Argininosuccinate Lyase Gene Research Articles

Effect of marker-free transgenic Chlamydomonas on the control of Aedes mosquito population and on plankton

BackgroundMore than half of the world’s population suffers from epidemic diseases that are spread by mosquitoes. The primary strategy used to stop the spread of mosquito-borne diseases is vector control. Interference RNA (RNAi) is a powerful tool for controlling insect populations and may be less susceptible to insect resistance than other strategies. However, public concerns have been raised because of the transfer of antibiotic resistance marker genes to environmental microorganisms after integration into the recipient genome, thus allowing the pathogen to acquire resistance. Therefore, in the present study, we modified the 3-hydroxykynurenine transaminase (3hkt) and hormone receptor 3 (hr3) RNAi vectors to remove antibiotic resistance marker genes and retain the expression cassette of the inverse repeat sequence of the 3hkt/hr3 target gene. This recombinant microalgal marker-free RNAi insecticide was subsequently added to the suburban water in a simulated-field trial to test its ability to control mosquito population.MethodsThe expression cassette of the 3hkt/hr3 inverted repeat sequence and a DNA fragment of the argininosuccinate lyase gene without the ampicillin resistance gene were obtained using restriction enzyme digestion and recovery. After the cotransformation of Chlamydomonas, the recombinant algae was then employed to feed Aedes albopictus larvae. Ten and 300 larvae were used in small- and large-scale laboratory Ae.albopictus feeding trials, respectively. Simulated field trials were conducted using Meishe River water that was complemented with recombinant Chlamydomonas. Moreover, the impact of recombinant microalgae on phytoplankton and zooplankton in the released water was explored via high-throughput sequencing.ResultsThe marker-free RNAi-recombinant Chlamydomonas effectively silenced the 3hkt/hr3 target gene, resulting in the inhibition of Ae. albopictus development and also in the high rate of Ae. albopictus larvae mortality in the laboratory and simulated field trials. In addition, the results confirmed that the effect of recombinant Chlamydomonas on plankton in the released water was similar to that of the nontransgenic Chlamydomonas, which could reduce the abundance and species of plankton.ConclusionsThe marker-free RNAi-recombinant Chlamydomonas are highly lethal to the Ae. albopictus mosquito, and their effect on plankton in released water is similar to that of the nontransgenic algal strains, which reduces the abundance and species of plankton. Thus, marker-free recombinant Chlamydomonas can be used for mosquito biorational control and mosquito-borne disease prevention.Graphical

Variants associated with urea cycle disorders in Japanese patients: Nationwide study and literature review.

Urea cycle disorders (UCDs) are inherited metabolic diseases that lead to hyperammonemia with variable clinical manifestations. Using data from a nationwide study, we investigated the onset time, gene variants, clinical manifestations, and treatment of patients with UCDs in Japan. Of the 229 patients with UCDs diagnosed and/or treated between January 2000 and March 2018, identified gene variants and clinical information were available for 102 patients, including 62 patients with ornithine transcarbamylase (OTC) deficiency, 18 patients with carbamoyl phosphate synthetase 1 (CPS1) deficiency, 16 patients with argininosuccinate synthetase (ASS) deficiency, and 6 patients with argininosuccinate lyase (ASL) deficiency. A total of 13, 10, 4, and 5 variants in the OTC, CPS1, ASS, and ASL genes were respectively identified as novel variants, which were neither registered in ClinVar databases nor previously reported. The onset time and severity in patients with UCD could be predicted based on the identified gene variants in each patient from this nationwide study and previous studies. This genetic information may help in predicting the long-term outcome and determining specific treatment strategies such as liver transplantation in patients with UCDs.

Recombinant Bacillus caldovelox Arginase Mutant (BCA-M) Induces Apoptosis, Autophagy, Cell Cycle Arrest and Growth Inhibition in Human Cervical Cancer Cells.

