The role of orotic acid measurement in routine newborn screening for urea cycle disorders.

Urea cycle disorders (UCDs), including OTC deficiency (OTCD), are life-threatening diseases with a broad clinical spectrum. Early diagnosis and initiation of treatment based on a newborn screening (NBS) test for OTCD with high specificity and sensitivity may contribute to reduction of the significant complications and high mortality. The efficacy of incorporating orotic acid determination into routine NBS was evaluated. Combined measurement of orotic acid and citrulline in archived dried blood spots from newborns with urea cycle disorders and normal controls was used to develop an algorithm for routine NBS for OTCD in Israel. Clinical information and genetic confirmation results were obtained from the follow-up care providers. About 1147986 newborns underwent routine NBS including orotic acid determination, 25 of whom were ultimately diagnosed with a UCD. Of 11 newborns with OTCD, orotate was elevated in seven but normal in two males with early-onset and two males with late-onset disease. Orotate was also elevated in archived dried blood spots of all seven retrospectively tested historical OTCD patients, only three of whom had originally been identified by NBS with low citrulline and elevated glutamine. Among the other UCDs emerge, three CPS1D cases and additional three retrospective CPS1D cases otherwise reported as a very rare condition. Combined levels of orotic acid and citrulline in routine NBS can enhance the detection of UCD, especially increasing the screening sensitivity for OTCD and differentiate it from CPS1D. Our data and the negligible extra cost for orotic acid determination might contribute to the discussion on screening for proximal UCDs in routine NBS.

Challenges in diagnosing and managing adult patients with urea cycle disorders.

Urea cycle disorders (UCD) are a group of rare inherited metabolic conditions of amino acid catabolism caused by an enzyme deficiency within the hepatic ammonia detoxification pathway. The presentation of these disorders ranges from life-threatening intoxication in the neonate to asymptomatic status in adults. Late-onset UCDs can present for the first time in adulthood and may mimic other causes of acute confusion or psychiatric diseases, and are often associated with neurological symptoms. Late-onset UCDs may become apparent during periods of metabolic stress such as rapid weight loss, gastric bypass surgery, chronic starvation or the postpartum period. Early diagnosis is critical for effective treatment and to prevent long-term complications of hyperammonemia. The challenges of management of adults include for example: (a) poor compliance to dietary and medical treatment which can result in recurrent hospital admissions; (b) severe neurological dysfunction; (c) the management of pregnancy and the postpartum period; and (d) access to multidisciplinary care peri-operatively. In this review, we highlight a number of challenges in the diagnosis and management of adult patient with late-onset UCDs and suggest a systematic management approach.

Clinical outcomes and the mutation spectrum of the OTC gene in patients with ornithine transcarbamylase deficiency.

Ornithine transcarbamylase (OTC) deficiency is an X-linked inborn error of the urea cycle that leads to the accumulation of ammonia, resulting in neurological deficits. This study was performed to describe the clinical outcomes, biochemical features and molecular spectra of patients with OTC deficiency. A total of 49 patients from 47 unrelated Korean pedigrees were included who were diagnosed with OTC deficiency based on biochemical findings and molecular analyses. Patient clinical features,biochemical findings and molecular data were analyzed retrospectively. Males with neonatal-onset phenotype presented with seizure or altered mentality (n=20). Biochemical findings showed high blood ammonia (1132.5±851.6 µmol l (− 1)) and urineorotic acid (1840.7±1731.3 mmol mol(− 1) Cr) levels. There were also five males with late-onset disease who presented with vomiting, irritability and seizure at age 8.2±9.4 years old (range, 0.6­20 years). Symptomatic females presented with vomiting,seizure, and altered mentality at age 3.5±3.5 years (range, 0.2­12.8 years; n=24). More males with the late-onset form and symptomatic females displayed mild hyperammonemia and orotic aciduria compared with those showing a neonatal phenotype (P<0.05). Molecular analysis identified 37 different mutations (22 missense, 5 large deletions, 4 small deletions, 1 insertion,3 nonsense and 2 splice sites) from all 49 patients; the mutations were dispersed throughout all coding exons. In Korean patients with OTC deficiency, mutations in OTC are genetically heterogeneous. Male patients with the neonatal-onset phenotype showed poor outcomes because of severe hyperammonemia. Early diagnosis and interventions for hyperammonemia can provide more favorable prognosis.

Cross-sectional observational study of 208 patients with non-classical urea cycle disorders.

