An Exon-Specific Small Nuclear U1 RNA (ExSpeU1) Improves Hepatic OTC Expression in a Splicing-Defective spf/ash Mouse Model of Ornithine Transcarbamylase Deficiency.

OTC splicing mutations are generally associated with the severest and early disease onset of ornithine transcarbamylase deficiency (OTCD), the most common urea cycle disorder. Noticeably, splicing defects can be rescued by spliceosomal U1snRNA variants, which showed their efficacy in cellular and animal models. Here, we challenged an U1snRNA variant in the OTCD mouse model (spf/ash) carrying the mutation c.386G > A (p.R129H), also reported in OTCD patients. It is known that the R129H change does not impair protein function but affects pre-mRNA splicing since it is located within the 5' splice site. Through in vitro studies, we identified an Exon Specific U1snRNA (ExSpeU1O3) that targets an intronic region downstream of the defective exon 4 and rescues exon inclusion. The adeno-associated virus (AAV8)-mediated delivery of the ExSpeU1O3 to mouse hepatocytes, although in the presence of a modest transduction efficiency, led to increased levels of correct OTC transcripts (from 6.1 ± 1.4% to 17.2 ± 4.5%, p = 0.0033). Consistently, this resulted in increased liver expression of OTC protein, as demonstrated by Western blotting (~3 fold increase) and immunostaining. Altogether data provide the early proof-of-principle of the efficacy of ExSpeU1 in the spf/ash mouse model and encourage further studies to assess the potential of RNA therapeutics for OTCD caused by aberrant splicing.

Ex Vivo Enteroids Recapitulate In Vivo Citrulline Production in Mice.

Background: The endogenous production of arginine relies on the synthesis of citrulline by enteral ornithine transcarbamylase (OTC). Mutations in the gene coding for this enzyme are the most frequent cause of urea cycle disorders. There is a lack of correlation between in vivo metabolic function and DNA sequence, transcript abundance, or in vitro enzyme activity. Objective: The goal of the present work was to test the hypothesis that enteroids, a novel ex vivo model, are able to recapitulate the in vivo citrulline production of wild-type (WT) and mutant mice. Methods: Six-week-old male WT and OTC-deficient mice [sparse fur and abnormal skin (spf-ash) mutation] were studied. Urea and citrulline fluxes were determined in vivo, and OTC abundance was measured in liver and gut tissue. Intestinal crypts were isolated and cultured to develop enteroids. Ex vivo citrulline production and OTC abundance were determined in these enteroids. Results: Liver OTC abundance was lower (mean ± SE: 0.16 ± 0.01 compared with 1.85 ± 0.18 arbitrary units; P < 0.001) in spf-ash mice than in WT mice, but there was no difference in urea production. In gut tissue, OTC was barely detectable in mutant mice; despite this, a lower but substantial citrulline production (67 ± 3 compared with 167 ± 8 µmol · kg-1 · h-1; P < 0.001) was shown in the mutant mice. Enteroids recapitulated the in vivo findings of a very low OTC content accompanied by a reduced citrulline production (1.07 ± 0.20 compared with 4.64 ± 0.44 nmol · µg DNA-1 · d-1; P < 0.001). Conclusions: Enteroids recapitulate in vivo citrulline production and offer the opportunity to study the regulation of citrulline production in a highly manipulable system.

Activity of the liver enzyme ornithine carbamoyltransferase (OTC) in blood: LC-MS/MS assay for non-invasive diagnosis of ornithine carbamoyltransferase deficiency.

