Clinical manifestations

Presentation and course

The classic presentation of argininosuccinic acidemia is as a catastrophic illness in the first week of life. Typically, the affected neonate is born after an uncomplicated full-term pregnancy with normal labor, delivery, and Apgar scores. Clinical symptoms develop in the first week of life as “poor suck,” hypotonia, vomiting, lethargy, and hyperventilation, progressing to coma (18). During hyperammonemic coma, increased intracranial pressure with neurologic findings, including increased deep tendon reflexes, papilledema, and decorticate or decerebrate posturing are usually found. Seizures are a late complication and follow alterations in consciousness. Epileptiform activity consists mainly of multiareal spikes, spike-waves, or sharp-and-slow-wave activity. There may also be sustained monorhythmic theta activity (74).

Cases of childhood-onset disease with a partial enzyme deficiency have been reported (68). In these cases, symptoms generally develop later in childhood and include anorexia and food intolerance, developmental delay, and behavioral changes (32). Hyperammonemic crises in affected individuals have been precipitated by high-protein meals, viral infections, medication (eg, valproate), trauma, anesthesia (05), and surgery (54). Late-onset patients are being increasingly reported in current publications. They may have been underreported previously. Late-onset patients who never develop hyperammonemic crises, but predominantly present with intellectual disability or other neurologic symptoms, often remain undiagnosed for months or years (37). In addition, patients with a putatively benign phenotype have been identified by newborn screening, demonstrating the clinical variability of this disease (78; 52).

In addition to hyperammonemic coma in the newborn period and recurrent hyperammonemic episodes later in childhood, affected children develop a specific abnormality of the hair termed "trichorrhexis nodosa." Nodules appear on the hair shaft and the hair is friable. A generalized erythematous maculopapular skin rash may also appear in this disorder. Both conditions are associated with arginine deficiency and respond to arginine supplementation (19). Chronic hepatomegaly (8 to 10 cm below the costal margin) has been reported, both in patients treated with protein restriction and in those receiving arginine supplementation. Pathologic examination shows modest fatty infiltration and fibrosis. Liver function tests can be normal in some patients except during hyperammonemic crises (84), but in other patients, are chronically and progressively abnormal. Liver abnormalities are observed mainly in patients with the severe early-onset form of the disease (72). Hypertension due to impaired systemic nitric oxide (NO) production may be an underrecognized feature of this disease (66; 15). Hypertension was shown to respond dramatically to treatment with an organic nitrate (56). In a mouse model, argininosuccinic acidemia was lethal in the neonatal period and showed a biochemical phenotype identical to the human disease (64).

Grioni and colleagues reported that 6 of 11 patients with argininosuccinic acidemia developed epilepsy following a seizure-free interval after onset of the disease (28). Abnormal neuroimaging findings in argininosuccinic aciduria are frequent and consist of parenchymal infarcts, focal white matter hyperintensity, cortical or cerebral atrophy, and nodular hypertopia. Although proton spectroscopy of basal ganglia indicated a significant decrease of N-acetylaspartate and choline in patients with early-onset disease, spectroscopy of white matter was characterized by a concomitant decrease of creatine and increased concentrations of guanidinoacetate in both early- and late-onset patients (11).

An international study on urea cycle disorders demonstrated that some adolescent and adult patients developed chronic renal failure (39). Because the onset of chronic renal failure does not correlate with the severity of neurologic symptoms in this cohort, different pathomechanisms should be considered for renal and neurologic disease manifestations. This notion is supported by a longitudinal study performed by Baruteau and colleagues, who investigated frequency and pattern of neurologic and systemic disease manifestations in a cohort of 56 patients. Extra-neurologic symptoms, including persistent rise in plasma alanine aminotransferase activity usually associated with hepatomegaly, transient or persistent hypokalemia, and chronic diarrhea, were more frequent in patients with an early-onset as opposed to the late-onset or screened cohort. In contrast, the pattern and severity of the neurologic phenotype was equally distributed between the early- and late-onset group and did not correlate with the severity of hyperammonemia and plasma argininosuccinic acid concentrations, implicating alternative underlying pathomechanisms (eg, cerebral NO-deficiency, secondary creatine deficiency) for the neurologic impairment in argininosuccinic aciduria (11).

Prognosis and complications

Prior to the development of alternate pathway therapy, virtually all children with neonatal-onset argininosuccinic acidemia died in infancy (69). Most died in the newborn period, and the remainder succumbed to intercurrent hyperammonemic episodes or protein malnutrition. Although a significant number of neonates may still succumb during neonatal hyperammonemic coma (12), patients with argininosuccinate lyase deficiency have the lowest risk of neonatal mortality among all urea cycle disorders. A metaanalysis demonstrated that overall 91% of symptomatic patients with this disease survive the first year of life, whereas only 81% of patients with neonatal disease manifestation survive the neonatal period (20).

