Amyloidogenic Propensity of Metabolites in the Uric Acid Pathway and Urea Cycle Critically Impacts the Etiology of Metabolic Disorders.

Novel insights into the etiology of metabolic disorders have recently been uncovered through the study of metabolite amyloids. In particular, inborn errors of metabolism (IEMs), including gout, Lesch-Nyhan syndrome (LNS), xanthinuria, citrullinemia, and hyperornithinemia-hyperammonemia-homocitrullinuria (HHH) syndrome, are attributed to the dysfunction of the urea cycle and uric acid pathway. In this study, we endeavored to understand and mechanistically characterize the aggregative property exhibited by the principal metabolites of the urea cycle and uric acid pathway, specifically hypoxanthine, xanthine, citrulline, and ornithine. Employing scanning electron microscopy (SEM), transmission electron microscopy (TEM), and atomic force microscopy (AFM), we studied the aggregation profiles of the metabolites. Insights obtained through molecular dynamics (MD) simulation underscore the vital roles of π-π stacking and hydrogen bonding interactions in the self-assembly process, and thioflavin T (ThT) assays further corroborate the amyloid nature of these metabolites. The in vitro MTT assay revealed the cytotoxic trait of these assemblies, a finding that was substantiated by in vivo assays employing the Caenorhabditis elegans (C. elegans) model, which revealed that the toxic effects were more pronounced and dose-specific in the case of metabolites that had aged via longer preincubation. We hence report a compelling phenomenon wherein these metabolites not only aggregate but transform into a soft, ordered assembly over time, eventually crystallizing upon extended incubation, leading to pathological implications. Our study suggests that the amyloidogenic nature of the involved metabolites could be a common etiological link in IEMs, potentially providing a unified perspective to study their pathophysiology, thus offering exciting insights into the development of targeted interventions for these metabolic disorders.

Discovery of a Carbamoyl Phosphate Synthetase 1-Deficient HCC Subtype With Therapeutic Potential Through Integrative Genomic and Experimental Analysis.

BACKGROUND AND AIMS: Metabolic reprogramming plays an important role in tumorigenesis. However, the metabolic types of different tumors are diverse and lack in-depth study. Here, through analysis of big databases and clinical samples, we identified a carbamoyl phosphate synthetase 1 (CPS1)-deficient hepatocellular carcinoma (HCC) subtype, explored tumorigenesis mechanism of this HCC subtype, and aimed to investigate metabolic reprogramming as a target for HCC prevention. APPROACH AND RESULTS: A pan-cancer study involving differentially expressed metabolic genes of 7,764 tumor samples in 16 cancer types provided by The Cancer Genome Atlas (TCGA) demonstrated that urea cycle (UC) was liver-specific and was down-regulated in HCC. A large-scale gene expression data analysis including 2,596 HCC cases in 7 HCC cohorts from Database of HCC Expression Atlas and 17,444 HCC cases from in-house hepatectomy cohort identified a specific CPS1-deficent HCC subtype with poor clinical prognosis. In vitro and in vivo validation confirmed the crucial role of CPS1 in HCC. Liquid chromatography-mass spectrometry assay and Seahorse analysis revealed that UC disorder (UCD) led to the deceleration of the tricarboxylic acid cycle, whereas excess ammonia caused by CPS1 deficiency activated fatty acid oxidation (FAO) through phosphorylated adenosine monophosphate-activated protein kinase. Mechanistically, FAO provided sufficient ATP for cell proliferation and enhanced chemoresistance of HCC cells by activating forkhead box protein M1. Subcutaneous xenograft tumor models and patient-derived organoids were employed to identify that blocking FAO by etomoxir may provide therapeutic benefit to HCC patients with CPS1 deficiency. CONCLUSIONS: In conclusion, our results prove a direct link between UCD and cancer stemness in HCC, define a CPS1-deficient HCC subtype through big-data mining, and provide insights for therapeutics for this type of HCC through targeting FAO.

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.

Biomarkers for liver disease in urea cycle disorders.