BACKGROUND: Liver enzymes are released from hepatocytes into circulation and their activity can be measured in the blood. We examined whether the plasma activity of the liver enzyme ornithine carbamoyltransferase, determined by a novel liquid chromatography-mass spectrometry (LC-MS/MS) assay, could be utilized for the detection of OTC deficiency (OTCD), an X-linked inborn error of the urea cycle. METHODS: The plasma ornithine carbamoyltransferase (OTC) activity was assayed in the reverse reaction using isotopically labeled citrulline-d4 as a substrate and by determination of the product, ornithine-d4, by LC-MS/MS analysis. RESULTS: The plasma OTC activity in the controls was in the range of 111-658 pkat/L (n=49, median 272 pkat/L), and the activity increased linearly with serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities in patients with hepatopathy. The OTC activity was subsequently determined in 32 individuals carrying mutations in the OTC gene, and OTC/ALT and OTC/AST ratios were calculated to account for the degree of hepatopathy, which is a common finding in OTCD. The OTC/ALT ratio enabled clear differentiation of OTCD hemizygotes (n=11, range 0-69×10-6) from controls (504-3440×10-6). This ratio also enabled the detection of 11 of 12 symptomatic heterozygotes (range 38-794×10-6), while this marker did not allow for reliable differentiation of asymptomatic heterozygotes (n=9) from controls. CONCLUSIONS: LC-MS/MS assay of plasma OTC activity enabled the detection of all hemizygous and the majority of symptomatic heterozygous OTCD patients in the tested cohort. This study demonstrates that non-invasive assay of enzymes expressed predominantly in the liver could be used as an alternative approach for diagnosing inborn errors of metabolism.

Impact of enzyme activity assay on indication in liver transplantation for ornithine transcarbamylase deficiency.

There are no objective and concrete guidelines for the management of Ornithine transcarbamylase deficiency (OTCD). Based on previous findings, we hypothesized that patients with OTCD have a low Ornithine transcarbamylase (OTC) activity in the liver, and therefore it would be better to determine the appropriate indications and optimal timing for liver transplantation (LT) based on the OTC activity. However, few data have so far been accumulated on the OTC activity in cases that are indicated for LT. The purpose of the present study was to clarify the OTC activity in cases that were indicated for LT. This study involved thirteen children with OTCD (8 males and 5 females) who underwent LT, and two females with OTCD who did not require LT. The OTC activity of the neonatal onset type ranged from 0% to 7.2%, while that of the late onset type who underwent LT ranged from 4.4% to 18.7%. The OTC activity of the late onset type which did not require LT was 33-38% based on a preoperative needle liver biopsy. Some late onset patients that underwent LT, showed an activity that was as low as that observed in the neonatal onset cases. This is the first report to show the results of measuring the OTC activity for serial OTCD cases indicated for LT. OTC activity might be an indicator to determine the indications for and the timing of LT in the late onset type, however, further investigations are necessary.

Preclinical evaluation of a clinical candidate AAV8 vector for ornithine transcarbamylase (OTC) deficiency reveals functional enzyme from each persisting vector genome.

Ornithine transcarbamylase deficiency (OTCD), the most common and severe urea cycle disorder, is an excellent model for developing liver-directed gene therapy. No curative therapy exists except for liver transplantation which is limited by available donors and carries significant risk of mortality and morbidity. Adeno-associated virus 8 (AAV8) has been shown to be the most efficient vector for liver-directed gene transfer and is currently being evaluated in a clinical trial for treating hemophilia B. In this study, we generated a clinical candidate vector for a proposed OTC gene therapy trial in humans based on a self-complementary AAV8 vector expressing codon-optimized human OTC (hOTCco) under the control of a liver-specific promoter. Codon-optimization dramatically improved the efficacy of OTC gene therapy. Supraphysiological expression levels and activity of hOTC were achieved in adult spf(ash) mice following a single intravenous injection of hOTCco vector. Vector doses as low as 1×10(10) genome copies (GC) achieved robust and sustained correction of the OTCD biomarker orotic aciduria and clinical protection against an ammonia challenge. Functional expression of hOTC in 40% of liver areas was found in mice treated with a low vector dose of 1×10(9) GC. We suggest that the clinical candidate vector we have developed has the potential to achieve therapeutic effects in OTCD patients.

Disruption of OTC promoter-enhancer interaction in a patient with symptoms of ornithine carbamoyltransferase deficiency.

