Next-generation sequencing detection of a novel mutation (D145G) in argininosuccinic aciduria

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Next-generation sequencing detection of a novel mutation (D145G) in argininosuccinic aciduria and evidence for variable splicing of the human argininosuccinate lyase gene


Argininosuccinic aciduria (ASA; OMIM 207900) is a rare autosomal recessive heterogeneous urea cycle disorder (UCD), which leads to the accumulation of argininosuccinic acid in the blood and urine. We report a heterozygous novel mutation in exon 6 of argininosuccinate lyase (ASL) by next generation sequencing, resulting in the formation of an alternative transcript with the deletion of exon 6 using RT-PCR-detected exon-trapping. Another identified heterozygous mutation (R456W) was reported in an Italian patient previously. This is the first report of a novel mutation with aberrant splicing and the formation of an alternative transcript causing ASA. This study also demonstrates the value of next-generation sequencing in the identification of novel mutations and molecular diagnosis in future of these families.


Argininosuccinic aciduria (ASA; OMIM 207900) is an inborn error of the urea cycle caused by mutations in the argininosuccinate lyase (ASL) gene that cleaves argininosuccinate to fumarate and arginine. The prevalence of ASA is 1 in 70,000 live births and is the second most common urea cycle disorder (UCD) (1).

The UCD results from defects in the metabolism of waste nitrogen from the breakdown of protein and other nitrogen-containing molecules. UCD patient accumulates the nitrogen in the form of ammonia, which is a highly toxic substance and is not removed from the body. Six urea cycle defects have been identified. The inherited deficiencies of five catalytic enzymes (CPS1, OTC, ASS1, ASL, ARG) and a cofactor-producing enzyme (NAGS) result in UCD. The deficiencies of all enzymes are inherited in an autosomal recessive manner except for OTC that is X-linked (2).

The clinical presentation of patients with ASA is variable. Generally, the disease has two forms, a severe neonatal form and a late onset form. The severe neonatal form is characterized by hyperammonemia within the first few days of life with poor feeding, vomiting, lethargy, and seizures, with subsequent progression to coma. The late onset form manifests late in infancy or in childhood, it presents less severe symptoms with mental retardation, intermittent ataxia, episodic hyperammonaemia and longer survival (3).

The clinical diagnosis is confirmed by measuring ammonia and ASA levels in plasma (4). The human ASL gene spans approximately 17kb and comprises 17 exons (the coding region starts at exon 2), and is located on chromosome 7cen-q11.2 (5). Previous studies have revealed splice alterative transcripts of ASL appearing in all investigated cells and tissues mainly involves deletions of exon 2 and 7 (6-8).

Until now, the number of reported mutations is still quite small. Our study aims to identify the mutation in ASL gene by next generation sequencing on a Chinese pedigree with ASA and analyze the underlying molecular defects. A custom-made capture array (NimbleGen, Roche, USA) was designed to capture six USD genes (CPS1, OTC, ASS1, ASL, ARG, NAGS), SLC25A13 encoding a protein called citrin and SLC25A15 encoding mitochondrial ornithine transporter. Both citrin and mitochondrial ornithine transporter play important role in the urea cycle.

Materials and Methods


The patient is a 4-year-old Chinese boy, and at 6 days of age he was brought to the hospital with jaundice, poor feeding, vomiting and without crying. At 7 days of age, he was admitted to the hospital with neonatal sepsis, neonatal conjunctivitis and neonatal jaundice. Then, the patient presented lethargy, poor response, eyeballs with hyperemia and had a large number of purulent secretions. Then the patient was treated with fasting, fluid infusion and intravenous antibiotics for anti-infection. After one day (8 days of age) to the hospital, conditions became worse with hypotonia, mild lethargy and poor activation. Biochemical analysis revealed hyperammonaemia (456μmol/L, normal range 18-72μmol/L), after one day the serum ammonia was still high (568μmol/L). He was treated with amikacin and arginine, after 3 days, the serum ammonia decreased to 51μmol/L, on the 9 days after admitted to the hospital, the level of serum ammonia was 21μmol/L and then he was brought out of the hospital.

After leaving the hospital, he was followed regularly with low-protein diet, oral arginine tablets, sodium benzoate band daily intake of protein is no more than 1.5g/Kg per day, the serum ammonia was controlled no more than 80μmol/L. Currently, the patient demonstrates mild motor retardation and normal physical development.

He has an elder sister two years older than he who presents a normal condition. The pedigree is shown in Figure 1, which demonstrating a pattern suggestive of autosomal recessive inheritance.

Sample collection and DNA extraction

Following informed consent, the patient and his parents, elder sister’s peripheral blood (3ml) were collected and anticoagulated with EDTA. Total DNA was extracted using the QIAamp DNA extraction kit (Qiagen, Hilden, Germany) as the instruction described.

Targeted next generation sequencing

DNA samples were sequenced using Microarray-based next generation sequencing. We designed a custom array from Roche NimbleGen (Madison, USA) to capture exons (including the 10bp flanking either side of each) of 2181 genes known to be associated with 561 common genetic diseases. Genomic DNA was fragmented ranging from 200-300bp. Primers and adapters were then ligated to the purified DNA fragments to construct the library. Library was amplified by PCR and hybridized to the capture array. Samples were then sequenced on Illumina HiSeq2000 Analyzers (Illumina, San Diego, USA) for 90 cycles to generate paired-end reads. Image analysis and base calling were performed using the Illumina Pipeline.