Urea cycle disorders (UCDs) are inherited disorders of ammonia detoxification often regarded as mainly of relevance to pediatricians. Based on an increasing number of case studies it has become obvious that a significant number of UCD patients are affected by their disease in a non-classical way: presenting outside the newborn period, following a mild course, presenting with unusual clinical features, or asymptomatic patients with only biochemical signs of a UCD. These patients are surviving into adolescence and adulthood, rendering this group of diseases clinically relevant to adult physicians as well as pediatricians. In preparation for an international workshop we collected data on all patients with non-classical UCDs treated by the participants in 20 European metabolic centres. Information was collected on a cohort of 208 patients 50% of which were ≥ 16 years old. The largest subgroup (121 patients) had X-linked ornithine transcarbamylase deficiency (OTCD) of whom 83 were female and 29% of these were asymptomatic. In index patients, there was a mean delay from first symptoms to diagnosis of 1.6 years. Cognitive impairment was present in 36% of all patients including female OTCD patients (in 31%) and those 41 patients identified presymptomatically following positive newborn screening (in 12%). In conclusion, UCD patients with non-classical clinical presentations require the interest and care of adult physicians and have a high risk of neurological complications. To improve the outcome of UCDs, a greater awareness by health professionals of the importance of hyperammonemia and UCDs, and ultimately avoidance of the still long delay to correctly diagnose the patients, is crucial.

[Analysis of ornithine transcarbamylase gene mutations in three boys affected with late-onset ornithine transcarbamylase deficiency].

OBJECTIVE: To identify the types of OTC gene mutations in three male patients with late onset ornithine transcarbamylase deficiency (OTCD, MIM #311250). METHODS: Genomic DNA was extracted from peripheral blood leukocytes. The 10 exons and their flanking sequences of the OTC gene were amplified with polymerase chain reaction and subjected to direct DNA sequencing. RESULTS: Based on DNA sequence analysis, all of the three patients have carried OTC gene mutations. Patients 1 and 2 were both hemizygous for mutation c.586G> A(p.D196N). A novel mutation c.800G> C(p.S267T) were confirmed in patient 3. CONCLUSION: p.S267T mutation has affected the conserved amino acid motif of the OTC protein, and is therefore a pathogenic mutation.

Ornithine transcarbamylase deficiency diagnosed in pregnancy.

Urea cycle enzymes deficiencies are rare metabolic disorders. Ornithine transcarbamylase (OTC) deficiency is the most common type. The syndrome results from a deficiency of the mitochondrial enzyme OTC which catalyses the conversion of ornithine and carbamoyl phosphate to citrulline. It shows X-linked inheritance and typically remains asymptomatic until late infancy or early childhood. The severity of the symptoms depends on the age of the patient and the duration of hyperammonemia. Female heterozygotes are more difficult to diagnose. They suffer from hyperammonemic periods which can be triggered by trauma, infections, surgery, childbirth, parenteral nutrition, and by the initiation of sodium valproate therapy. The prognosis of OTC deficiency is better for those with an onset after infancy, but morbidity from brain damage does not appear to be linked to the number of episodes of hyperammonemia that have occurred. However, early diagnosis and prompt initiation of ammonia-lowering treatment are essential for survival of these patients. This case presents a patient who was diagnosed with OTC deficiency following mental confusion during pregnancy.

Urea cycle disorders in Argentine patients: clinical presentation, biochemical and genetic findings.

BACKGROUND: The incidence, prevalence, and molecular epidemiology of urea cycle disorders (UCDs) in Argentina remain underexplored. The present study is the first to thoroughly assess the clinical and molecular profiles of UCD patients examined at a single reference center in Argentina. RESULTS: Forty-nine UCD cases were collected. About half (26/49, 53%) manifested neonatally with classical presentation and had a high mortality (25/26, 96%). Ornithine transcarbamylase deficiency (OTCD) was the most common UCD (26 patients). Argininosuccinate synthetase deficiency (ASSD) was detected in 19 cases, while argininosuccinate lyase deficiency (ASLD) was diagnosed in 4 cases. Molecular genetic analysis revealed 8 private OTC mutations and two large deletion/duplication events in the OTC gene. Most mutations in the ASS1 and ASL genes were recurrent missense changes, and four alterations were novel. The clinical outcome of our UCD cohort was poor, with an overall mortality of 57% (28/49 cases), and a 28% (6/21) disability rate among the survivors. CONCLUSIONS: Most patients in our case series showed severe neonatal onset, with high morbidity/mortality. We detected in total 19 mutations, most of them recurrent and of high frequency worldwide. Noteworthy, we highlight the presence of a geographic cluster with high prevalence of a point mutation in the ASS1 gene. This study suggests that these disorders may be more frequent than commonly assumed, and stresses the need for increased awareness amongst health professionals and greater availability of diagnostic tools for accurate identification, early diagnosis, and timely treatment.

