Clinical Diagnosis

Argininosuccinic aciduria (ASA) results from deficiency of the enzyme argininosuccinate lyase (ASL), the fourth step in the urea cycle, in which argininosuccinic acid is cleaved to produce arginine and fumarate.

The two most common forms of ASL deficiency are severe neonatal form and late-onset form.

Neonatal onset ASL deficiency is suspected in infants who present in the first week of life with hyperammonemia manifest as increasing lethargy, somnolence, refusal to feed, vomiting, tachypnea, and respiratory alkalosis.

The presentation is indistinguishable from that of other disorders of the proximal urea cycle (i.e., carbamoyl-phosphate synthetase I deficiency, ornithine transcarbamylase deficiency, and argininosuccinate synthase deficiency), except that hepatomegaly is more commonly observed in infants with ASL deficiency.

Late onset ASL deficiency can manifest as:

• Episodic hyperammonemia triggered by acute infection or stress or by non-compliance with dietary restrictions and/or medication

• Cognitive impairment or learning disabilities in the absence of any documented hyperammonemic episodes

• Normal

ASL deficiency can also present with long-term complications not commonly observed in other urea cycle disorders, including:

• Liver involvement. Hepatomegaly; elevated transaminases; liver fibrosis or cirrhosis

• Neurocognitive deficits. ADHD, developmental disability, learning disability, and seizures that may be independent of hyperammonemia

• Trichorrhexis nodosa. Coarse and brittle hair that breaks easily. See figure at dermatlas.med.jhmi.edu (linked with permission from Dermatlas).

• Hypertension. Possibly occurring in late childhood and adolescence, in the absence of primary renal vascular disease or an endocrine disorder

• Hypokalemia. The cause is poorly understood; may be secondary to renal wasting.

Testing

Newborn screening. All 50 states in the US include ASL deficiency in their newborn screening programs [National Newborn Screening Status Report].

Citrulline, assayed by tandem mass spectroscopy, is the metabolite used for detection of ASL deficiency in newborn screening programs. Elevation of citrulline can also be seen with citrullinemia type 1 (ASS deficiency), citrullinemia type 2 (citrin deficiency) and pyruvate carboxylase deficiency; hence, confirmation of the diagnosis of ASL deficiency rests on demonstration of elevated plasma or urine concentration of argininosuccinic acid or its anhydride compounds.

Plasma ammonia concentration

• In the severe forms of ASL deficiency, the initial plasma ammonia concentration (before treatment) may be greater than 1000 µmol/L, though typically elevations are in the ranges of few hundreds. See Table 1.

• In the milder neonatal and adult forms of ASL deficiency, a lower plasma ammonia concentration may be seen (adult upper limit of normal: <26 µmol/L).

Plasma quantitative amino acid analysis. See Table 2.

Urinary analysis

• Orotic acid excretion is typically normal (0.3-2.8 mmol/mol of creatinine); however, orotic aciduria may be observed [Gerrits et al 1993, Brosnan & Brosnan 2007].

• Argininosuccinic acid is significantly elevated. Urinary concentration of argininosuccinate is typically greater than 10,000 µmol/g of creatinine on urine amino acid analysis [Ficicioglu et al 2009] (normal range 0-1 µmol/L).

Argininosuccinate lyase (ASL) enzyme activity. ASL enzyme activity can be measured in cell homogenates from a flash-frozen liver biopsy or, more conveniently, from skin fibroblasts or red blood cells by one of two methods:

• Forward reaction: addition of the substrate argininosuccinate followed by flurometric measurement of the product fumarate [Kleijer et al 2002]

• Reverse reaction: addition of the substrates ¹⁴C fumarate and unlabelled arginine followed by assay of the product ¹⁴C argininosuccinate

Note: The residual in vitro ASL enzyme activity does not seem to correlate with the clinical severity [Ficicioglu et al 2009].

The citrulline incorporation test is an indirect method of assessing the ASL enzyme activity in fibroblast cell cultures. Incorporation of ¹⁴C from L-[ureido-¹⁴C]citrulline and ³H from L-[3,4,5-³H(N)]-leucine into acid-precipitable material has been shown in at least one longitudinal study to be more sensitive in detecting residual ASL enzyme activity than the direct ASL enzyme activity assay performed on cell lysates and, hence, correlates better with phenotype [Ficicioglu et al 2009]. However, because the citrulline incorporation test involves a skin biopsy and because the differences in results of this test compared to the results of the direct ASL enzyme assay were not significant, it is debated whether this test is of value in determining the prognosis in a given individual with ASL deficiency.

