Clinical heterogeneity, genotype, and neurological outcomes in six Palestinian patients with ornithine transcarbamylase deficiency

Abstract

Background

Ornithine transcarbamylase deficiency (OTCD) is the most common urea cycle disorder. This X-linked condition, mapped to Xp21.1, results from a deficiency of the mitochondrial enzyme ornithine transcarbamylase, which catalyzes a critical step in ureagenesis. Disease severity ranges from a complete enzymatic block, presenting as neonatal hyperammonemic coma with high mortality, to partial deficiencies that can cause life-threatening hyperammonemia at any age.

Patients and methods

This report describes six patients diagnosed with OTCD, with ages ranging from 8 days to 17 years at the time of diagnosis. The onset of symptoms occurred at different ages, from just 2 days to 17 years. Two male patients experienced severe neonatal courses and, unfortunately, died from hyperammonemic coma shortly after birth. In contrast, four other patients (three males and one female) presented with symptoms later in life. Molecular analysis identified pathogenic variants in the OTC gene, which included four missense variants and one in-frame deletion. Notably, a recurrent variant was found in two individuals from unrelated families.

Results

The cohort comprised six patients with two distinct clinical presentations. Two hemizygous males presented neonatally and progressed to severe hyperammonemic coma within the first days of life. The other four patients (3 males, 1 female) exhibited a late-onset form, with symptom onset ranging from infancy to 17 years. Their phenotypes were highly variable, encompassing acute hyperammonemic episodes and a chronic neurological course characterized by speech and motor delay, muscle cramps, and persistent toe-walking. Genetic analysis revealed five distinct pathogenic or likely pathogenic variants (four missense substitutions and one in-frame deletion). These were identified by whole-exome sequencing in five patients and by targeted Sanger sequencing in one patient.

Conclusions

This study highlights the molecular and phenotypic heterogeneity of OTCD and explores the correlation between genotype and phenotype. The manifestation of the disease in a heterozygous female suggests that skewed X-inactivation may serve as a potential pathogenic mechanism. The variation in age at symptom onset, alongside the genetic diversity observed, underscores the continuum between neonatal and late-onset OTCD.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12887-026-06673-1.

Introduction

OTCD (OMIM #311250) is the most common urea cycle disorder. It is inherited as an X-linked trait, and the gene encoding ornithine transcarbamylase (OTC) is located on Xp21.1 [1]. OTC is a mitochondrial enzyme that catalyzes the conversion of ornithine and carbamoyl phosphate to citrulline [2]. Infants with a complete enzyme deficiency typically present in the neonatal period with life-threatening hyperammonemic coma. Those with a partial deficiency can develop hyperammonemia at any age, which carries a 10% mortality risk and significant potential for developmental disabilities. The disease has two main phenotypes: a severe neonatal form, most common in males, characterized by complete enzyme blockade and often fatal coma in the first week of life; and a milder late-onset form, seen in most heterozygous females and some males, involving partial enzyme activity. All symptoms arise from the neurotoxic effects of hyperammonemia, which can lead to brain edema, coma, and death [3].

Specific OTC gene variants may influence both the time of symptom onset and maximum blood ammonia concentrations. The mutational spectrum of OTCD primarily consists of single-base substitutions, while small and large deletions or insertions make up a minority of cases [4]. Females who are heterozygous for severe variants in the OTC gene may be asymptomatic, mildly affected, or severely affected. This variability is likely attributable to a complex interplay of dietary, environmental, hormonal, and other unidentified factors [5]. The survival rate of symptomatic females varies across different studies. Some research indicates a survival rate of 80–90%, while others report a much lower rate of around 15%. The higher mortality rate has been linked to increased occurrences of hyperammonemic episodes, more severe cases of hyperammonemia, and more significant hepatic dysfunction [6].

Patients with late-onset OTCD can exhibit symptoms at any age from infancy to adulthood. Hemizygous males with partial enzyme deficiency, along with approximately 20% of heterozygous females, fall into this category. Interestingly, more than 80% of heterozygous females remain asymptomatic throughout their lives. The clinical manifestations of late-onset OTCD are primarily due to hyperammonemia and can include neurological, psychiatric, and gastrointestinal symptoms, particularly affecting the liver. These symptoms are often triggered by external stressors such as infections, prolonged fasting, certain medications, and pregnancy [7].

In adults, OTCD can present as altered mental status, potentially leading to hyperammonemic coma, or it may cause a sudden onset of psychiatric symptoms, such as abnormal behavior triggered by high protein intake, even in the absence of prior catabolic stress [8, 9]. Additionally, hyperammonemic coma in this age group can occur after steroid administration, as glucocorticoids induce a catabolic effect by enhancing protein turnover, and as acute cerebral edema associated with severe hyperammonemia triggered by a previous episode of gastroenteritis [10, 11]. In infants, the neonatal-onset form is particularly severe due to a complete loss of enzyme activity. This condition is primarily associated with hemizygous variants in males, while females are rarely affected [12].