With our recent success in developing a recombinant human arginase drug against broad-spectrum cancer cell lines, we have explored the potential of a recombinant Bacillus caldovelox arginase mutant (BCA-M) for human cervical cancer treatment. Our studies demonstrated that BCA-M significantly inhibited the growth of human cervical cancer cells in vitro regardless of argininosuccinate synthetase (ASS) and argininosuccinate lyase (ASL) expression. Drug susceptibilities correlate well with the expressions of major urea cycle genes and completeness of L-arginine regeneration pathways. With the expressions of ASS and ASL genes conferring resistance to L-arginine deiminase (ADI) which is undergoing Phase III clinical trial, BCA-M offers the advantage of a broader spectrum of susceptible cancer cells. Mechanistic studies showed that BCA-M inhibited the growth of human cervical cancer cells by inducing apoptosis and cell cycle arrest at S and/or G2/M phases. Our results also displayed that autophagy served as a protective mechanism, while the growth inhibitory effects of BCA-M could be enhanced synergistically by its combination to the autophagy inhibitor, chloroquine (CQ), on human cervical cancer cells.

From genotype to phenotype: Early prediction of disease severity in argininosuccinic aciduria.

Argininosuccinic aciduria (ASA) is an inherited urea cycle disorder and has a highly variable phenotypic spectrum ranging from individuals with lethal hyperammonemic encephalopathy, liver dysfunction, and cognitive deterioration, to individuals with a mild disease course. As it is difficult to predict the phenotypic severity, we aimed at identifying a reliable disease prediction model. We applied a biallelic expression system to assess the functional impact of pathogenic argininosuccinate lyase (ASL) variants and to determine the enzymatic activity of ASL in 58 individuals with ASA. This cohort represented 42 ASL gene variants and 42 combinations in total. Enzymatic ASL activity was compared with biochemical and clinical endpoints from the UCDC and E-IMD databases. Enzymatic ASL activity correlated with peak plasma ammonium concentration at initial presentation and with the number of hyperammonemic events (HAEs) per year of observation. Individuals with ≤9% of enzymatic activity had more severe initial decompensations and a higher annual frequency of HAEs than individuals above this threshold. Enzymatic ASL activity also correlated with the cognitive outcome and the severity of the liver disease, enabling a reliable severity prediction for individuals with ASA. Thus, enzymatic activity measured by this novel expression system can serve as an important marker of phenotypic severity.

Developmental changes in the utilization of citrulline by neonatal pigs.

Developmental changes in the renal expression and activity of argininosuccinate synthase (ASS1) and argininosuccinate lyase (ASL), enzymes that use citrulline for the production of arginine, have been reported. Thus, the ability of neonates, and especially premature neonates, to produce arginine may be compromised. To determine the utilization of citrulline in vivo, we measured renal expression of ASS1 and ASL and conducted citrulline compartmental and noncompartmental kinetics using [15N]citrulline in pigs of five different ages (from 10 days preterm to 5 wk of age). The tracer was given in substrate amounts to also test the ability of neonatal pigs to use exogenous citrulline. Preterm and term pigs at birth had lower ASS1 and ASL expression than older animals, which was reflected in the longer half-life of citrulline in the neonatal groups. The production and utilization of citrulline by 1-wk-old pigs was greater than in pigs of other ages, including 5-wk-old animals. Plasma citrulline concentration was not able to capture these differences in citrulline production and utilization. In conclusion, the developmental changes in renal ASS1 and ASL gene expression are reflected in the ability of the pigs to use citrulline. However, it seems that there is an excess capacity to use citrulline at all ages, including during prematurity, since the bolus dose of tracer did not result in an increase in endogenous citrulline. Our results support the idea that citrulline supplementation in neonatal, including premature, pigs is a viable option to increase arginine availability.

Identification of mutations in Malaysian patients with argininosuccinate lyase (ASL) deficiency