Although neonatal mortality has decreased, morbidity remains high in survivors of neonatal hyperammonemic coma. Over 75% have mental retardation, and there is frequently comorbidity with cerebral palsy, seizure disorder, and visual deficits (55; 73). There appears to be a correlation between intellectual function and duration of hyperammonemic coma. Children in neonatal hyperammonemic coma for less than 3 days have a far better outcome than those in coma for longer periods of time (55). Cognitive outcome also is better in prospectively treated infants with complete defects and in children with partial defects (47). However, increasing evidence has been gathered that neurologic disease is progressive over time with increasing frequency of cognitive impairment despite early therapeutic intervention (52; 01; 11).

Clinical vignette

A 17-month-old girl, the product of a 36-week gestation, was born to a gravida 1, para 2, 22-year-old mother. There was no consanguinity. The child breast fed normally until 60 hours of age, when she started to refuse feedings and became lethargic. Nevertheless, she was discharged on the third day of life. At home, she was difficult to arouse and had gasping respirations. At 90 hours of age, she had a seizure and a respiratory arrest. She was readmitted to the hospital, where she was intubated and treated for sepsis. She remained in stage IV coma, being unresponsive to painful stimuli. The plasma ammonium concentration, determined on the fifth day of life, was 500 µM. Plasma amino acids showed a large peak of argininosuccinic acid, and its 2 anhydrides and citrulline concentration was also elevated. The child was treated with peritoneal dialysis and intravenous arginine. The ammonium concentration fell to 250 µM within 4 hours of initiation of therapy. However, she did not recover from coma until 9 days of age. Computerized tomography of the brain showed a grade IV intraventricular hemorrhage. She was discharged at 2 months of age on a protein-restricted diet and arginine supplementation. The child subsequently developed myoclonic seizures and spastic quadriplegia. Cognitive development remained extremely delayed. She had no further hyperammonemic episodes until 17 months of age, when she developed vomiting and lethargy during an upper respiratory tract infection. The mother elected not to take her to the hospital, and she died at home.

Biological basis

Etiology and pathogenesis

Argininosuccinic acidemia is caused by a partial or complete deficiency of argininosuccinate lyase (EC 4.3.2.1), also called argininosuccinase, a cytosolic enzyme in the urea cycle that catalyzes the cleavage of argininosuccinic acid into arginine and fumarate (16). The majority of mutations are private and missense mutations (08).

Argininosuccinic acidemia is inherited as an autosomal recessive trait. The deficient enzyme is expressed in multiple tissues. The ASL gene is located on chromosome 7q11.21 (58; 71). The enzyme is a homotetramer of 50-kD subunits. There is evidence of multiple allelic mutations and intraallelic complementation in this disorder, indicating extensive genetic heterogeneity (75; 80; 08), however, some mutations appear at a higher frequency than others and may represent hot spots with higher susceptibility for alteration (46; 08). A founder mutation causing neonatal-onset disease has been found in patients from Saudi Arabia (03). Another founder mutation was identified in the Finnish population (08). The presence of cross-reacting material in all the mutants is consistent with expression of the mutant enzyme (09). The multiple mutations may account for the observed heterogeneity in this disease at the clinical level (31). Erez and colleagues demonstrated that argininosuccinate lyase is not only an enzyme with catalytic function that leads to arginine production by the urea cycle, but it is also part of a multi-protein complex that includes endothelial nitric oxide synthase and is critically important for systemic nitric oxide synthesis (26). Erez and colleagues also showed that dysfunction of the enzyme not only leads to endogenous arginine deficiency, but it hampers the ability of the cell to use extracellular arginine for nitric oxide synthesis. Because arterial hypertension is not frequently reported, it remains to be determined whether this complication is still underreported or whether impaired systemic nitric oxide production is restricted to patients with specific mutations.

