Argininosuccinic Aciduria Research Articles

Chronic liver disease and impaired hepatic glycogen metabolism in argininosuccinate lyase deficiency.

BACKGROUNDLiver disease in urea cycle disorders (UCDs) ranges from hepatomegaly and chronic hepatocellular injury to cirrhosis and end-stage liver disease. However, the prevalence and underlying mechanisms are unclear.METHODSWe estimated the prevalence of chronic hepatocellular injury in UCDs using data from a multicenter, longitudinal, natural history study. We also used ultrasound with shear wave elastography and FibroTest to evaluate liver stiffness and markers of fibrosis in individuals with argininosuccinate lyase deficiency (ASLD), a disorder with high prevalence of elevated serum alanine aminotransferase (ALT). To understand the human observations, we evaluated the hepatic phenotype of the AslNeo/Neo mouse model of ASLD.RESULTSWe demonstrate a high prevalence of elevated ALT in ASLD (37%). Hyperammonemia and use of nitrogen-scavenging agents, 2 markers of disease severity, were significantly (P < 0.001 and P = 0.001, respectively) associated with elevated ALT in ASLD. In addition, ultrasound with shear wave elastography and FibroTest revealed increased echogenicity and liver stiffness, even in individuals with ASLD and normal aminotransferases. The AslNeo/Neo mice mimic the human disorder with hepatomegaly, elevated aminotransferases, and excessive hepatic glycogen noted before death (3-5 weeks of age). This excessive hepatic glycogen is associated with impaired hepatic glycogenolysis and decreased glycogen phosphorylase and is rescued with helper-dependent adenovirus expressing Asl using a liver-specific (ApoE) promoter.CONCLUSIONOur results link urea cycle dysfunction and impaired hepatic glucose metabolism and identify a mouse model of liver disease in the setting of urea cycle dysfunction.TRIAL REGISTRATIONThis study has been registered at ClinicalTrials.gov (NCT03721367, NCT00237315).FUNDINGFunding was provided by NIH, Burroughs Wellcome Fund, NUCDF, Genzyme/ACMG Foundation, and CPRIT.

Earwax: A potentially useful medium to identify inborn errors of metabolism?

Earwax was investigated as a source to identify patients' different inborn errors of metabolism (IEMs). Acylcarnitines, amino acids, and guanidino metabolites were measured from 28 treated patients with 11 different metabolic disorders including 3 organic acidaemias, 2 fatty acid oxidation defects, 6 amino acid disorders, and 1 peroxisomal abnormality. On the basis of the ratio of different acylcarnitine species relative to free carnitine, isovaleric acidaemia, methylmalonic acidaemia, and long‐chain hydroxyacylCoA dehydrogenase deficiency could be discriminated from the other disorders. For amino acids, neither creatinine nor alternative amino acid proved suitable reference standards against which results could be expressed. However, argininosuccinate and alloisoleucine were present in significantly elevated concentrations in two patients with argininosuccinate lyase deficiency and two patients with branched‐chain ketoacid dehydrogenase deficiency. This study has raised the potential of earwax for investigation of IEMs and may also have role in postmortem investigations. In view of its limited invasiveness, earwax also may have a role as a material to monitor treatment responses and compliance in patients with IEMs.

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.

Intrahepatic Administration of Human Liver Stem Cells in Infants with Inherited Neonatal-Onset Hyperammonemia: A Phase I Study

Previous studies have shown that human liver stem-like cells (HLSCs) may undergo differentiation in vitro into urea producing hepatocytes and in vivo may sustain liver function in models of experimentally induced acute liver injury. The aim of this study was to assess the safety of HLSCs intrahepatic administration in inherited neonatal-onset hyperammonemia. The study was approved by the Agenzia Italiana del Farmaco on favorable opinion of the Italian Institute of Health as an open-label, prospective, uncontrolled, monocentric Phase I study (HLSC 01–11, EudraCT-No. 2012–002120-33). Three patients affected by argininosuccinic aciduria (patient 1) and methylmalonic acidemia (patients 2 and 3) and included in the liver transplantation list were enrolled. In all patients, HLSCs were administered by percutaneous intrahepatic injections (once a week for two consecutive weeks) within the first months of life. The first patient received 125,000 HLSCs x gram of liver/dose while the other two patients received twice this dose. No immunosuppression was administered since HLSCs possess immunomodulatory activities. None of the patients experienced infections, hyperammonemia decompensation, or other adverse events during the whole observation period. No donor specific antibodies (DSA) against HLSCs were detected. Patients were metabolic stable despite an increase (~30%) in protein intake. Two patients underwent liver transplantation after 19 and 11 months respectively, and after explantation, the native livers showed no histological alterations. In conclusion, percutaneous intrahepatic administration of HLSCs was safe in newborn with inherited neonatal-onset hyperammonemia. These data pave the way for Phase II studies in selected inherited and acquired liver disorders.