Cross-sectional multicenter study of patients with urea cycle disorders in the United States.

Inherited urea cycle disorders comprise eight disorders (UCD), each caused by a deficiency of one of the proteins that is essential for ureagenesis. We report on a cross-sectional investigation to determine clinical and laboratory characteristics of patients with UCD in the United States. The data used for the analysis was collected at the time of enrollment of individuals with inherited UCD into a longitudinal observation study. The study has been conducted by the Urea Cycle Disorders Consortium within the Rare Diseases Clinical Research Network (RDCRN) funded by the National Institutes of Health. One-hundred eighty-three patients were enrolled into the study. Ornithine transcarbamylase (OTC) deficiency was the most frequent disorder (55%), followed by argininosuccinic aciduria (16%) and citrullinemia (14%). Seventy-nine percent of the participants were white (16% Latinos), and 6% were African American. Intellectual and developmental disabilities were reported in 39% with learning disabilities (35%) and half had abnormal neurological examination. Sixty-three percent were on a protein restricted diet, 37% were on Na-phenylbutyrate and 5% were on Na-benzoate. Forty-five percent of OTC deficient patients were on L-citrulline, while most patients with citrullinemia (58%) and argininosuccinic aciduria (79%) were on L-arginine. Plasma levels of branched-chain amino acids were reduced in patients treated with ammonia scavenger drugs. Plasma glutamine levels were higher in proximal UCD and in neonatal type disease. The RDCRN allows comprehensive analyses of rare inherited UCD, their frequencies and current medical practices.

Clinical onset and prognosis of Asian children with organic acidemias, as detected by analysis of urinary organic acids using GC/MS, instead of mass screening.

Organic acidemias (OAs) have been detected worldwide in symptomatic patients using gas chromatography mass spectrometry. We diagnosed 188 Asian cases of OAs by analysis of urinary organic acids and investigated their clinical onset and outcome. Methylmalonic acidemia (MMA) was most common (74 cases), followed by propionic acidemia (23 cases), ornitine transcarbamylase deficiency (22 cases), and multiple carboxylase deficiency (15 cases). For these 188 patients, onset was most frequent in the neonatal period or early infancy. Approximately 30% of the patients had a family history of similar symptoms or diseases. Although the outcome of OA patients varied, patients with early onset generally had poor outcomes despite early detection. Of the 45 MMA patients whose clinical data were available, 25 were clinically vitamin B12-responsive, while the remaining 20 were non-responsive. A favorable outcome was obtained in 7 of the 25 B12-responsive patients, and in only 3 of the 20 B12-nonresponsive patients. It was suggested that even in B12-responsive MMA cases, earlier detection and B12 therapy were needed to improve the prognosis. We concluded that detection of such patients at the presymptomatic stages using newborn mass screening is essential for prognosis improvement with OAs.

Evidence for mutational cis-acting factors affecting mutagenesis in the ornithine transcarbamylase gene.

Ornithine transcarbamylase (OTC; EC 2.1.3.3) is an urea cycle enzyme coded by a gene located at Xp21.1. The genetic deficiency is caused by a wide spectrum of pathological mutations, most of them occurring de novo. Using two (CA)n flanking markers of the OTC gene (DXS997 and DXS1068), we have defined the haplotypic background underlying 37 different mutational events and compared the results with a random sample of control chromosomes (N=141) from Iberia Peninsula. The allelic distribution of the (CA)n markers revealed significant differences between affected and non-affected chromosomes. One particular haplotypic combination can be considered as a risk factor for carrying OTC mutations, with a relative risk of 13.3 (95% confidence interval 2.89-61.5, p=1.5 x 10(-5)). Since most of pathogenic OTC mutations are short-lived or de novo, these findings strongly support the hypothesis that a specific haplotypic background confers a higher risk for mutation occurrence at this locus.

Magnetic resonance imaging in late-onset ornithine transcarbamylase deficiency.