BACKGROUND: Urea cycle disorders (UCDs) are among the most common inborn errors of liver metabolism. As therapies for hyperammonemia associated with urea cycle dysfunction have improved, chronic complications, such as liver disease, have become increasingly apparent in individuals with UCDs. Liver disease in UCDs may be associated with hepatic inflammation, hepatic fibrosis, portal hypertension, liver cancer and even liver failure. However, except for monitoring serum aminotransferases, there are no clear guidelines for screening and/or monitoring individuals with UCDs for liver disease. Thus, we systematically evaluated the potential utility of several non-invasive biomarkers for liver fibrosis in UCDs. METHODS: We evaluated grey-scale ultrasonography, liver stiffness obtained from shear wave elastography (SWE), and various serum biomarkers for hepatic fibrosis and necroinflammation, in a cohort of 28 children and adults with various UCDs. RESULTS: Overall, we demonstrate a high burden of liver disease in our participants with 46% of participants having abnormal grey-scale ultrasound pattern of the liver parenchyma, and 52% of individuals having increased liver stiffness. The analysis of serum biomarkers revealed that 32% of participants had elevated FibroTest™ score, a marker for hepatic fibrosis, and 25% of participants had increased ActiTest™ score, a marker for necroinflammation. Interestingly, liver stiffness did not correlate with ultrasound appearance or FibroTest™. CONCLUSION: Overall, our results demonstrate the high overall burden of liver disease in UCDs and highlights the need for further studies exploring new tools for identifying and monitoring individuals with UCDs who are at risk for this complication. TRIAL REGISTRATION: This study has been registered in ClinicalTrials.gov (NCT03721367).

A randomized trial to examine the impact of food on pharmacokinetics of 4-phenylbutyrate and change in amino acid availability after a single oral administration of sodium 4-phenylbutyrarte in healthy volunteers.

Urea cycle disorders (UCDs), inborn errors of hepatocyte metabolism, result in the systemic accumulation of ammonia to toxic levels. Sodium 4-phenylbutyrate (NaPB), a standard therapy for UCDs for over 20 years, generates an alternative pathway of nitrogen deposition through glutamine consumption. Administration during or immediately after a meal is the accepted use of NaPB. However, this regimen is not based on clinical evidence. Here, an open-label, single-dose, five-period crossover study was conducted in healthy adults to investigate the effect of food on the pharmacokinetics of NaPB and determine any subsequent change in amino acid availability. Twenty subjects were randomized to one of four treatment groups. Following an overnight fast, NaPB was administered orally at 4.3 g/m2 (high dose, HD) or 1.4 g/m2 (low dose, LD) either 30 min before or just after breakfast. At both doses, compared with post-breakfast administration, pre-breakfast administration significantly increased systemic exposure of PB and decreased plasma glutamine availability. Pre-breakfast LD administration attenuated plasma glutamine availability to the same extent as post-breakfast HD administration. Regardless of the regimen, plasma levels of branched-chain amino acids (BCAA) were decreased below baseline in a dose-dependent manner. In conclusion, preprandial oral administration of NaPB maximized systemic exposure of the drug and thereby its potency to consume plasma glutamine. This finding may improve poor medication compliance because of the issues with odor, taste, and pill burden of NaPB and reduce the risk of BCAA deficiency in NaPB therapy.

Identification of rare variants causing urea cycle disorders: A clinical, genetic, and biophysical study.

Urea cycle disorders (UCDs) are a group of rare metabolic conditions characterized by hyperammonemia and a broad spectrum of phenotypic severity. They are caused by the congenital deficiency in the eight biomolecules involved in urea cycle. In the present study, five cases of UCD were recruited and submitted to a series of clinical, biochemical, and genetic analysis with a combination of high throughput techniques. Moreover, in silico analysis was conducted on the identified missense genetic variants. Various clinical and biochemical indications (including profiles of amino acids and urinary orotic acids) of UCD were manifested by the five probands. Sequence analysis revealed nine diagnostic variants, including three novel ones, which caused Argininosuccinic aciduria (ASA) in one case, Carbamoyl phosphate synthetase 1deficiency (CPS1D) in two cases, Ornithine transcarbamylase deficiency (OTCD) in one case, and Citrin deficiency in 1case. Results of in silico biophysical analysis strongly suggested the pathogenicity of each the five missense variants and provided insight into their intramolecular impacts. In conclusion, this study expanded the genetic variation spectrum of UCD, gave solid evidence for counselling to the affected families, and should facilitate the functional study on the proteins in urea cycle.

