Urea cycle disorder
The urea cycle is a cycle of biochemical reactions which produce urea ((NH2)2CO) from ammonia (NH3), e.g. converts highly toxic ammonia to urea for excretion.
Urea cycle disorders (UCD) result from inherited deficiencies in the six enzymes of the urea cycle pathway (CPS1, OTC, ASS1, ASL, ARG, and NAGS) 1. These enzymes can be classified as follows:
Five catalytic enzymes:
- Carbamoylphosphate synthetase I (CPS1)
- Ornithine transcarbamylase (OTC)
- Argininosuccinic acid synthetase (ASS1)
- Argininosuccinic acid lyase (ASL)
- Arginase (ARG)
A cofactor-producing enzyme:
- N-acetyl glutamate synthetase (NAGS)
Urea cycle disorders are a group of genetic disorders caused by a mutation that results in a deficiency of one of the six enzymes in the urea cycle (Table). The urea cycle involves a series of biochemical steps in which nitrogen, a waste product of protein metabolism, is removed from the blood and converted to a compound called urea in the blood. In urea cycle disorders, where the crucial enzymes of the urea cycle are deficient or absent, the nitrogen accumulates in the form of highly toxic ammonia resulting in the state of hyperammonemia. Increased levels of blood ammonia can cause irreversible brain damage, coma, and death.
Figure: The urea cycle is a complex biochemical reaction that produces urea from the highly toxic ammonia and is performed in the five following consecutive biochemical steps:
Step 1: HCO3 + NH4 + 2ATP = Carbamyl phosphate + 2ADP + Pi
Step 2: Carbamyl phosphate + Ornithine = citruline + Pi
Step 3: Citruline + Aspartate + ATP = Argininosuccinate + AMP + PPi
Step 4: Argininosuccinate = Arginine + Fumarate
Step 5: Arginin + H2O = Ornithine + Urea
Severe deficiency or absence of activity of any of the first four enzymes (CPS1, OTC, ASS, ASL) in the urea cycle or the cofactor producer (NAGS) results in the accumulation of ammonia and other precursor metabolites during the first few days of life 1. Infants with a severe urea cycle disorder, normal at birth, rapidly develop cerebral edema and the related signs of lethargy, anorexia, respiratory distress, seizures, neurologic abnormalities, and finally coma. Some forms of urea cycle disorders have a milder phenotype, due to the milder deficiencies of related enzymes. In arginase deficiency (ARG), ammonia accumulation may be triggered by illness or stress at any time of life.
Children with severe urea cycle disorders typically show symptoms after the first hours of life. Major clinical features in affected infants are vomiting, problems with feeding, and increasing lethargy. Very soon after birth affected infants show signs of seizures, hypotonia, and respiratory distress. Later symptoms may include frequent episodes of vomiting, especially following high-protein meals, lethargy and delirium, and finally, if the condition is undiagnosed and untreated, hyperammonemic coma or death may occur.
Arginase deficiency (hyperargininemia, ARG deficiency) is not typically characterized by rapid-onset hyperammonemia, however, some individuals present earlier with more severe symptoms. Affected individuals develop progressive spasticity and can also develop tremor, ataxia, and choreoathetosis. Growth is also affected. The ARG gene is an oncogene, related to ABL (protooncogene encodes a cytoplasmic and nuclear protein tyrosine kinase) in DNA sequences from human plasma. The ARG/ABL1 genes are implicated in processes of cell differentiation, cell division, cell adhesion, and stress response. Among the 44 disease-causing mutations identified in the ARG gene so far, the majority are missense mutations such as: homozygous c.612C>T (R21X); c.703G>A (G235R), small deletions (c.262del4bp) and insertions (IVS4-2A>G; c.647_648ins32 exon 6), as well as many others.