Note: The two types of ASL enzyme activity assays are available only at few specialized laboratories; this limited availability precludes their widespread use in clinical settings.

Molecular Genetic Testing

Gene. ASL is the only gene in which mutations are known to be associated with ASL deficiency.

Clinical testing

• Sequence analysis. Sequence analysis of the coding region of ASL detects mutations in about 90% of individuals with the clinical and biochemical diagnosis of ASL deficiency. Pathogenic mutations include nonsense and missense mutations, insertions, deletions, and those affecting mRNA splicing. Mutations are scattered throughout the gene; however; exons 4, 5, and 7 appear to be mutational hotspots [Linnebank et al 2002, Trevisson et al 2007].

• Deletion/duplication analysis. Though array comparative genomic hybridization-based (aCGH) assays have recently become available to detect deletions or duplications of ASL, no data are available on the frequency of these rearrangements in ASL deficiency. The authors suggest that aCGH be performed in patients with a clinical and/or biochemical phenotype of ASL deficiency who have only one mutation identified by sequence analysis [Authors, personal observation].

• Targeted mutation analysis. The ASL c.1153C>T variant is associated with residual ASL enzyme activity as measured by the incorporation of [¹⁴C]citrulline into proteins [Kleijer et al 2002]. This mutation accounts for approximately 60% of mutations in the Finnish population. [Keskinen et al 2008].

Table 3. Summary of Molecular Genetic Testing Used in Argininosuccinate Lyase (ASL) Deficiency

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Gene Symbol	Test Method	Mutations Detected	Mutation Detection Frequency by Test Method 1	Test Availability
ASL	Sequence analysis	Sequence variants 2	~90%	Clinical
	Deletion / duplication analysis 3	Exonic or whole-gene deletions	Unknown
	Targeted mutation analysis	c.1153C>T	100% for targeted variant

Test Availability refers to availability in the GeneTests™ Laboratory Directory. GeneReviews designates a molecular genetic test as clinically available only if the test is listed in the GeneTests Laboratory Directory by either a US CLIA-licensed laboratory or a non-US clinical laboratory. GeneTests does not verify laboratory-submitted information or warrant any aspect of a laboratory's licensure or performance. Clinicians must communicate directly with the laboratories to verify information.

1. The ability of the test method used to detect a mutation that is present in the indicated gene

2. Examples of mutations detected by sequence analysis may include small intragenic deletions/insertions and missense, nonsense, and splice site mutations.

3. Testing that identifies deletions/duplications not readily detectable by sequence analysis of genomic DNA; a variety of methods including quantitative PCR, long-range PCR, multiplex ligation-dependent probe amplification (MLPA), or targeted array GH (gene/segment-specific) may be used. A full array GH analysis that detects deletions/duplications across the genome may also include this gene/segment. See array GH.

Interpretation of test results. For issues to consider in interpretation of sequence analysis results, click here.

Testing Strategy

To confirm/establish the diagnosis in a proband

• Elevated plasma ammonia concentration (>150 µmol/L, sometimes up to ≥2000-3000 µmol/L), elevated plasma citrulline concentration (usually 200-300 µmol/L), and elevated argininosuccinic acid in the plasma or urine establish the diagnosis of ASL deficiency.

• ASL molecular genetic testing and assay of ASL enzyme activity help in confirmation of diagnosis but are not required to establish the diagnosis in the presence of characteristic biochemical findings. In the presence of equivocal biochemical findings, molecular genetic testing and enzyme activity are required for the diagnosis.

Carrier testing for at-risk relatives requires prior identification of the disease-causing mutations in the family.

Note: Carriers are heterozygotes for this autosomal recessive disorder and are not at risk of developing the disorder.

Prenatal diagnosis and preimplantation genetic diagnosis (PGD) for at-risk pregnancies can be accomplished by molecular genetic testing if the mutations in ASL are known [Häberle & Koch 2004].

Prenatal diagnosis for at-risk pregnancies can also be accomplished by biochemical testing/assay of ASL enzyme activity if the responsible mutations in the family are not known. Elevated levels of argininosuccinic acid in the amniotic fluid can reliably detect an affected fetus [Kamoun et al 1995, Kleijer et al 2006]. Analysis of ASL enzyme activity by direct methods in chorionic villus tissue or amniocytes or indirect methods such as ¹⁴C-citrulline incorporation in uncultured chorionic villus samples has been successful [Kleijer et al 2002, Pijpers et al 1990].

