[Identification of a homozygous ASS1 mutation in a child with citrullinemia type â with high-melting curve method].

OBJECTIVE: To carry out rapid genetic diagnosis for a child affected with citrullinemia type Ⅰ. METHODS: Peripheral venous blood samples were obtained from the two-day-old child and his parents as well as 100 healthy controls. Serum ammonia and citrulline was determined by biochemical test and tandem mass spectrometry. Sixteen pairs of primers were designed for high-resolution melting (HRM) analysis of all exons and adjacent intronic sequences of the ASS1 gene in the proband, parents and healthy controls. Suspected mutations were confirmed by DNA sequencing, while the mRNA transcripts of the ASS1 gene were determined by reverse transcription (RT)-PCR. Functional impact of the mutation sites was predicted with PolyPhen-2 and SIFT Blink software. RESULTS: Blood ammonia and citrulline of the proband have respectively reached 286 µmol/L and 487.69 µmol/L, which far superseded the normal values. HRM analysis and DNA sequencing have identified in the child a homozygous c.380G>A (p.R127Q) mutation in exon 6 of the ASS1 gene, in addition with a homozygous IVS8+60G>A substitution in intron 8, while his parents were heterozygous carriers for both mutations. RT-PCR assay indicated that the IVS8+60G>A mutation did not result in abnormal splicing of the ASS1 gene transcripts. Bioinformatic analysis suggested that the site for p.R127Q was conserved among 45 species of vertebrates and may play a crucial role in citrulline metabolism. CONCLUSION: The severe urea cycle disorder in the proband was probably due to the compound homozygous R127Q and IVS8+60G>A mutations of the ASS1 gene.

Modelling urea-cycle disorder citrullinemia type 1 with disease-specific iPSCs.

Citrullinemia type 1 (CTLN1) is a urea cycle disorder (UCD) caused by mutations of the ASS1 gene, which is responsible for production of the enzyme argininosuccinate synthetase (ASS), and classically presented as life-threatening hyperammonemia in newborns. Therapeutic options are limited, and neurological sequelae may persist. To understand the pathophysiology and find novel treatments, induced pluripotent stem cells (iPSCs) were generated from a CTLN1 patient and differentiated into hepatocyte-like cells (HLCs). CTLN1-HLCs have lower ureagenesis, recapitulating part of the patient's phenotype. l-arginine, an amino acid clinically used for UCD treatment, improved this phenotype in vitro. Metabolome analysis revealed an increase in tricarboxylic acid (TCA) cycle metabolites in CTLN1, suggesting a connection between CTLN1 and the TCA cycle. This CTLN1-iPSC model improves the understanding of CTLN1 pathophysiology and can be used to pursue new therapeutic approaches.

Kinetic mutations in argininosuccinate synthetase deficiency: characterisation and in vitro correction by substrate supplementation.

BACKGROUND: Citrullinemia type 1 is an autosomal-recessive urea cycle disorder caused by mutations in the ASS1 gene and characterised by increased plasma citrulline concentrations. Of the ∼90 argininosuccinate synthetase (ASS) missense mutations reported, 21 map near the substrate (aspartate or citrulline) binding site, and thus are potential kinetic mutations whose decreased activities could be amenable to substrate supplementation. This article aims at characterising these 21 ASS mutations to prove their disease-causing role and to test substrate supplementation as a novel therapeutic approach. METHODS: We used an Escherichia coli expression system to study all potentially kinetic ASS mutations. All mutant enzymes were nickel-affinity purified, their activity and kinetic parameters were measured using tandem mass spectrometry and their thermal stability using differential scanning fluorimetry. Structural rationalisation of the effects of these mutations was performed. RESULTS: Of the characterised mutants, 13 were totally inactive while 8 exhibited decreased affinity for aspartate and citrulline. The activity of these eight kinetic mutations could be rescued to ∼10-99% of the wild-type using high l-aspartate concentrations. CONCLUSIONS: Substrate supplementation raised in vitro the activity of eight citrullinemia type 1 mutations with reduced affinity for aspartate. As a direct translation of these results to the clinics, we propose to further evaluate the use of oxaloacetate, a nitrogen-free aspartate precursor and already available medical food (anti-ageing and brain stimulating, not considered as a drug by the US Food and Drug Administration), in patients with citrullinemia type 1 with decreased aspartate affinity. Although only patients with kinetic mutations would benefit, oxaloacetate could offer a safe novel treatment.

Functional analysis of novel splicing and missense mutations identified in the ASS1 gene in classical citrullinemia patients.