Argininosuccinate lyase (ASL) deficiency impairs the function of the urea cycle that detoxifies blood ammonia in the body. Mutation that occurs in the ASL gene is the cause of occurrence of ASL deficiency (ASLD). This deficiency causes hyperammonemia, hepatopathy and neurodevelopmental delay in patients. In this study, the clinical characteristics and molecular analysis of 10 ASLD patients were presented. 8 patients were associated with severe neonatal onset, while the other 2 were associated with late onset. Molecular analysis of ASL gene identified four new missense variants, which were c.778C>T, p.(Leu260Arg), c.1340G>C, p.(Ser447Thr), c.436C>G, p.(Arg146Gly) and c.595C>G, p.(Leu199Val) and four reported missense variants, which were c.638G>A, p.(Arg213Gln); c.556C>T, p.(Arg186Trp), c.578G>A, p.(Arg193Gln) and c.436C>G, p.(Arg146Trp). In silico servers predicted all new and reported variants as disease-causing. Structural examination exhibited that all pathogenic variants affected the stability of the tetrameric ASL structure by disturbing the bonding pattern with the neighboring residues. ConclusionThis study revealed the genetic heterogeneity among Malaysian ASL patients. This study has also expanded the mutational spectrum of the ASL.

Genetic diagnosis of a Chinese pedigree affected with neonatal argininosuccinic aciduria

To explore the genetic basis of a neonate with argininosuccinic aciduria (ASA). A neonate with lethargy and food refusal was admitted. The patient had myoclonus, myasthenia, uroschesis, irregular breathing and paroxysmal ventricular tachycardia, and died at 75 hours after birth. Laboratory test showed marked increase in blood ammonia (1249.8 μmol/L). Peripheral blood samples of the patient, her parents and sister were collected and subjected to trio whole-exome sequencing. Whole-exome sequencing revealed that the patient has carried compound heterozygous mutations of the argininosuccinate lyase (ASL) gene, namely c.425(exon5)_c.426(exon5) insAGCTCCCAGCT (p.Thr142Thrfs*37) and c.626(exon8)delT (p.Leu209Argfs*42). The patient was diagnosed as ASA caused by ASL gene mutations. Her parents and her elder sister were heterozygous carriers of the above mutations and had a normal phenotype. ASA is a severe congenital genetic metabolic disease and can manifest as onset of hyperammonemia in neonates. The clinical diagnosis is difficult and ASL gene testing may be helpful.

Functional Characterization of Argininosuccinate Lyase Gene Variants by Mini-Gene Splicing Assay.

ObjectiveArgininosuccinate lyase (ASL) gene mutations account for argininosuccinic aciduria (ASA). This study aimed to design a minigene construct of ASL gene in order to investigate the impact of variants on splicing.MethodsThe peripheral blood samples were collected from the family members, and genomic DNA was extracted for gene diagnosis using the total exon sequencing method. The novel mutation gene was cloned into pEGFP-C1 vector, and the pathogenicity of the mutation was examined in cultured cells in vitro.ResultsThe clinical diagnosis of the proband as ASA was clear. Two pathogenic mutations, c.281G>T (p.Arg94Leu) and c.208-15 T>A were detected in the ASL gene, and the two mutations had not been reported. The minigene expression in vitro confirmed that c.208-15 T>A could cause aberrant splicing, resulting in the retention of 13 bp in intron 2 to exon 3.ConclusionTwo new pathogenic mutations of ASL gene, c.208-15 T>A and c.281G>T, were found in an ASA family, which enriches the mutational profile of the ASL gene and provides a basis for genetic diagnosis of ASA. Minigenes are optimal approaches to determine whether the intron mutation can cause aberrant splicing.

A simple dual-inducible CRISPR interference system for multiple gene targeting in Corynebacterium glutamicum

The development of CRISPR interference (CRISPRi) technology has dramatically increased the pace and the precision of target identification during platform strain development. In order to develop a simple, reliable, and dual-inducible CRISPRi system for the industrially relevant Corynebacterium glutamicum, we combined two different inducible repressor systems in a single plasmid to separately regulate the expression of dCas9 (anhydro-tetracycline-inducible) and a given single guide RNA (IPTG-inducible). The functionality of the resulting vector was demonstrated by targeting the l-arginine biosynthesis pathway in C. glutamicum. By co-expressing dCas9 and a specific single guide RNA targeting the 5′-region of the argininosuccinate lyase gene argH, the specific activity of the target enzyme was down-regulated and in a l-arginine production strain, l-arginine formation was shifted towards citrulline formation. The system was also employed for down-regulation of multiple genes by concatenating sgRNA sequences encoded on one plasmid. Simultaneous down-regulated expression of both argH and the phosphoglucose isomerase gene pgi proved the potential of the system for multiplex targeting. The system can be a promising tool for further pathway engineering in C. glutamicum. Cumulative effects on targeted genes can be rapidly evaluated avoiding tedious and time-consuming traditional gene knockout approaches.