In Palestine, OTCD is not involved in newborn screening. In resource-limited settings, it often results in fatal or severely disabling conditions due to late diagnosis and a lack of access to essential, life-saving medications and dietary therapy. With the limited resources, management primarily focuses on preventing crises, relying heavily on the immense resilience of families.

The principles of management are: emergency treatment of hyperammonemia with high-calorie intake; provision of glucose and intralipids during the catabolic state; and administration of intravenous sodium phenylacetate, sodium benzoate, and arginine. On an emergent basis, ammonia is filtered from the blood via hemodialysis and hemofiltration [2, 8]. Long-term treatment consists of a low-protein diet, arginine and/or citrulline supplementation, and nitrogen scavengers, such as sodium benzoate and/or sodium phenylbutyrate [2].

We report on six patients with OTCD, illustrating the broad spectrum of this disorder. The age of symptom onset varied widely, from 2 days to 17 years. A severe neonatal course was observed in two male patients who died from severe hyperammonemic coma within days of birth. In contrast, four patients (three males and one female) had the later-onset form, presenting with heterogeneous clinical manifestations. Molecular analysis identified pathogenic variants in the OTC gene, including four missense mutations and one in-frame deletion. Notably, one variant was recurrent, identified in two individuals from unrelated families.

Materials and methods

This study presents a retrospective analysis of six patients with OTCD from two tertiary care centers in Palestine, representing the first reported cohort of this condition in the region. The cohort comprised five males and one female, with ages ranging from 8 days to 17 years at diagnosis. OTCD was confirmed in all patients through clinical phenotyping, urine organic acid analysis, plasma amino acid profiling, and molecular genetic testing. Data were extracted from medical records and supplemented by information from parents or patients during follow-up visits at the metabolic clinic. All patients received a standardized emergency protocol for hyperammonemia, which included intravenous glucose with electrolytes, a 24–48 h protein restriction, and the administration of sodium benzoate, arginine, and Intralipids to halt catabolism. Clinical examinations were performed at diagnosis, with four patients undergoing further assessment during follow-up. Notably, two male patients succumbed to the disease at 8 and 11 days of age (Table 1). To our knowledge, this represents the first cohort of patients with OTCD reported in Palestine.

Table 1.

Clinical manifestations, biochemical findings, and neurological outcomes of the patients enrolled in the study

#	Sex	Age at onset of symptoms	Age at diagnosis	Consanguinity	Clinical manifestations	Birth history	Initial Serum Ammonia µmol/L (RR 30–85)	Plasma Gln µmol/L (RR 254–823)	Plasma Cit µmol/L (RR 10–45)	Urine orotic acid	AST U/L (RR 10–60)	ALT U/L (RR 5–55)	PT/ INR	PTT	Age at last visit and Neurological outcome
1	Male Deceased	3 days	9 days	First cousins	Poor feeding, respiratory distress, lethargy, coma, hypotension requiring mechanical ventilation, liver dysfunction, renal failure, coagulopathy.	Uneventful, term vaginal delivery. BW 3900 g	1770 increased to 7390	4765	0	Massive excretion	290	197	76/6.5	99	Died at the age of 11 days due to progressively rising ammonia levels, coagulopathy, and multiorgan dysfunction
2	Male Deceased	2 days	8 days	No	Poor feeding, lethargy, rapidly progressing coma	Uneventful, term vaginal delivery. BW 4000 g	1000	NA	NA	NA	NA	NA	NA	NA	Died at the age of 8 days at a hospital in Gaza due to progressively rising ammonia levels
3	Male	4 months	5 mo	No	Vomiting, poor feeding, lethargy, coma. Subsequent five episodes of lethargy, altered mental status, and hyperammonemia. After the initial presentation, he experienced 5 other episodes of symptomatic hyperammonemia without progression to coma.	Uneventful, C/S delivery due to previous C/S BW 3200 g	470	505	3	Moderate excretion	34	41	25/2	46	5 years. Attends kindergarten. Normal development, cognition, and behavior. Normal neurological exam
4	Male	1 year	8 years	No	Speech and motor delay, easy fatigability, muscle cramps, elevated CPK, and ptosis at around the age of 2 years. Poor school performance. Persistent toe-walking and brisk deep tendon reflexes.	Uneventful, term vaginal delivery. BW 3400 g	113	800	9	Mild	42	24	11.4/1	42	13 years. Poor school performance. Learning disability. Persistent toe-walking. Brisk DTRs, positive Gower’s sign
5	Female	2 years	7 years	No	Recurrent vomiting, hypoactivity, lethargy, and altered mental status. After the initial presentation, she experienced other 4 episodes of symptomatic hyperammonemia without progression to coma.	Uneventful, term vaginal delivery. BW 3800 g	368	700	8	Mild	87	66	15/1.2	35	She underwent a liver transplantation at the age of 13½ years. After the liver transplant, she has shown normal development and behavior, and cognition. Normal neurological exam. Her school performance remains suboptimal.
6	Male	17 years	17 years	No	Irritability, poor concentration, headache, hallucinations, recurrent vomiting, lethargy progressing to coma.	Uneventful, term vaginal delivery. BW 3200 g	236	1013	16	Mild excretion	18	55	18.7/ 1.37	34	At the age of 18.5 years. Normal behavior. Normal cognition. Normal physical and neurological examination