In a female patient with signs of ornithine carbamoyltransferase deficiency (OTCD), the only variation found was a heterozygous single nucleotide substitution c.-366A>G. Determination of transcription start sites of human OTC 95, 119 and 169 bp upstream of the initiation codon located the variation upstream of the 5'-untranslated region. We predicted the human promoter and enhancer elements from homology with rat and mouse, performed function analysis of both regulatory regions and assessed the impact of the promoter variation in functional studies using dual luciferase reporter assay. Our data indicate that: (i) Full transcriptional activity of human OTC promoter depends on an upstream enhancer, as do the rodent promoters. (ii) The promoter variation c.-366A>G does not affect the function of the promoter alone but it disrupts the interaction of the promoter with the enhancer. (iii) The promoter-enhancer interaction contributes to tissue specific expression of OTC in the liver. We conclude that mutations in the regulatory regions of OTC can lead to OTCD and should be included in genetic testing.

Adult-onset ornithine transcarbamylase (OTC) deficiency unmasked by the Atkins' diet.

BACKGROUND & AIMS: Late-onset symptoms of urea-cycle disorder may lead to a life-threatening disease which is often undetected. We report the clinical and metabolic manifestations of acute hyperammonemic encephalopathy in a 47-year-old asymptomatic man with ornithine transcarbamylase (OTC) deficiency. The hyperammonemic encephalopathy was unmasked by a high-protein Atkins diet. METHODS: Genetic analysis of the patient's family, 89 unrelated Ashkenazi Jewish and 50 unrelated Europeans subjects was performed using polymerase chain reaction amplification and DNA sequencing of the OTC gene. RESULTS: Treatment with hemodialysis, provision of adequate calories to prevent catabolism, and protein elimination for 24h followed by protein restriction and ammonia scavenging medications effectively lowered the patient's plasma ammonia level and resulted in full recovery. Genetic analysis of the OTC gene revealed a novel hemizygous missense mutation in exon 5 (c.477T>G), leading to an isoleucine-to-methionine substitution in codon 159 (Ile159Met). Further genetic analysis of the patient's family yielded the mutation in many of them, although findings were negative in 89 unrelated Ashkenazi Jewish and 50 unrelated Europeans subjects. CONCLUSIONS: This is the first reported case of an adult urea-cycle defect unmasked by the Atkins diet. Measurements of serum ammonia level must be part of the basic work-up in all patients presenting with encephalopathy of unknown origin even in the absence of liver dysfunction. Awareness of this important association can contribute to prompt diagnosis and life-saving treatment. Correct diagnosis is also important to prevent future recurrences and to provide genetic counselling for family members.

An exon 1 deletion in OTC identified using chromosomal microarray analysis in a mother and her two affected deceased newborns: implications for the prenatal diagnosis of ornithine transcarbamylase deficiency.

We describe the outcome of two consecutive pregnancies with a clinical presentation of ornithine transcarbamylase (OTC) deficiency (OTCD) without a molecular diagnosis. A 119kb deletion on Xp11.4 including the OTC gene was detected in the mother. The same deletion was identified in the blood spots from deceased male newborns. In patients with a clinical and biochemical presentation of OTCD and negative OTC sequencing, whole genome or targeted chromosomal microarray analysis (CMA) with coverage of the OTC and neighboring genes should be performed as a reflex test.

Complete deletion of ornithine transcarbamylase gene confirmed by CGH array of X chromosome.

Ornithine transcarbamylase deficiency is an X-linked semidominant trait that is the most frequent inborn error of the urea cycle. Three hundred and fifty different mutations, including mostly point mutations and a small proportion of large rearrangements have been reported. Conventional molecular diagnosis is highly reliable for point mutations but can miss gross rearrangements. We describe a contiguous gene syndrome involving the RPGR, OTC and TM4SF2 genes in a male patient with severe neonatal OTC deficiency identified by the conventional molecular approach. Molecular characterization was ascertained by X chromosome CGH array and confirmed by MLPA. Complete deletion of the OTC gene led to absent OTC enzymatic activity in liver and to a severe clinical phenotype. The maternal phenotype, characterized by less severe hyperammonaemic crises associated with neurological impairment would result from a deficient but not null OTC activity due to random X chromosome inactivation in the liver. Our cases are similar toothers described presenting with OTC deficient phenotype in which OTC and contiguous genes are affected. Clinical expression would be conditioned by complete OTC deficiency in males and by X chromosome inactivation in females, leading to compensation by the normal allele in tissues such as blood or muscle but not sufficiently in liver. The application of high-resolution genetic techniques allows the characterization of causative mutations such as large deletions in order to guide genetic counselling and prenatal diagnosis.