Note: It is the policy of GeneReviews to include in GeneReviews™ chapters any clinical uses of testing available from laboratories listed in the GeneTests™ Laboratory Directory; inclusion does not necessarily reflect the endorsement of such uses by the author(s), editor(s), or reviewer(s).

Genetically Related (Allelic) Disorders

No phenotypes other than those discussed in this GeneReview are known to be associated with mutations in ASL.

Natural History

The clinical presentation of argininosuccinate lyase (ASL) deficiency is variable. The two most common forms are a severe neonatal onset form and a late onset form.

The clinical presentation of the severe neonatal onset form, which is indistinguishable from that of other urea cycle disorders, is characterized by hyperammonemia within the first few days after birth. Newborns typically appear healthy for the first 24 hours but within the next few days develop vomiting, lethargy, and hypothermia and refuse to accept feeds [Brusilow & Horwich 2009]. Tachypnea and respiratory alkalosis are early findings. Failure to recognize and treat the defect in ureagenesis leads to worsening lethargy, seizures, coma, and death. The findings of hepatomegaly and trichorrhexis nodosa (coarse and friable hair) at this early stage are the only clinical findings that may suggest the diagnosis of ASL deficiency [Brusilow & Horwich 2009].

In contrast, the manifestations of the late onset form range from episodic hyperammonemia (triggered by acute infection or stress or by non-compliance with dietary restrictions and/or medication) to cognitive impairment, behavioral abnormalities, and/or learning disabilities in the absence of any documented episodes of hyperammonemia [Brusilow & Horwich 2009].

While manifestations secondary to hyperammonemia are common to all urea cycle disorders, many individuals with ASL deficiency present with a more complex clinical phenotype. The increased incidence of (1) neurocognitive deficiencies; (2) hepatitis, cirrhosis; (3) trichorrhexis nodosa; and (4) systemic hypertension are unique to ASL deficiency. All these manifestations appear to be unrelated to the severity or duration of hyperammonemic episodes [Saudubray et al 1999, Mori et al 2002, Ficicioglu et al 2009].

Neurocognitive deficiencies. In a cross-sectional study of individuals with a urea cycle disorder (UCD), it was observed that persons with ASL deficiency had a significant increase in disabilities and neurologic abnormalities compared to persons with OTC deficiency [Tuchman et al 2008].

Individuals with ASL deficiency also had an increased incidence of attention deficit hyperactivity disorder (ADHD), developmental disability (intellectual disability, behavioral abnormalities, and/or learning disability), and seizures compared to persons with all other UCD [Tuchman et al 2008]. Though neurocognitive deficits are more common in ASL deficiency than in other UCDs, they are not universally present; many individuals with ASL deficiency who are treated with protein restriction and supplemental arginine have normal cognition and development [Widhalm et al 1992, Ficicioglu et al 2009].

The increasing reliance on newborn screening programs for early diagnosis of ASL deficiency allows the evaluation of early treatment on disease progression, especially in the late onset form.

• Ficicioglu et al [2009] reported the long-term outcome of 13 infants diagnosed between age four and six weeks by newborn screening programs. All had low ASL enzyme activity; in spite of optimal therapy with protein restriction and arginine supplementation, four of 13 had learning disability, three had mild developmental delay, three had seizures, and six had an abnormal EEG including abnormal sharp irregular background activity, frequent bilateral paroxysms, and increased slow wave activity.

• In a separate cohort of 17 individuals with ASL deficiency diagnosed by newborn screening in Austria, IQ was average/above average in 11 (65%), low average in five (29%), and in the mild intellectual disability range in one (6%). Four had an abnormal EEG without evidence of clinical seizures [Mercimek-Mahmutoglu et al 2010]. The overall favorable outcomes in persons in this cohort may be attributable not only to early dietary and therapeutic interventions but also to the high proportion of persons with very mild disease.

Liver disease in individuals with ASL deficiency also appears to be independent of the defect in ureagenesis. The spectrum of hepatic involvement ranges from hepatomegaly to elevations of liver enzymes to severe liver fibrosis [Billmeier et al 1974, Mori et al 2002, Tuchman et al 2008]. Liver involvement has been noted even in individuals treated with protein restriction and arginine supplementation who had not experienced significant hyperammonemia [Mori et al 2002, Mercimek-Mahmutoglu et al 2010]. At present no biochemical or molecular features help predict liver dysfunction in people with ASL deficiency. Given the potential direct toxicity of argininosuccinate on hepatocytes, lowering of the argininosuccinate levels in plasma (a reflection of its production by the liver) may have potential benefit, though this has not yet been proven.

