1.Clinical and biochemical findings of patients with OTCD.
Patient 1 was a 2-year and 3-month old girl admitted to our hospital for evaluating the causes of acute liver failure. She was the first child of non-consanguineous parents. A week prior to admission, the child developed vomiting, and abnormally increased serum ALT (1316U/L, reference range: 5-40U/L) and AST (833 U/L, reference range: 8-40U/L). Upon referral to our hospital, laboratory investigations revealed elevated levels of ALT (1176U/L, reference range:9-50U/L), AST (550U/L, reference range: 15-40U/L) and ammonia (256mmol/L, reference range: 9-33mmol/L). The International Normalized Ratio (INR) was 1.55 (reference range:0.8–1.2). Plasma L-glutamine was 19.22 µmol/L (reference range:1–70µmol/L) and citrulline was 36.71 µmol/L (reference range:6–45 µmol/L). Urine organic acid analysis was performed 5 days after the treatment of hepatoprotective drugs and the uracil was 119.19 mmol/L (reference range:0-7mmol/L) (Table 1). The results of electroencephalography and brain magnetic resonance imaging (MRI) showed normal. Abdominal ultrasound demonstrated slightly enhanced liver echo. Based on above clinical findings, urea cycle defect was suspected.WES revealed a novel heterozygous frameshift mutation in the OTC gene (c.617dupT, p.M206fs*19) (Table 2). PCR-Sanger sequencing confirmed the presence of this mutation in the patient, and this mutation was absent in her mother (Fig. 1A). In addition to protein restriction, L-ornithine L-aspartate and L-arginine were included in her treatments. During hospitalization and after discharge, she never experienced any mental symptom and metabolic decompensation. At the last follow-up visit when the patient was 3.5 years old, she had entered kindergarten.
Patient 2 was a 3-year and 4-month old girl and was the first child of non-consanguineous parents. She was noted hyperactivity and sleep-wake disturbances from 2 months ago before admission. 4 days before admission, vomiting occurred once after eating in the kindergarten, and after that, her spirit was poor and speech was unclear. The biochemical examination report at that time showed the elevation of transaminase (ALT 903U/L, reference range:7-40U/L and AST 425.2U/L, reference range:13-35U/L). Computed tomography (CT) of the head demonstrated no abnormalities. Upon hospitalization, laboratory investigations revealed elevated ammonia level (192 µmol/L; reference range 9–33 µmol/L) with transaminitis (ALT583U/L, reference range:9-50U/L and AST 61U/L, reference range:15-40U/L). The INR was 1.79 (reference range:0.8–1.2). Plasma citrulline was 34.85 µmol/L (reference range:7–36 µmol/L). Urine organic acid analysis was performed on the second day of admission, the uracil was 185.06 mg/mg. Cr (reference range:0-5.5 mg/mg. Cr) and urinary orotic acid 294.93 mg/mg. Cr (reference range:0-1.5 mg/mg. Cr) (Table 1). After the treatment with protein restriction diet, liver protection and blood hypoammonia drugs, investigations revealed a peak ammonia level of 373 µmol/L. Brain MRI showed swelling of bilateral frontal, temporal, parietal and insular gyrus suggesting metabolic encephalopathy (Fig. 2A). In addition to drug treatment, continuous renal replacement therapy (CRRT) was performed to reduce blood ammonia. After two times of CRRT, the child ' s consciousness gradually improved and could communicate easily. After discharge, the patient has taken hepatoprotective and hypoammonia drugs and the plasma ammonia levels remained at about 100µmol/L. WES revealed a novel heterozygous splicing variant in the OTC gene (c.664-1G > C) (Table 2). Using PCR-Sanger sequencing, this variant was not present in her mother (Fig. 1B).
Patient 3 was a 3-year and 1-month old boy who was a product of non-consanguineous parents. There was no family history of intellectual impairment or metabolic disorder. 2 days period to admission, he was noted to be sleepy. At admission, his plasma ammonia level was 149 µmol/L (reference range :9–33 µmol/L), and his ALT level was 85U/L (reference range:9-50U/L) (Table 1). After the oral administration of citrulline and arginine, the plasma ammonia level decreased to 43 µmol/L (reference range:9–33 µmol/L). He was not detected blood amino acids or urinary organic acids. DNA studies revealed a single nucleotide change from A to G, c.365A > G, was detected in exon 4 (Table 2). The nucleotide change resulted in a missense mutation, p. Glu122Gly, which has previously been reported[13]. Using PCR-Sanger sequencing, this variant was also present in his asymptomatic mother (Fig. 1C).
Patient 4 was a 1-year and 10-month old girl and was the first child of non-consanguineous parents with an uneventful perinatal period. Her first hospitalization in May 2015 was due to liver dysfunction and astasia. Upon referral to our hospital, laboratory investigations showed ALT 477U/L (reference range:9-50U/L) and AST 203U/L (reference range:15-40U/L). Additional investigations revealed an elevated ammonia level of 272 µmol/L (reference range 9–33 µmol/L) with unremarkable plasma amino acid analyses. DNA studies revealed a single nucleotide change from C to T, c.67C > T, was detected in exon 1(Table 2). The nucleotide change resulted in a nonsense mutate on with a predicted stop codon at codon 23, p. Arg23*, which has previously been reported [14](Fig. 1D). Since OTC was diagnosed, the patient had been treated conservatively until she was 2.5 years old. Due to increasingly severe neurologic disturbances, the patient received liver transplantation at her third hospital stay. Results of liver biopsy showed multifocal hepatic steatosis and infiltration of inflammatory cells in hepatic portal area (Fig. 3A and 3B). Brain MRI showed increased signal in bilateral basal ganglia and brain atrophy when she was 5.5 years old (Fig. 2B). She took long-term immunosuppressants after surgery. Unfortunately, this patient died of severe pneumonia at 6 years and 3 months old.
Patient 5 was a 4 years old boy who was the first child of non-consanguineous parents. There was no family history of seizure, intellectual impairment, metabolic disorder or sudden death. He was in good health until 2 days prior to admission, when he developed vomiting, drowsiness, fever and seizure. He became comatose and was adopted mechanical ventilation half a day before admission. MRI of the brain revealed increased signal intensity in bilateral basal ganglia (Fig. 2C). Electroencephalography demonstrated at low voltage performance at bilateral cerebral hemispheres. Upon hospitalization, his physical examination revealed a deep coma state, Glasgow coma scale was 3 points, and had a weak reflection of bilateral pupil. Laboratory investigations revealed elevated ammonia levels (1263µmol/L, reference range 18–72µmol/L) and transaminitis (ALT 64 U/L, reference range:0-38U/L, AST 82U/L, reference range:0-38U/L). The INR was 1.7(reference range:0.8–1.2). Plasma citrulline was 3.83 µmol/L (reference range:4–30 µmol/L). After nearly 3 months treatment of mechanical ventilation, medication and blood purification, the child was still in deep coma with dilated pupil and unstable hemodynamics. Finally, his parents decided to give up continuing treatment. WES revealed a hemizygous mutation in the OTC gene (c.119G > A). The nucleotide change resulted in a missense mutation, p. Arg40His, which has previously been reported [15](Table 2). Using PCR-Sanger sequencing, this variant was also present in his asymptomatic mother (Fig. 1E).