Chapter 33 Inborn Errors of Urea Synthesis
Inherited urea cycle disorders represent a devastating group of inborn errors of metabolism that are associated with hyperammonemic encephalopathy and high mortality and morbidity rates. They comprise deficiencies in any of the six enzymes and two amino acid transporters involved in urea synthesis (Figure 33-1). Accordingly, these disorders are named as follows (estimated prevalence rates are given) [Brusilow and Maestri, 1996; Tuchman, 1992; Yamanouchi et al., 2002]:
These disorders are inherited as autosomal-recessive traits, except for ornithine transcarbamylase deficiency, which is X-linked. Because of the absence of mass newborn screening for these disorders, the true incidence of urea cycle disorders is unknown. Based on case reports and questionnaires about referred patients, the combined prevalence of all urea cycle disorders is estimated to be 1 per 8200 [Brusilow and Maestri, 1996].
Other than in arginase deficiency, infants with a complete deficiency of a urea cycle enzyme (N-acetylglutamate synthase, CPS I, ornithine transcarbamylase, argininosuccinate synthetase, or argininosuccinate lyase) commonly present in the newborn period with hyperammonemic coma. Despite aggressive treatment that relies primarily on hemodialysis, the mortality rate in infancy has been reported to approximate 50 percent [Maestri et al., 1999], and as demonstrated by our previous studies, virtually all of the survivors are left with developmental disabilities [Msall et al., 1984; Krivitzky et al., 2009]. Patients with late-onset disease (those with partial enzyme deficiencies, including ornithine transcarbamylase-deficient female heterozygotes) may present at any age with hyperammonemic crises that carry a 10 percent mortality rate and a significant risk of intellectual disabilities [Batshaw et al., 1986]. Even asymptomatic ornithine transcarbamylase-deficient heterozygotes have been shown to have mild cognitive deficits [Batshaw et al., 1980; Gyato et al., 2004].
The Urea Cycle
Dietary protein, on average, contains approximately 16 percent nitrogen. More than 90 percent of the nitrogen that is not used for anabolic processes normally is metabolized and excreted as urea. Therefore, substantial urea synthesis capacity (approximately 16 g per day in adults) is required [Linder, 1985]. With a deficiency of one of the urea cycle enzymes, an insufficient amount of urea will be formed, and nitrogen in the form of ammonia will accumulate. Accumulation in brain causes altered mental status and encephalopathy.
The urea cycle was proposed by Hans Krebs and Kurt Henseleit in 1932, and was the first cyclic pathway elucidated. Six enzymes, one co-factor, and two transporters are necessary for optimal urea cycle activity (see Figure 33-1). The clinically most important co-factor is N-acetylglutamate, which is formed from acetyl coenzyme A (acetyl-CoA) and glutamate in a reaction catalyzed by N-acetylglutamate synthase. N-acetylglutamate activates the first enzyme of the urea cycle, carbamyl phosphate synthetase I, which uses adenosine triphosphate, bicarbonate, and glutamine or ammonia to synthesize carbamyl phosphate, contributing the first atom of waste nitrogen to the cycle. Carbamyl phosphate synthetase I is expressed in periportal hepatocytes and intestinal mucosa epithelial cells [Ryall et al., 1985]. It is the most abundant protein in liver mitochondria, accounting for 20 percent of the mitochondrial matrix protein [Lusty, 1978]. The enzyme consists of a single polypeptide with a molecular weight of 165,000 and approximately 1500 amino acid residues [Haraguchi et al., 1991].