Hypertransaminasemia in childhood as a marker of genetic liver disorders.

BACKGROUND: The widespread use of routine biochemical assays has led to increased incidental findings of hypertransaminasemia. We aimed to evaluate the prevalence of different causes of raised aminotransferase levels in children referred to a university department of pediatrics. METHODS: We investigated 425 consecutive children (age range, 1-18 years) with isolated hypertransaminasemia. All patients had raised aminotransferase levels on at least two occasions in the last month before observation. Cases due to major hepatotropic viruses were excluded. RESULTS: During the first 6 months of observation, 259 children showed normalized liver enzymes. Among the remaining 166 patients with hypertransaminasemia lasting for more than 6 months, 75 had obesity-related liver disease; 51, genetic disorders; 7, autoimmune hepatitis; 5, cholelithiasis; 3, choledochal cyst; and 3, celiac disease. Among the 51 children with genetic disorders, 18 had Wilson disease; 14, muscular dystrophy; 4, alpha-1-antitrypsin deficiency; 4, Alagille syndrome; 4, hereditary fructose intolerance; 3, glycogen storage disease (glycogenosis IX); 2, ornithine transcarbamylase deficiency; and 2, Shwachman's syndrome. In 22 children, the hypertransaminasemia persisted for more than 6 months in the absence of a known cause. CONCLUSIONS: Genetic disease accounted for 12% of cases of isolated hypertransaminasemia observed in a tertiary pediatric department. A high level of suspicion is desirable for an early diagnosis of these disorders, which may present with isolated hypertransaminasemia and absence of typical clinical signs.

Genotype-Phenotype Correlations in Ornithine Transcarbamylase Deficiency: A Mutation Update.

Ornithine transcarbamylase (OTC) deficiency is an X-linked trait that accounts for nearly half of all inherited disorders of the urea cycle. OTC is one of the enzymes common to both the urea cycle and the bacterial arginine biosynthesis pathway; however, the role of OTC has changed over evolution. For animals with a urea cycle, defects in OTC can trigger hyperammonemic episodes that can lead to brain damage and death. This is the fifth mutation update for human OTC with previous updates reported in 1993, 1995, 2002, and 2006. In the 2006 update, 341 mutations were reported. This current update contains 417 disease-causing mutations, and also is the first report of this series to incorporate information about natural variation of the OTC gene in the general population through examination of publicly available genomic data and examination of phenotype/genotype correlations from patients participating in the Urea Cycle Disorders Consortium Longitudinal Study and the first to evaluate the suitability of systematic computational approaches to predict severity of disease associated with different types of OTC mutations.

Functional characterization of the spf/ash splicing variation in OTC deficiency of mice and man.