Biochemically, the principal finding is hyperammonemia, argininosuccinic acidemia, and argininosuccinic aciduria. Plasma ammonium concentrations are 10- to 100-fold normal during hyperammonemic crisis, and the plasma argininosuccinic acid peak is large (normally undetectable) (14). It should be noted that this peak may be missed in the amino acid analysis, as it can overlie the peak for the essential amino acids leucine or isoleucine. However, the presence of the 2 anhydrides of argininosuccinic acid in areas of the chromatogram where homocystine and GABA are found, aids in the diagnosis. Argininosuccinic acid is also detectable by tandem mass spectrometry, and markedly increased argininosuccinic acid is readily identifiable in the urine. Other plasma amino acid abnormalities include 3-fold to 10-fold elevated concentrations of citrulline (100 to 300 µmol/L), elevated glutamine (a storage form of ammonia), and decreased arginine (the product of argininosuccinate lyase). Additionally, urinary orotic acid excretion can be increased (06). The diagnosis can be confirmed by measuring argininosuccinate lyase in erythrocytes or fibroblasts and by molecular genetic testing.

A model that predicts phenotypic severity in individuals with argininosuccinic acidemia has been developed, which integrates genotype-specific data with biochemical and clinical endpoints in affected individuals. In this model, residual enzymatic ASL activity not only correlates with the clinical severity of the hyperammonemic episode at initial manifestation, but also predicts the frequency and severity of future hyperammonemic decompensations and neurologic outcomes. This model highlights that in vitro residual enzyme activity is a robust biomarker that differentiates between severe (ASL activity < 8.7%) and attenuated phenotypes (ASL activity ≥8.7%) (82). In neonates dying of hyperammonemic coma, neuropathologic changes involve prominent cerebral edema and generalized neuronal cell loss (43). In survivors of prolonged neonatal hyperammonemic coma, changes observed on neuroimaging studies obtained months later include ventriculomegaly with increased sulcal markings, bilateral symmetrical low-density white matter defects, and diffuse atrophy with sparing of the cerebellum (55). Neuropathology in those children who subsequently died was consistent with the neuroimaging findings and included ulegyria, cortical atrophy with ventriculomegaly, and prominent cortical neuronal loss (53; 21).

The mechanism of the ammonia-induced brain damage remains unclear. Ammonia is physiologically detoxified in astrocytes by glutamate dehydrogenase and glutamine synthetase. The accumulation of ammonia and glutamine has a number of potentially toxic effects on the brain, including depletion of intermediates of cell energy metabolism and organic osmolytes, altered amino acid and neurotransmitter concentrations (eg, glutamate and GABA), increased extracellular potassium concentrations (32; 23; 13; 85; 22; 44; 81; 83; 63), potentially altered water transport through aquaporin 4 channels (44), and oxidative and nitrosative stress due to increased free radical production and increased nitric oxide synthesis (60). More than 1 mechanism is probably active. It is unclear whether argininosuccinic acid accumulation contributes to brain injury. Patients who have never had a hyperammonemic crisis can exhibit intellectual disability and seizures, thereby supporting this possibility (27; 25; 56). Further study is needed to determine the pathologic role of the elevated concentrations of argininosuccinic acid in the brains of patients with this disorder, especially because liver transplant does not fully correct the argininosuccinic acidemia and abnormalities in brain metabolism (66; 50; 59). It has been hypothesized that cerebral accumulation of argininosuccinate may be facilitated by limited efflux of di- and tricarboxylic acids across the blood-brain barrier causing intracerebral entrapment of these metabolites (38).

In addition to the potential neurotoxic effect of argininosuccinic acid, nitric oxide deficiency may be a cause of tissue damage in argininosuccinic acidemia. As mentioned, argininosuccinate lyase is thought to be part of a multi-protein complex that includes nitric oxide synthase and is critical for nitric oxide synthesis. Patients with argininosuccinic aciduria are deficient in arginine and NO production; deficient NO production can be another cause of the brain damage (26; 56). Cerebral nitrosative stress due to reactive nitrogen species has been demonstrated as an additional underlying cause of neurotoxicity in argininosuccinic aciduria (10). It has been shown that ASL deficiency is associated with decreased catecholamine synthesis due to impaired tyrosine hydroxylase activity in the nucleus locus coeruleus as well as dopaminergic neurons and the substantia nigra, which leads to the formation of tyrosine aggregates and an elevation of alpha-synuclein, thereby promoting abnormal stress response, seizure sensitivity, as well as motor and neurocognitive deficits (41; 42).

Liver enlargement and elevated liver enzyme in serum are frequently observed in neonatal-onset cases. Hepatic pathology studies show diffuse microvesicular steatosis, periportal nuclear glycogen, and variable portal fibrosis with occasional portal to portal bridging (07). The mechanism for liver damage is unclear and could potentially be caused by argininosuccinate or its derivatives. Anecdotal reports suggest that treatment with ammonia scavenging drugs reduce liver size and liver enzyme activities in serum in some patients.

