Urea Cycle Disorders Panel

The main function of the urea cycle is the management and elimination of waste nitrogen, which arises from the catabolism of protein and other nitrogen-containing compounds (Ah Mew et al. 2015; Kölker et al. 2016). Defects in any of the genes encoding the protein components of the urea cycle lead to urea cycle disorders (UCDs). These disorders are characterized by a classic triad of hyperammonemia, encephalopathy and respiratory alkalosis (Shchelochkov et al. 2015). Biochemically, in addition to hyperammonemia, UCD patients are found to have altered levels of blood glutamine, glutamate, alanine, citrulline, arginine, ornithine, and/or argininesuccinic acid, as well as altered urinary orotic acid, citrulline, arginine, argininosuccinic acid, lysine, and/or homocitrulline. The specific metabolite profile varies with each type of UCD (Mönch 2015; Ah Mew et al. 2015).

The age of onset of a UCD is dependent upon the amount of residual activity of the affected protein and the severity and duration of hyperammonemia episodes. Severe protein defects are associated with neonatal onset UCDs, whereas defects that result in some residual protein activity are typically associated with later-onset, more mild forms of UCD that may first become evident at any age (Batshaw et al. 2014; Ah Mew et al. 2015; Waisbren et al. 2016).

Patients with severe neonatal-onset UCDs tend to appear clinically normal for the first ~24 hours of life, after which they begin to become symptomatic, typically presenting initially with feeding refusal, vomiting and lethargy. Without quick recognition of the hyperammonemic state and subsequent treatment, the patient’s symptoms may rapidly progress to multi-organ failure, worsening lethargy, seizures, coma, and possibly death (Mönch 2015; Ah Mew et al. 2015; Kölker et al. 2016).

In patients that present after the neonatal period, clinical symptoms are variable and tend to be episodic. Such episodes can be triggered by a wide variety of changes, such as excessive protein intake, illness, stress, surgery, prolonged fasting, and childbirth (Mönch 2015; Ah Mew et al. 2015). These patients typically present with loss of appetite, vomiting, lethargy, and behavioral abnormalities such as irritability, disorientation, agitation, inattention, sleep disorders, hallucinations and psychosis (Mönch 2015; Ah Mew et al. 2015; Shchelochkov et al 2015; Kölkher et al 2016).Some may also present with delayed motor and intellectual development as well as muscular hypotonia (Mönch 2015). Later-onset patients have higher levels of residual protein activity than those with severe UCDs (Mönch 2015).

The clinical symptoms observed in UCD patients are primarily linked directly to the hyperammonemia that develops as a result of the protein deficiency (Mönch 2015; Ah Mew et al. 2015). Long-term studies have shown that nearly all surviving patients have some degree of developmental disability, which has been correlated with the number, severity and duration of hyperammonemic episodes (Waisbren et al. 2016). Treatment of acute symptoms in UCD patients is aimed primarily at normalizing plasma ammonia concentrations (Ah Mew et al. 2015). This is typically accomplished via dialysis, reducing overall dietary protein intake, providing supplemental arginine and/or cirulline, and pharmacological treatment with nitrogen scavengers such as sodium benzoate and sodium phenylbutyrate (Mönch 2015; Ah Mew et al. 2015; Shchelochkov et al. 2015). Dietary control and liver transplantation are long-term treatment options (Mönch 2015; Ah Mew et al. 2015; Shchelochkov et al. 2015).

Urea cycle disorders (UCDs) are caused by defects in any of six enzymes that participate directly in the urea cycle, which are encoded by the ASL, ASS1, ARG1, CPS1, OTC and NAGS genes. In addition, UCDs can also be caused by defects one of two transporter proteins that transport metabolites necessary for the urea cycle into or out of the mitochondria. These transporter proteins are encoded by the SLC25A13 and SLC25A15 genes. Lastly, the OAT gene encodes the enzyme ornithine aminotransferase. Although OAT deficiency is not classified as a UCD, we have included OAT in this gene panel because OAT deficiency may lead to a biochemical profile similar to a UCD (Mitchell et al. 1988; Peltola et al. 2002). Looking only at UCDs due to defects in the ASL, ASS1, ARG1, CPS1, OTC and NAGS genes, the overall prevalence of urea cycle disorders has been estimated to be between 1:8,000 to 1:45,000 in the United States. It is thought that the true prevalence is likely higher due to the difficulty in diagnosing individuals with partial UCDs (Batshaw et al. 2014; Mönch 2015; Ah Mew et al. 2015; Waisbren et al. 2016).