Serendipita indica modulates expression of arginine metabolism genes during colonization of Arabidopsis thaliana roots

Abstract Endophytic fungus Serendipita indica belongs to the order Sebacinales. It is an extremely versatile root endophyte colonizing more than 150 different plant species including the model plant Arabidopsis thaliana. Colonization of S. indica triggers enhanced growth, early flowering, increase in seed content, alteration of secondary metabolite pathways and adaption to different biotic and abiotic stresses. This is why this fungus got ample attention by the plant researchers. In this study, we report modulation in expression of argininosuccinate synthase (AS), argininosuccinate lyase (AL) and arginase 2 (ARGAH2) genes in A. thaliana as a result of S. indica colonization. Similarly, expression of GUS reporter gene driven by AS and ARGAH2 promoters in roots was altered. AS, AL and ARGAH2 genes were down-regulated at early stage of root colonization i.e at 3 days post inoculation (dpi), with a subsequent up-regulation at later stages (7 and 14 dpi). Other arginine catabolism genes, Arginine decarboxylase-1 (ADC1), Arginine decarboxylase-2 (ADC2) and Pyrroline-5-carboxylate reductase (P5CR) were strongly up-regulated at later stages of S. indica colonization. Our findings show significant changes in expression of several important genes in arginine metabolic pathway upon S. indica colonization. These changes are very specific and strongly depend on the colonization phase of the fungus. Keywords: AL; ARGAH1; ARGAH2; AS; Arginine metabolism; GUS; Quantitative RT-PCR; Serendipita indica http://dx.doi.org/10.19045/bspab.2018.700203

Recurrent encephalopathy and hyperammonemia in new born: case of arginosuccinic aciduria

Urea cycle disorders result from defects in the metabolism of waste nitrogenous compounds derived from the breakdown of proteins and other nitrogen-containing molecules. Argininosuccinic aciduria is a rare autosomal recessive heterogeneous urea cycle disorder, which leads to the accumulation of argininosuccinic acid in the blood and urine. It is caused by defect in the argininosuccinate lyase (ASL) gene, which regulates the breakdown of argininosuccinate to fumarate and arginine in the urea cycle. They are a heterogeneous group of disorders which are associated with hyperammonemia resulting in severe neurological dysfunction like encephalopathy, seizures, developmental and psychomotor delay. The severe central nervous system dysfunction is by alteration of amino acid and neurotransmitters pathways and interference with normal cerebral energy metabolism and oxidative stress. Ammonium toxicity to the brain provokes irreversible damage due to cortical atrophy, edema, and demyelination, resulting in seizures, coma, and even death. We report such a case of urea cycle defect in a newborn that presented with recurrent encephalopathy with hyperammonemia precipitated by minor infections. Detailed investigations including genetic analysis lead to the diagnosis of argininosuccinic aciduria.

Adeno-associated viral gene therapy corrects a mouse model of argininosuccinic aciduria

Argininosuccinic aciduria (ASA) is the second most common genetic disorder affecting the urea cycle. The disease is caused by deleterious mutations in the gene encoding argininosuccinate lyase (ASL); total loss of ASL activity results in severe neonatal onset of the disease, which is characterized by hyperammonemia within a few days of birth that can rapidly progress to coma and death. The long-term complications of ASA, such as hypertension and neurocognitive deficits, appear to be resistant to the current treatment options of dietary restriction, arginine supplementation, and nitrogen scavenging drugs. Treatment-resistant disease is currently being managed by orthotopic liver transplant, which shows variable improvement and requires lifetime immunosuppression. Here, we developed a gene therapy strategy for ASA aimed at alleviating the symptoms associated with urea cycle disruption by providing stable expression of ASL protein in the liver. We designed a codon-optimized human ASL gene packaged within adeno-associated virus serotype 8 (AAV8) as a vector for targeted delivery to the liver. To evaluate the therapeutic efficacy of this approach, we utilized a murine hypomorphic model of ASA. Neonatal administration of AAV8 via the temporal facial vein extended survival in ASA hypomorphic mice, although not to wild-type levels. Intravenous injection into adolescent hypomorphic mice led to increased survival and body weight and correction of metabolites associated with the disease. Our results demonstrate that AAV8 gene therapy is a viable approach for the treatment of ASA.