Free Radical Scavengers Prevent Argininosuccinic Acid-Induced Oxidative Stress in the Brain of Developing Rats: a New Adjuvant Therapy for Argininosuccinate Lyase Deficiency?

Tissue accumulation and high urinary excretion of argininosuccinate (ASA) is the biochemical hallmark of argininosuccinate lyase deficiency (ASLD), a urea cycle disorder mainly characterized by neurologic abnormalities, whose pathogenesis is still unknown. Thus, in the present work, we evaluated the in vitro and in vivo effects of ASA on a large spectrum of oxidative stress parameters in brain of adolescent rats in order to test whether disruption of redox homeostasis could be involved in neurodegeneration of this disorder. ASA provoked in vitro lipid and protein oxidation, decreased reduced glutathione (GSH) concentrations, and increased reactive oxygen species generation in cerebral cortex and striatum. Furthermore, these effects were totally prevented or attenuated by the antioxidants melatonin and GSH. Similar results were obtained by intrastriatal administration of ASA, in addition to increased reactive nitrogen species generation and decreased activities of superoxide dismutase, glutathione peroxidase, and glutathione S-transferase. It was also observed that melatonin and N-acetylcysteine prevented most of ASA-induced in vivo pro-oxidant effects in striatum. Taken together, these data indicate that disturbance of redox homeostasis induced at least in part by high brain ASA concentrations per se may potentially represent an important pathomechanism of neurodegeneration in patients with ASLD and that therapeutic trials with appropriate antioxidants may be an adjuvant treatment for these patients.

ASL Metabolically Regulates Tyrosine Hydroxylase in the Nucleus Locus Coeruleus

SummaryPatients with germline mutations in the urea-cycle enzyme argininosuccinate lyase (ASL) are at risk for developing neurobehavioral and cognitive deficits. We find that ASL is prominently expressed in the nucleus locus coeruleus (LC), the central source of norepinephrine. Using natural history data, we show that individuals with ASL deficiency are at risk for developing attention deficits. By generating LC-ASL-conditional knockout (cKO) mice, we further demonstrate altered response to stressful stimuli with increased seizure reactivity in LC-ASL-cKO mice. Depletion of ASL in LC neurons leads to reduced amount and activity of tyrosine hydroxylase (TH) and to decreased catecholamines synthesis, due to decreased nitric oxide (NO) signaling. NO donors normalize catecholamine levels in the LC, seizure sensitivity, and the stress response in LC-ASL-cKO mice. Our data emphasize ASL importance for the metabolic regulation of LC function with translational relevance for ASL deficiency (ASLD) patients as well as for LC-related pathologies.

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.

Orphan designation: Sodium benzoate,Sodium phenylacetate, Treatment of argininosuccinic aciduria

Orphan designation: Sodium benzoate,Sodium phenylacetate, Treatment of argininosuccinic aciduria

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.

Rapid quantification of underivatized alloisoleucine and argininosuccinate using mixed-mode chromatography with tandem mass spectrometry

Plasma elevations of the amino acids alloisoleucine and argininosuccinic acid (ASA) are pathognomonic for maple syrup urine disease and argininosuccinate lyase deficiency, respectively. Reliable detection of these biomarkers is typically achieved using methods with tedious sample preparations or long chromatographic separations, and many published amino acid assays report poor specificity and/or sensitivity for one or both of these compounds. This report describes a novel liquid chromatography tandem mass spectrometry (LC-MS/MS) method that provides rapid quantification of alloisoleucine and ASA in human plasma. The basis of this method is a mixed-mode solid phase separation that achieves baseline resolution of alloisoleucine from isobaric interferents without the use of derivatization or ion pairing agents. The inject-to-inject time is 6 min including elution, column washing and re-equilibration. Validation studies demonstrate excellent limits of quantification (1 μmol/L), linearity (r = 0.999 from 1 to 250 μmol/L), accuracy (bias = −3.8% and −10.1%), and inter-assay imprecision (CV < 8.06%) for plasma analyses. Data from long-term clinical application confirms chromatographic consistency equivalent to more traditional reversed-phase or HILIC based columns. Additional matrix studies indicate low suppression (<10%) for a wide range of amino acids and compatibility with other matrixes such as blood spot analyses. Finally, analysis of our first 257 clinical specimens demonstrates high analytic specificity and sensitivity, allowing the detection of subtle but clinically relevant elevations of alloisoleucine and ASA that may be missed by other less sensitive methods. In conclusion, the novel LC-MS/MS method reported here overcomes a number of the challenges associated with alloisoleucine and ASA quantification. Combining this approach with published incomplete amino acid quantification methods allows, for the first time, a rapid and comprehensive LC-MS/MS analysis of underivatized amino acids without the use of ion pairing agents.