Trichorrhexis nodosa (see figure) is characterized by nodular swellings of the hair shaft accompanied by frayed fibers and loss of cuticle. About half of individuals with ASL deficiency have an abnormality of the hair manifest as dull, brittle hair surrounded by areas of partial alopecia [Fichtel et al 2007]. Normal hair contains 10.5% arginine by weight; hair that is deficient in arginine as a result of ASL deficiency is weak and tends to break. Thus, this clinical feature responds to arginine treatment.

Hypertension. Recently, it has been noted that hypertension is over-represented in persons with ASL deficiency [Brunetti-Pierri et al 2009]. Usually no secondary causes of hypertension are detected, suggesting that this finding is related to the primary metabolic defect.

Electrolyte imbalances. Some individuals develop electrolyte imbalances such as hypokalemia [Author, personal observation]. The hypokalemia is observed even in individuals who are not treated with sodium phenylbutyrate. The etiology is unclear; increased renal wasting has been suggested.

Genotype-Phenotype Correlations

Data are insufficient to infer any genotype-phenotype correlations.

Direct correlation between the clinical phenotype and residual ASL enzyme activity has been difficult to establish most probably as a result of the limited sensitivity of the enzyme assay (see Testing, Argininosuccinate lyase enzyme activity).

Prevalence

The estimated incidence is 1:70,000 live births [Brusilow & Horwich 2009].

Evaluations Following Initial Diagnosis

To establish the extent of disease and needs of an individual diagnosed with argininosuccinate lyase (ASL) deficiency the following evaluations are recommended:

• Complete neurocognitive evaluation

• Evaluation for evidence of hepatic involvement including hepatomegaly, hepatitis, and signs of liver failure

• Plotting of the systolic and diastolic blood pressure on the centile charts based on age and stature

Treatment of Manifestations

Treatment involves rapid control of hyperammonemia during metabolic decompensations and long-term management to help prevent episodes of hyperammonemia and long-term complications.

During acute hyperammonemic episodes severe enough to cause neurologic symptoms, treatment includes the following [Urea Cycle Disorders Conference Group 2001] (see ):

• Discontinuing oral protein intake

• Supplementing oral intake with intravenous lipids, glucose and intravenous insulin if needed (with close monitoring of blood glucose) to promote anabolism.

• Intravenous nitrogen scavenging therapy. A loading dose of 600 mg/kg L-arginine-HCL and 250 mg/kg each of sodium benzoate and sodium phenylacetate in 25 to 35 mL/kg of 10% dextrose solution given intravenously over a 90-minute period is recommended. This is followed by a sustained intravenous infusion of 600 mg/kg L-arginine-HCL and 250 mg/kg each of sodium benzoate and sodium phenylacetate over a 24-hour period. When available, plasma concentrations of ammonia scavenging drugs should be monitored to avoid toxicity. In the absence of drug levels, a serum anion gap of greater than 15 mEq/L and an anion gap that has risen more than 6 mEq/L could indicate drug accumulation and increased risk for toxicity.

Ammonia levels usually normalize with therapy; however failure to decrease ammonia levels with medical therapy mandates prompt institution of hemodialysis.

Hemodialysis is the preferred method for rapid reduction of ammonia in patients who do not respond to nitrogen scavenging therapy. Continuous arteriovenous hemodialysis (CAVHD) or continuous venovenous hemodialysis (CVVHD) with flow rates greater than 40 to 60 mL/min is optimal. Some centers use extracorporeal membrane oxygenation (ECMO) with hemodialysis. Although this combination of techniques provides very high flow rates (170-200 mL/min) and rapidly reduces ammonia levels, morbidity is greater because of the need for surgical vascular access. Nitrogen scavenging therapy needs to be continued during hemodialysis. It is the authors’ policy to continue nitrogen scavenging therapy for 12-24 hours after the patient has been stabilized and is able to accept enteral feeds and medications [Author, personal observation].

Prevention of Primary Manifestations

Dietary restriction of protein and dietary supplementation with arginine are the mainstays of long-term management.

Diet. Lifelong dietary management is necessary and requires the services of a metabolic nutritionist. The recommended daily allowance (RDA) for dietary protein is higher than the minimum needed for normal growth and, hence, most children with a urea cycle disorder (UCD) can receive less than the RDA of protein and still maintain adequate growth. To achieve this end [Brusilow & Horwich 2009]:

• Plasma concentrations of ammonia, branched chain amino acids, and arginine should be maintained within normal ranges.

