemia–homocitrullinuria (HHH) syndrome, a disorder clinically characterized by neurological symptoms, whose pathophysiology is practically unknown. In the present study, we investigated the in vitro effect of Orn, Hcit and Oro on important parameters of energy metabolism in brain of 30-day-old Wistar rats since mitochondrial abnormalities have been observed in the affected patients. We first verified that Orn and Hcit significantly inhibited the citric acid cycle (inhibition of CO 2 synthesis from [1- 14C] acetate, as well as aconitase and α-ketoglutarate dehydrogenase activities), the aerobic glycolytic pathway (reduced CO 2 production from [U- 14C] glucose) and moderately the electron transfer flow (inhibitory effect on complex I–III). Hcit, but not Orn, was also able to significantly inhibit the mitochondrial creatine kinase activity. Furthermore, this inhibition was prevented by GSH, suggesting a possible role of reactive species oxidizing critical thiol groups of the enzyme. In contrast, the other enzyme activities of the citric acid cycle and of the electron transfer chain, as well as synaptic Na +,K +-ATPase were not altered by either Orn or Hcit at concentrations as high as 5.0 mM. Similarly, Oro did not interfere with any of the tested parameters. Taken together, these data strongly indicate that Orn and Hcit compromise brain energy metabolism homeostasis and Hcit also interferes with cellular ATP transfer and buffering. It is therefore suggested that Orn and especially Hcit may be involved in the neurological damage found in patients affected by HHH syndrome.

The Human and Mouse SLC25A29 Mitochondrial Transporters Rescue the Deficient Ornithine Metabolism in Fibroblasts of Patients With the Hyperornithinemia-Hyperammonemia-Homocitrullinuria (HHH) Syndrome

The hyperornithinemia-hyperammonemia-homocitrullinuria (HHH) syndrome is a disorder of the urea cycle (UCD) and ornithine degradation pathway caused by mutations in the mitochondrial ornithine transporter (ORNT1). Unlike other UCDs, HHH syndrome is characterized by a less severe and variable phenotype that we believe may, in part, be due to genes with redundant function to ORNT1, such as the previously characterized ORNT2 gene. We reasoned that SLC25A29, a member of the same subfamily of mitochondrial carrier proteins as ORNT1 and ORNT2, might also have overlapping function with ORNT1. Here, we report that both the human and mouse SLC25A29, previously identified as mitochondrial carnitine/acyl-carnitine transporter-like, when overexpressed transiently also rescues the impaired ornithine transport in cultured HHH fibroblasts. Moreover, we observed that, in the mouse, the Slc25a29 message is more significantly expressed in the CNS and cultured astrocytes when compared with the liver and kidney. These results suggest a potential physiologic role for the SLC25A29 transporter in the oxidation of fatty acids, ornithine degradation pathway, and possibly the urea cycle. Our results show that SLC25A29 is the third human mitochondrial ornithine transporter, designated as ORNT3, which may contribute to the milder and variable phenotype seen in patients with HHH syndrome.

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

Hyperornithinemia-hyperammonemia-homocitrullinuria (HHH) syndrome is an autosomal recessive disorder of the urea cycle. With the exception of the French-Canadian founder effect, no common mutation has been detected in other populations. In this study, we collected 16 additional HHH cases and expanded the spectrum of SLC25A15/ORC1 mutations. Eleven novel mutations were identified including six new missense and one microrearrangement. We also measured the transport properties of the recombinant purified proteins in reconstituted liposomes for four new and two previously reported missense mutations and proved that the transport activities of these mutant forms of ORC1 were reduced as compared with the wild-type protein; residual activity ranged between 4% and 19%. Furthermore, we designed three-dimensional (3D)-modeling of mutant ORC1 proteins. While modeling the changes in silico allowed us to obtain new information on the pathomechanisms underlying HHH syndrome, we found no clear-cut genotype-phenotype correlations. Although patient metabolic alterations responded well to low-protein therapy, predictions concerning the long-term evolution of HHH syndrome remain uncertain. The preference for a hepatic rather than a neurological presentation at onset also continues, largely, to elude us. Neither modifications in oxidative metabolism-related energy, such as those expected in different mtDNA haplogroups, nor sequence variants in SLC25A2/ORC2 seem to be crucial. Other factors, including protein stability and function, and ORC1-ORC2 structural interactions should be further investigated.

Hyperornithinemia‐Hyperammonemia‐Homocitrullinuria Syndrome (HHH) Presenting With Acute Fulminant Hepatic Failure

We report on two Aboriginal patients with the hyperornithinemia-hyperammonemia-homocitrullinuria (HHH) syndrome. Both presented with acute hepatic failure with severe hypertransaminasemia and coagulopathy, prompting evaluation for emergent liver transplantation. The diagnosis of HHH syndrome was based on the presence of typical metabolic abnormalities. A protein-restricted diet and L-arginine or L-citrulline supplementation were immediately started, with rapid normalization of liver function test results and other biochemical abnormalities. Molecular analysis of the SLC25A15 gene showed that the two patients were homozygous for the common French Canadian mutation (F188Delta). The diagnosis of HHH syndrome should be considered in patients with unexplained fulminant hepatic failure. There does not appear to be a genotype-phenotype correlation for this presentation, inasmuch as the only other reported patient presenting with this picture had two different point mutations. Early identification and prompt treatment of these patients is crucial to avoid liver transplantation and can be life saving.