Abbreviations # Number, Gln Glutamine, Cit Citrulline, RR Reference range, C/S Cesarean section, AST Aspartate Aminotransferase, ALT Alanine Aminotransferase, PT Prothrombin Time, INR International Normalized Ratio, PTT Partial Thromboplastin Time, CPK Creatine Phosphokinase, DTR Deep Tendon Reflex, BW Birth Weight

Sample collection

Blood samples were collected from all six patients, and DNA extraction was performed using Qiagen Flexigene Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The concentration and purity of the extracted DNA were confirmed using a Nanodrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Informed consent for molecular genetic analysis was obtained from all participants.

Whole exome sequencing (WES)

WES was performed at the Molecular Genetics Laboratory, Istishari Arab Hospital (Ramallah, Palestine) on DNA samples from patients 1,2,4,5, and 6 (Table 2). Library preparation was carried out using TruSeq Capture Exome Kit (Illumina). This kit provides coverage of 45 Mb of exonic content. The probe set was designed to enrich 214,405 exons, ensuring > 98% of targeted bases covered at a minimum depth of 20×. After sequencing on NextSeq 500, data were uploaded onto our server, and reads were aligned to the reference human genome (hg19) using the BWA aligner. Prior to variant calling by GATK (Genome Analysis Toolkit), mapped reads (BAM format) went through preprocessing steps by removing PCR duplicates, realigning around indels, and recalibrating base quality. WES-based copy-number variants (CNVs) were called using an in-house bioinformatics detection pipeline. Variant annotation was performed using ANNOVAR, followed by a multi-step filtering strategy to prioritize variants relevant to the patients’ phenotypes. Variants with a sequencing depth < 5× were excluded, and common polymorphisms were filtered out using minor allele frequency (MAF) thresholds of > 1% in gnomAD (v2.1.1) and > 5% in our in-house exome/genome database. The analysis focused on coding regions and the flanking ± 10 intronic nucleotides. Variants predicted to exert a high or moderate functional impact—including missense, nonsense, frameshift, and splice-site variations—were retained. Synonymous or deep intronic variants were excluded unless previously reported to alter splicing or be disease-associated. The remaining variants were assessed for pathogenicity using multiple in-silico prediction algorithms, including PolyPhen-2, SIFT, AlphaMissense, and CADD.

Table 2.

The genetic variants identified in the patients enrolled in the study, along with their in-silico predictions

