Introduction

Ornithine transcarbamylase (OTC) deficiency (OMIM #311250) is the most common urea cycle disorder caused by mutations in the OTC gene located on chromosome Xp21, resulting in hyperammonemia that causes neurological deficits.1 OTC catalyzes the formation of citrulline and inorganic phosphate from carbamyl phosphate and ornithine in the urea cycle.2 Its clinical signs and symptoms are caused by the toxic effects of hyperammonemia on the brain, leading to coma, cerebral edema and, in severe cases, death.2 When clinically suspected, the diagnosis of OTC deficiency can be made biochemically based on a low blood citrulline level and increased urinary excretion of orotic acid.1

OTC deficiency can be classified into two groups: a neonatal-onset phenotype that completely abolishes enzyme activity resulting from null alleles and a later onset phenotype with residual enzyme activity.1 Hemizygous males with the neonatal-onset form present with a hyperammonemic coma that often leads to death in the first week of life; those individuals who recover from coma can still be neurologically handicapped as a consequence of hyperammonemic encephalopathy.1 By contrast, most heterozygous females and some male patients manifest milder late-onset phenotypes with partial enzyme activity.3 Mutations that result in residual enzymatic activity confer a milder phenotype, with the first clinical presentation occurring sometime between infancy and adulthood.

As of March 2015, 457 OTC mutations have been reported (http://www.hgmd.org/). Most of the mutations that cause OTC deficiency consist of single-base pair substitutions that cause missense or nonsense mutations (301/457, 65.9%), whereas a smaller proportions consist of small deletions or insertions (57/457, 12.5%), splice site mutations (50/457, 10.9%), large deletions (41/457, 9.0%), complex rearrangements (6/457, 1.3%) or regulatory mutations (2/457, 0.4%).

Our research group has previously reported clinical features, biochemical findings and molecular analyses of OTC in a total of 33 patients with OTC deficiency in Korea.4, 5, 6, 7 These studies included a small number of patients and did not include the long-term outcomes of all subjects. Hence, this present study was undertaken to analyze the clinical outcomes and biochemical features of Korean patients with OTC deficiency and to better characterize the molecular spectrum of mutations of the OTC gene among these cases.

Subjects and methods

Patients

A total of 49 patients (25 males, 24 females) from 47 independent pedigrees were included in the current investigation. All patients were born at term with an uneventful delivery. The diagnosis of OTC deficiency was based on the biochemical findings and molecular analyses of the OTC gene. The clinical phenotypes of all patients, such as age at diagnosis, presenting symptoms and developmental outcomes, as well as biochemical findings, including plasma ammonia, serum glutamate, glutamine, citrulline and urine orotic acid levels, were reviewed retrospectively. Molecular defects in the OTC gene were also analyzed (see below). Written informed consent was obtained from all subjects or from their parents. The study protocol was approved by the Institutional Review Board of Asan Medical Center.

Molecular analysis of the OTC gene

Genomic DNA was extracted from peripheral blood leukocytes using a QuickGene DNA blood kit (Fujifilm, Tokyo, Japan). A total of 10 coding exons and exon–intron boundaries of the OTC gene were amplified by PCR with customized primers. PCR products were directly sequenced with the same primers using a BigDye Terminator v3.0 Cycle Sequencing Ready Kit (Applied Biosystems, Foster City, CA, USA). Sequencing results were compared with the established human OTC sequences (National Center for Biotechnology Information Accession No. NM_000531.5).

Multiplex ligation-dependent probe amplification analysis was performed for patients in whom no OTC mutations were identified by direct sequencing using the OTC MLPA kit (P079; MRC Holland, Amsterdam, The Netherlands) in accordance with the manufacturer’s protocol. The products were separated by electrophoresis and the data were analyzed using an ABI3130x1 Genetic Analyzer (Applied Biosystems). The peak height of each probes was analyzed using GeneMarker software version 1.70 (SoftGenetics, State College, PA, USA).

Statistical analyses

Statistical analyses were performed using SPSS for Windows version 21.0 (SPSS, Chicago, IL, USA). The age at onset and biochemical parameters in neonatal-onset males, late-onset males and symptomatic females were analyzed using the Kruskal–Wallis test. Clinical outcomes were classified as normal development, developmental delay or death; the outcomes of the three groups were analyzed using Fisher’s exact test. Kaplan–Meier curves of the estimated survival rate for each group were generated. P-values <0.05 were considered to be statistically significant.