Hyperornithinemia–hyperammonemia–homocitrullinuria syndrome with stroke-like imaging presentation: Clinical, biochemical and molecular analysis

Hyperornithinemia-hyperammonemia-homocitrullinuria (HHH) syndrome is an autosomal recessive disorder caused by mutations in ORNT1 gene that encodes a mitochondrial ornithine transporter. It has variable clinical presentations with episodic hyperammonemia, liver dysfunction, and chronic neurological manifestations. In this work, we report the findings of HHH syndrome in 3 Saudi siblings. The 4-year-old proband presented with recurrent Reye-like episodes, hypotonia, and multiple stroke-like lesions on brain MRI. Biochemical and molecular analysis confirmed that she had HHH syndrome. She significantly improved on protein restriction and sodium benzoate. Her two older siblings have milder phenotypes with protein intolerance and learning problems. In comparison to their sister, their homocitrulline and orotic acid were only mildly elevated even before treatment. The three patients were homozygous for a novel mutation in ORNT1 with a Gly220Arg change. In view of the CNS lesions, which initially were felt to be suggestive of MELAS, we sequenced the entire mtDNA genome and no potential pathogenic mutations were detected. Analysis of ORNT2 did not provide explanation of the clinical and biochemical variability. This work presents a yet unreported CNS involvement pattern, notably multiple supratentorial stroke-like lesions in association with HHH syndrome. Moreover, it illustrates considerable clinical/biochemical correlation, and describes a novel mutation. We suggest including HHH syndrome in the differential diagnosis of patients found to have stroke-like lesions on brain MRI.

Equimolar Ammonia Interference in Potassium Measurement on the Osmetech OPTI Critical Care Analyzer

Ammonia is a toxic byproduct of amino acid metabolism, and increased blood concentrations of ammonia are associated with severe encephalopathy (1). In mammals, ammonia is detoxified in the liver via formation of urea (2). Hyperammonemia can result from hepatic failure, enzymatic deficiencies of the urea cycle or defects in ornithine transport [e.g., HHH syndrome (hyperornithinemia, hyperammonemia, hyperhomocitrullinuria)], or it may be secondary to other organic acidopathies (3). The hyperammonemia observed in methylmalonic acidemia is thought to arise because accumulated propionyl CoA interferes with formation of N -acetylglutamate, an obligatory activator of carbamyl phosphate synthase, the initial step in urea synthesis (4). The Osmetech (Roswell, GA) OPTI Critical Care Analyzer (CCA) is a point-of-care instrument used to monitor electrolytes and blood gases; at our institution it is used to monitor critically ill patients during transport from outside …

A novel R275X mutation of the SLC25A15 gene in a Japanese patient with the HHH syndrome

The hyperornithinemia–hyperammonemia–homocitrullinuria (HHH) syndrome (MIM 238970) is an autosomal recessive metabolic disorder caused by a deficiency of the mitochondrial ornithine transporter, one of the urea cycle components. Mutations in the SLC25A15 gene have been coupled to the HHH syndrome. We describe a Japanese female patient with the HHH syndrome due to a novel homozygous R275X SLC25A15 mutation and male sibling who presumably carried the same mutation. He exhibited slowly progressive deterioration with seizures, a gait disturbance due to polyneuropathy, episodic confusion, and died of acute encephalopathy at 34 years of age while the proband exhibited moderate mental retardation, seizures, mild spastic paraplegia, and deafness without neurological deterioration for more than 20 years. The clinical features of previously documented patients with the homozygous SLC25A15 mutation demonstrated that genotype did not simply correlate with clinical severity. The phenotypic variability might depend on other factors, such as dietary and other genetic ones.

Hyperornithinemia, hyperammonemia, and homocitrullinuria syndrome with evidence of mitochondrial dysfunction due to a novel SLC25A15 ( ORNT1) gene mutation in a Palestinian family

Hyperornithinemia, hyperammonemia, and homocitrullinuria (HHH) syndrome is caused by mutations in the SLC25A15 ( ORNT1) gene encoding the mitochondrial ornithine transporter, but the mechanism of pathogenesis of the encephalopathy, spastic paraparesis and hepatopathy remains undetermined. HHH syndrome was diagnosed in a 2-year-old Palestinian boy with developmental delay and seizures, and subsequently in his 13-year-old brother with developmental delay. Direct sequencing of the PCR products of SLC25A15 exon amplifications revealed that both brothers were homozygous for a novel 446G deletion in exon 3 as well as for a 760A>T (I254L) polymorphism in exon 5, which is downstream of a premature termination codon produced by the frameshift resulting from the 446G deletion. The index patient had elevated liver enzymes as well as hyperalaninemia, lactic acidemia with an elevated lactate to pyruvate ratio, and increased urinary excretion of lactate, glutarate and Krebs cycle intermediates. These findings are indicative of mitochondrial dysfunction and are in accordance with ultrastructural studies showing increased numbers of large and bizarre mitochondria in liver, muscle, leukocytes and fibroblasts of some HHH patients. Neurologic and hepatic manifestations are characteristic of some primary mitochondrial disorders. Secondary mitochondrial dysfunction may contribute to the pathogenesis of these same features in HHH syndrome.

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

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.