#	Sex	Variant	Testing method	Confirmation by Sanger	Zygosity	In-Silico prediction tools	Allele frequency in gnomAD v4.1.0	ClinVar Classification	ACMG Classification	Variant Type	Family segregation	Variant Citations
1	M	NM_000531.6:c.626 C > T p.Ala209Val rs72558417	WES	No	Hemizygous	PolyPhen: Uncertain SIFT: Benign. MutationTaster: Deleterious CADD:25.2. Alphafold:0.314	ND	Pathogenic	Pathogenic	nonsynonymous	Not performed	Previously reported: (Zhou Q et al. 2020, Martín-Hernández E et al. 2014, Kurihara A  et al. 2003, Tuchman M et al. 2002, Popowska E  et al. 1999, Tuchman M et al. 1998, García-Pérez MA et al. 1997, Matsuda I  et al. 1997, Gilbert-Dussardier B et al. 1996, García-Pérez MA et al. 1995)
2	M	NM_000531.6:c.77G > A p.Arg26Gln rs68031618	WES	Yes	Hemizygous	PolyPhen: Benign SIFT: Tolerated MutationTaster: Polymorphism CADD:23.8. Alphafold:0.091	ND	Likely pathogenic	Likely pathogenic	nonsynonymous	The mother is heterozygous for the variant	Previously reported: (Shao Y et al. 2017, Choi JH et al. 2015, Magesh R et al. 2014, Kim YM et al. 2013, Buratti E et al. 2007, Arranz JA et al. 2007, Kim GH et al. 2006, Zhang MQ et al. 1998, Grompe M et al. 1991, Grompe M et al. 1989)
3	M	NM_000531.6:c.817_819del p.Glu273del rs72558452	Sanger sequencing	Yes	Hemizygous	PolyPhen: NA SIFT: NA MutationTaster: NA CADD: NA Alphafold NA	0.000004175	Likely pathogenic	Likely pathogenic	in-frame deletion	The mother is a wildtype for the variant	Previously reported: ( Santamaria R et al. 2022, Gobin-Limballe S et al. 2021, Toquet S et al. 2021, Ducich N et al. 2020, Martín-Hernández E et al. 2014, Arranz JA  et al. 2007, McCullough BA et al. 2000, Schultz RE et al. 2000, Ségues B et al. 1996)
4	M	NM_000531.6:c.622G > A p.Ala208Thr rs72558416	WES	Yes	Hemizygous	Polyphen: Uncertain SIFT: Uncertain MutationTaster: Deleterious CADD:24.8 Alphafold:0.4	0.000007438	Pathogenic	Pathogenic	nonsynonymous	The mother is heterozygous for the variant	Previously reported: ( Toquet S et al. 2021, Lu D et al. 2020, Silvera-Ruiz SM  et al. 2019, Sánchez AI et al. 2017, Bijvoet GP et al. 2016, Gascon-Bayarri J et al. 2015, Martín-Hernández E et al. 2014, Cavicchi C et al. 2014, Schultz RE et al. 2000, Ausems MG et al. 1997)
5	F	NM_000531.6:c.482 A > G p.Asn161Ser rs72556271	WES	Yes	Heterozygous	Polyphen: Uncertain SIFT: Uncertain. MutationTaster: Deleterious CADD:27.6 Alphafold:0.153	ND	Pathogenic	Pathogenic	Nonsynonymous	The parents are wildtype.	Previously reported: ( Adam MP et al. 2022, Anderson AA et al. 2020, Chongsrisawat V et al. 2018, Shao Y  et al. 2017, Choi JH et al. 2015, Lipskind S  et al. 2011, Kim GH  et al. 2006, Bisanzi S et al. 2002, Rapp B  et al. 2001, Tuchman M et al. 1996)
6	M	NM_000531.6:c.622G > A p.Ala208Thr rs72558416	WES	Yes	Hemizygous	Polyphen: Uncertain SIFT: Uncertain MutationTaster: Deleterious CADD:24.8 Alphafold:0.4	0.000007438	Pathogenic	Pathogenic	Nonsynonymous	The mother is heterozygous for the variant.	Previously reported: ( Toquet S et al. 2021, Lu D et al. 2020, Silvera-Ruiz SM  et al. 2019, Sánchez AI et al. 2017, Bijvoet GP et al. 2016, Gascon-Bayarri J et al. 2015, Martín-Hernández E et al 2014, Cavicchi C et al. 2014, Schultz RE et al. 2000, Ausems MG et al 1997)

Abbreviations # number, WES Whole exome sequencing, ND Not detected, NA Not Applicable

Sanger sequencing

The OTC gene sequencing was performed for patient 3 (Table 2). Touchdown PCR was conducted in a 25 µL reaction mixture containing 50 ng of genomic DNA, 12.5 µL of Hot-Start Taq mix (PCRbio), 1 µL of forward primer (10 µM), and 1 µL of reverse primer (10 µM), with nuclease-free water added to reach the final volume. Separate PCR reactions were performed for each of the 10 exons of the gene using exon-specific primers (Supplementary Table 1) designed to flank the corresponding exon boundaries. The cycling conditions included an initial denaturation at 95 °C for 2 min, followed by 3 cycles of denaturation at 95 °C for 30 s, annealing at 63 –55 °C (descending by 3 °C) for 30 s, and extension at 72 °C for 30 s, concluding with a final extension at 72 °C for 10 min. PCR products were analyzed by electrophoresis on a 2% agarose gel stained with ethidium bromide to confirm the presence and size of the amplified fragments. Bands were visualized under UV light and compared to a DNA ladder (Invitrogen). Amplified products were purified using the Eppic Fast kit (A&A Biotechnology) to remove excess primers and nucleotides. Sequencing reactions were performed using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) according to the manufacturer’s protocol. Sequencing was carried out on an Applied Biosystems 3500 DNA Analyzer.

Results

Patients

We report on six patients (five males and one female) from six distinct families across five regions of the West Bank and Gaza Strip. Among these, patient #1 was born to consanguineous parents, whereas the remaining patients were offspring of a non-consanguineous marriage. The age of symptom onset exhibited considerable variability, ranging from 2 days to 17 years. The comprehensive clinical, biochemical, and neurological phenotypes of each patient are summarized in Table 1.

Two male patients (#1 and #2) presented with severe hyperammonemic coma at ages 3 days and 2 days, respectively; both patients subsequently succumbed at ages 9 days and 8 days.

Patient #1 was a male born to first-cousin parents, delivered at term after an uneventful pregnancy and labor. His birth weight (BWT) was 3900 g. He presented to a distant hospital with symptoms of poor feeding, respiratory distress, and lethargy, which rapidly progressed to a coma.