Gene delivery corrects N-acetylglutamate synthase deficiency and enables insights in the physiological impact of L-arginine activation of N-acetylglutamate synthase.

The urea cycle protects the central nervous system from ammonia toxicity by converting ammonia to urea. N-acetylglutamate synthase (NAGS) catalyzes formation of N-acetylglutamate, an essential allosteric activator of carbamylphosphate synthetase 1. Enzymatic activity of mammalian NAGS doubles in the presence of L-arginine, but the physiological significance of NAGS activation by L-arginine has been unknown. The NAGS knockout (Nags-/-) mouse is an animal model of inducible hyperammonemia, which develops hyperammonemia without N-carbamylglutamate and L-citrulline supplementation (NCG + Cit). We used adeno associated virus (AAV) based gene transfer to correct NAGS deficiency in the Nags-/- mice, established the dose of the vector needed to rescue Nags-/- mice from hyperammonemia and measured expression levels of Nags mRNA and NAGS protein in the livers of rescued animals. This methodology was used to investigate the effect of L-arginine on ureagenesis in vivo by treating Nags-/- mice with AAV vectors encoding either wild-type or E354A mutant mouse NAGS (mNAGS), which is not activated by L-arginine. The Nags-/- mice expressing E354A mNAGS were viable but had elevated plasma ammonia concentration despite similar levels of the E354A and wild-type mNAGS proteins. The corresponding mutation in human NAGS (NP_694551.1:p.E360D) that abolishes binding and activation by L-arginine was identified in a patient with NAGS deficiency. Our results show that NAGS deficiency can be rescued by gene therapy, and suggest that L-arginine binding to the NAGS enzyme is essential for normal ureagenesis.

Glycerol phenylbutyrate efficacy and safety from an open label study in pediatric patients under 2 months of age with urea cycle disorders.

BACKGROUND/AIMS: Neonatal onset Urea cycle disorders (UCDs) can be life threatening with severe hyperammonemia and poor neurological outcomes. Glycerol phenylbutyrate (GPB) is safe and effective in reducing ammonia levels in patients with UCD above 2 months of age. This study assesses safety, ammonia control and pharmacokinetics (PK) of GPB in UCD patients below 2 months of age. METHODS: This was an open-label study in UCD patients aged 0 - 2 months, consisting of an initiation/transition period (1 - 4 days) to GPB, followed by a safety extension period (6 months to 2 years). Patients presenting with a hyperammonemic crisis (HAC) did not initiate GPB until blood ammonia levels decreased to below 100 µmol/L while receiving sodium phenylacetate/sodium benzoate and/or hemodialysis. Ammonia levels, PK analytes and safety were evaluated during transition and monthly during the safety extension for 6 months and every 3 months thereafter. RESULTS: All 16 patients with UCD (median age 0.48 months, range 0.1 to 2.0 months) successfully transitioned to GPB within 3 days. Average plasma ammonia level excluding HAC was 94.3 µmol/L at baseline and 50.4 µmol/L at the end of the transition period (p = 0.21). No patient had a HAC during the transition period. During the safety extension, the majority of patients had controlled ammonia levels, with mean plasma ammonia levels lower during GPB treatment than baseline. Mean glutamine levels remained within normal limits throughout the study. PK analyses indicate that UCD patients <2 months are able to hydrolyze GPB with subsequent absorption of phenylbutyric acid (PBA), metabolism to phenylacetic acid (PAA) and conjugation with glutamine. Plasma concentrations of PBA, PAA, and phenylacetylglutamine (PAGN) were stable during the safety extension phase and mean plasma phenylacetic acid: phenylacetylglutamine ratio remained below 2.5 suggesting no accumulation of GPB. All patients reported at least 1 treatment emergent adverse event with gastroesophageal reflux disease, vomiting, hyperammonemia, diaper dermatitis (37.5% each), diarrhea, upper respiratory tract infection and rash (31.3% each) being the most frequently reported. CONCLUSIONS: This study supports safety and efficacy of GPB in UCD patients aged 0 -2 months who cannot be managed by dietary protein restriction and/or amino acid supplementation alone. GPB undergoes intestinal hydrolysis with no accumulation in this population.