The Citrulline Recycling Pathway Sustains Cardiovascular Function in Arginine-Depleted Healthy Mice, but Cannot Sustain Nitric Oxide Production during Endotoxin Challenge

The recycling of citrulline by argininosuccinate synthase 1 (ASS1) and argininosuccinate lyase (ASL) is crucial to maintain arginine availability and nitric oxide (NO) production. Pegylated arginine deiminase (ADI-PEG20) is a bacterial enzyme used to deplete circulating arginine. The goal of this research was to test the hypothesis that citrulline is able to sustain intracellular arginine availability for NO production in ADI-PEG20 arginine-depleted mice. Six- to 8-wk-old male C57BL/6J mice injected with ADI-PEG20 (5 IU) or saline (control) were used in 4 different studies. Arginine, citrulline, and NO kinetics were determined by using stable isotopes in unchallenged (study 1) and endotoxin-challenged (study 2) mice. Blood pressure was determined by telemetry for 6 d after ADI-PEG20 administration (study 3), and vasomotor activity and ASS1 and ASL gene expression were determined in mesenteric arteries collected from additional mice (study 4). ADI-PEG20 administration resulted in arginine depletion (<1 compared with 111±37 µmol/L) but in greater plasma citrulline concentrations (900±123 compared with 76±8 µmol/L; P<0.001) and fluxes (402±17 compared with 126±4 µmol ⋅ kg-1 ⋅ h-1; P<0.001) compared with controls. Endotoxin-challenged ADI-PEG20-treated mice produced less NO than controls (13±1 compared with 27±2 µmol ⋅ kg-1 ⋅ h-1; P<0.001). No differences (P>0.50) were observed for cardiovascular variables (heart rate, blood pressure) between ADI-PEG20-treated and control mice. Furthermore, no ex vivo vasomotor differences were observed between the 2 treatments. ADI-PEG20 administration resulted in greater gene expression of ASS1 (∼3-fold) but lower expression of ASL (-30%). ADI-PEG20 successfully depleted circulating arginine without any effect on cardiovascular endpoints in healthy mice but limited NO production after endotoxin challenge. Therefore, the citrulline recycling pathway can sustain local arginine availability independently from circulating arginine, satisfying the demand of arginine for endothelial NO production; however, it is unable to do so when a high demand for arginine is elicited by endotoxin.

Two Novel Mutations in the Argininosuccinate Lyase Gene in Iranian Patients and Literature Review

Introduction: The argininosuccinate lyase (ASL) gene encodes argininosuccinate lyase (ASL), which is one of the six enzymes of the urea cycle that detoxifies blood ammonia. Argininosuccinate lyase deficiency impairs the function of the urea cycle and causes hyperammonemia, neurodevelopmental delay and hepatopathy. Case Presentation: Here we report two patients with argininosuccinate aciduria. They were treated with peritoneal dialysis and scavenger drugs. Molecular genetic testing showed two novel homozygous mutations, c.146T > G (p.Leu49Arg) in exon 3 and c.1144-1G > C in intron 15 of the ASL gene. Conclusions: This report intends to underline the importance of pediatricians being aware of the existence of a metabolic disease in any ill neonate. Diagnosis of urea cycle disorders is particularly important because of availability of effective treatment options.