Argininosuccinate lyase deficiency (ASL deficiency) can also present with rapid-onset hyperammonemia in the neonatal period indistinguishable from other urea cycle disorders. The major presenting symptoms are vomiting, lethargy, hypothermia, and poor feeding. The estimated incidence of ASL deficiency is 1:70,000 newborns 5. ASL enzyme cleaves argininosuccinic acid to produce arginine and fumarate in the fourth step of the urea cycle (Figure). Among more than 140 disease-causing mutations, the majority are missense, and some large deletions and insertions have also been reported. Some of the mutations include: the IVS5+1G-A splice site mutation, missense mutations R385C, V178M, R379C, Q286R and others. Variant c.1153C>T is associated with residual ASL enzyme activity and accounts for approximately 60% of pathogenic variants in the Finnish population 9. Two founder variants are present in people of Arab ancestry from the Kingdom of Saudi Arabia: the c.1060C>T change that results in a premature stop codon is responsible for approximately 50% of the ASL pathogenic variants in this population 10 and the c.346C>T variant.
Citrullinemia type I (ASS1 deficiency) is characterized by severe hyperammonemia, progressive lethargy, poor feeding, vomiting and signs of increased intracranial pressure, with variable time of onset, from neonatal to later stages of life. Citrullinemia type I has been estimated to occur in 1:57,000 births 5. Citrullinemia type I results from a deficiency of the enzyme argininosuccinate synthase, the third step in the urea cycle (Figure), in which citrulline is condensed with aspartate to form arginosuccinic acid.
Affected individuals are able to incorporate some waste nitrogen into urea cycle intermediates, which makes treatment slightly easier than in the other UCDs. The ASS1 gene encodes argininosuccinate synthetase-1, a cytosolic urea cycle enzyme mainly expressed in hepatocytes, but also in most other body tissues. More than 110 mutations are distributed throughout the ASS1 gene, and it is usually difficult to predict the phenotype based on genotype. Seven variants are associated with severe disease; three of them (p.Arg304Trp, c.421-2A>G, and p.Gly390Arg) account for the majority of citrullinemia type I 6. In the homozygous state p.Gly362Val is associated with mild or no clinical symptoms, as is compound heterozygosity for c.[323G>T];[970+5G>A] 7. Fifty percent of individuals with non-classic presentations were found to be homozygous for one of the following three missense variants: p.Trp179Arg, p.Val263Met, or p.Gly362Val 8.
Carbamoylphosphate synthetase I deficiency (CPS1 deficiency) is the most severe of the urea cycle disorders, with an estimated incidence of 1 in 1,300,000 2, 3, 1. Individuals with complete CPS1 deficiency rapidly develop life-threatening hyperammonemia in the newborn period. Carbamoyl phosphate synthetase I is the rate-limiting enzyme that catalyzes the first committed step of the hepatic urea cycle by synthesizing carbamoyl phosphate from ammonia, bicarbonate, and 2 molecules of ATP. CPS1 is expressed in the liver and in epithelial cells of the intestinal mucosa. More than 250 mutations causing urea cycle disorders have been reported in the CPT1 gene. The majority of these mutations are missense, splicing, small deletions and insertions, but a few large deletions/duplications have also been reported 4.
Ornithine transcarbamylase deficiency (OTC deficiency) is a severe neonatal-onset urea cycle disorder that affects mostly males. Affected boys are normal at birth but soon after birth they develop acute neonatal encephalopathy with hyperventilation and hypothermia. OTC deficiency is thought to be the most common urea cycle defect with the estimated prevalence of OTC deficiency was one in 14,000 live births 11. Ornithine carbamoyltransferase is a nuclear-encoded mitochondrial matrix enzyme that catalyzes the second step of the urea cycle (Figure). More than 450 mutations were reported for the OTC gene, and more than half of these are missense mutations (55%) 12. About 13% of reported pathogenic variants affect splicing of OTC, and an additional 12% of reported variants are structural rearrangements 12, 13.
NAGS deficiency is characterized by symptoms which mimic those of CPS1 deficiency, as CPS1 is rendered inactive in the absence of NAGS (Figure). N-acetylglutamate synthase is a mitochondrial enzyme that catalyzes the formation of N-acetylglutamate (NAG), an essential allosteric activator of carbamoyl phosphate synthase I, the first and rate-limiting enzyme in the urea cycle 1. Patients with NAGS deficiency develop hyperammonemia because CPS1 is inactive without NAGS. Mutations in the NAGS gene are distributed throughout the reading frame of the gene, and so far more than 25 missense, splicing mutations and small deletions have been reported as disease-causing mutations in the NAGS gene.