Direct Sequencing for ASL

Exon 6 and exon 17 of the ASL gene were amplified using 200ng of genomic DNA, 1μM each of primers (ASL-exon6-F5’- GGCTCCTCAGGGAAGCAACA-3’,ASL-exon6-R5’-AGTTCTGGGATGCCCCTGTC-3’,ASL-exon17-F5’-AAGTGAGCCTGGGTGCCTGG-3’,ASL-exon16-R5’-CGAAAGCCCAGCAACGAGG-3’), 0.25mM of dNTP, and 1U Taq polymerase in 1×buffer with anneal temperature at 64 and 67 degree separately.PCR products were purified using QIA PCR purification kit (Qiagen, Crawley, UK).

RT-PCR based exon trapping analysis of the novel mutation

To study the function of the novel mutation in exon 6, we performed exon trapping studies in vitro (9). We amplified a genomic fragment contains exon 6, intron6 and exon 7 with primer F5’-CCGTGTTGTCCCAACCTTGA-3’ and R5’-GGGCTGTGCTAGAGGGGA-3’ in the patient and in a normal individual respectively. The products were cloned into the pSPL3 exon trapping vector (Invitrogen, Carlsbad, CA). Wild type (WT) and mutant plasmids were then transfected into the COS7 cell line using lipo2000 (Invitrogen, Carlsbad, CA) respectively. After culture for 48 hours, total RNA was extracted from cell line using Trizol (TaKaRa, Dalian, China). 5μg RNA was reverse transcribed to cDNA in a total volume of 20μl with superscript II RNAse H-reverse transcriptase and oligo-dT priming (TaKaRa, Dalian, China). cDNA was amplified with vector primers SD6

(5’-TCTGAGTCACCTGGACAACC-3’) and SA2 (5’-ATCTCAGTGGTATTTGTGAGC-3’), PCR products were separated on a 2% TBE agarose gel. After purification, amplification products were characterized by direct sequencing.


Two mutations identified in the patient

Next-generation sequencing totally identified ten variants located in four different genes in the patient (Table S1), in those there are two heterozygous mutations, one is a novel mutation: c.434A>G located in exon6, another mutation c.1366C>T has been reported previously (3), located in exon 17. The patient’s father was heterozygous for c.434A>G and his mother was heterozygous for c.1366C>T. His elder sister was heterozygous for c.434A>G. We validated the results of next-generation sequencing by direct sequencing (Fig.2a-b).

We also found the novel mutation c.434A>G in the patient results in the original dinucleotide from AT to GT, same as the splice donor site. This mutation position is also located in the end of exon 6, 12bp upstream to the true splice donor site (Fig.2a). In general, splice donor site mutations are expected to disturb normal splicing (10).

Novel mutation c.434A>G abolishes normal splicing and causes aberrant splicing

We performed an in vitro exon-trapping assay to analyze the effects of c.434A>G at the transcript level. cDNA products gel electrophoresis are shown in Figure.3a. The band of lane2 is mutated cDNA product named ASL-M with length at 350bp approximately. Two bands of lane 3 are products of WT cDNA named ASL-N-1 and ASL-N-2 separately. After direct sequencing, the sequence of ASL-M is absolutely same as ASL-N-2 with aberrant splicing of exon6 (Fig.3b). The sequence result of ASL-N-1 showing normal sequence without splicing (Fig.3c). We drew a schematic diagram to describe the novel mutation c.434A>G led to the skipping of exon 6. Functional analysis of this mutation demonstrates that the substitution of nucleotide (c.434A>G) leads to the complete loss of exon 6.


In conclusion, we identified two compound heterozygous mutations in ASL using next generation sequencing, confirming the clinical diagnosis of ASA. In vitro exon-trapping study demonstrates the novel mutation c.434A>G completely abolishes normal splicing of exon 6. The biological impact of shortened transcript remains unknown. One thing is certain if shortened transcripts are translated, the corresponding polypeptides might be misfolded.

Previous report has described another mutation c.1366C>T (R456W) involves a conserved arginine in the terminal alpha helix of the protein. Substitution with tryptophan is predicted to cause a displacement and to shift the position of glutamine454 (3). Even though we cannot be certain that our model exactly suitable in vivo, our data indicate that the two mutations in two strands could not translate normal protein, which has a severe effect on the function of ASL.

We should attach molecular analysis importance to the diagnosis of ASA. Although determination of ASL activity in cultured fibroblasts or erythrocytes is a reliable method to confirm the diagnosis, it is complex and not widely available in most of the labs worldwide. However, molecular analysis is more feasible and efficient. Therefore, we recommend next generation sequencing technologies to diagnosis ASA and also amplify the number of reported mutations involved.

Overall, this is the first report of a pathogenic mutation with aberrant splicing and the formation of an alternative transcript with exon 6 trapping causing ASA. This study also demonstrates the value of next-generation sequencing in the identification of novel mutations and molecular diagnosis in future of these families.

Competing interests

The authors declare that they have no competing interests.


The authors thank the patient and her family members who participated in this study.


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