CPS1: Looking at an ancient enzyme in a modern light.

The mammalian urea cycle (UC) is responsible for siphoning catabolic waste nitrogen into urea for excretion. Disruptions of the functions of any of the enzymes or transporters lead to elevated ammonia and neurological injury. Carbamoyl phosphate synthetase 1 (CPS1) is the first and rate-limiting UC enzyme responsible for the direct incorporation of ammonia into UC intermediates. Symptoms in CPS1 deficiency are typically the most severe of all UC disorders, and current clinical management is insufficient to prevent the associated morbidities and high mortality. With recent advances in basic and translational studies of CPS1, appreciation for this enzyme's essential role in the UC has been broadened to include systemic metabolic regulation during homeostasis and disease. Here, we review recent advances in CPS1 biology and contextualize them around the role of CPS1 in health and disease.

Severity-adjusted evaluation of newborn screening on the metabolic disease course in individuals with cytosolic urea cycle disorders.

OBJECTIVE: The implementation of newborn screening (NBS) programs for citrullinemia type 1 (CTLN1) and argininosuccinic aciduria (ASA) is subject to controversial debate. The aim of this study was to assess the impact of NBS on the metabolic disease course and clinical outcome of affected individuals. METHODS: In 115 individuals with CTLN1 and ASA, we compared the severity of the initial hyperammonemic episode (HAE) and the frequency of (subsequent) HAEs with the mode of diagnosis. Based on a recently established functional disease prediction model, individuals were stratified according to their predicted severe or attenuated phenotype. RESULTS: Individuals with predicted attenuated forms of CTLN1 and ASA were overrepresented in the NBS group, while those with a predicted severe phenotype were underrepresented compared to individuals identified after the manifestation of symptoms (SX). Identification by NBS was associated with reduced severity of the initial HAE both in individuals with predicted severe and attenuated phenotypes, while it was not associated with lower frequency of (subsequent) HAEs. Similar results were obtained when including some patients diagnosed presymptomatically (i.e. prenatal testing, and high-risk family screening) in this analysis. CONCLUSION: Since one of the major challenges of NBS outcome studies is the potential overrepresentation of individuals with predicted attenuated phenotypes in NBS cohorts, severity-adjusted evaluation of screened and unscreened individuals is important to avoid overestimation of the NBS effect. NBS enables the attenuation of the initial HAE but does not affect the frequency of subsequent metabolic decompensations in individuals with CTLN1 and ASA. Future long-term studies will need to evaluate the clinical impact of this finding, especially with regard to mortality, as well as cognitive outcome and quality of life of survivors.

The Application of Next-Generation Sequencing (NGS) in Neonatal-Onset Urea Cycle Disorders (UCDs): Clinical Course, Metabolomic Profiling, and Genetic Findings in Nine Chinese Hyperammonemia Patients.

During Jan. 2016-Dec. 2019, nine Chinese patients from eight unrelated families were diagnosed with neonatal-onset UCDs by targeted panel sequencing or whole-exome sequencing (WES). Their clinical manifestations, biochemical features, 180-day-age outcomes, and molecular genetic characteristics were reviewed retrospectively. NGS-based tests revealed 7 patients diagnosed with ornithine transcarbamylase deficiency (OTCD) and 2 with carbamoylphosphate synthetase I deficiency (CPS1D). The spectrum of the clinical presentation of nine affected individuals progressed from unspecific symptoms like poor feeding to somnolence, coma, and death. All patients presented with an acute hyperammonemia. The most robust metabolic pattern in OTCD was hyperglutaminemic hyperammonemia with high concentration of urine orotic acid, and it was reported in six patients. Of ten variants found on the OTC gene and CPS1 gene, 3 were novel: (c.176T>C (p.L59P)) in the OTC gene, c.2938G>A (p.G980S) and c.3734T>A (p.L1245H) in the CPS1 gene. There was a high mortality rate of 77.78% (7/9) for all the defects combined. An OTC-deficient male and a CPS1-deficient female survived from episodes of hyperammonemia. Although prompt recognition of UCD and the use of alternative pathway therapy in addition to provision of appropriate nutrition and dialysis improved survival, the overall outcomes for the neonatal-onset type are poor in China.