Early diagnosis of urea cycle disorders is essential for successful patient outcome. Treatment should include dialysis and hemofiltration for the acute severe hyperammonemia, intravenous administration of arginine hydrochloride and nitrogen scavenger drugs in order to allow alternative pathway excretion of excess nitrogen. Restriction of protein intake can reduce the amount of nitrogen. In addition, clinical trials are in progress, with the aim to test the new potential therapeutics for the treatment of urea cycle disorders (trials with developing drugs such as HPN-100; BUPHENYL®; NaPBA; Sodium [1,2-13C]-Acetate and many others 1).
CENTOGENE offers full gene sequencing and deletion/duplication analysis for the Urea cycle disorder panel (genes: ARG1, ASL, ASS1, CPS1, NAGS, OTC).
An overview of urea cycle disorder associated genes
|chr. locus||Enzym||Associated/allelic disorders|
|7q11.21||Argininosuccinate lyase||Argininosuccinic aciduria (207900)|
|9q34.11||Argininosuccinate synthase||Citrullinemia (215700)|
|2q34||Carbamoyl-phosphate synthase||Carbamoylphosphate synthetase I deficiency (237300); |
Neonatal pulmonary hypertension(615371)
|17q21.31||N-acetylglutamate synthase||N-acetylglutamate synthase deficiency (237310)|
|Xp11.4||Ornithine carbamoyltransferase||Ornithine transcarbamylase deficiency (311250)|
The differential diagnosis of urea cycle-related disorders – depending on the major symptoms in the initial case – includes the following diseases:
Inborn errors of metabolism:
- Organic acidemias (propionic acidemia and methylmalonic acidemia)
- Tyrosinemia type 1
- Mitochondrial disorders
- Fatty acid oxidation disorders
Diseases of the liver and biliary tract:
- Herpes simplex virus infection
- Vascular bypass of the liver
- Biliary atresia
- Acute liver failure
Effect of medications:
- Valproic acid
- 5-pentanoic acid
CENTOGENE offers an advanced, fast and cost-effective strategy to test large NGS panels and diagnose complex phenotypes based on PCR-free whole genome sequencing and NGS technology. This approach offers an unparalleled advantage by reducing amplification/capture biases and providing sequencing of the entire gene with more uniform coverage.
To confirm/establish the diagnosis, CENTOGENE offers the following testing strategy for urea cycle disorders using NGS Panel Genomic targeted towards this specific phenotype:
Step 1: Whole genome sequencing from a single filter card. The sequencing covers the entire gene (coding region, exon/intron boundaries, intronic and promoter) for all the genes included in the Urea cycle disorders panel. Copy Number Variants analysis derived from NGS data is also included.
Step 2: If no mutation is identified after analysis of the Urea cycle disorders panel, we further recommend continuing the bioinformatics analysis of the data using whole genome sequencing to cover those genes which are either implicated in an overlapping phenotype or could be involved in a similar pathway but are not strongly clinically implicated based on the current information in literature.
The following individuals are candidates for urea cycle disorders testing:
- Individuals with a family history of urea cycle disorders and presentation of the most common symptoms
- Individuals without a positive family history, but with symptoms resembling urea cycle disorders
- Individuals with a negative but suspected family history of urea cycle disorders, in order to perform proper genetic counseling.
Sequencing, deletion/duplication of the panel genes should be performed in all individuals suspected of having urea cycle disorders and suspected phenotypes. In parallel, other genes reported to be related with this clinical phenotype should also be analyzed for the presence of mutations, due to the overlap in many clinical features caused by those particular genes.
Confirmation of a clinical diagnosis through genetic testing can allow for genetic counseling and may direct medical management. Genetic counseling can provide a patient and/or family with the natural history of the urea cycle disorders and related disorders identify at-risk family members, provide disease risks as well as appropriate referral for patient support and/or resources.
More information on CENTOGENE´s Urea cycle disorder panel can be found in our genetic test catalogue.