Clinical Description of Urea Cycle Disorders
N-Acetylglutamate Synthase Deficiency
Inherited N-acetylglutamate synthase (NAGS) deficiency leads to hyperammonemia by causing a secondary deficiency of CPS I. This disorder has been reported in approximately two dozen patients to date [Bachmann et al., 1982; Elpeleg et al., 2002; Caldovic et al. 2007], but is likely to be underdiagnosed because of the lack of specific biochemical markers and the physiologically low abundance of NAGS in the liver. NAGS deficiency is inherited as an autosomal-recessive disorder and has a phenotype that is similar to that found in CPS I deficiency. It is characterized by hyperammonemia in the newborn period or later in life, and can be fatal or lead to mental retardation and other developmental disabilities. Plasma amino acid analysis usually demonstrates an increased level of glutamine and reduced or absent levels of citrulline. Urinary orotic acid level is normal or low. NAGS activity in the liver has a variable degree of deficiency (ranging from undetectable to normal residual activity) that is unresponsive to l-arginine. Because enzyme analysis requires large amounts of liver tissue and may not be entirely reliable, analysis of genomic DNA for mutations in the N-acetylglutamate synthase gene is the best diagnostic method [Morizono et al., 2004]. Treatment of NAGS deficiency used to consist of a low-protein diet and use of ammonia-scavenging drugs (Table 33-1). A more specific therapy using N-carbamyl-l-glutamate is now possible because N-carbamyl-l-glutamate has been approved for the treatment of NAGS deficiency. N-carbamyl-l-glutamate is a stable structural analog of N-acetylglutamate and can substitute for it in the activation of CPS I.
Carbamyl Phosphate Synthetase I Deficiency
CPS I deficiency was first reported in 1969 [Hommes et al., 1969]. This mitochondrial urea cycle enzyme should be distinguished from a similar cytosolic enzyme, carbamyl phosphate synthetase 2, which is involved in de novo pyridine synthesis. This disorder can manifest with hyperammonemic coma in the newborn period or later in childhood. Biochemically, the principal findings are hyperammonemia, an increased level of plasma glutamine, and reduced level or absence of citrulline on plasma amino acid analysis. Urinary orotic acid level is normal or low. Patients with neonatal-onset disease generally demonstrate less than 5 percent normal CPS I activity in liver, whereas those with late-onset disease have higher residual activity [Qureshi et al., 1986]. Like most of the other urea cycle disorders, CPS I deficiency does not arise from a common mutation [Funghini et al., 2003; Summar, 1998]. Therapy consists of dialysis and/or intravenous alternate pathway therapy during severe hyperammonemic episodes, and low-protein diet and oral alternate pathway therapy for chronic treatment (see Table 33-1). Patients with the neonatal-onset form who survive the initial crisis generally require liver transplantation for long-term survival.
Ornithine Transcarbamylase Deficiency
Ornithine transcarbamylase deficiency was first reported in 1962 in two girls, aged 20 months and 6 years, who were found to have hyperammonemia associated with episodic vomiting, delirium, stupor, failure to thrive, and mental retardation [Russell et al., 1962]. The mothers of the children were sisters, and both demonstrated evidence of a similar but less marked metabolic disorder. Both girls died before the age of 8 years.
Several years later, this disorder also was identified in males. The delayed recognition in males was attributable to the almost total lack of enzyme activity and rare survival of affected males beyond the newborn period. The reason for this difference between males and females subsequently was determined to be related to its X-linked inheritance pattern, with the occurrence of symptomatic females explained by skewed X chromosome inactivation [Ricciuti et al., 1976]. The classic presentation of OTC deficiency in hemizygous males is as a catastrophic illness in the first week of life. In symptomatic female heterozygotes and in males with partial OTC deficiency, symptoms rarely present in the newborn period. Only one case of lethal OTC deficiency in a female neonate has been reported [Klosowski et al., 1998]. In partial deficiencies, age at presentation after the newborn period covers a wide spectrum, with development of hyperammonemic episodes in infancy in some patients, in later childhood in others, and not until adulthood in still others [Ahrens et al., 1996; Ausems et al., 1997; McCullough et al., 2000]. These patients generally have 5–30 percent of normal OTC activity in liver on in vitro measurement. Biochemically, the principal findings are hyperammonemia, hyperglutaminemia, reduced levels or complete absence of citrulline in plasma, and increased urinary orotic acid level.