Results

Clinical characteristics of OTC-deficient cases at the time of presentation

The clinical phenotypes and biochemical findings at presentation, and molecular analysis of the OTC gene are summarized in Tables 1,2,3 according to neonatal-onset males, late-onset males and symptomatic females. All 20 males in our cohort with a neonatal-onset phenotype presented with seizure or altered mental function at the age of 4.3±2.1 days (range, 2–10 days). Only one patient (subject 14) was diagnosed immediately after birth in the presymptomatic period for screening of high-risk family members because his two male siblings died during the neonatal period. Mutations in OTC were verified in the mothers in 14 (70%) of these patients. Biochemical findings revealed high blood ammonia (1132.5±851.6 μmol l−1), serum glutamine (2009.8±963.5 μmol l−1; normal 246–1182) and glutamate (625.0±1175.2 μmol l−1; normal 32–140) levels and profuse urine orotic acid excretion (1840.7±1731.3 mmol mmol−1 Cr; normal 0.3–6.0) levels (Table 3).

Table 1 Clinical, biochemical features and molecular analysis of the OTC gene in male subjects with a neonatal-onset OTC deficiency
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Table 2 Clinical, biochemical features and molecular analysis of the OTC gene in male subjects with late-onset OTC deficiency
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Table 3 Clinical, biochemical features and molecular analysis of symptomatic female subjects with mutations in the OTC gene
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The five males from four independent pedigrees with a late-onset phenotype presented with vomiting, irritability, seizure or drowsy mental status resulting from hyperammonemia at the age of 8.2±9.5 years old (range, 0.6–20 years). These cases were confirmed to have OTC deficiency at the age of 8.6±9.5 years old (range, 0.8–20 years). An OTC mutation was documented in the mothers of three (3/5, 60%) of these cases. Biochemical findings of subjects with a late-onset phenotype demonstrated mild hyperammonemia (163.8±100.4 μmol l−1; range, 40–258), moderately high serum glutamine (706±231.7 μmol l−1; range, 462–923) and glutamate (435.7±537.6 μmol l−1; range, 88–1331) levels and orotic aciduria (128.1±266.8 mmol mmol−1 Cr; range, 0–605) compared with those cases showing a neonatal-onset phenotype (P<0.05), except for serum glutamate levels (Table 4). Among the five late-onset male patients in our series, two patients (subjects 24 and 25 in Table 2) were siblings who had the same mutation, but the older brother (subject 25) had no clinical abnormalities; the other sibling (subject 24) manifested altered consciousness and hyperammonemia (258 μmol l−1) at the age of 18 years old and recovered after continuous renal replacement therapy, suggesting that phenotypic heterogeneity existed within this family.5 Subject 25 was diagnosed in the presymptomatic stage by screening for OTC deficiency in a high-risk family. Prophylactic sodium benzoate and sodium phenylbutyrate were initiated when he was confirmed to have OTC deficiency.

Table 4 Comparison of clinical and biochemical features in male subjects with a neonatal- or late-onset phenotype and symptomatic females
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The 24 symptomatic females with heterozygous mutations in OTC presented with vomiting, seizure and altered mentality at the age of 3.5±3.5 years old (range, 0.2–12.8 years). Laboratory findings demonstrated mild hyperammonemia (350.1±146.8 μmol l−1; range, 157–850), mild elevation of serum glutamine (996.0±390.7 μmol l−1; range, 334–1754), glutamate (250.8±314.1 μmol l−1; range, 40–1269) and urine orotic acid (83.9±74.4 mmol mol−1 Cr; range, 11.5–293) levels. Notably, in a female heterozygote (subject 28), hyperammonemia (285 μmol l−1) was triggered by a traffic accident at the age of 12.1 years old. Subject 45 (Table 3) was a female sibling of a male patient (subject 13 in Table 1) who showed a neonatal-onset phenotype and shared the same mutation, suggesting phenotypic heterogeneity associated with the same mutation.

The mutational spectrum of the OTC gene

Molecular analysis identified 37 different mutations (22 missense mutations (59.5%), 5 large deletions (13.5%), 4 small deletions (10.8%), 3 nonsense mutations (8.1%), 2 splice site mutations (5.4%) and 1 insertion (2.7%)) among the 49 patients in our series. These mutations were dispersed throughout all coding exons and did not exhibit any domain-specificity as depicted in Figure 1. The p.R26Q, p.H214Y, p.A140P, p.R141Q and complete deletion of OTC mutations were found in two unrelated probands, respectively. The c.664_667delinsAC (p.G222Tfs*2) mutation was identified in two siblings (subjects 13 and 45). Among the 37 mutations, six have not been reported elsewhere (p.L57P, deletion of exons 6–10, deletion of exon 10, deletion of exon 2, deletion of exon 5 and c.799_800insA). The p.L57P variant was predicted to be deleterious by PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2/) and SIFT (http://sift.jcvi.org/), and was not found in the 1000Genomes (http://browser.1000genomes.org/) and dbSNP (http://www.ncbi.nlm.nih.gov/snp/) databases.

