Citrullinemia, Type I via the ASS1 Gene

  • Summary and Pricing
  • Clinical Features and Genetics
  • Citations
  • Methods
  • Ordering/Specimens
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Test Code Test Copy GenesIndividual Gene PriceCPT Code Copy CPT Codes
553 ASS1$840.00 81406 Add to Order
Targeted Testing

For ordering targeted known variants, please proceed to our Targeted Variants landing page.

Turnaround Time

The great majority of tests are completed within 18 days.

Clinical Sensitivity

Test sensitivity should be very high in the newborn with clinical symptoms of ammonium intoxication and a plasma amino acid profile suggestive of argininosuccinate synthetase deficiency. Gao et al. (2003) reported 2 alleles in 100% of the 38 patients studied, and Häberle et al. (2003) reported 2 alleles in 100% of the 21 patients (from 17 families) they studied. Quinonez and Thoene (2016) report a clinical sensitivity of ~96%.

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Deletion/Duplication Testing via aCGH

Test Code Test Copy GenesIndividual Gene PriceCPT Code Copy CPT Codes
600 ASS1$990.00 81479 Add to Order
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Turnaround Time

The great majority of tests are completed within 20 days.

Clinical Sensitivity

Although the clinical sensitivity of this array test is expected to be relatively low, at least four gross deletions have been reported in the ASS1 gene (Human Gene Mutation Database).

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Clinical Features

Urea cycle defects are characterized by hyperammonemia, encephalopathy and respiratory alkalosis (Brusilow and Horwich 2014). Eight clinical disorders have been described involving defective urea cycle enzymes or transporter proteins: N-acetylglutamate synthase deficiency, carbamoyl phosphate synthetase I deficiency, ornithine transcarbamolase deficiency, argininosuccinate synthetase deficiency (also known as citrullinemia type I), argininosuccinate lyase deficiency, arginase deficiency, citrin deficiency (also known as citrullinemia type II) and hyperornithinemia-hyperammonemia-homocitrullinemia (HHH) syndrome (Ah Mew et al. 2015).

Type I citrullinemia presents in the neonate following protein intake with massively elevated serum citrulline levels resulting in hyperammonemia. Patients exhibit lethargy, refusal to feed, vomiting, and tachypnea or stroke (Häberle et al. 2003; Ah Mew et al. 2015; Quinonez and Thoene 2016). Untreated, argininosuccinate synthetase deficiency can lead to increased intracranial pressure, increased neuromuscular tone, seizures, loss of consciousness, and death (Quinonez and Thoene 2016). A milder adult-onset form of the disease has also been described (Häberle et al. 2003; Martín-Hernández et al. 2014). Clinical signs of the milder form include recurrent lethargy, somnolence, mental retardation, and chronic or recurrent hyperammonemia. Treatment includes a protein restricted diet and sodium benzoate to scavenge ammonia (Quinonez and Thoene 2016).


Type I or classic citrullinemia is an autosomal recessive disorder. Pathogenic variants in the ASS1 gene cause both the severe neonatal and milder adult onset forms of citrullinemia type I (Häberle et al. 2003; Martín-Hernández et al. 2014). The ASS1 gene encodes argininosuccinate synthetase, a urea cycle enzyme that converts citrulline and aspartate to argininosuccinate. Missense variants are the predominant type of disease-causing variants in ASS1, though nonsense, splicing, small deletions and insertions and gross deletions have been reported as well (Gao et al. 2003; Martín-Hernández et al. 2014; Human Gene Mutation Database). Pathogenic variants are spread throughout the gene and most reported variants appear to be private. However, a few variants have been more commonly reported, such as c.421-2A>G (common in Japanese and Korean patients), c.535T>C (p.Trp179Arg), c.910C>T (p.Arg304Trp), c.970G>A (p.Gly324Ser) and c.1168G>A (p.Gly390Arg) (Engel et al. 2009; Woo et al. 2014).

Type II Citrullinemia, a multisystem disorder, is caused by pathogenic variants in the SLC25A13 gene and has clinically and biochemically distinct features (Kobayashi et al. 2014). Testing for both types of citrullinemia is available individually as well as via our Urea Cycle Disorders NextGen Sequencing (NGS) panel.

Testing Strategy

This test involves bidirectional Sanger sequencing using genomic DNA of all coding exons of the ASS1 gene plus ~10 bp of flanking non-coding DNA on each side. This test also includes coverage for the intronic variant designated as c.174-1119G>A. We will also sequence any single exon (Test #100) or pair of exons (Test #200) in family members of patients with known mutations or to confirm research results. 