More than 340 different point mutations and polymorphisms have been found in OTC-deficient patients, and no mutations are prevalent [McCullough et al., 2000; Tuchman et al., 2002]. In about 80 percent of affected families, prenatal diagnosis using DNA techniques is possible [Grompe et al., 1991]. When the mutation has been identified, carrier testing and prenatal diagnosis can be offered to the family. With severe OTC deficiency being an X-linked lethal disease, the calculated probability for the mother of an affected male to be a carrier is 2/3 or 66 percent, and the probability that the patient has a de novo mutation is 1/3 or 33 percent. The identification of common intragenic polymorphisms allows tracking of the mutant allele, even when the deleterious mutation is unknown [Plante and Tuchman, 1998]. Although most families have point mutations, 8 percent of families have large deletions of one or more exons, 10 percent have small deletions or insertions of a few basepairs, 18 percent have splice site mutations, and in about 20 percent no mutation can be found. In about half of those where no mutation could be found, large deletions involving the OTC locus were detected using microarray technology [Shchelochkov et al., 2009]. There are several disease genes very close to the OTC locus, including the Duchenne muscular dystrophy gene and the chronic granulomatous disease gene. Patients with these large deletions may thus have other severe genetic diseases at the same time, making them extremely difficult to manage [Deardorff et al., 2008]. Amongst those patients with point mutations, mutations causing neonatal disease affect amino acid residues that are in the interior of the enzyme, especially around the active site, whereas those associated with late-onset and milder phenotypes tend to be located on the surface of the protein [Tuchman et al., 1998].
Heterozygote detection of OTC deficiency is important both to identify at-risk family members and to offer prenatal diagnosis. It can be accomplished by either molecular studies or, in cases where no mutation was found, by provocation testing. The best provocation testing is an allopurinol load [Hauser et al., 1990]. This test has replaced the protein loading test, which can precipitate a hyperammonemic episode. The allopurinol load (300 mg given orally to adults) leads to increased excretion of orotic acid, reaching 10–20 times control values in 90 percent of OTC heterozygotes [Hauser et al., 1990]. Approximately 15 percent of OTC-deficient heterozygous females will become symptomatic during their lifetime [Batshaw et al., 1986]. (Heterozygotes for other urea cycle disorders are asymptomatic.) Therapy for the neonatal-onset form of OTC deficiency consists of dialysis and administration of intravenous ammonia scavenger drugs, followed by maintenance on a low-protein diet and long-term alternative pathway therapy (see Table 33-1). Patients with the neonatal-onset form who survive the initial crisis generally require liver transplantation.
Citrullinemia
Citrullinemia was first reported in 1962 [McMurray et al., 1962]. Its name derives from the marked elevation of citrulline in blood of affected persons. This disorder also has been called citrullinuria because of the increased excretion of citrulline in urine, and argininosuccinic acid (argininosuccinate) synthetase deficiency to denote its enzyme deficit. Heterogeneity is seen clinically, biochemically, and at the molecular level. Two distinct forms have been reported: neonatal/childhood-onset citrullinemia (type I; with diminished levels of argininosuccinate synthetase in all organs) and citrullinemia type II or citrin deficiency, an adult-onset citrullinemia that is in some but not all cases preceeded by neonatal cholestasis and decreased synthetic function of the liver (caused by a defect in citrin) [Saheki et al., 1987].
Biochemically, the principal findings are hyperammonemia, citrullinemia, and citrullinuria. Citrulline levels generally are elevated 50–100-fold [normal levels less than 50 μmol/L] [Batshaw et al., 1981]. Urinary orotic acid levels also may be increased but less so than in OTC deficiency or argininemia. Citrullinemia is inherited as an autosomal-recessive trait. The gene has been localized to the q34 region of chromosome 9, and the nucleotide coding sequence and deduced amino acid sequence for the enzyme are known [Gao et al., 2003]. To date, more than 50 mutations have been identified; some involve single base changes in the coding sequence, and others involve skipping of an exon in the messenger RNA (mRNA) due to abnormal splicing. Most patients appear to be compound heterozygotes of two different mutations. In neonatal-onset cases, argininosuccinate synthetase activity in liver is less than 5 percent of normal, whereas in childhood-onset cases, 10–25 percent residual activity is seen [Brusilow and Horwich, 2001].
Therapy for citrullinemia (type I) consists of dialysis during severe hyperammonemic crises, followed by low-protein diet and long-term alternative pathway therapy (see Table 33-1). After the initial crisis, patients are in general more stable and easier to manage than patients with more proximal defects (CPS I and OTC deficiencies).