We examined brain magnetic resonance imaging (MRI) in a cohort of seven patients with ornithine transcarbamylase deficiency (OTCD), and correlated MRI findings with clinical manifestations. Seven patients with OTCD, aged 3-27 years, all with a missense mutation, were involved in the study. We classified the OTCD patients clinically into four stages. MR study was performed with a 1.5-T superconducting magnet during asymptomatic periods. MRI revealed white matter lesions in two patients with an advanced clinical stage, i.e. T1 and T2 prolongated round lesions in the deep white matter and posterolateral angle of the lateral ventricle in one patient; small foci of T2 and T1 prolongation in the subcortical white matter in another. Parenchymal lesions, and cerebral and cerebellar atrophy were not found in the other five patients. MRI might be normal in the early stage of the disease, and progress in proportion to the clinical stage of OTCD. OTCD should be considered as a differential diagnosis of small foci in the white matter in children.

Recombinant human arginase inhibits proliferation of human hepatocellular carcinoma by inducing cell cycle arrest.

Human hepatocellular carcinoma (HCC) has an elevated requirement for arginine in vitro, and pegylated recombinant human arginase I (rhArg-PEG), an arginine-depleting enzyme, can inhibit the growth of arginine-dependent tumors. While supplementation of the culture medium with ornithine failed to rescue Hep3B cells from growth inhibition induced by rhArg-PEG, citrulline successfully restored cell growth. The data support the roles previously proposed for ornithine transcarbamylase (OTC) in the arginine auxotrophy and rhArg-PEG sensitivity of HCC cells. Expression profiling of argininosuccinate synthetase (ASS), argininosuccinate lyase (ASL) and OTC in 40 HCC tumor biopsy specimens predicted that 16 of the patients would be rhArg-sensitive, compared with 5 who would be sensitive to arginine deiminase (ADI), another arginine-depleting enzyme with anti-tumor activity. Furthermore, rhArg-PEG-mediated deprivation of arginine from the culture medium of different HCC cell lines produced cell cycle arrests at the G(2)/M or S phase, possibly mediated by transcriptional modulation of cyclins and/or cyclin dependent kinases (CDKs). Based on these results, together with further validation of the in vivo efficacy of rhArg-PEG against HCC, we propose that the application of rhArg-PEG alone or in combination with existing chemotherapeutic drugs may represent a specific and effective therapeutic strategy against HCC.

Paternal transmission and slow elimination of mutant alleles associated with late-onset ornithine transcarbamylase deficiency in male patients.

In ten families with late-onset ornithine transcarbamylase (OTC) deficiency in male patients, three mutant alleles-R40H, R277W, and Y55D-were identified. In a total of 20 informative parent-offspring pairs, father-to-daughter transmission and mother-to-offspring transmission occurred in five (25%) and 15 (75%), respectively, indicating that paternal transmission contributes substantially to the pool of these mutant alleles. Relative reproductive fitness of males and females carrying the mutant alleles was calculated to be 0.49 and 0.89, respectively. Comparison of the life span of the mutant alleles, estimated on the basis of these fitness values with those associated with classic phenotype (neonatal onset) in which reproductive fitness of male patients was nil, revealed that mutant alleles associated with the late-onset phenotype were eliminated more slowly. This would allow the late-onset phenotype mutant alleles to be retained more frequently in a population than those associated with classic phenotype. Although heterozygous females carrying the late-onset phenotype mutant alleles were generally asymptomatic, one female carrying the R40H allele died after a hyperammonemic episode at the age of 18 years. Such heterozygous females should be alerted to possible hyperammonemic crisis.

Diagnosis, symptoms, frequency and mortality of 260 patients with urea cycle disorders from a 21-year, multicentre study of acute hyperammonaemic episodes.

AIM: A large longitudinal interventional study of patients with a urea cycle disorder (UCD) in hyperammonaemic crisis was undertaken to amass a significant body of data on their presenting symptoms and survival. METHODS: Between 1982 and 2003, as part of the FDA approval process, data were collected on patients receiving an intravenous combination of nitrogen scavenging drugs (Ammonul sodium phenylacetate and sodium benzoate (10%, 10%)) for the treatment of hyperammonaemic crises caused by urea cycle disorders. RESULTS: A final diagnosis of a UCD was made for 260 patients, representing 975 episodes of hospitalization. Only 34% of these patients presented within the first 30 days of life and had a mortality rate of 32%. The most common presenting symptoms were neurological (80%), or gastrointestinal (33%). This cohort is the largest collection of patients reported for these diseases and the first large cohort in the United States. CONCLUSION: Surprisingly, the majority (66%) of patients with heritable causes of hyperammonaemia present beyond the neonatal period (>30 days). Patients with late-onset presenting disorders exhibited prolonged survival compared to the neonatal-presenting group.