A clinical trial of ammonia scavenger treatment combined with either low-dose arginine supplementation or high-dose arginine supplementation demonstrated benefit of an ammonia scavenger combined with low-dose arginine treatment in ameliorating liver damage (57).

Epidemiology

The prevalence of argininosuccinic acidemia has been estimated at approximately 1 in 70,000 in the United States (66). However, large-scale observational trials in the United States and Europe, as well as implementation of argininosuccinic acidemia in the newborn screening programs in the United States, have demonstrated that the incidence might be as low as 1 in 220,000 newborns (70).

Prevention

Argininosuccinic acid concentrations in amniotic fluid, as well as [¹⁴C]citrulline incorporation into proteins of chorionic villi, have been used for prenatal diagnosis in at-risk families (35; 49; 36). Preimplantation genetic diagnosis in couples at risk is now a possibility (30), and prenatal DNA diagnosis was made using a single fetal nucleated erythrocyte isolated from maternal blood (76). In affected families, prospective treatment of this disorder from birth has been associated with an improved outcome (24; 47). Newborn screening by tandem mass spectrometry using blood spots allows preclinical diagnosis of this condition and early treatment (04). However, it still remains to be elucidated whether newborn screening is beneficial for patients with argininosuccinic acidemia, especially with regard to cognitive function. Some patients may already present with a neonatal metabolic crisis before the results of newborn screening are available; other patients identified by newborn screening may have a benign disease course (52). The group of symptomatic individuals who initially exhibited symptoms after the newborn period, ie, the late-onset group, may derive the greatest benefit from newborn screening (61). A severity-adjusted analysis for the cytosolic urea cycle disorders, citrullinemia type 1 and argininosuccinic academia, demonstrated that early identification by newborn screening (and subsequent early implementation of therapy) resulted in reduced severity of the initial hyperammonemic episodes for individuals with severe and attenuated phenotypes (62). Intriguingly, the effect was most pronounced for the attenuated disease group because individuals identified by newborn screening exhibited plasma ammonium concentrations within the normal range (62).

Differential diagnosis

A number of inborn errors of metabolism have a similar clinical presentation to argininosuccinic acidemia, especially in the newborn period. These include other urea cycle disorders and amino acidopathies, congenital lactic acidoses, defects in fatty acid oxidation, and organic acidurias. In addition, a number of acquired conditions, including transient hyperammonemia of the newborn, sepsis, intracranial hemorrhage, and cardiorespiratory disorders can present with a similar symptom complex. Differentiation depends on identifying hyperammonemia associated with argininosuccinic acidemia.

In older children and adults, a number of acquired disorders can also present with hyperammonemia, including liver disease, Reye syndrome, drug toxicity, and hepatotoxins. Again, the presence of marked elevations of argininosuccinic acid in blood and urine should make the diagnosis obvious.

Diagnostic workup

Plasma ammonium and plasma and urinary amino acids should be obtained in suspected cases. The principal biochemical feature of this disorder is hyperglutaminergic hyperammonemia, argininosuccinic acidemia, and argininosuccinic aciduria. Citrulline concentrations are also elevated, but are much lower than those found in citrullinemia. Urinary orotic acid concentrations may also be mildly elevated. The detection of elevated argininosuccinate and citrulline in dried blood spots by tandem mass spectrometry allows early identification during the newborn period (04).

Management

For a detailed discussion of treatment see the current published guidelines for urea cycle disorders (29).

Long-term treatment of argininosuccinic acidemia relies on the principles of restricting nitrogen intake and stimulating an alternate pathway for waste nitrogen excretion. Nitrogen restriction involves the use of a high-caloric, low-protein diet. The approach to stimulating an alternate pathway of waste nitrogen excretion involves the use of arginine supplementation (18). The rationale for using arginine is as follows. Argininosuccinic acid contains both waste nitrogen atoms destined for excretion as urea. Furthermore, it has a renal clearance rate equal to the glomerular filtration rate. Thus, provided it is continuously synthesized and excreted, argininosuccinic acid should serve as an efficient substitute for urea as a waste nitrogen product. However, deficient activity of argininosuccinate lyase prevents the synthesis of arginine, leading to decreased availability of ornithine for argininosuccinic acid synthesis. A solution would be to provide adequate arginine to prime the cycle. The new pathway for waste nitrogen excretion would then start at arginine and end in argininosuccinic acid excretion. For each mole of arginine given, 2 moles of waste nitrogen would be removed as argininosuccinic acid. Two to 4 moles/kg per day (0.4 to 0.7 g/kg per day) of L-arginine free base are thought to be sufficient to stabilize ammonium concentrations when combined with protein restriction (12). However, as mentioned above, 1 clinical trial has shown efficacy of phenylbutyrate treatment combined with low-dose arginine compared to the combined treatment with high-dose arginine in ameliorating liver damage (57). Therefore, the daily arginine dosage should not exceed 2 mmol/kg per day (29).