Citrullinemia Type II or Citrin Deficiency
Citrullinemia type II was identified first in Japan but the mutation has been traced back to China. Citrin deficiency is caused by mutations in a gene encoding a previously unknown calcium-dependent mitochondrial membrane protein named citrin (SLC25A13) [Kobayashi et al., 2003]. This inner mitochondrial membrane carrier enables the exchange of matrix aspartate for cytosolic glutamate across the inner mitochondrial membrane [Palmieri et al., 2001]. Plasma ammonia levels are less severely elevated during acute episodes than in other urea cycle disorders, and citrulline levels are elevated up to 20-fold [Kobayashi et al., 1993]. It has been noted that serum pancreatic secretory trypsin inhibitor also is increased and may be useful as a diagnostic marker for the disorder [Kobayashi et al., 1993].
Citrullinemia type II, citrin deficiency, manifests in adulthood with cyclical bizarre behavior patterns (aggression, irritability, hyperactivity), dysarthria, seizures, motor weakness, and coma. Dementia and hepatomegaly eventually develop. Cases of hepatocellular carcinoma also have been reported in affected persons [Hagiwara et al., 2003]. In retrospect, many of the patients have had symptoms since childhood that suggested hyperammonemia, including recurrent episodes of vomiting, lethargy, and irritability [Okeda et al., 1989]. Treatment generally relies on alternate pathway therapy (arginine and phenylbutyrate) [Imamura et al., 2003]; however, liver transplantation is becoming a more common practice in the treatment of this disorder because of the possible liver complications [Ikeda et al., 2001; Yazaki et al., 2004].
More recently, a neonatal-onset form of citrin deficiency has been identified; this form is associated with intrahepatic cholestasis [Tamamori et al., 2002; Tazawa et al., 2001]. Affected infants have multiple metabolic abnormalities, including aminoacidemia, galactosemia, hypoproteinemia, hypoglycemia, and cholestasis. Treatment usually is by high-protein/low-carbohydrate diet, and symptoms often disappear within a year [Saheki et al., 2004]. A few children, however, have a severe form of the disorder with liver damage and tyrosinemia that necessitate liver transplantation. Hyperammonemia is not a major component of this disorder.
Argininosuccinicacidemia
Argininosuccinicacidemia was first described in 1958 by Allan et al. [1958]. Its name derives from the marked elevation of argininosuccinic acid in blood of affected persons. This disorder also has been called argininosuccinicaciduria (ASA) because of the increased excretion of argininosuccinic acid in urine, and argininosuccinate lyase deficiency to denote the underlying enzyme deficiency. In addition to hyperammonemic coma in the newborn period and recurrent hyperammonemic episodes later in childhood, a specific abnormality of the hair termed trichorrhexis nodosa develops in affected children. Nodules appear on the hair shaft, and the hair is friable. A generalized erythematous maculopapular skin rash also may appear in this disorder. Both conditions are associated with arginine deficiency and respond to arginine supplementation [Brusilow and Horwich, 2001].
Chronic marked hepatomegaly has been reported in patients managed with protein restriction and in those receiving arginine supplementation, but this finding is not universal. Pathologic examination reveals modest fatty infiltration and fibrosis. Results of liver function tests frequently are abnormal, especially during hyperammonemic crises, and patients may develop cirrhosis [Zimmermann et al., 1986]. Why some patients with ASA develop cirrhosis and others do not is not understood.
The gene for ASL has been localized to the long arm of chromosome 7 [Kleijer et al., 2002]. The deficient enzyme, a homotetramer of 50-kilodalton (kDa) subunits, is expressed in multiple tissues, including the brain. Evidence of multiple allelic mutations and intragenic complementation, indicating extensive genetic heterogeneity, is characteristic of this disorder [Yu et al., 2001]. The frequency of some mutations, however, is higher than that of others; such mutations may occur at “hot spots” with higher susceptibility for alteration [Linnebank et al., 2002]. The multiple mutations may account for some of the observed heterogeneity in this disease at the clinical level.
