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.

ASL mRNA-LNP Therapeutic for the Treatment of Argininosuccinic Aciduria Enables Survival Benefit in a Mouse Model.

Argininosuccinic aciduria (ASA) is a metabolic disorder caused by a deficiency in argininosuccinate lyase (ASL), which cleaves argininosuccinic acid to arginine and fumarate in the urea cycle. ASL deficiency (ASLD) leads to hepatocyte dysfunction, hyperammonemia, encephalopathy, and respiratory alkalosis. Here we describe a novel therapeutic approach for treating ASA, based on nucleoside-modified messenger RNA (modRNA) formulated in lipid nanoparticles (LNP). To optimize ASL-encoding mRNA, we modified its cap, 5' and 3' untranslated regions, coding sequence, and the poly(A) tail. We tested multiple optimizations of the formulated mRNA in human cells and wild-type C57BL/6 mice. The ASL protein showed robust expression in vitro and in vivo and a favorable safety profile, with low cytokine and chemokine secretion even upon administration of increasing doses of ASL mRNA-LNP. In the ASLNeo/Neo mouse model of ASLD, intravenous administration of the lead therapeutic candidate LNP-ASL CDS2 drastically improved the survival of the mice. When administered twice a week lower doses partially protected and 3 mg/kg LNP-ASL CDS2 fully protected the mice. These results demonstrate the considerable potential of LNP-formulated, modified ASL-encoding mRNA as an effective alternative to AAV-based approaches for the treatment of ASA.

Late-onset argininosuccinic aciduria in a 72-year-old man presenting with fatal hyperammonemia.

Argininosuccinate lyase deficiency (ASLD, MIM #207900) is an inherited urea cycle disorder. There are mainly two clinical forms, an acute neonatal form which manifests as life-threatening hyperammonemia, and a late-onset form characterised by polymorphic neuro-cognitive or psychiatric presentation with transient hyperammonemia episodes. Here, we report a late-onset case of ASLD in a 72-year-old man carrying a homozygous pathogenic variant in the exon 16 of the ASL gene, presenting for the first time with fatal hyperammonemic coma. This case report shows the need to systematically carry out an ammonia assay when faced with an unexplained coma.

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.

Clinical and genetic analysis of five Chinese patients with urea cycle disorders.

BACKGROUND: The urea cycle plays a key role in preventing the accumulation of toxic nitrogenous waste products, including two essential enzymes: ornithine transcarbamylase (OTC) and argininosuccinate lyase (ASL). Ornithine transcarbamylase deficiency (OTCD) results from mutations in the OTC. Meanwhile, argininosuccinate lyase deficiency (ASLD) is caused by mutations in the ASL. METHODS: Blood tandem mass spectrometric analysis and urea organic acidemia screening were performed on five Chinese cases, including three OTCD and two ASLD patients. Next-generation sequencing was then used to make a definite diagnosis, and the related variants were validated by Sanger sequencing. RESULTS: The five patients exhibited severe clinical symptoms, with abnormal biochemical analysis and amino acids profile. Genetic analysis revealed two variants [c.77G>A (p.Arg26Gln); c.116G>T (p.Gly39Val)] in the OTC, as well as two variants [c.1311T>G (p.Tyr437*); c.961T>A (p.Tyr321Asn)] in the ASL. Conservation analysis showed that the amino acids of the two novel mutations were highly conserved in different species and were predicted to be possibly damaging with several in silico prediction programs. 3D-modeling analysis indicated that the two novel missense variants might result in modest distortions of the OTC and ASL protein structures, respectively. CONCLUSIONS: Two novel variants expand the mutational spectrums of the OTC and ASL. All the results may contribute to a better understanding of the clinical course and genetic characteristics of patients with urea cycle disorders.

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

BACKGROUND: The 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. RESULTS: Forty-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. CONCLUSIONS: Most 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.

Refeeding syndrome in a young woman with argininosuccinate lyase deficiency.

A severely chronically protein and calorie restricted young woman with argininosuccinate lyase deficiency developed transient refeeding syndrome (RFS) and hyperammonemia after modest diet liberalization following initiation of glycerol phenylbutyrate (GPB). The patient required IV supportive care and supplementation with potassium, magnesium and calcium. She is now doing well on GPB and an appropriate maintenance diet. Susceptibility to RFS should be considered in chronically nutritionally restricted patients with metabolic disorders after liberalization of diet.