Theoretically, the efficacy of arginine supplementation may be limited by a relative deficiency in aspartate; this substance normally combines with citrulline to form argininosuccinic acid. It has been suggested that a supplement of citrate salt (2 mmol/kg per day) may prevent this from occurring by repletion of aspartate stores through the production of oxaloacetate (33; 65).

In some patients, occasionally additional alternate pathway therapy may be required; in particular with arginine supplementation, plasma arginine concentrations should be kept below 2 mmol/kg per day. Nitrogen scavengers, such as sodium benzoate and sodium or glycerol phenylbutyrate, are used for this purpose. Benzoate is a compound that is conjugated with glycine to form hippurate that is cleared by the kidney at 5-fold the glomerular filtration rate (40). Phenylacetate, which is formed from phenylbutyrate by the liver, conjugates with glutamine to form phenylacetylglutamine, a substance that is excreted by the kidney. Over 40% of total waste nitrogen can be excreted as phenylacetylglutamine (17). Commonly used starting doses are either 250 mg/kg per day (or 5 g/m2 per day) each of benzoate and phenylbutyrate (29); however, doses must be adjusted to the individual patient. In general, antiepileptic treatment with valproate should be avoided, if possible.

Enzyme replacement through liver transplantation has been attempted. In children with urea cycle defects who have received orthotopic liver transplants, the procedure has improved the metabolic abnormalities and permitted a normal protein intake in most patients (77; 50; 59). However, there have been reports of at least 2 deaths from graft-versus-host disease, and long-term immunosuppression is required in all patients. Most importantly, liver transplantation does not correct the defect in tissues other than liver.

Early identification and treatment of intercurrent hyperammonemic episodes is essential, both because treatment is more effective at lower ammonium concentrations and because neurologic outcome appears to be a function of duration of severe hyperammonemia (55). Anticipatory management of hyperammonemia is often feasible in a previously diagnosed case. Increases in ammonium concentrations have been found to lag by days to weeks behind elevations in glutamine (48). Therefore, periodic measurement of plasma amino acids (that include glutamine) and ammonium may permit adjustment of therapy before clinical symptoms appear. When the patient is asymptomatic and biochemical abnormalities are detected, the patient usually responds to lowering nitrogen intake or increasing the doses of ammonia scavenging agents.

If symptoms of vomiting and lethargy become evident and ammonium concentrations are increased, intermittent metabolic emergency treatment is indicated (29). This involves hospitalization with reduction or transiently complete elimination of protein and the intravenous administration of glucose-enriched saline, intravenous L-arginine-HCl, benzoate, and phenylacetate. In severe hyperammonemic encephalopathy, extracorporeal detoxification with hemofiltration/hemodialysis should be initiated rapidly. For details, please read the international recommendations for urea cycle disorders (29).

Cysteamine treatment in vitro increases the residual enzyme activity of transfected cells with cysteine for arginine mutations. Because 12% of all known pathogenic genetic variations of argininosuccinate lyase deficiency cause a cysteine for arginine change, it has been hypothesized that cysteamine, an orphan drug for cystinosis, may be a novel therapeutic option for some patients in the future (34). However, clinical trials are required to test the therapeutic safety and efficacy of this approach.

Special considerations

Pregnancy

Women carrying affected offspring have not routinely experienced complications during pregnancy, and the children initially appear well born at term. Three women with mild (non-neonatal-onset) argininosuccinic acidemia have gone through pregnancy without complications. There were no clear teratogenic effects in their infants (79; 51).

Anesthesia

There has been 1 report of hyperammonemia induced by enflurane in argininosuccinic aciduria (05). It is prudent to use anesthetics with low toxicity to the liver. However, surgery requires the stopping of oral medication and is associated with a catabolic stress, and both of these may induce hyperammonemia. Therefore, it is important to continue arginine therapy intravenously until the patient is able to accept oral medication. The patient should also receive adequate glucose and fat to prevent catabolism. One report describes the perioperatively uneventful use of midazolam, s-ketamine, fentanyl, and isoflurane with local injection of ropivacaine, along with intravenous infusion of glucose and alternate pathway drugs in 2 siblings with severe OTC deficiency (67).