Glucose-dependent partitioning of arginine to the urea cycle protects ß-cells from inflammation.

Chronic inflammation is linked to diverse disease processes, but the intrinsic mechanisms that determine cellular sensitivity to inflammation are incompletely understood. Here, we show the contribution of glucose metabolism to inflammation-induced changes in the survival of pancreatic islet ß-cells. Using metabolomic, biochemical and functional analyses, we investigate the protective versus non-protective effects of glucose in the presence of pro-inflammatory cytokines. When protective, glucose metabolism augments anaplerotic input into the TCA cycle via pyruvate carboxylase (PC) activity, leading to increased aspartate levels. This metabolic mechanism supports the argininosuccinate shunt, which fuels ureagenesis from arginine and conversely diminishes arginine utilization for production of nitric oxide (NO), a chief mediator of inflammatory cytotoxicity. Activation of the PC-urea cycle axis is sufficient to suppress NO synthesis and shield cells from death in the context of inflammation and other stress paradigms. Overall, these studies uncover a previously unappreciated link between glucose metabolism and arginine-utilizing pathways via PC-directed ureagenesis as a protective mechanism.

Neonatal factors related to survival and intellectual and developmental outcome of patients with early-onset urea cycle disorders.

PURPOSE: We aimed to identify prognostic factors for survival and long-term intellectual and developmental outcome in neonatal patients with early-onset urea cycle disorders (UCD) experiencing hyperammonaemic coma. METHODS: We retrospectively analysed ammonia (NH3) and glutamine levels, electroencephalogram and brain images obtained during neonatal coma of UCD patients born between 1995 and 2011 and managed at a single centre and correlated them to survival and intellectual and developmental outcome. RESULTS: We included 38 neonates suffering from deficiencies of argininosuccinate synthetase (ASSD, N = 12), ornithine transcarbamylase (OTCD, N = 10), carbamoylphosphate synthetase 1 (CPSD, N = 7), argininosuccinate lyase (ASLD, N = 7), N-acetylglutamate synthase (NAGS, N = 1) or arginase (ARGD, N = 1). Symptoms occurred earlier in mitochondrial than in cytosolic UCD. Sixty-eight percent of patients survived, with a mean (standard deviation-SD) follow-up of 10.4 (5.3) years. Mortality was mostly observed in OTCD (N = 7/10) and CPSD (N = 4/7) patients. Plasma NH3 level during the neonatal period, expressed as area under the curve, but not glutamine level was associated with mortality (p = .044 and p = .610). 62.1% of the patients had normal intellectual and developmental outcome. Intellectual and developmental outcome tended to correlate with UCD subtype (p = .052). No difference in plasma NH3 or glutamine level during the neonatal period among developmental outcomes was identified. EEG severity was linked to UCD subtypes (p = .004), ammonia levels (p = .037), duration of coma (p = .043), and mortality during the neonatal period (p = .020). Status epilepticus was recorded in 6 patients, 3 of whom died neonatally, 1 developed a severe intellectual disability while the 2 last patients had a normal development. CONCLUSION: UCD subtypes differed by survival rate, intellectual and developmental outcome and EEG features in the neonatal period. Hyperammonaemia expressed as area under the curve was associated with survival but not with intellectual and developmental outcome whereas glutamine was not associated with one of these outcomes. Prognostic value of video-EEG monitoring and the association between status epilepticus and mortality should be assessed in neonatal hyperammonaemic coma in further studies.

Proteomic Profiling of the First Human Dental Pulp Mesenchymal Stem/Stromal Cells from Carbonic Anhydrase II Deficiency Osteopetrosis Patients.