Chronic liver involvement in urea cycle disorders.

The increased survival of urea cycle disorders (UCDs) patients has led the attention to clinical manifestations that characterize the long-term disease course. Acute and chronic liver disease have been anecdotally reported since the very first description of UCDs. However, a detailed analysis of long-term liver involvement in large patient cohorts is still needed. Chronic liver damage in UCDs has probably a multifactorial origin, but the specific underlying mechanisms of liver disease have not yet been well elucidated. In this study, we report on chronic liver involvement and on associated metabolic abnormalities in a large cohort of 102 UCD patients, followed by two reference centers in Italy. Chronic liver involvement was observed in over 60% of UCDs patients, and comparison between individual diseases showed a significant higher frequency in argininosuccinate lyase deficiency (ASLD) and in hyperornithinemia-hyperammonemia-homocitrullinemia (HHH) syndrome with elevation of transaminases and of gamma-GT in ASLD, and of alpha-fetoprotein in HHH syndrome. Also, consistent with a chronic hepatic dysfunction, ultrasound examination revealed more pronounced abnormalities in ASLD and in HHH syndrome, when compared to other UCDs. Our study highlights in a large UCDs patients' cohort that chronic liver disease is a common finding in UCDs, often with a distinct phenotype between different diseases. Furthers studies are needed to elucidate the specific involvement of different metabolic pathways in the pathogenesis of liver dysfunction in UCDs.

Urea cycle disorders in Argentine patients: clinical presentation, biochemical and genetic findings

BackgroundThe incidence, prevalence, and molecular epidemiology of urea cycle disorders (UCDs) in Argentina remain underexplored. The present study is the first to thoroughly assess the clinical and molecular profiles of UCD patients examined at a single reference center in Argentina.ResultsForty-nine UCD cases were collected. About half (26/49, 53%) manifested neonatally with classical presentation and had a high mortality (25/26, 96%). Ornithine transcarbamylase deficiency (OTCD) was the most common UCD (26 patients). Argininosuccinate synthetase deficiency (ASSD) was detected in 19 cases, while argininosuccinate lyase deficiency (ASLD) was diagnosed in 4 cases. Molecular genetic analysis revealed 8 private OTC mutations and two large deletion/duplication events in the OTC gene. Most mutations in the ASS1 and ASL genes were recurrent missense changes, and four alterations were novel. The clinical outcome of our UCD cohort was poor, with an overall mortality of 57% (28/49 cases), and a 28% (6/21) disability rate among the survivors.ConclusionsMost patients in our case series showed severe neonatal onset, with high morbidity/mortality. We detected in total 19 mutations, most of them recurrent and of high frequency worldwide. Noteworthy, we highlight the presence of a geographic cluster with high prevalence of a point mutation in the ASS1 gene. This study suggests that these disorders may be more frequent than commonly assumed, and stresses the need for increased awareness amongst health professionals and greater availability of diagnostic tools for accurate identification, early diagnosis, and timely treatment.

Orphan designation: Sodium benzoate, Treatment of argininosuccinic aciduria

Orphan designation: Sodium benzoate, Treatment of argininosuccinic aciduria

Orphan designation: Glyceryl tri-(4-phenylbutyrate), Treatment of argininosuccinic aciduria

Orphan designation: Glyceryl tri-(4-phenylbutyrate), Treatment of argininosuccinic aciduria

Resting energy expenditure in argininosuccinic aciduria and in other urea cycle disorders.

No data are available on the specific energy needs of patients affected with Urea Cycle disorders (UCD) and especially argininosuccinic aciduria (ASA). In our experience, ASA patients tend to develop central adiposity and hypertriglyceridemia when treated with apparently adequate energy intake, while the other UCD do not. The aim of this study was to evaluate anthropometric parameters, body composition, risk of metabolic syndrome (MS) and resting energy expenditure (REE), both by indirect calorimetry (IC) and predictive equations, in UCD patients. Hypertension (5/13), pathological waist circumference-to-height ratio (WtHr) (6/13), hypertriglyceridemia (12/13), reduced HDL cholesterol (12/13), and MS (5/13) were found in ASA group. In the ASA cohort, the mean and median IC-REE were 88% of what was predicted by Food and Agriculture Organization of the United Nations and Harris-Benedict equations. The "other UCD" cohort did not show hypertension, dyslipidaemia nor MS; IC-REE was similar to the REE predicted by equations. A significant difference was seen for the presence of hypertension, dyslipidaemia, pathological WtHr, MS and IC-REE/predictive equations-REE in the two cohorts. ASA patients have a risk of overfeeding if their energy requirement is not assessed individually with IC. Excessive energy intake might increase the cardiovascular risk of ASA patients. We suggest to test ASA individuals with IC every year if the patient is sufficiently collaborative. We speculate that most of the features seen in ASA patients might depend on an imbalance of Krebs cycle. Further studies are needed to verify this hypothesis.