Initial serum ammonia levels were extremely high at 1770 µmol/L (reference range: 30–85), along with high plasma glutamine, and undetectable plasma citrulline. Qualitative urine organic acid analysis showed massive excretion of orotic acid. Additionally, liver transaminases were elevated, and coagulation profiles were abnormal (Table 1). Despite aggressive treatment—including mechanical ventilation, intravenous sodium benzoate and arginine each at a dose of 700 mg/kg/day, vitamin K, fresh frozen plasma, and inotropic support, serum ammonia levels continued to rise, reaching 4765 µmol/L. Unfortunately, he died at the age of 11 days due to progressive hyperammonemic coma, coagulopathy, and multiorgan dysfunction. Hemodialysis and hemofiltration are not accessible for newborns in Palestine. WES identified a pathogenic hemizygous missense variant in the OTC gene (Table 2). The family chose not to undergo genetic testing for the mother and did not attend genetic counseling at the clinic.

Patient #2 was a full-term male, born via uncomplicated vaginal delivery to non-consanguineous parents in Gaza, with a birth weight of 4000 g. At two days of age, he presented with poor feeding and lethargy, which rapidly progressed to coma. Laboratory investigation revealed a markedly elevated serum ammonia level. Although plasma amino acid and urine organic acid analyses were not available, a definitive diagnosis of OTCD was established by WES, which identified a pathogenic hemizygous missense variant in the OTC gene (Table 2). The infant’s clinical course deteriorated, and he died at eight days of age in a Gaza hospital from complications of a hyperammonemic coma. Details about the genetic testing of other family members and the emergency treatment of hyperammonemia were lacking.

Patients #3, #4, #5, and #6 presented with the late-onset form of the disease.

Patient #3 was a male born at term via cesarean section due to a previous cesarean delivery, following an uneventful pregnancy and labor, BWT 3200 g. He first presented at 4 months old with symptoms including vomiting, poor feeding, and lethargy, which progressed to coma. Initial tests revealed a high serum ammonia level, normal plasma glutamine, and low plasma citrulline. A qualitative urine organic acid analysis indicated moderate excretion of orotic acid. Liver transaminases were normal, while the coagulation profile showed elevated prothrombin time (PT) and INR. Serum zinc level was normal at 80 µg/dL, within the reference range of 65 to 122 µg/dL. Sanger sequencing identified a hemizygous in-frame deletion c.817_819delGAG p.Glu273del in the OTC gene (Table 2). The mother was a wildtype for the variant. The patient was initially treated at another hospital with intravenous glucose infusion, oral sodium benzoate, and arginine (250 mg/kg/day each) with a low-protein diet (1 g/kg/day). Over two days, serum ammonia level decreased, and neurological status improved. The patient experienced five hyperammonemia episodes, presenting as lethargy and altered mental status, each managed with intravenous glucose, a protein-restricted diet, oral sodium benzoate, and arginine. Hemodialysis or hemofiltration was not required. During follow-up at the metabolic clinic, the patient continued on oral sodium benzoate (250 mg/kg/day) and arginine (200 mg/kg/day), divided into three doses, with a low-protein diet of 1–1.5 g/kg/day. During his last hospitalization at 4½ years old, his plasma ammonia level was measured at 140 µmol/L, while both liver transaminases and the coagulation profile were within normal ranges. At his final follow-up visit at the metabolic clinic, conducted when he was 5 years old, he was attending kindergarten, adhering to his treatment plan, and showed normal development, behavior, and results from physical and neurological examinations.

Patient #4 was a male born at term to non-consanguineous parents following an uncomplicated pregnancy and delivery, with a birth weight of 3,400 g. His early development was globally delayed; he achieved initial speech and motor milestones at approximately 1.5 years and began walking independently at 2 years of age. Around this time, he developed a constellation of symptoms including muscle weakness, cramps, and easy fatigability. These symptoms were notably worse in the evenings and were accompanied by ptosis. Initial laboratory investigation revealed a mild elevation in serum creatine phosphokinase (360 U/L). At age 3, he was hospitalized for further evaluation. Paradoxically, the physical examination during this admission documented normal activity without fatigue, brisk deep tendon reflexes, and normal muscle power and tone. Electrophysiological studies, including electromyography and nerve conduction velocity, were within normal limits.