Osteopetrosis is a hereditary disorder characterized by sclerotic, thick, weak, and brittle bone. The biological behavior of mesenchymal cells obtained from osteopetrosis patients has not been well-studied. Isolated mesenchymal stem/stromal cells from dental pulp (DP-MSSCs) of recently extracted deciduous teeth from osteopetrosis (OP) patients and healthy controls (HCs) were compared. We evaluated whether the dental pulp of OP patients has a population of MSSCs with similar multilineage differentiation capability to DP-MSSCs of healthy subjects. Stem/progenitor cells were characterized using immunohistochemistry, flow cytometry, and proteomics. Our DP-MSSCs were strongly positive for CD44, CD73, CD105, and CD90. DP-MSSCs obtained from HC subjects and OP patients showed similar patterns of proliferation and differentiation as well as gene expression. Proteomic analysis identified 1499 unique proteins with 94.3% similarity in global protein fingerprints of HCs and OP patients. Interestingly, we observed subtle differences in expressed proteins of osteopetrosis disease-related in pathways, including MAPK, ERK 1/2, PI3K, and integrin, rather than in the stem cell signaling network. Our findings of similar protein expression signatures in DP-MSSCs of HC and OP patients are of paramount interest, and further in vivo validation study is needed. There is the possibility that OP patients could have their exfoliating deciduous teeth banked for future use in regenerative dentistry.

Corticospinal tract damage in HHH syndrome: a metabolic cause of hereditary spastic paraplegia.

BACKGROUND: Hyperornithinemia-hyperammonemia-homocitrullinuria (HHH) syndrome is a rare disorder of urea cycle characterized by progressive pyramidal and cerebellar dysfunction, whose pathophysiology is not yet fully understood. Here we describe the spectrum of the long fibers involvement in HHH syndrome, attempting a correlation between clinical, electrophysiological and neuro-radiological data. METHODS: Nine HHH patients were longitudinally evaluated by clinical examination, neurophysiological assessment including motor (MEPs), somato-sensory evoked potentials (PESS) and nerve conduction velocity (NCV), brain and spinal cord MRI RESULTS: All patients had pyramidal dysfunction and 3/9 an overt spastic paraplegia. Mild to moderate cerebellar signs were found in 7/9, intellectual disability in 8/9. At lower limbs, MEPs resulted abnormal in 7/8 patients and PESS in 2/8; peripheral sensory-motor neuropathy was found in 1/9. MRI documented atrophic changes in supra-tentorial brain regions in 6/9 patients, cerebellum in 6/9, spinal cord in 3/7. CONCLUSIONS: A predominant corticospinal dysfunction is evident in HHH syndrome, along with milder cerebellar signs, intellectual disability of variable degree and rare peripheral neuropathy. Phenotypical similarities with other disorders affecting the urea cycle (argininemia and pyrroline-5-carboxylate synthetase deficiency) suggest possible common mechanisms contributing in the maintenance of the corticospinal tract integrity. HHH syndrome phenotype largely overlaps with complex Hereditary Spastic Paraplegias (HSPs), in the list of which it should be included, emphasizing the importance to screen all the unsolved cases of HSPs for metabolic biomarkers.

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.

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.

The Therapeutic Hypothermia in Treatment of Hyperammonemic Encephalopathy due to Urea Cycle Disorders and Organic Acidemias.

BACKGROUND: Hyperammonemic encephalopathy in newborns with urea cycle disorders and certain organic acidurias can cause severe brain injury, coma and death. Standard therapy includes protein restriction, nitrogen-scavenging drugs, prevention of catabolism and hemodialysis. Neuroprotective hypothermia as part of the treatment has been reported only 3 times. It has been suggested that mild systemic hypothermia can contribute to better neurological outcomes in hyperammonemic encephalopathy. However, the limited experience precludes accurate conclusions on safety and efficacy. METHODS: Whole body therapeutic hypothermia was included in the standard treatment of hyperammonemic encephalopathy in 4 neonates with urea cycle disorder or organic aciduria. RESULTS: Two patients survived the initial crisis. One patient has a developmental quotient of 0.8, while the other shows severe developmental delay. The cooling protocol had to be discontinued in 3 patients due to the otherwise untreatable complications (hypotension and hemorrhage). CONCLUSION: The efficacy and safety of therapeutic hypothermia in the treatment of neonatal hyperammonemic encephalopathy depend on various factors, requiring further evaluation.