• Serum plasma total protein and prealbumin levels should be maintained within the low normal ranges.

• Plasma glutamine concentration should be maintained at less than 1000 µmol/L if possible [normal range for individuals ages two to 18 years is 266-746 µmol/L].

Some of the correlations between compliance with the prescribed diet and outcome are contradictory. Although in some patients dietary therapy along with arginine supplementation have been shown to reverse the abnormalities of hair, to improve cognitive outcome, and to reverse abnormalities on EEG [Coryell et al 1964, Kvedar et al 1991, Ficicioglu et al 2009], in many dietary therapy has not been shown to influence the outcome of liver disease or cognitive impairment [Mori et al 2002, Mercimek-Mahmutoglu et al 2010].

Arginine base supplementation: The doses of arginine base routinely recommended are 400-700 mg/kg/day in persons weighing less than 20 kg and 8.8-15.4 g/m²/day in those weighing more than 20 kg. Supplementation with arginine base helps replenish this amino acid (which is deficient in persons with ASL deficiency) and promote excretion of nitrogen through the urea cycle as argininosuccinate. Arginine base is preferred for long-term chronic treatment as the chronic use of arginine hydrochloride may lead to hyperchloremic acidosis.

Arginine base supplementation has been shown to reverse the hair changes; however, its efficacy in preventing the chronic complications is not known. While evidence suggests that arginine base supplementation may prevent metabolic decompensations in those with severe early-onset disease, long-term follow up of persons identified through newborn screening programs did not detect a difference in outcomes between those who were supplemented with arginine base and those who were not [Batshaw et al 2001, Ficicioglu et al 2009, Mercimek-Mahmutoglu et al 2010]. As the renal clearance of argininosuccinic acid is high, increasing its production through arginine supplementation effectively increases waste nitrogen disposal, thereby decreasing the risk of hyperammonemia. However, because of the theoretic risk of argininosuccinic acid toxicity on hepatocytes, reducing the amount of supplemental arginine by initiating nitrogen scavenging therapy may have merits.

Oral nitrogen scavenging therapy: Patients who have had frequent metabolic decompensations or episodes of elevated ammonia despite being on a protein-restricted diet and arginine base supplementation are candidates for oral nitrogen scavenging therapy, an alternative pathway therapy in which sodium benzoate and sodium phenyl butyrate stimulate the excretion of nitrogen in the form of hippuric acid and phenylacetylglutamine, respectively [Batshaw et al 2001]. The dose of sodium phenyl butyrate is 400-600 mg/kg/day for persons weighing up to 20 kg and 9-13 g/m²/day for those weighing more than 20 kg; the dose of sodium benzoate is 250-500 mg/kg/day.

Orthotopic liver transplantation (OLT). Long-term correction of ASL deficiency in the liver can be accomplished by OLT [Lee & Goss 2001] which has resulted in “biochemical cure” [Robberecht et al 2006, Marble et al 2008, Newnham et al 2008]. However, OLT does not correct the arginine deficiency or elevation of argininosuccinic acid at the tissue level, two abnormalities thought to account for the long-term complications of ASL deficiency. Thus, the authors have recommended OLT only in patients with recurrent hyperammonemia or metabolic decompensations that are resistant to conventional medical therapy, or in patients who develop cirrhosis with associated metabolic decompensations [Author, personal observations].

Prevention of Secondary Complications

Salt restriction and use of antihypertensives are indicated in those with elevated blood pressure. Antihypertensives may be tried although their efficacy has not been established.

Electrolyte (potassium) supplementation is appropriate when indicated.

Surveillance

Regular monitoring of the concentration of plasma amino acids to identify deficiency of essential amino acids as well as impending hyperammonemia is indicated. The appropriate intervals for monitoring depend on the clinical scenario, but need to be more frequent in neonates and in those with frequent metabolic decompensations. The authors prefer to evaluate neonates every one to two weeks, infants between age two months and one year every one to three months, and children older than age two years every three to four months.

Early signs of impending hyperammonemic episodes in older individuals include mood changes, headache, lethargy, nausea, vomiting, refusal to feed, ankle clonus, and elevated plasma concentrations of glutamine, alanine, and glycine. Plasma glutamine concentration may rise 48 hours in advance of increases in plasma ammonia concentration in such individuals.

Follow up in a metabolic clinic with a qualified metabolic dietician and clinical biochemical geneticist is preferred when possible.