The patient was first evaluated in the metabolic clinic at 8 years of age. Clinical assessment at this time revealed a positive Gower’s sign and brisk deep tendon reflexes, predominantly in the lower limbs. Biochemical investigations showed a mild excretion of orotic acid on qualitative urine organic acid analysis. Plasma amino acid analysis indicated a low citrulline level with a normal glutamine concentration. Liver transaminases and coagulation profiles were unremarkable. Serum zinc level was normal at 85 µg/dL. A definitive diagnosis was established by WES, which identified a hemizygous pathogenic missense variant (c.622G > A p.Ala208Thr) in the OTC gene (Table 2). His unaffected mother and sister were confirmed as heterozygous. They did not exhibit any symptoms of encephalopathy and continued to follow a normal diet. Both had normal serum ammonia levels. Orotic acid was not measured. The other brother and two sisters were wildtype for the variant. Management was initiated with oral sodium benzoate at adose of 250 mg/kg/day, arginine at a dose of 150 mg/kg/day in 3 divided doses, and a low-protein diet. He did not require intravenous treatment throughout the course of his disease. At his most recent follow-up at 13 years of age, he had poor school performance, exhibited persistent toe-walking, and occasional muscle cramps. Notably, his parents reported no history of symptomatic hyperammonemic episodes.

Patient #5 was a term female infant born to non-consanguineous parents after an uncomplicated pregnancy and delivery, with a birth weight of 3,400 g. Her initial presentation occurred at one year of age with symptoms of acute hyperammonemia, including recurrent vomiting, lethargy, hypoactivity, and altered mental status; however, her condition did not progress to coma. Diagnostic tests revealed a significant hyperammonemia with mild elevations in liver transaminases and a normal coagulation profile. Plasma glutamine was within normal limits. Urine organic acid analysis demonstrated mild orotic aciduria, prompting further genetic analysis. WES identified a heterozygous, pathogenic missense variant, confirming the diagnosis of OTCD (Table 2). The mother was wildtype for the variant. Management involved a protein-restricted diet, along with oral sodium benzoate and arginine at a dosage of 250 mg/kg/day each, divided into three daily doses. Intravenous sodium benzoate and arginine were not required. Following the initial episode, the patient experienced four additional hyperammonemic crises, each characterized by lethargy and somnolence but without progression to coma. She was monitored regularly in the metabolic clinic. At her most recent follow-up at 13 years of age, she was attending the 7th grade, adhering to her medical regimen, and demonstrating normal physical, neurological, and behavioral development. To achieve definitive metabolic control, the patient underwent successful orthotopic liver transplantation at 13.5 years of age. Post-transplant, she remains in good health, consumes an unrestricted diet without the need for ammonia-scavenging medications, and maintains an excellent quality of life. Serum zinc level was normal at 95 µg/dL.

Patient #6 A 17-year-old male was born to non-consanguineous parents after an uncomplicated pregnancy and delivery, with a birth weight of 3,200 g. He was referred to our hospital due to the sudden onset of irritability, recurrent vomiting, headache, poor concentration, hallucinations, and lethargy, which progressed to coma within hours. At the age of 2, he was diagnosed with minimal-change nephrotic syndrome through a renal biopsy. Frequent relapses characterized his clinical course. He had been treated with prednisone at a dose of 25 mg every other day, along with mycophenolic acid (2 tablets daily), before the onset of his current symptoms.

Initial laboratory tests revealed elevated serum ammonia levels, elevated plasma glutamine, and normal plasma citrulline levels. A qualitative urine organic acid analysis showed mild excretion of orotic acid. Serum transaminases and coagulation profile results were normal (see Table 1). A definitive diagnosis was made through whole exome sequencing (WES), which identified a hemizygous pathogenic missense variant (c.622G > A p.Ala208Thr) in the OTC gene. This variant is the same as the one found in patient #4, although the two families are unrelated and come from different governorates (Table 2). The patient’s unaffected mother was confirmed to be heterozygous; she remained asymptomatic, maintained a normal diet, and had a normal serum ammonia level. Orotic acid was not measured. The patient’s two brothers and two sisters were also asymptomatic, but genetic testing was not conducted for any of them. The brain MRI conducted during the episode of hyperammonemia was normal.

Emergency treatment for hyperammonemia included mechanical ventilation, intravenous glucose with electrolyte support, halting protein intake, and administering intravenous sodium benzoate and arginine (250 mg/kg/day each). Parenteral intralipids were provided for anabolism. After 48 h, the patient recovered from coma without the need for hemodialysis or hemofiltration. He gradually tolerated a low-protein diet with oral sodium benzoate (250 mg/kg/day in three doses) and arginine (150 mg/kg/day in 3 divided doses). At his 18.5-year follow-up at the metabolic clinic, he was compliant with therapy, showed normal cognition, behavior, and had unremarkable physical and neurological examination.

Discussion

OTCD (OMIM #311250) is the most common urea cycle disorder, caused by pathogenic variants in the OTC gene. The disorder follows an X-linked inheritance pattern and results in impaired ammonia detoxification due to deficient hepatic OTC enzyme activity. Residual enzyme activity correlates with age at first symptoms, initial peak plasma ammonia, frequency of metabolic decompensations, and also predicts mortality [13].