The spf/ash mouse model of ornithine transcarbamylase (OTC) deficiency, a severe urea cycle disorder, is caused by a mutation (c.386G>A; p.R129H) in the last nucleotide of exon 4 of the Otc gene, affecting the 5' splice site and resulting in partial use of a cryptic splice site 48 bp into the adjacent intron. The equivalent nucleotide change and predicted amino acid change is found in OTC deficient patients. Here we have used liver tissue and minigene assays to dissect the transcriptional profile resulting from the "spf/ash" mutation in mice and man. For the mutant mouse, we confirmed liver transcripts corresponding to partial intron 4 retention by the use of the c.386+48 cryptic site and to normally spliced transcripts, with exon 4 always containing the c.386G>A (p.R129H) variant. In contrast, the OTC patient exhibited exon 4 skipping or c.386G>A (p.R129H)-variant exon 4 retention by using the natural or a cryptic splice site at nucleotide position c.386+4. The corresponding OTC tissue enzyme activities were between 3-6% of normal control in mouse and human liver. The use of the cryptic splice sites was reproduced in minigenes carrying murine or human mutant sequences. Some normally spliced transcripts could be detected in minigenes in both cases. Antisense oligonucleotides designed to block the murine cryptic +48 site were used in minigenes in an attempt to redirect splicing to the natural site. The results highlight the relevance of in depth investigations of the molecular mechanisms of splicing mutations and potential therapeutic approaches. Notably, they emphasize the fact that findings in animal models may not be applicable for human patients due to the different genomic context of the mutations.

A pilot study of in vivo liver-directed gene transfer with an adenoviral vector in partial ornithine transcarbamylase deficiency.

Ornithine transcarbamylase deficiency (OTCD) is an inborn error of urea synthesis that has been considered as a model for liver-directed gene therapy. Current treatment has failed to avert a high mortality or morbidity from hyperammonemic coma. Restoration of enzyme activity in the liver should suffice to normalize metabolism. An E1- and E4-deleted vector based on adenovirus type 5 and containing human OTC cDNA was infused into the right hepatic artery in adults with partial OTCD. Six cohorts of three or four subjects received 1/2 log-increasing doses of vector from 2 x 10(9) to 6 x 10(11) particles/kg. This paper describes the experience in all but the last subject, who experienced lethal complications. Adverse effects included a flu-like episode and a transient rise in temperature, hepatic transaminases, thrombocytopenia, and hypophosphatemia. Humoral responses to the vector were seen in all research subjects and a proliferative cellular response to the vector developed in apparently naive subjects. In situ hybridization studies showed transgene expression in hepatocytes of 7 of 17 subjects. Three of 11 subjects with symptoms related to OTCD showed modest increases in urea cycle metabolic activity that were not statistically significant. The low levels of gene transfer detected in this trial suggest that at the doses tested, significant metabolic correction did not occur.

Lymphocyte mRNA analysis of the ornithine transcarbamylase gene in Italian OTCD male patients and manifesting carriers: identification of novel mutations.

A new simple, non-invasive method using ornithine transcarbamylase (OTC) mRNA isolated from peripheral blood (PBL) or lymphoblastoid cell lines has been performed. This approach based on reverse transcription and nested PCR to obtain a double strand PBL OTC cDNA allowed the identification of genetic lesions in five Italian families affected by OTC deficiency (OTCD). In the PBL OTC mRNA two new mutations, T262K and W265L, have been detected in three unrelated male OTCD patients with mild symptoms. One known mutation, T264A, has been identified in one manifesting carrier. The known mutation E310X, detected on genomic DNA of another manifesting carrier, failed to be detected in her PBL OTC mRNA because of the presence of a STOP codon. All mutations have been confirmed in the patients' and their relatives' genomic DNA. In three patients the mutations have also been confirmed in the mRNA isolated from frozen liver biopsy. The T262K amino acid substitution has been detected in a male's PBL OTC mRNA at homozygous state while a heterozygous pattern has been detected at the genomic DNA level, suggesting that the patient is a somatic mosaic for this mutation. Here we show that PBL OTC mRNA analysis is useful to detect genetic lesions in male and female OTCD patients.

Mutations and polymorphisms in the human ornithine transcarbamylase gene.