A gene-within-a-gene Cas9/sgRNA hybrid construct enables gene editing and gene replacement strategies in Chlamydomonas reinhardtii

Previous studies demonstrated highly inefficient gene editing in C. reinhardtii using conventional Cas9 and sgRNA genes (only 1 editing event using >1.5×109 initial cells). Design and testing of a hybrid gene-within-a-gene construct (composed of a Cas9 gene containing an artificial intron with an inserted sgRNA gene) demonstrated that such constructs were functional both in tobacco cells and C. reinhardtii cells. In tests with C. reinhardtii, approximately one in every ~3×107 initial cells contained an edited version of the targeted FKB12 gene (i.e., an average of ~3 colonies with an edited FKB12 gene per electroporation using 108 initial cells). Lack of an intact Cas9/intron-sgRNA gene in cells carrying either of two different edited genes strongly suggested that editing was due to transient expression of the Cas9/intron-sgRNA gene and the likely toxicity of long-term expression of Cas9 in C. reinhardtii cells. Co-transformation of the arginine-requiring mutant, arg7-8, with Cas9/intron-sgRNA constructs and appropriately designed synthetic, 80 nucleotide ssDNAs complementary to the argininosuccinate lyase (ARG) gene led to successful homologous recombination or nucleotide replacement and production of arginine prototrophs. As a practical application, a similar ssDNA oligonucleotide targeting the acetolactate synthase (ALS) gene and an appropriate Cas9/intron-sgRNA construct was used to create cells resistant to the herbicide, sulfometuron methyl.

Reversal of endothelial dysfunction in aorta of streptozotocin-nicotinamide-induced type-2 diabetic rats by S-Allylcysteine.

Dietary measures and plant-based therapies as prescribed by native systems of medicine have gained attraction among diabetics with claims of efficacy. The present study investigated the effects of S-Allylcysteine (SAC) on body weight gain, glucose, insulin, insulin resistance, and nitric oxide synthase in plasma and argininosuccinate synthase (AS) and argininosuccinate lyase (ASL), lipid peroxides and antioxidant enzymes in aorta of control and streptozotocin-nicotinamide (STZ-NA)-induced diabetic rats. Changes in body weight, glucose, insulin, insulin resistance, and antioxidant profiles of aorta and mRNA expressions of nitric oxide synthase, AS, and ASL were observed in experimental rats. SAC (150mg/kg b.w) showed its therapeutic effects similar to gliclazide in decreasing glucose, insulin resistance, lipid peroxidation, and increasing body weight; insulin, antioxidant enzymes, and mRNA levels of nitric oxide synthase, argininosuccinate synthase, and argininosuccinate lyase genes in STZ-NA rats. Histopathologic studies also revealed the protective nature of SAC on aorta. In conclusion, garlic and its constituents mediate the anti-diabetic potential through mitigating hyperglycemic status, changing insulin resistance by alleviating endothelial dysregulation in both plasma and tissues.

Neonatal hyperammonemia and citrullinemia caused by argininosuccinate lyase gene mutations

Objective To analyze the characteristics of neonatal hyperammonemia and citrullinemia caused by argininosuccinate lyase (ASL) gene mutations, and to have a better understanding of this disease. Methods A neonatal patient with the onset of hyperammonemia and citrullinemia admitted to the Department of Pediatrics of Peking University First Hospital on April 2, 2014, was retrospectively studied. Peripheral blood leukocyte DNA of the patient and his parents was collected to detect ASS1, ASL and SLC25A13 gene mutations. The literature related to neonatal hyperammonemia, citrullinemia and argininosuccinic aciduria was reviewed. Results The baby in this case appeared lethargic, had weaker crying and food refusal since three days after birth, and analysis of blood amino acid found a marked increase in blood ammonia (1 332 μmol/L) and a significant rise in citrulline (759.12 μmol/L). Sanger sequencing detection revealed compound heterozygous mutations in the ASL gene (c.434 A>G, c.857A>C) and this c.857A>C mutation was the first reported case in China. This case of hyperammonemia and citrullinemia was confirmed as argininosuccinic aciduria caused by ASL gene mutations. A protein-limited diet and the treatment of arginine and L-carnitine were given. His blood ammonia decreased to normal level and there was a significant improvement in physical and intellectual progress at five months old. Unfortunately, he had an intestinal infection when he was over five months old and the blood ammonia level tested in the local hospital was 480 μmol/L. Gradually there was a disturbance of consciousness, then coma, and he finally died after active rescue in the local hospital. Conclusions Hyperammonemia and citrullinemia in neonates are likely to be argininosuccinic aciduria and a gene mutation test may be helpful for diagnosis. Key words: Hyperammonemia; Citrullinemia; Argininosuccinate lyase; Argininosuccinic aciduria; Mutation; Infant, newborn