BACKGROUND: Classical citrullinemia (CTLN1) is an inborn error of the urea cycle caused by reduced/abolished activity of argininosuccinate synthetase due to mutations in the ASS1 gene. To determine the pathogenicity of novel variants detected in patients is often a huge challenge in molecular diagnosis. The purpose of our study was to characterize novel ASS1 gene mutations identified in CTLN1 patients. METHODS: Exon trapping assay with pSPL3 was used to confirm splice aberrations while bioinformatics structural analysis predicted the possible effects of missense mutations. RESULTS: Novel donor site (c.174+1G>A) and missense (p.V141G) mutations were detected in a patient exhibiting a biochemical phenotype only. The splice mutation provoked exon skipping hence the truncated product. The mutation p.V141G, is predicted to disturb a hydrophobic pocket in the ATP binding domain in the ASS. Both mutations are predicted to lower binding of ATP. The second patient presented with early onset neonatal citrullinemia marked by an elevated biochemical profile and a clinical phenotype. Analysis revealed a donor site (c.773+1G>A) mutation leading to both exon skipping and intron retention. Subsequent introduction of premature stop codons would result in severely truncated products likely to be degraded. A previously reported R265C is predicted to distort the citrulline binding site. CONCLUSIONS: Three novel mutations are reported in this study. They expand the spectrum of genetic pathology underlying CTLN1. Overall this study provides new insight of CTLN1 and illustrates a comprehensive protocol investigating inborn errors of metabolism at the molecular level.

[Genetic analysis of ASS1, ASL and SLC25A13 in citrullinemia patients].

OBJECTIVE: To detect potential mutations of Y9ASS1, ASL and SLC25A13 genes in four patients manifesting citrullinemia. METHODS: Genomic DNA was extracted from peripheral blood leukocytes. Exons and their flanking sequences of the three genes were amplified with polymerase chain reaction and subjected to direct DNA sequencing. RESULTS: Based on DNA sequence analysis, one case was diagnosed with argininosuccinate synthetase deficiency, and the mutation type (ASS1 gene) was c.236C>T (p.S79F) + c.431C>G (p.P144R). Two cases were diagnosed with argininosuccinic aciduria (ASL gene), and their gene mutations were c.434A>G (p.D145G) + c.1366C>T (p.R456W) and c.331C>T (p.R111W) + IVS8+2insT, respectively. A thirteen months boy who carried a heterozygous 851del4 mutation (SLC25A13 gene) was diagnosed with citrullinemia adult-onset type II. CONCLUSION: Through analysis of relevant pathogenic genes, four patients have been diagnosed.

Citrullinemia type I, classical variant. Identification of ASS-p~G390R (c.1168G>A) mutation in families of a limited geographic area of Argentina: a possible population cluster.

OBJECTIVE: Citrullinemia type I (CTLN1) is an urea cycle defect caused by mutations in the argininosuccinate synthetase gene. We report the first identification in Argentina of patients with CTLN1 in a limited geographic area. DESIGN AND METHODS: Molecular analysis in patient/relatives included PCR, sequencing and restriction enzyme assay. RESULTS: The studied families showed the same mutation: ASS~p.G390R, associated with the early-onset/severe phenotype. CONCLUSION: We postulate a possible population cluster. A program to know the carrier frequency in that population is in progress.

The role of molecular testing and enzyme analysis in the management of hypomorphic citrullinemia.

Expanded newborn screening detects patients with modest elevations in citrulline; however it is currently unclear how to treat these patients and how to counsel their parents. In order to begin to address these issues, we compared the clinical, biochemical, and molecular features of 10 patients with mildly elevated citrulline levels. Three patients presented with clinical illness whereas seven came to attention as a result of expanded newborn screening. One patient presented during pregnancy and responded promptly to IV sodium phenylacetate/sodium benzoate and arginine therapy with no long-term adverse effects on mother or fetus. Two children presented with neurocognitive dysfunction, one of these responded dramatically to dietary protein reduction. ASS enzyme activity was not deficient in all patients with biallelic mutations suggesting this test cannot exclude the ASS1 locus in patients with mildly elevated plasma citrulline. Conversely, all symptomatic patients who were tested had deficient activity. We describe four unreported mutations (p.Y291S, p.R272H, p.F72L, and p.L88I), as well as the common p.W179R mutation. In silico algorithms were inconsistent in predicting the pathogenicity of mutations. The cognitive benefit in one patient of protein restriction and the lack of adverse outcome in seven others restricted from birth, suggest a role for protein restriction and continued monitoring to prevent neurocognitive dysfunction.

Pregnancy in a healthy woman with untreated citrullinemia.

We report the clinical and biochemical data on a second successful pregnancy in a woman with citrullinemia due to argininosuccinate synthetase deficiency (CTLN1). Despite very elevated plasma and urine citrulline and little or no measurable argininosuccinate synthetase enzyme activity on cultured skin fibroblasts, this 29-year-old woman, who was identified through newborn screening, has remained asymptomatic throughout her life. Mutation analysis has recently revealed that she is a compound heterozygote for a known and a novel mutation (IVS15-1G > C and K310Q, respectively). Many newborn screening programs have recently been expanded to include citrullinemia and numerous asymptomatic hypercitrullinemic infants and children have been identified. It is now important to define prognostic indicators that will help with treatment decisions and genetic counseling for these patients. This patient, as the only citrullinemic adult who has been followed prospectively, contributes important information in this regard. In addition, her child was unaffected by the high citrulline levels demonstrated in amniotic fluid and breast milk suggesting that citrulline is not teratogenic. Although pregnancy is an important risk factor for women with CTLN1, it appears that females with citrullinemia can have normal pregnancy outcomes, as long as metabolic crisis is avoided.