Annual measurement of blood pressure using the appropriate-sized cuff and plotting the centile values for age and stature is indicated.

Periodic evaluation of liver function tests may be necessary; the appropriate frequency is unknown.

Periodic evaluation of serum electrolytes is appropriate; the frequency unknown.

Agents/Circumstances to Avoid

Avoid the following:

• Excess protein intake

• Large boluses of protein or amino acids

• Less than recommended intake of protein

• Prolonged fasting or starvation

• Obvious exposure to communicable diseases

• Valproic acid

• Intravenous steroids

• Hepatotoxic drugs in those with hepatic involvement

Evaluation of Relatives at Risk

Evaluation of at-risk sibs (either by molecular genetic testing if the family-specific mutations are known or by biochemical testing) shortly after birth can reduce morbidity by permitting early diagnosis and treatment of those who are affected.

See Genetic Counseling for issues related to testing of at-risk relatives for genetic counseling purposes.

Therapies Under Investigation

Search ClinicalTrials.gov for access to information on clinical studies for a wide range of diseases and conditions. Note: There may not be clinical trials for this disorder.

Registries

Contact information for voluntary patient registries is provided by GeneReviews staff.

Urea Cycle Disorders Consortium Registry
Children's National Medical Center
Phone: 815-333-4014
Email: jseminar@cnmc.org
Web: rarediseasesnetwork.epi.usf.edu

Other

Genetics clinics, staffed by genetics professionals, provide information for individuals and families regarding the natural history, treatment, mode of inheritance, and genetic risks to other family members as well as information about available consumer-oriented resources. See the GeneTests Clinic Directory.

See Consumer Resources for disease-specific and/or umbrella support organizations for this disorder. These organizations have been established for individuals and families to provide information, support, and contact with other affected individuals.

Mode of Inheritance

Argininosuccinate lyase (ASL) deficiency is inherited in an autosomal recessive manner.

Risk to Family Members

Parents of a proband

• The parents of an affected individual are obligate heterozygotes (i.e., carriers of one mutant allele).

• Heterozygotes (carriers) are asymptomatic.

Sibs of a proband

• At conception, each sib of an affected individual has a 25% chance of being affected, a 50% chance of being an asymptomatic carrier, and a 25% chance of being unaffected and not a carrier.

• Once an at-risk sib is known to be unaffected, the risk of his/her being a carrier is 2/3.

• Heterozygotes (carriers) are asymptomatic.

• Though the phenotypic manifestations can be variable, in families with one child with the severe neonatal onset form subsequent children are likely to have the severe neonatal onset form. In contrast, the phenotype of late-onset forms associated with partial ASL enzyme activity is variable.

Offspring of a proband

• The offspring of an individual with ASL deficiency are obligate heterozygotes (carriers) for an ASL disease-causing mutation.

• Unless an individual with ASL deficiency has children with an affected individual or a carrier, his/her offspring will be obligate heterozygotes (carriers) for a disease-causing ASL mutation.

Other family members. Each sib of the proband’s parents is at a 50% risk of being a carrier.

Carrier Detection

Carrier testing for at-risk family members is possible if the disease-causing mutations in the family have been identified.

Carrier testing is not available by biochemical methods.

Related Genetic Counseling Issues

See Management, Evaluation of Relatives at Risk for information on evaluating at-risk relatives for the purpose of early diagnosis and treatment.

Family planning

• The optimal time for determination of genetic risk, clarification of carrier status, and discussion of the availability of prenatal testing is before pregnancy.

• It is appropriate to offer genetic counseling (including discussion of potential risks to offspring and reproductive options) to young adults who are affected or at risk of being affected, or are carriers or at risk of being carriers.

DNA banking is the storage of DNA (typically extracted from white blood cells) for possible future use. Because it is likely that testing methodology and our understanding of genes, mutations, and diseases will improve in the future, consideration should be given to banking DNA of affected individuals. See for a list of laboratories offering DNA banking.

Prenatal Testing

Molecular genetic testing. Prenatal diagnosis for pregnancies at increased risk is possible by analysis of DNA extracted from fetal cells obtained by amniocentesis usually performed at about 15 to 18 weeks’ gestation or chorionic villus sampling (CVS) at approximately ten to 12 weeks’ gestation. The disease-causing mutations in the family must have been identified before prenatal molecular genetic testing can be performed.

Note: Gestational age is expressed as menstrual weeks calculated either from the first day of the last normal menstrual period or by ultrasound measurements.