Ornithine transcarbamylase (OTC) deficiency, an X-linked, semidominant disorder, is the most common inherited defect in ureagenesis resulting in hyperammonemia. The previous two mutation updates for the OTC gene were published in 1993 and 1995 and included 36 and 30 mutations respectively. This comprehensive update contains a compilation of 244 mutations including 13 polymorphisms. Twenty-four of the mutations are reported here for the first time. Forty-two percent of the disease-causing mutations are associated with acute neonatal hyperammonemia; 21% were found in patients with late onset disease and approximately 37% were found in manifesting heterozygous females, most of which are presumed to confer a neonatal phenotype in hemizygous males. Also included are residual enzyme activities and residual incorporation of ammonium nitrogen into urea and results of expression studies for a small proportion of the mutations. Most mutations in the OTC gene are "private" and are distributed throughout the gene with paucity of mutation in the sequence encoding the leader peptide (exon 1 and beginning of exon 2) and in exon 7. Almost all mutations in consensus splicing sites confer a neonatal phenotype. Thirteen polymorphisms have been found, several of which are useful for allele tracking in patients in whom the mutation can't be found. Even with sequencing of the entire reading frame and exon/intron boundaries, only about 80% of the mutations are detected in patients with proven OTC deficiency. The remaining probably occur within the introns or in regulatory domains.

Identification of seven novel missense mutations, two splice-site mutations, two microdeletions and a polymorphic amino acid substitution in the gene for ornithine transcarbamylase (OTC) in patients with OTC deficiency.

Ornithine transcarbamylase (OTC) deficiency, a X-linked disorder, is the most frequent inborn error of the urea cycle. Point mutations and small deletions/insertions in the OTC gene are responsible for the majority of the cases and have a "private" character with little recurrence. We report on eleven pathological changes in the OTC gene sequence detected in three males with mild clinical presentations, and in eight symptomatic females. All of these mutations are novel. Only one mutation affects a CpG mutational hot spot, whereas all but one of the mutations caused an abnormal SSCP of the corresponding PCR-amplified exon. Two mutations occurring in females involved one or two base deletions in codons 196 and 330, respectively, causing frameshift changes and premature termination. Another two mutations in a female and a male affected acceptor splice sites at bases -1 and -3 of the intron 6/exon 7 and intron 9/exon 10 junctions, respectively. All other mutations were point changes causing the simple amino acid substitutions (M1I, I160S, L191F, M206I, L301F, P305H and L341P), although the mutation M1I may abolish translation of the OTC polypeptide. This mutation coexisted in a female patient with the change T333A that appears to be a previously unreported polymorphism. The three male patients but only four of the eight female patients inherited the mutation.

Distinctly abnormal brain metabolism in late-onset ornithine transcarbamylase deficiency.

OBJECTIVE: To assess alterations in brain metabolites in patients with late-onset ornithine transcarbamylase deficiency (OTCD). METHODS: Six unrelated, asymptomatic Japanese late-onset OTCD patients were analyzed by proton MRS ((1)HMRS) using a point-resolved spectroscopy technique (repetition and echo times, 5000 and 30 ms). Localized spectra for the centrum semiovale were acquired and absolute metabolite concentrations were calculated using an LCModel. RESULTS: Compared with age-matched controls, N-acetylaspartate and creatine concentrations were normal in all patients. The glutamine (Gln) plus glutamate concentration was increased in four patients, which progressed in proportion to the clinical stage. myo-inositol (mI) could not be detected in five symptomatic patients. A decreased choline (Cho) concentration was detected in two clinically severe patients. (1)HMRS after liver transplantation in one patient revealed the normalization of all metabolites. CONCLUSION: These findings suggest progression of neurochemical events in OTCD, i.e., mI depletion and Gln accumulation followed by Cho depletion, which is reverse of that in hepatic encephalopathy, i.e., Cho depletion followed by mI depletion and Gln accumulation.

[Molecular characterization of a new mutation E122G of human ornithine transcarbamylase gene].

OBJECTIVE: To determine the molecular basis of late onset ornithine transcarbamylase (OTC) deficiency in a Chinese family of Han nationality and the exon sequences of OTC gene of this patient. METHODS: Polymerase chain reaction-single strand conformation polymorphism and direct sequencing were used to identify the mutation type. RESULTS: A missense mutation E122G in the conserved residue of exon 4 was identified which is unreported before. CONCLUSION: The E122G mutation in human OTC gene may cause late onset OTC deficiency.