Indications for Test

A plasma ammonia concentration of 150 μmol/L or higher, associated with a normal anion gap and a normal serum glucose concentration is a strong indication for the presence of a urea cycle defect (Ah Mew et al. 2015). Plasma citrulline levels can differentiated between defects in proximal urea cycle enzymes (low citrulline; OTC and carbamoyl phosphate synthetase) from distal enzymes (high citrulline; argininosuccinate synthetase, argininosuccinate lyase, and arginase). Elevated plasma citrulline and absent argininosuccinate is indicative of absent or reduced argininosuccinate synthetase activity. In the mild form, plasma citrulline levels may be near the upper limit of normal. Family members of patients known to have ASS1 variants are also good candidates for this test, and we will sequence the ASS1 gene to determine carrier status.


Official Gene Symbol OMIM ID
ASS1 603470
Inheritance Abbreviation
Autosomal Dominant AD
Autosomal Recessive AR
X-Linked XL
Mitochondrial MT


Name Inheritance OMIM ID
Citrullinemia Type I AR 215700


Genetic Counselors
  • Ah Mew N. et al. 2015. Urea Cycle Disorders Overview. In: Pagon RA, Adam MP, Ardinger HH, Bird TD, Dolan CR, Fong C-T, Smith RJ, and Stephens K, editors. GeneReviews(®), Seattle (WA): University of Washington, Seattle. PubMed ID: 20301396
  • Brusilow S.W. and Horwich A.L. 2014. Urea Cycle Enzymes. Online Metabolic & Molecular Bases of Inherited Disease, New York, NY: McGraw-Hill.
  • Engel K. et al. 2009. Human Mutation. 30: 300-7. PubMed ID: 19006241
  • Gao H.Z. et al. 2003. Human Mutation. 22: 24-34. PubMed ID: 12815590
  • Häberle J. et al. 2003. Molecular Genetics and Metabolism. 80: 302-6. PubMed ID: 14680976
  • Human Gene Mutation Database (Bio-base).
  • Kobayashi K. et al. 2014. Citrin Deficiency. In: Pagon RA, Adam MP, Ardinger HH, Bird TD, Dolan CR, Fong C-T, Smith RJ, and Stephens K, editors. GeneReviews(®), Seattle (WA): University of Washington, Seattle. PubMed ID: 20301360
  • Martín-Hernández E. et al. 2014. Orphanet Journal of Rare Diseases. 9: 187. PubMed ID: 25433810
  • Quinonez S.C. et al. 2016. Citrullinemia Type I. In: Pagon RA, Adam MP, Ardinger HH, Bird TD, Dolan CR, Fong C-T, Smith RJ, and Stephens K, editors. GeneReviews(®), Seattle (WA): University of Washington, Seattle. PubMed ID: 20301631
  • Woo H.I. et al. 2014. Clinica Chimica Acta; International Journal of Clinical Chemistry. 431: 1-8. PubMed ID: 24508627
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Bi-Directional Sanger Sequencing

Test Procedure

Nomenclature for sequence variants was from the Human Genome Variation Society (  As required, DNA is extracted from the patient specimen.  PCR is used to amplify the indicated exons plus additional flanking non-coding sequence.  After cleaning of the PCR products, cycle sequencing is carried out using the ABI Big Dye Terminator v.3.0 kit.  Products are resolved by electrophoresis on an ABI 3730xl capillary sequencer.  In most cases, sequencing is performed in both forward and reverse directions; in some cases, sequencing is performed twice in either the forward or reverse directions.  In nearly all cases, the full coding region of each exon as well as 10 bases of non-coding DNA flanking the exon are sequenced.

Analytical Validity

As of February 2018, we compared 26.8 Mb of Sanger DNA sequence generated at PreventionGenetics to NextGen sequence generated in other labs. We detected only 4 errors in our Sanger sequences, and these were all due to allele dropout during PCR. For Proficiency Testing, both external and internal, in the 14 years of our lab operation we have Sanger sequenced roughly 14,300 PCR amplicons. Only one error has been identified, and this was an error in analysis of sequence data.

Our Sanger sequencing is capable of detecting virtually all nucleotide substitutions within the PCR amplicons. Similarly, we detect essentially all heterozygous or homozygous deletions within the amplicons. Homozygous deletions which overlap one or more PCR primer annealing sites are detectable as PCR failure. Heterozygous deletions which overlap one or more PCR primer annealing sites are usually not detected (see Analytical Limitations). All heterozygous insertions within the amplicons up to about 100 nucleotides in length appear to be detectable. Larger heterozygous insertions may not be detected. All homozygous insertions within the amplicons up to about 300 nucleotides in length appear to be detectable. Larger homozygous insertions may masquerade as homozygous deletions (PCR failure).

Analytical Limitations

In exons where our sequencing did not reveal any variation between the two alleles, we cannot be certain that we were able to PCR amplify both of the patient’s alleles. Occasionally, a patient may carry an allele which does not amplify, due for example to a deletion or a large insertion. In these cases, the report contains no information about the second allele.

Similarly, our sequencing tests have almost no power to detect duplications, triplications, etc. of the gene sequences.