Biochemical testing. The concentration of argininosuccinate in the amniotic fluid can be measured between 15 and 18 weeks of gestation. Because of limited data, the sensitivity of the test is not known. However, because argininosuccinate is not detectable in amniotic fluid under normal conditions and because ASL deficiency is the only disorder that causes elevation of argininosuccinate, the finding of increased concentrations of argininosuccinate in the amniotic fluid is diagnostic of ASL deficiency. In the authors’ experience there is complete concordance between the presence of argininosuccinic acid in the amniotic fluid and decreased ASL enzyme activity on cultured amniocytes [Author, personal observation].

ASL enzyme activity can be measured in uncultured fetal tissue obtained by chorionic villus sampling (CVS) at approximately ten to 12 weeks' gestation or cultured amniocytes obtained by amniocentesis usually performed at approximately 15 to 18 weeks' gestation. Note: Inadequate data preclude calculation of the sensitivity of this test method; however, when the molecular defect in a family is unknown, assay of ASL enzyme activity can be used for prenatal diagnosis.

Preimplantation genetic diagnosis (PGD) may be available for families in which the disease-causing mutations have been identified. For laboratories offering PGD, see .

Note: It is the policy of GeneReviews to include in GeneReviews™ chapters any clinical uses of testing available from laboratories listed in the GeneTests™ Laboratory Directory; inclusion does not necessarily reflect the endorsement of such uses by the author(s), editor(s), or reviewer(s).

Molecular Genetic Pathogenesis

It is unlikely that elevated plasma ammonia is the only toxic compound in ASL deficiency because neurocognitive delays, liver fibrosis, and hypertension have been described even in affected individuals with no documented episodes of hyperammonemia [Ficicioglu et al 2009, Mercimek-Mahmutoglu et al 2010]. In addition, these clinical features are unique to ASL deficiency and are not seen in other urea cycle disorders, supporting the hypothesis that the phenotype in ASL deficiency is likely attributable to a combination of the increase in argininosuccinic acid together with the additional roles of the enzyme ASL in generating endogenous arginine in various tissues outside the liver.

Arginine is a semi-essential amino acid. The sources of arginine are exogenous from the diet and endogenous from the breakdown of proteins and synthesis from citrulline [Wu & Morris 1998]. In healthy adults the level of endogenous synthesis generates sufficient arginine so that under usual circumstances it is not necessary to obtain it through exogenous sources. However, in situations such as catabolic stress or dysfunction of the kidneys or small intestine, endogenous arginine production is not commensurate with metabolic requirements and arginine becomes an essential amino acid (i.e., it must be provided exogenously).

The liver, the major site of arginine metabolism, rapidly converts arginine generated in the urea cycle to urea and ornithine. Thus, under normal conditions the liver does not contribute to the circulating pool of arginine.

Approximately 60% of net synthesis of arginine in adult mammals occurs in the kidney, where citrulline is extracted from the blood and converted to arginine by the action of the enzymes argininosuccinate synthetase (ASS) and ASL, which are localized within the proximal tubules [Windmueller & Spaeth 1981]. However, many other tissues and cell types also contain both these enzymes for generating arginine from citrulline [Mori & Gotoh 2004]. In ASL deficiency, arginine becomes an essential amino acid because all cells and tissues are deficient in the enzyme ASL.

Arginine serves as the precursor for the synthesis of urea, nitric oxide, polyamines, proline, glutamate, creatine, and agmatine (Figure 1). Thus, in contrast to the one enzyme (ASL) that produces arginine; four enzymes use arginine as substrate: arginine decarboxylase ADC, arginase, nitric oxide synthase (NOS), and glycine amidinotransferase. Nitric oxide is the most studied of the arginine metabolites. With deficiency of the ASL enzyme and the resulting deficiency in the amino acid arginine, one could hypothesize that there would also be deficiency of nitric oxide and other metabolites for which it is a precursor.

Figure

Figure 1. Metabolic fates of arginine

Arginine is derived from dietary sources, protein catabolism, or endogenous synthesis. The arginine-citrulline cycle is responsible for regeneration of arginine in various tissues. Arginine serves (more...)

In support of this hypothesis, a hypomorphic mouse model of Asl deficiency was recently reported to demonstrate a multi-organ dysfunction including hypertension consistent with systemic NO deficiency [Erez et al 2011]. The Asl-deficient mice have decreased NO production as evidenced by a significant decrease in S-nitrosylation and/or nitrite in heart and other tissues. Moreover, dynamic measurements of metabolite fluxes using stable isotopes in individuals with ASL deficiency reveal a decreased NO flux even with adequate arginine supplementation [Erez A et al 2011]. These results were further explained by the finding that there is a structural requirement of ASL for channeling of arginine into a protein complex necessary for NO production, in addition to its catalytic requirement for the synthesis of endogenous arginine [Erez et al 2011].