NGS in argininosuccinic aciduria detects a mutation (D145G) which drives alternative splicing of ASL: a case report study

BackgroundArgininosuccinic aciduria (ASAuria; OMIM 207900) is a rare autosomal recessive heterogeneous urea cycle disorder, which leads to the accumulation of argininosuccinic acid in the blood and urine. We aimed to perform genetic test to the patient and help clinician to diagnose precisely.Case presentationIn this study, we use next generation sequencing (NGS) and exon trapping to analysis the family members. We identified compound heterozygous mutations of the argininosuccinate lyase (ASL) gene in a Chinese Han ASAuria patient. The c.434A>G (p.(D145G)) mutation in exon 5 was shown by exon trapping to select for the formation of an alternative transcript deleted for exon 5. The c.1366C>T (p.(R456W)) mutation had been previously reported in an Italian patient.ConclusionsThis is the first report of a missense mutation driving alternative splicing which results in the loss of exon 5 in ASAuria. This study also demonstrates the value of NGS in the identification of mutations and molecular diagnosis for ASAuria families.Electronic supplementary materialThe online version of this article (doi:10.1186/s12881-016-0273-7) contains supplementary material, which is available to authorized users.

Effect of Cysteamine on Mutant ASL Proteins with Cysteine for Arginine Substitutions

Cysteamine is used to treat cystinosis via the modification of cysteine residues substituting arginine in mutant proteins. We investigated the effect of cysteamine on mutant argininosuccinate lyase (ASL), the second most common defect in the urea cycle. In an established mammalian expression system, 293T cell lysates were produced after transfection with all known cysteine for arginine mutations in the ASL gene (p.Arg94Cys, p.Arg95Cys, p.Arg168Cys, p.Arg379Cys, and p.Arg385Cys), allowing testing of the effect of cysteamine over 48 h in the culture medium as well as for 1 h immediately prior to the enzyme assay. Cysteamine at low concentrations showed no effect on 293T cell viability, ASL protein expression, or ASL activity when applied during cell culture. However, incubation of transfected cells with 0.05 mM cysteamine immediately before the enzyme assay resulted in increased ASL activity of p.Arg94Cys, p.Arg379Cys, and p.Arg385Cys by 64, 20, and 197 %, respectively, and this result was significant (p < 0.01). Cell lysates carrying p.Arg385Cys and treated with cysteamine recover enzyme activity that is similar to the untreated designed mutation p.Arg385Lys, providing circumstantial evidence for the assumed cysteamine-induced change of a cysteine to a lysine analogue. Since 12 % of all known genotypes in ASL deficiency are affected by a cysteine for arginine mutation, we conclude that the potential of cysteamine or of related substances as remedy for this disease should be investigated further.

The yeast ζ-crystallin/NADPH:quinone oxidoreductase (Zta1p) is under nutritional control by the target of rapamycin pathway and is involved in the regulation of argininosuccinate lyase mRNA half-life.

The yeast ζ-crystallin (Zta1p) is a quinone oxidoreductase belonging to the ζ-crystallin family, with activity in the reduction of alkenal/alkenone compounds. Various biological functions have been ascribed to the members of this protein family, such as their ability to interact specifically with AU-rich sequences in mRNA, and thus they have been proposed to act as AU-rich element-binding proteins (AREBPs). In this study, we evaluated the specificity of Zta1p for RNA versus DNA by means of a novel nonisotopic method for the in vitro quantitative detection of protein · RNA complexes. Through comparative transcriptomic analysis, we found that the lack of Zta1p negatively affects the expression of a group of genes involved in amino acid biosynthesis, the argininosuccinate lyase (ARG4) gene being one of them. Here, we propose that Zta1p participates in the post-transcriptional regulation of ARG4 expression by increasing the ARG4 mRNA half-life. In addition, expression of the ζ-crystallin gene (ZTA1) is itself regulated by nutrient availability through the general amino acid control and target of rapamycin pathways. Our results shed new light on the ζ-crystallin family members from yeast to humans as stress response proteins with a bifunctional role in the detoxification of alkenal and alkenone compounds, and the regulation of gene expression.