Figure 1
figure 1

Mutational spectrum of the OTC gene in Korean patients with ornithine transcarbamylase (OTC) deficiency. Molecular analysis identified 37 different mutations among the 49 patients. These mutations were dispersed throughout all coding exons without domain-specificity.

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A total of 18 different mutations were identified in males with a neonatal-onset phenotype (Figure 1). Of these, 75% (15 subjects) of the mutations were inherited from their mother (Table 1). Most have been previously reported to cause a neonatal phenotype,2, 4, 5, 8, 9, 10, 11 except for the two novel large deletion mutations (deletion of exon 10 and deletion of exons 6–10). In five subjects with a late-onset phenotype from four unrelated families, four mutations were identified: p.G100R, p.R277W, p.V323M and c.867-3T>C (Figure 1). The mutations were inherited in three subjects (60%) from two families. The p.G100R and p.V323M mutations were found to have residual enzyme activities, which ranged from 17 to 23%,4 and the p.R277W and c.867-3T>C mutations have also been reported to be associated with late-onset disease.5, 12

We observed 21 different mutations in symptomatic heterozygous females in our series. Seven (29.2%) females’ mutations were documented to be inherited from their mother. The p.R26Q, p.R141Q, p.Y183C, p.H214Y and p.R320* mutations were also identified in male patients with neonatal-onset phenotype in our cohort. The p.N47I, p.A140P, p.N161S, p.T178M, p.M206R, p.K221N, c.717+3A>G and complete deletion of OTC mutation have been previously reported in patients with neonatal onset.2, 13, 14, 15, 16, 17 We found only one manifesting female heterozygote who had a mutation that was associated with late-onset disease in males (p.R129H).18 Two mutations (p.T44I and p.A237D) have only been previously reported in female heterozygotes by our group.4 This finding suggests that most mutations found in symptomatic female heterozygotes are presumed to confer a neonatal phenotype in hemizygous males, whereas mutations conferring a late-onset type in males are rarely observed in manifesting heterozygous females.3

Clinical outcomes

The clinical outcomes of all subjects with OTC deficiency are summarized in Figure 2. Patients were treated with protein restriction with special formula, arginine (100–150 mg kg−1 per day), or citrulline (150–200 mg kg−1 per day) and alternative pathway therapy that included sodium benzoate (250 mg kg−1 per day) and sodium phenylbutyrate (250 mg kg−1 per day). In Figure 3, 11 (11/20, 45%) neonatal-onset male patients died because of hyperammonemic crisis in the neonatal period or infancy, and seven males exhibited severe neurological deficits. Although four of our patients with neonatal onset (subjects 9, 13, 19 and 20) underwent continuous renal replacement therapy, one (subject 20) died of hyperammonemic encephalopathy at 2 months of age, and two (subjects 9 and 13) displayed developmental delay. Subject 2 developed a cataract in the left eye and lens dislocation in the right eye at 7 months of age, and then underwent lens removal at 8 months of age. Subject 24 with late-onset disease underwent continuous renal replacement therapy at the time of diagnosis because of altered consciousness. He recovered completely without any neurological defects.

Figure 2
figure 2

Clinical outcomes of the study subjects with ornithine transcarbamylase (OTC) deficiency. Male patients with the neonatal-onset phenotype showed poor outcomes with neurological deficit or death because of severe hyperammonemia, whereas most females exhibited normal development without neurological sequelae. Two males with neonatal phenotype and one heterozygous female who underwent liver transplantation showed normal development without neurologic sequelae.

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Figure 3
figure 3

Survival rates of male subjects with neonatal onset or late onset and symptomatic females. The survival rate of neonatal-onset disease was significantly lower (45%) compared with those of late-onset disease (100%) and symptomatic females (91.7%; both, P<0.001).

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Most females (14/24, 58.3%) in our series exhibited normal development without neurological sequelae. The remaining eight heterozygous female patients experienced neurological sequelae, such as developmental delay. Two female heterozygotes, subjects 27 and 42, died at 2.3 and 5.5 years of age, respectively. Interestingly, one heterozygous female (subject #26 in Table 3) was diagnosed with 21-hydroxylase deficiency at 1 month of age because of a salt-losing phenomenon and hyperkalemia. Molecular testing of the CYP21A2 gene identified the compound heterozygous mutations c.[293-13A>G];[955C>T] (p.[splicing];[Q319*]) in this individual. At the age of 2.2 years, she experienced recurrent vomiting and a drowsy mentality and was subsequently diagnosed with OTC deficiency by biochemical and molecular analysis. This subject has since been under treatment with sodium beozoate, sodium phenylbutyrate and arginine with a low-protein diet and has shown normal development.19

Liver transplantation was performed in two males in our cohort with neonatal phenotype (subjects 14 and 19) during infancy and one heterozygous female (subject 38) at age 2 years; these patients showed normal development without neurologic sequelae. Patients were classified into neonatal onset, late-onset males and symptomatic females. Their outcomes, including normal development, developmental delay and death, were examined using a Fisher’s exact test. Overall, we found that the outcomes of the three groups were significantly different (P<0.001), indicating that an earlier onset age was associated with poorer developmental outcomes.