It is important to note that the depletion of arginine as substrate for nitric oxide synthesis has the effect of increasing free radical production because of the uncoupling of NOS [Pignitter et al 2006]. Increase in free radical production results in tissue damage, with the brain being sensitive to both direct damage as well as an indirect damage caused by increases in intracellular free Ca²⁺ and, possibly, release of excitatory amino acids. Free radicals could also interact with argininosuccinic acid to form guanidinosuccinic acid (GSA), a known cellular and neuronal toxin [D'Hooge et al 1992, Aoyagi et al 2001, Aoyagi 2003].

Normal allelic variants. Human ASL consists of 16 exons. The number of exons is sometimes given as 17 with the first encoding only for the 5’ UTR [Trevisson et al 2007]. The presence of another partial sequence on chromosome 22 was assumed to be a pseudogene but later found to code for Ig-λ like mRNA [Linnebank et al 2002, O'Brien et al 1986]. Recently, a pseudogene Ψ ASL2 was located on chromosome 7, approximately 3 Mb upstream of ASL. The pseudogene includes intron 2, exon 3, and part of intron 3 of ASL [Trevisson et al 2007].

Pathologic allelic variants. More than 90 pathologic allelic variants have been described. The enzymatic activity of ASL requires assembly of four ASL monomers to form a homotetramer. Hence, the phenotypic consequences of a specific mutation are dependent on the mutation on the other allele and their ability to complement one another. The pathogenic mutations include nonsense and missense mutations, insertions, deletions, and those affecting mRNA splicing. Mutations are scattered throughout the gene; however, exons 4, 5, and 7 appear to be mutational hotspots [Linnebank et al 2002, Trevisson et al 2007].

• The ASL c.1153C>T variant, the founder mutation in the Finnish population, is associated with residual ASL enzyme activity as measured by the incorporation of [¹⁴C]citrulline into proteins [Kleijer et al 2002].

• Two founder mutations 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 mutations in this population [Al-Sayed et al 2005].

  • c.346C>T is common in this population; the proportion of individuals with this mutation is not known.

Normal gene product. ASL cDNA encodes a deduced protein of 464 amino acids with a predicted molecular mass of 52 kd. Argininosuccinate lyase is a cytosolic homotetramer of 208 kd. It catalyzes the cleavage of argininosuccinic acid to arginine and fumarate, an essential step in urea production. ASL is the only enzyme in the body that is capable of generating arginine.

Abnormal gene product. The argininosuccinate lyase enzyme is inactive or absent as a result of mutations preventing the formation of the tetramer itself or of its active sites. Residual enzyme activity varies with intragenic complementation.

Recently, yeast-based functional complementation assays were used to assess the pathogenicity of ASL mutant alleles observed in affected individuals [Trevisson et al 2009]. This method can detect low levels of residual ASL enzyme activity and assess the effect of allelic complementation. This model demonstrated that mutant ASL alleles typically found in affected individuals with late onset ASL deficiency had either high residual ASL enzyme activity or two mutant alleles that exhibited complementation [Yu & Howell 2000, Trevisson et al 2009]. Although this assay does not assess the effects of genetic background on ASL enzyme activity, it provides insight into how different allelic combinations may affect the clinical phenotype.

The recent findings of a structural requirement for ASL in addition to its catalytic function could in future allow for further correlation of ASL mutations and their phenotypic consequences — potentially with important therapeutic implications.

Published Guidelines/Consensus Statements

1. Urea Cycle Disorders Conference Group. Consensus statement from a conference for the management of patients with urea cycle disorders. (pdf) Available online. 2001. Accessed 1-31-2012.

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Acknowledgments

SCSN is supported by the National Urea Cycle Disorders Foundation and the Linked Clinical Research Center of the Osteogenesis Imperfecta Foundation.

AE is supported by the NIH (DK081735). AE was an awardee of the National Urea Cycle Disorders Foundation fellowship.

BL is supported by the NIH (GM090310 and HD061221).

Revision History

• 2 February 2012 (ae) Revision: author edits to Molecular Genetic Pathogenesis and Abnormal gene product

• 3 February 2011 (me) Review posted live

• 31 August 2010 (ae) Original submission