With the exception of OTC, all the genes in this panel are associated with autosomal recessive UCDs. OTC is located on the X chromosome and is inherited in an X-linked recessive manner. OTC deficiency is generally associated with a severe phenotype in males, although heterozygous female carriers may also present with hyperammonemic episodes requiring treatment, as well as cognitive impairment (Lichter-Knoecki et al. 2016).

Massively parallel sequencing plus Sanger confirmation will detect the vast majority of sequence variants known to cause UCDs. It should be noted, however, that large deletions or duplications which are most likely not detectable via sequencing have been reported in several genes in this panel.

Please see individual test descriptions for additional information on the molecular biology of each gene.

Among patients with clinical and biochemical features consistent with a urea cycle disorder (UCD), clinical sensitivity of this gene sequencing test is expected to be quite high. Martín-Hernández et al. (2014) reported an observational study of 104 patients diagnosed with urea cycle disorders. Among their patient cohort, 67 were diagnosed with OTC deficiency, 22 with ASS1 deficiency, 10 with ASL deficiency, 2 with ARG1 deficiency, 2 with CPS1 deficiency, and 1 with NAGS deficiency. The SLC2513, SLC25A15 and OAT genes were not included in their analysis.

For patients diagnosed enzymatically with each of the individual UCDs, clinical sensitivity of direct sequencing is expected to be high. For the ASL, ASS1, ARG1, OAT, NAGS, SLC25A13 and SLC25A15 genes, reported pathogenic allele detection ranges from ~95-100% (Uchino et al. 1995; Cardoso et al. 1999; Salvi et al. 2001; Linnebank et al. 2002; Gao et al. 2003; Häberle et al. 2003; Dimmock et al. 2009; Camacho and Rioseco-Camacho 2012; Carvalho et al. 2012; Song et al. 2013; Kobayashi et al. 2014; Sancho-Vaello et al. 2016). Sensitivity for the CPS1 and OTC genes is expected to be between ~80-90% as large copy number variants are more common in these two genes (Yamaguchi et al. 2006; Kurokawa et al. 2007; Funghini et al. 2012; Choi et al. 2015; Human Gene Mutation Database).

Overall, large (exonic level, usually multi-exon) deletions and duplications have been documented in the ASL, ARG1, OAT, NAGS and SLC25A15 genes, but appear to be relatively uncommon (Human Gene Mutation Database).

Large deletions or duplications have been reported as a somewhat more common cause of disease in the ASS1, CPS1, OTC and SLC25A13 genes. Thus far, 4 gross deletions have been reported in the ASS1 gene, 9 gross deletions in the CPS1 gene, and 2 gross deletions and 4 gross insertions in the SLC25A13 gene (Human Gene Mutation Database).

Lichter-Knoecki et al. (2016) report that deletions and duplications account for ~5-10% of causative alleles in the OTC gene, with nearly 50 large copy number variants reported in the literature (Human Gene Mutation Database).

This test is performed using Next-Gen sequencing with additional Sanger sequencing as necessary.

This panel provides 100% coverage of all coding exons of the genes plus 10 bases of flanking noncoding DNA in all available transcripts along with other non-coding regions in which pathogenic variants have been identified at PreventionGenetics or reported elsewhere. We define coverage as ≥20X NGS reads or Sanger sequencing. PGnome panels typically provide slightly increased coverage over the PGxome equivalent. PGnome sequencing panels have the added benefit of additional analysis and reporting of deep intronic regions (where applicable).

In addition to the regions described above, this testing includes coverage of the following variants that reside in untranslated or deep intronic regions: ARG1 c.306-611T>C, ASS1 c.175-1119G>A, CPS1 c.4102-239A>G, NAGS c.-3064C>A, OAT c.190+313C>G and the OTC variants c.-366A>G, c.540+265G>A, c.867+1126A>G and c.1005+1091C>G.

Dependent on the sequencing backbone selected for this testing, discounted reflex testing to any other similar backbone-based test is available (i.e., PGxome panel to whole PGxome; PGnome panel to whole PGnome).