Biochemically, the principal findings are elevated citrulline level, hyperammonemia, argininosuccinicacidemia, and argininosuccinicaciduria. The plasma argininosuccinic acid peak is large (normally it is undetectable) [Batshaw et al., 1981]. Of note, this peak may be missed, because it can overlie the peak for leucine or isoleucine. The presence of the two anhydrides of argininosuccinic acid, however, in areas of the chromatogram where homocystine and gamma-aminobutyric acid (GABA) normally are found, aids in the diagnosis. Markedly increased levels of argininosuccinic acid also are readily identifiable in the urine. Additionally, citrulline levels are increased 3–10-fold (to 100 to 300 μmol/L).
The diagnosis can be confirmed by measuring ASL in erythrocytes or fibroblasts, although this is rarely needed. In cerebrospinal fluid, elevated concentrations of argininosuccinic acid and its anhydrides, as well as pyrimidines (pseudouridine and uridine), have been found [Gerrits et al., 1993]. After the initial hyperammonemic crisis and establishment of the diagnosis, recommended treatment for argininosuccinicacidemia consists of a low-protein diet and l-arginine supplementation (see Table 33-1). Many metabolic physicians are also using ammonia scavengers in their treatment regimen.
Argininemia
Argininemia, or hyperargininemia, was first described in 1969 by Terheggen and colleagues [Terheggen et al., 1969]; it is caused by a deficiency of arginase 1. Its name derives from the marked elevation of arginine in the blood of affected persons. Argininemia presents differently from all of the other congenital urea cycle disorders. It usually appears as a progressive neurologic disorder, rather than as an acute encephalopathy [Cederbaum et al., 1979; Prasad et al., 1997].
Clinical symptoms do not classically start with hyperammonemic coma in infancy. In one report, however, a 2-month-old child with recurrent vomiting, persistent jaundice, and hepatomegaly (with associated cirrhosis) was diagnosed as having hyperargininemia [Braga et al., 1997]. Another patient presented with cerebral edema and growth retardation [Harrington et al., 2000]. More commonly, development for the first few years of life appears to be normal, although a detailed history often reveals evidence of protein aversion (often with anorexia, vomiting, and irritability) and some developmental delay. The disease runs a chronic course, but with acute episodes of ataxia, behavioral disturbances, vomiting, lethargy, and seizures. Such episodes often are precipitated by intercurrent viral illnesses [Grody et al., 1993]. Associated biochemical abnormalities usually include moderately elevated plasma ammonia levels of 3–4 times normal and plasma arginine levels of greater than 5 times normal, often exceeding 1000 μmol/L (normal is less than 120 μmol/L).
The unique feature of this disorder is the development of progressive muscle weakness, tremor, and spasticity (diplegia or quadriplegia) [Prasad et al., 1997; Scheuerle et al., 1993]. Mental retardation and growth failure also commonly are evident by childhood, and glaucoma has been reported [Sacca et al., 1996]. Affected children generally do not succumb to hyperammonemic coma and therefore have a longer life span than those affected by proximal urea cycle disorders.
Arginase 1 is found in liver and red blood cells. By contrast, renal arginase (arginase 2) is found in the mitochondrial matrix and differs from the liver type of enzyme in biochemical, molecular, and antigenic properties [Cederbaum et al., 2004]. Arginase 2 also is found in the small intestine and the brain, and its levels have been found to be elevated in patients with argininemia [Iyer et al., 1998]. It is possible that the presence of arginase 2 in hyperargininemia provides some degree of protection from nitrogen accumulation, resulting in less severe hyperammonemic episodes than in other urea cycle disorders. The gene for liver arginase has been localized to chromosome band 6q23 [Sparkes et al., 1986]. Available evidence suggests multiple point mutations and microdeletions in this disorder, indicating extensive genetic heterogeneity [Vockley et al., 1996], and many affected persons are compound heterozygotes. Correlation between the severity of the mutation and the degree of clinical symptoms has been shown [Uchino et al., 1998].
The principal biochemical finding is markedly elevated plasma levels of arginine. Arginine plus ornithine, aspartate, threonine, glycine, and methionine levels are elevated in cerebrospinal fluid [Cederbaum et al., 1982]. In addition, a generalized dibasic aminoaciduria (argininuria, lysinuria, cystinuria, ornithinuria) is present. Urinary excretion of orotic acid and guanidine compounds also is markedly increased [Marescau et al., 1990]. The diagnosis can be confirmed by measuring arginase 1 activity in erythrocytes.