This cohort describes the clinical diversity, genetic findings, and neurological outcomes of six patients with OTCD. Two male infants with hemizygous OTC variants presented with neonatal-onset disease, developed severe hyperammonemic coma, and died during the neonatal period, consistent with published reports. The other four patients—three hemizygous males and one heterozygous female—had late-onset OTCD with heterogeneous presentations, ranging from recurrent hyperammonemic encephalopathy to chronic features such as speech and motor delays, muscle cramps, Gower’s sign, and elevated creatine phosphokinase (CPK) without symptomatic hyperammonemia. The female patient experienced recurrent hyperammonemic episodes marked by vomiting, lethargy, and altered mental status, but did not progress to coma. Patient 6 presented at 17 years with hyperammonemic coma; his course was complicated by coexisting minimal-change nephrotic syndrome treated with tapering prednisone and mycophenolic acid. He recovered and achieved a normal neurological outcome (see Table 1). Following oral sodium benzoate and arginine therapy, all patients except patient 4 had normal neurological outcomes. Patient 4 demonstrated poor school performance, persistent toe walking, and a Gower sign but never developed hyperammonemic episodes.

The clinical variability seen in this cohort is consistent with findings in the literature, highlighting the diverse age of onset, clinical severity, and neurological outcomes among patients with OTCD. This variability is crucial for the genetic counseling of families with affected patients, particularly for those whose mothers carry the heterozygous variant.

Molecular analysis of the six patients revealed a diverse set of pathogenic variants within the OTC gene. All patients carried known pathogenic or likely pathogenic variants, representing four missense substitutions and one in-frame deletion. WES was performed for five patients, while targeted Sanger sequencing was used for one (patient# 3) (Fig. 1). Five distinct variants were identified (Table 2). The c.626 C > T p.Ala209Val variant identified in patient 1 results in the substitution of alanine by valine at codon 209, adjacent to a residue critical for enzyme stability and function. Garcia-Perez et al. 1995 reported experimental evidence indicating severely reduced OTC enzyme activity in patient cells with this variant [14]. The c.77G > A p.Arg26Gln variant found in patient #2 alters a residue near the donor splice site, potentially affecting both splicing and protein structure. This variant was previously reported as a disease-causing variation for OTCD [15–17]. The c.817_819delGAG p.Glu273del variant detected in patient 3 results in a non-repeat in-frame deletion of a single glutamic acid residue at position 273 of the ornithine transcarbamylase protein. This deletion occurs in a conserved region of the gene and is expected to alter protein structure or function. Literature sources supporting the pathogenicity of this variant include [18–22]. It is extremely rare in gnomAD and occurred de novo as confirmed by parental testing. The p.Ala208Thr variant (c.622G > A) was observed in two unrelated male patients (patients 4 and 6) and in their heterozygous unaffected mothers, consistent with X-linked inheritance; it affects a highly conserved residue and has been previously reported in patients with adult and neonatal onset of OTCD [10, 23–27]. The p.Asn161Ser variant (c.482 A > G) in patient 5 lies within the carbamoyl-phosphate binding domain, disrupting a conserved site essential for catalytic activity. Functional studies cited in the literature [28] demonstrate a deleterious impact on enzyme function. In our cohort, this variant was identified as a de novo event in patient 5, a heterozygous female who manifested symptomatic disease. Although OTCD is classically described as an X-linked disorder affecting hemizygous males, heterozygous females may also develop symptomatic disease, potentially due to variable patterns of X-chromosome inactivation. The severity among females ranges widely, from asymptomatic carriers with normal ammonia metabolism to severe neonatal-onset hyperammonemia comparable to that seen in affected males. Skewed X-inactivation favoring the mutant allele in liver tissue can markedly reduce functional OTC enzyme levels, resulting in metabolic decompensation, especially under catabolic stress, high protein intake, infection, or postpartum periods [29].

Fig. 1.

Partial Sanger sequencing chromatogram of exon 8 in the OTC gene from patient 3, demonstrating an in-frame deletion of codon 273 (c.817_819delGAG, p.Glu272del). A Patient sequence showing the three-base-pair deletion. B Corresponding wild-type control sequence confirming the absence of this deletion. The red boxes indicate the nucleotide region affected by the deletion in the patient and the corresponding intact sequence in the control

All identified variants were absent from 1,600 ethnically matched control samples and exhibit very low or no frequency in public population databases (gnomAD, 1000 Genomes, ESP). In silico predictions (PolyPhen-2, SIFT, MutationTaster) consistently indicated a deleterious effect on protein function. Structural modeling using AlphaMissense (Fig. 2) demonstrated that p.Ala209Val, p.Arg26Gln, and p.Ala208Thr produced no major alterations in overall protein folding, whereas p.Asn161Ser and p.Glu273del caused detectable local conformational distortions within functionally critical domains, supporting their pathogenicity. All patients presented clinically during the first two years of age, except for patient #6, whose onset at 17 years likely reflects partial residual enzyme activity associated with the p.Ala208Thr variant. This activity allowed sufficient baseline function until metabolic stress occurred. The recurrence of substitutions at residues Ala208 and Ala209 suggests a mutational hotspot within exon 6, encoding a region critical for enzyme stability and catalytic function. The identification of these variants in hemizygous males, the detection of heterozygous unaffected mothers, and the presence of de novo hemizygous and heterozygous genetic variations in the male patient 3 and the female patient 5, respectively, are consistent with the expected X-linked inheritance pattern of OTCD. Schematic representation of the OTC gene showing the locations of variants identified in this study is shown in (Fig. 3).