In most cases, only the indicated exons and roughly 10 bp of flanking non-coding sequence on each side are analyzed. Test reports contain little or no information about other portions of the gene, including many regulatory regions.

In nearly all cases, we are unable to determine the phase of sequence variants. In particular, when we find two likely causative mutations for recessive disorders, we cannot be certain that the mutations are on different alleles.

Our ability to detect minor sequence variants, due for example to somatic mosaicism is limited. Sequence variants that are present in less than 50% of the patient’s nucleated cells may not be detected.

Runs of mononucleotide repeats (eg (A)n or (T)n) with n >8 in the reference sequence are generally not analyzed because of strand slippage during PCR and cycle sequencing.

Unless otherwise indicated, the sequence data that we report are based on DNA isolated from a specific tissue (usually leukocytes). Test reports contain no information about gene sequences in other tissues.

Deletion/Duplication Testing via Array Comparative Genomic Hybridization

Test Procedure

Equal amounts of genomic DNA from the patient and a gender matched reference sample are amplified and labeled with Cy3 and Cy5 dyes, respectively. To prevent any sample cross contamination, a unique sample tracking control is added into each patient sample. Each labeled patient product is then purified, quantified, and combined with the same amount of reference product. The combined sample is loaded onto the designed array and hybridized for at least 22-42 hours at 65°C. Arrays are then washed and scanned immediately with 2.5 µM resolution. Only data for the gene(s) of interest for each patient are extracted and analyzed.

Analytical Validity

PreventionGenetics' high density gene-centric custom designed aCGH enables the detection of relatively small deletions and duplications within a single exon of a given gene or deletions and duplications encompassing the entire gene. PreventionGenetics has established and verified this test's accuracy and precision.

Analytical Limitations

Our dense probe coverage may allow detection of deletions/duplications down to 100 bp; however due to limitations and probe spacing this cannot be guaranteed across all exons of all genes. Therefore, some copy number changes smaller than 100-300 bp within a targeted large exon may not be detected by our array.

This array may not detect deletions and duplications present at low levels of mosaicism or those present in genes that have pseudogene copies or repeats elsewhere in the genome.

aCGH will not detect balanced translocations, inversions, or point mutations that may be responsible for the clinical phenotype.

Breakpoints, if occurring outside the targeted gene, may be hard to define.

The sensitivity of this assay may be reduced when DNA is extracted by an outside laboratory.

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Ordering Options

myPrevent - Online Ordering
  • The test can be added to your online orders in the Summary and Pricing section.
  • Once the test has been added log in to myPrevent to fill out an online requisition form.
  • A completed requisition form must accompany all specimens.
  • Billing information along with specimen and shipping instructions are within the requisition form.
  • All testing must be ordered by a qualified healthcare provider.


(Delivery accepted Monday - Saturday)

  • Collect 3 ml -5 ml (5 ml preferred) of whole blood in EDTA (purple top tube) or ACD (yellow top tube). For Test #500-DNA Banking only, collect 10 ml -20 ml of whole blood.
  • For small babies, we require a minimum of 1 ml of blood.
  • Only one blood tube is required for multiple tests.
  • Ship blood tubes at room temperature in an insulated container. Do not freeze blood.
  • During hot weather, include a frozen ice pack in the shipping container. Place a paper towel or other thin material between the ice pack and the blood tube.
  • In cold weather, include an unfrozen ice pack in the shipping container as insulation.
  • At room temperature, blood specimen is stable for up to 48 hours.
  • If refrigerated, blood specimen is stable for up to one week.
  • Label the tube with the patient name, date of birth and/or ID number.


(Delivery accepted Monday - Saturday)

  • Send in screw cap tube at least 5 µg -10 µg of purified DNA at a concentration of at least 20 µg/ml for NGS and Sanger tests and at least 5 µg of purified DNA at a concentration of at least 100 µg/ml for gene-centric aCGH, MLPA, and CMA tests, minimum 2 µg for limited specimens.
  • For requests requiring more than one test, send an additional 5 µg DNA per test ordered when possible.
  • DNA may be shipped at room temperature.
  • Label the tube with the composition of the solute, DNA concentration as well as the patient’s name, date of birth, and/or ID number.
  • We only accept genomic DNA for testing. We do NOT accept products of whole genome amplification reactions or other amplification reactions.


(Delivery preferred Monday - Thursday)

  • PreventionGenetics should be notified in advance of arrival of a cell culture.
  • Culture and send at least two T25 flasks of confluent cells.
  • Some panels may require additional flasks (dependent on size of genes, amount of Sanger sequencing required, etc.). Multiple test requests may also require additional flasks. Please contact us for details.
  • Send specimens in insulated, shatterproof container overnight.
  • Cell cultures may be shipped at room temperature or refrigerated.
  • Label the flasks with the patient name, date of birth, and/or ID number.
  • We strongly recommend maintaining a local back-up culture. We do not culture cells.
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