The mechanism responsible for the spasticity and cognitive deficits in argininemia is unknown but is unlikely to be the result of the generally moderate hyperammonemia. Arginine, its guanidine metabolites, and altered biogenic amines are candidate neurotoxins [Marescau et al., 1990]. Arginine is the substrate for nitric oxide synthetase, so overproduction of nitric oxide may play a role in neuropathology [Iyer et al., 1998]. It is theoretically possible that pharmacologic inhibitors of nitric oxide synthetase may be beneficial. Treatment with alternate pathway therapy appears to halt the progression of the spasticity, and botulinum toxin (Botox) and surgical tendon release may improve function.
Hyperornithinemia-Hyperammonemia-Homocitrullinuria Syndrome
HHH syndrome was first described in 1969 [Shih et al., 1969], and only about 50 cases have been reported in the literature, most being in the French-Canadian population in Quebec [Gjessing et al., 1986]. Clinical symptoms are similar to those in other urea cycle disorders but rarely develop in infancy [Zammarchi et al., 1997]. Spastic paraparesis also has been noted and occasionally coagulation disorders occur [Gallagher et al., 2001; Lemay et al., 1992; Salvi et al., 2001a, b]. Plasma ornithine concentrations are elevated, ranging from 400 to 600 mmol/L. Plasma lysine level typically is low, and urinary excretion of homocitrulline is increased. The high blood levels and urinary excretion of homocitrulline likely are the result of conversion of lysine to homocitrulline by ornithine transcarbamylase, in the absence of ornithine in mitochondria. The etiology of HHH syndrome begins with a mutation in the ornithine transporter gene, ORNT1, also called SLC25A15, whose product is a member of the solute mitrochondrial carrier protein family [Camacho et al., 1999]. This leads to decreased ornithine levels in mitochondria and secondary impairment of urea synthesis. The expression of ORNT2, an intronless gene or processed pseudogene, encoding a protein about 90 percent identical to OTNT 1, may explain the milder clinical signs and symptoms compared with those in CPS I and OTC deficiencies [Camacho et al., 2003]. Treatment of HHH syndrome involves protein restriction, phenylbutyrate, and citrulline supplementation.
Common Clinical Presentations of Urea Cycle Disorders
The classic presentation of a complete defect in the urea cycle (other than arginase) is as a catastrophic illness in the first week of life. Clinical manifestations appear between 24 and 72 hours of age, starting as a poor suck, hypotonia, vomiting, lethargy, and hyperventilation with rapid progression to coma and seizures. The electroencephalographic (EEG) pattern during hyperammonemic coma is one of low voltage with slow waves and asymmetric delta and theta waves. The tracing may demonstrate a burst suppression pattern, and the duration of the interburst interval may correlate with the height of ammonia levels [Clancy and Chung, 1991]. Neuroimaging studies reveal cerebral edema with small ventricles, flattening of cerebral gyri, and diffuse low density of white matter; evidence of intracranial hemorrhage also may be seen [Kendall et al., 1983].
Partial urea cycle enzyme deficiencies have a spectrum of presentations, with hyperammonemic episodes developing in infancy in some patients, in later childhood in others, and not until adulthood in still others. Symptoms may be delayed in onset with a mild deficiency or by dietary self-restriction – specifically, avoidance of meats, fish, eggs, milk, and other high-protein foods. Signs and symptoms in childhood include anorexia, ataxia, and behavioral abnormalities such as episodes of erratic behavior, acting out of character, irritability, cloudedness to frank mental status change, nocturnal restlessness, and attention-deficit and hyperactivity [Rowe et al., 1986]. In adults, signs and symptoms may mimic those of psychiatric or neurologic disorders, and include migraine-like headache, nausea, dysarthria, ataxia, confusion, hallucinations, and visual impairment (blurred vision, scotomas, lost vision) [Arn et al., 1990]. In a case report of late-onset OTC deficiency, the patient was a heterozygote who presented with a syndrome mimicking complex partial status epilepticus [Bogdanovic et al., 2000