Hereditary urea cycle diseases in Finland.

AIM: To estimate the incidence of urea cycle diseases (UCDs) in Finland and determine the course of the various disorders as well as the outcome. METHODS: The original data were collected in the years 1998-2001. The diagnoses made after 2001, as well as the current status of the patients, were updated by surveys in the spring of 2007. RESULTS: We found a total of 55 cases of UCDs in Finland by 2007: 30 cases of ornithine transcarbamylase (OTC) deficiency, 20 of argininosuccinate lyase (ASL) deficiency, 3 of carbamyl phosphate synthetase (CPS-I) deficiency, 1 of type 1 citrullinaemia and 1 of argininaemia. The estimated total incidence of UCDs was 1:39 000. The incidences of individual disorders were: OTC deficiency 1:62 000, ASL deficiency 1:144 000, CPS deficiency 1:539 000 and citrullinaemia 1:1 616 000. Eighteen (33%) of the patients with a diagnosis of UCD have died, most during their first hyperammonaemic crisis. One patient with OTC deficiency has had a liver transplant. Neurological symptoms of varying severity are common among these patients, particularly those with ASL deficiency. CONCLUSION: The first survey on the incidence of UCDs in Finland shows some differences in the occurrence rates compared to other countries. Hyperammonaemia, and the neurological symptoms caused by it, can be avoided in most patients with late-onset UCDs with a standard treatment. However, in patients with ASL deficiency, the development of neurological symptoms seems to be inevitable in spite of careful treatment and avoidance of hyperammonaemia.

Late-onset ornithine transcarbamylase deficiency in male patients: prognostic factors and characteristics of plasma amino acid profile.

BACKGROUND: The occurrence of male patients with ornithine transcarbamylase (OTC) deficiency during adolescence or in adulthood has now been recognized. The aim of this study was to determine the prognostic factors that affect the prognosis of life, to explore a basis for therapeutic strategy. METHODS: In 10 patients, nine of whom carried the R40H mutation and the other one carrying the Y55D mutation in the OTC gene, 32 demographic and laboratory data were first compared between survivors and non-survivors, using the unpaired t-test. The factors with significant difference were then subjected to multiple regression analysis. RESULTS: The factors that exhibited significant difference were: age at onset, concentration of plasma ammonium, blood pH, and concentrations of six amino acids in plasma. The multiple regression analysis then revealed concentrations of ammonium, leucine, lysine, isoleucine, phenylalanine, glutamine and proline to be significant prognostic factors. The amino acid profile in the 10 patients showed increases in glutamine, proline, lysine, valine and methionine, and decreases in serine, ornithine and arginine. There was an inverse correlation between the age at onset and the level of the residual hepatic OTC activity. CONCLUSION: The results implied that: (i) the plasma amino acid profile was unique, in comparison to other liver diseases; (ii) the plasma concentration of each of the (mentioned above) six amino acids was a significant predictor of prognosis; and (iii) suppression of protein catabolism, as suggested by the higher concentrations in isoleucine and leucine in the non-survivors, prevention of glutamine-induced brain edema, correction of alkalosis, and supplementation with ornithine or arginine may improve the prognosis of life.

How reliable is the allopurinol load in detecting carriers for ornithine transcarbamylase deficiency?

The allopurinol test aims to distinguish carriers and noncarriers for ornithine transcarbamylase (OTC) deficiency. We have evaluated the reliability of the test in at-risk females of known genotype. Results based on urine orotidine and/or orotic acid measurement were compared in terms of sensitivity and specificity. Retrospectively, we analysed the results of allopurinol tests in 42 women (22 confirmed heterozygotes and 20 noncarriers) from 23 pedigrees at risk of being carriers for OTC deficiency. Using a cut-off of 2 standard deviations above the mean of controls, the highest sensitivity (91%) was given by orotidine alone or in combination with orotic acid, but specificity was only 70% and 65%, respectively. We conclude that the value of the allopurinol test for detecting OTC carriers in at-risk females is limited. This needs to be recognized when counselling families. The test still has a role as a safe, quick, noninvasive screen of individuals at risk, but test results in possible carriers should be interpreted with caution. In the absence of other supportive evidence, confirmation by mutation analysis is required.