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.

Impact of Diagnosis and Therapy on Cognitive Function in Urea Cycle Disorders.

Individuals with urea cycle disorders (UCDs) often present with intellectual and developmental disabilities. The major aim of this study was to evaluate the impact of diagnostic and therapeutic interventions on cognitive outcomes in UCDs. This prospective, observational, multicenter study includes data from 503 individuals with UCDs who had comprehensive neurocognitive testing with a cumulative follow-up of 702 patient-years. The mean cognitive standard deviation score (cSDS) was lower in symptomatic than in asymptomatic (p < 0.001, t test) individuals with UCDs. Intellectual disability (intellectual quotient < 70, cSDS < -2.0) was associated with the respective subtype of UCD and early disease onset, whereas height of the initial peak plasma ammonium concentration was inversely associated with neurocognitive outcomes in mitochondrial (proximal) rather than cytosolic (distal) UCDs. In ornithine transcarbamylase and argininosuccinate synthetase 1 deficiencies, we did not find evidence that monoscavenger therapy with sodium or glycerol phenylbutyrate was superior to sodium benzoate in providing cognitive protection. Early liver transplantation appears to be beneficial for UCDs. It is noteworthy that individuals with argininosuccinate synthetase 1 and argininosuccinate lyase deficiencies identified by newborn screening had better neurocognitive outcomes than those diagnosed after the manifestation of first symptoms. Cognitive function is related to interventional and non-interventional variables. Early detection by newborn screening and early liver transplantation appear to offer greater cognitive protection, but none of the currently used nitrogen scavengers was superior with regard to long-term neurocognitive outcome. Further confirmation could determine these variables as important clinical indicators of neuroprotection for individuals with UCDs. ANN NEUROL 2019.

Clinical and genetic analysis of two children suspected for argininosuccinic aciduria

To analyze the clinical and genetic features of two children suspected for arginylsuccinuria aciduria. The patients were subjected to high-throughput sequencing using a gene panel. Both patients had high citrulline (87.37-156.10 μmol/L) measured by mass spectrometry/mass spectrometry (MS/MS) upon neonatal screening but had no symptoms. Two compound heterozygous variants of the ASL gene were detected in patient 1 (exon 6: c.467C>T inherited from her father and exon 7: c.556C>T inherited from her mother), among which c.556C>T is novel. Patient 2 had mental retardation and two full siblings who had died of hyperammonemia. Two compound heterozygosity variants of the ASL gene were detected (exon 3: c.281G>T inherited from his father and intron: c.208-15T>A inherited from his mother). Both were novel mutations. Variants of the ASL gene probably underlie the argininosuccinic aciduria in the two patients. Above findings have enriched the spectrum of ASL mutations.

An unusual association of P450 oxidoreductase Deficiency and Argininosuccinatelyase Deficiency

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Argininosuccinate neurotoxicity and prevention by creatine in argininosuccinate lyase deficiency: An in vitro study in rat three-dimensional organotypic brain cell cultures.

The urea cycle disorder (UCD) argininosuccinate lyase (ASL) deficiency, caused by a defective ASL enzyme, exhibits a wide range of phenotypes, from life-threatening neonatal hyperammonemia to asymptomatic patients, with only the biochemical marker argininosuccinic acid (ASA) elevated in body fluids. Remarkably, even without ever suffering from hyperammonemia, patients often develop severe cognitive impairment and seizures. The goal of this study was to understand the effect on the known toxic metabolite ASA and the assumed toxic metabolite guanidinosuccinic acid (GSA) on developing brain cells, and to evaluate the potential role of creatine (Cr) supplementation, as it was described protective for brain cells exposed to ammonia. We used an in vitro model, in which we exposed three-dimensional (3D) organotypic rat brain cell cultures in aggregates to different combinations of the metabolites of interest at two time points (representing two different developmental stages). After harvest and cryopreservation of the cell cultures, the samples were analyzed mainly by metabolite analysis, immunohistochemistry, and western blotting. ASA and GSA were found toxic for astrocytes and neurons. This toxicity could be reverted in vitro by Cr. As well, an antiapoptotic effect of ASA was revealed, which could contribute to the neurotoxicity in ASL deficiency. Further studies in human ASL deficiency will be required to understand the biochemical situation in the brain of affected patients, and to investigate the impact of high or low arginine doses on brain Cr availability. In addition, clinical trials to evaluate the beneficial effect of Cr supplementation in ASL deficiency would be valuable.