Application of mutation analysis for the previously uncertain cases of adult-onset type II citrullinemia (CTLN2) and their clinical profiles.

Type II citrullinemia (CTLN2) is characterized by a deficiency of argininosuccinate synthetase (ASS) in the liver. Mutation analysis of the SLC25A13 gene, which is responsible for CTLN2, provides a rapid and accurate diagnosis. We describe clinical, biochemical and histologic features of two patients, whose diagnosis was finally made by mutation analysis. They initially presented with symptoms related to hyperammonemia at 16 to 22 years of age. A patient had shown mental retardation and growth failure from early childhood. Laboratory findings including amino acids, were characteristic, such as elevated citrulline, arginine, and lysine concentrations, but definitive diagnosis had not been made. The patients died of liver cirrhosis and hepatoma at 31 and 34 years old, respectively. Fatty change in the hepatocytes was commonly observed in the autopsied specimens. ASS activity was decreased in the liver of both patients, and a concomitant decrease of arginase activity was found in one case. Investigation for the SLC25A13 mutation revealed that one patient was homozygous for IVS11 + 1G>A, and the other was compound heterozygote (851del4/S225X). Comparison of genetic, enzymatic and biochemical data among various cases of CTLN2 will be essential to understand the real nature of the disease.

Phenotype and genotype heterogeneity in Mediterranean citrullinemia.

We summarize the diagnosis, outcome, and molecular studies of five Mediterranean patients with citrullinemia: four neonatal classical forms and one subacute form, who also suffers from Down syndrome and presented with severe hepatic encephalopathy at age 7. Mutational analysis revealed three alleles with a common mutation and five new mutations: two Moroccan siblings are homozygous for G390R, the subacute form is compound heterozygous for G390R/G117D (new mutation), and the two other neonatal forms are compound heterozygous for four new mutations: V69A/E270Q and T119I(R108L)/?.

A nonsense mutation is responsible for the RNA-negative phenotype in human citrullinaemia.

Citrullinaemia is an inborn error of metabolism resulting from a deficiency of argininosuccinate synthetase. Previous studies of RNA of argininosuccinate synthetase of citrullinaemia patients using S1 nuclease analysis have identified a class of so-called RNA-negative alleles in which no stable mRNA can be detected. To investigate the nature of mutation responsible for such a phenotype, a compound heterozygous citrullinaemia carrying an RNA-negative allele and an allele with a 3' splice site mutation in intron 6 (IVS6-2A>G) was analysed. Using sequences of a DNA polymorphism and the IVS6-2A>G mutation as markers, approximately equal amounts of pre-mRNAs from allelic genes were detected suggesting that RNA-negative phenotype could not be the result of defect in transcription initiation. A C-to-T transition converting the CGA arginine codon at residue 279 to a TGA termination codon (R279X) was identified by cDNA sequencing. No accumulation of partially spliced pre-mRNAs containing introns immediately upstream and downstream of the nonsense mutation was observed. In addition, no mRNA species of abnormal size was detected when cDNA from the RNA-negative allele was analysed. Hence, there is no indication of nonsense-associated altered splicing (NAS). The most likely event responsible for the RNA-negative phenotype appears to be nonsense-mediated mRNA decay (NMD).

Neonatal presentation of adult-onset type II citrullinemia.

Adult-onset type II citrullinemia (CTLN2) is characterized by a liver-specific argininosuccinate synthetase deficiency caused by a deficiency of the citrin protein encoded by the SLC25A13 gene. Until now, however, no SLC25A13 mutations have been reported in children with liver diseases. We described three infants who presented as neonates with intrahepatic cholestasis associated with hypermethioninemia or hypergalactosemia detected by neonatal mass screening. DNA analyses of SLC25A13 revealed that one patient was a compound heterozygote for the 851de14 and IVS11+IG-->A mutations and two patients (siblings) were homozygotes for the IVS11+lG-->A mutation. These results suggested that there may be a variety of liver diseases related to CTLN2 in children.

Genomic structure of the adult-onset type II citrullinemia gene, SLC25A13, and cloning and expression of its mouse homologue.

Citrullinemia is an autosomal recessive disease characterized by an argininosuccinate synthetase (ASS) deficiency. Adult-onset type II citrullinemia (CTLN2) is a form of the disease that is defined by a quantitative decrease in ASS protein, but with normal kinetic properties. The gene causing CTLN2 (SLC25A13) was identified by positional cloning (from 7q21.3) and found to encode a putative calcium-dependent mitochondrial carrier protein. To facilitate mutation analysis, here we describe the intron-exon boundaries of the human SLC25A13 gene. We have also cloned and characterized the mouse homologue (Slc25a13), which is predicted to encode a protein of 676 amino acids with 96% amino acid identity to SLC25A13. RNA in situ hybridization analysis shows that Slc25a13 is expressed in the branchial arches, as well as the limb and tail buds, during mouse embryonic development (E10.5). At E13.5 expression of Slc25a13 is most predominant in epithelial structures, in addition to the forebrain, kidney, and liver.