Discussion

We here describe 49 patients with OTC deficiency from 47 unrelated Korean pedigrees. The severity of clinical symptoms differed based on the age at onset and hyperammonemia. The age at onset of male patients with a neonatal-onset phenotype was significantly earlier than males with late-onset disease and symptomatic female heterozygotes (P<0.001, Table 4). Male patients with the neonatal-onset form showed poor clinical outcomes with neurological deficit or death resulting from severe hyperammonemia compared with those with late-onset and symptomatic females (P<0.001, Fisher’s exact test). The late onset of male and female patients presented with diverse clinical features, which can hamper early diagnosis for the prevention of acute metabolic crash.

The survival rates of patients with neonatal onset, late onset and manifesting female heterozygotes were 45%, 100% and 91.7%, respectively, resulting in an overall survival rate of 73.5% (Figure 3). The mortality rate of males with neonatal-onset disease was relatively higher than that reported previously20 because most patients with neonatal-onset type manifesting severe hyperammonemia were identified at a local hospital and were transferred to a tertiary medical center late. Subsequently, therapeutic interventions, such as hemodialysis, continuous renal replacement therapy and toxic metabolite removal by alternative pathway therapy, were so delayed that most patients died before reaching 2 weeks of age, which occurred prior to commencing treatment or becoming neurologically crippled. In addition, continuous renal replacement therapy for newborns was not widely available in the early 1990s. Early diagnosis and proper intervention is critical for achieving good outcomes, as supported by the fact that two males in our present series with a neonatal-onset phenotype (subjects 14 and 19) who were diagnosed early and underwent liver transplantation showed normal development.

Our present study identified 37 different mutations in 47 independent pedigrees in patients with OTC deficiency. The mutations were evenly distributed throughout all coding exons of the OTC gene and were largely ‘private’.21 In patients with neonatal onset, these mutations have been predicted to abolish all enzyme activity, with late-onset cases showing residual OTC enzyme activity.3, 22, 23 It is known that most OTC mutations found in patients with neonatal presentation involve amino-acid residues that are located within the interior of the protein or at the active site, whereas those found in patients with late-onset disease involve an amino-acid residue located far from the active site or on the surface.21 Splice site mutations in non-consensus sequences have also been shown to cause partial loss of enzymatic activity.2

However, in the case of an OTC mutation, it is not always possible to predict phenotype based on the genotype, especially in late onset and symptomatic female groups in which a different age at onset and divergent clinical courses can be observed within a genotype.24 Phenotypic heterogeneity can also be observed in patients within the same family, and might result from a combination of genetic factors and environmental variables, such as infections or other causes of catabolic stress.21 Thus, the diagnosis of OTC deficiency should be considered in patients with hyperammonemia, orotic aciduria and low plasma citrulline levels regardless of their age and gender.1 More research is needed to better understand the genetic and environmental factors that influence the manifestation of partial deficiencies.

Most of our current symptomatic females harbored mutations that were also identified in male patients with neonatal-onset phenotype, as previously described.3 However, female patients who are heterozygous for an OTC mutation present a wide spectrum of clinical manifestations, from apparently normal-to-severe neonatal onset because of random X-chromosome inactivation in hepatocytes or the severity of the mutation.25 Female patients in one family can also exhibit different phenotypes with the same mutation of the OTC gene.26 Our current data are consistent with the previous findings showing that symptomatic females were associated with OTC mutations that caused a severe neonatal-onset phenotype,3 except for subject 30 in our current series with the p.R129H mutation, which had been previously identified in late-onset disease.18 It is possible that X-chromosome inactivation in peripheral blood cells might not fully reflect the inactivation pattern in liver cells. Therefore, highly skewed X inactivation in the liver in favor of the mutant allele could cause disease manifestation in female heterozygotes with the hypomorphic mutation p.R129H.27 As symptomatic females who were treated with medicines that activate pathways of waste-nitrogen excretion have fewer hyperammonemic episodes and a reduced risk of cognitive impairment,28 it is recommended to treat them to improve survival and stabilizing cognitive functioning.

In summary, we here report the clinical outcomes of a series of 49 Korean patients with OTC deficiency, and the mutational spectrum in the OTC gene. Our data indicate that most of the mutations in this population were private and genetically heterogeneous. Our male patients with a neonatal-onset phenotype showed poor outcomes, and we found that an early diagnosis and interventions for hyperammonemia can provide a more favorable prognosis. In addition, family screening and genetic counseling can also be an important aspect of disease prevention.