Fig. 2.

AlphaMissense structural modeling of the OTC protein. A Reference wild-type structure; B p.Ala209Val; C p.Arg26Gln; D p.Asn161Ser; E p.Ala208Thr; and F p.Glu272del mutant models. Variants p.Ala209Val, p.Arg26Gln, and p.Ala208Thr B, C, E show no major alteration in overall protein folding, whereas p.Asn161Ser and p.Glu272del D, F demonstrate local conformational distortions within functionally critical regions, supporting their predicted pathogenicity

Fig. 3.

Schematic representation of the OTC gene showing the locations of variants identified in this study. The diagram illustrates the exon–intron organization of the OTC gene (exons 1–10) based on its genomic structure. The five pathogenic variants identified among the patients included in this study are mapped to their respective exon positions. The clustering of variants within exon 6 indicates a potential mutational hotspot region critical for enzyme stability and catalytic function

Diagnosing and managing adult patients with OTCD and other urea cycle disorders is challenging. Patients may present acutely with altered consciousness, seizures, loss of appetite, or chronic symptoms like cognitive impairment, confusion, lethargy, dizziness, headaches, and cyclical vomiting. Symptoms can mimic encephalitis or drug intoxication. Gastrointestinal symptoms, such as nausea and protein aversion, may also occur. Neurological outcomes depend on the severity and duration of hyperammonemia, making early diagnosis crucial for better results. Notably, asymptomatic patients may have normal ammonia levels, so a high index of suspicion and additional biochemical and genetic testing are essential in such cases [30].

There are limited data on pregnancy safety for both symptomatic and asymptomatic patients with OTCD. The largest study (36 patients) reported substantial morbidity and mortality, especially among undiagnosed women: 11% of 20 diagnosed patients required ICU care versus 69% of 16 undiagnosed patients, with a 31% maternal mortality rate in the undiagnosed group. Another review found that 35 of 52 pregnant women with urea cycle disorders experienced acute metabolic decompensations, and seven died from hyperammonemic crises during pregnancy or postpartum [31].

This study’s limitations include the relatively small sample size, which may have restricted genotype-phenotype correlation assessment.

Conclusions

This study highlights the molecular and phenotypic heterogeneity of OTCD and explores the correlation between genotype and phenotype. The manifestation of the disease in a heterozygous female suggests that skewed X-inactivation may serve as a potential pathogenic mechanism. The variation in age at symptom onset, alongside the genetic diversity observed, underscores the continuum between neonatal and late-onset OTCD. These findings emphasize the importance of precise molecular characterization in predicting disease progression, informing genetic counseling, and optimizing patient management.

Abbreviations

OTC

Ornithine transcarbamylase

OTCD

Ornithine transcarbamylase deficiency

WES

Whole exome sequencing

Authors’ contributions

ID, FA, and HK: Concept and design. ID, FA, and HK: Drafting the manuscript. ID, FIA, and KH: Clinical examinations. FA, HK: WES study. FA, and HK: Sanger sequencing.

Funding

This study received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

Data availability

All *OTC* variants detected in this study, along with their corresponding ClinVar accession numbers, are listed in the Table below.

OTC Gene Variant (Transcript ID: NM_000531.6)	Protein Change	ClinVar Accession
c.626C>T	p.Ala209Val	SCV006309115.1
c.77G>A	p.Arg26Gln	SCV006561132.1
c.622G>A	p.Ala208Thr	SCV006059733.1
c.482A>G	p.Asn161Ser	SCV006059734.1
c.817_819delGAG	p.Glu273del	SCV006059735.1

Declarations

Ethics approval and consent to participate

The study was approved by the Institutional Review Board (IRB) at the Arab American University, Palestine (registration number at FWA IORG0011491) and by the Palestinian Health Research Council (PHRC/HC/ 518/19). The study adhered to the Declaration of Helsinki to this effect. Informed consent to participate was obtained from the parents or legal guardians of any participant under the age of 16.

Consent for publication

Informed consent was obtained from the enrolled individuals and/or their legal guardians for detailed clinical phenotyping, genetic studies, and publication of identifying information/images in online open-access publications.

Competing interests

The authors declare no competing interests.