RESUMO
BACKGROUND: Carbamoyl phosphate synthetase 1 (CPS1) deficiency (OMIM 237300), an autosomal recessive rare and severe urea cycle disorder, is associated with hyperammonemia and high mortality. METHODS: Herein we present 12 genetic variants identified in seven clinically well-characterized Chinese patients with CPS1 deficiency who were admitted to the Children's Medical Center of Peking University First Hospital from September 2014 to August 2023. RESULTS: Seven patients (two male and five female patients including two sisters) experienced symptoms onset between 2 days and 13 years of age, and they were diagnosed with CPS1 deficiency between 2 months and 20 years. Peak blood ammonia levels ranged from 160 to 1,000 µmol/L. Three patients showed early-onset CPS1 deficiency, with only one surviving after treatment with sodium phenylbutyrate, N-carbamoyl-L-glutamate, and liver transplantation at 4 months, showing a favorable outcome. The remaining four patients had late-onset CPS1 deficiency, presenting with mental retardation, psychiatric symptoms, and self-selected low-protein diets. Among the 12 CPS1 variants identified in these patients, 10 were novel, with all patients exhibiting compound heterozygosity for CPS1 mutant alleles. Seven variants (c.149T > C, c.616 A > T, c.1145 C > T, c.1294G > A, c.3029 C > T, c.3503 A > T, and c.3793 C > T) resulted in single amino acid substitutions. Three frameshift variations (c.2493del, c.3067dup, and c.3241del) were identified, leading to enzyme truncation. One mutation (c.3506_3508del) caused an in-frame single amino acid deletion, while another (c.2895 + 2T > C) resulted in aberrant splicing. CONCLUSIONS: Except for two known variants, all other variants were identified as novel. No hotspot variants were observed among the patients. Our data contribute to expanding the mutation spectrum of CPS1.
Assuntos
Carbamoil-Fosfato Sintase (Amônia) , Doença da Deficiência da Carbamoil-Fosfato Sintase I , Humanos , Feminino , Masculino , Doença da Deficiência da Carbamoil-Fosfato Sintase I/genética , Carbamoil-Fosfato Sintase (Amônia)/genética , Lactente , Criança , Pré-Escolar , Adolescente , Recém-Nascido , China , Mutação , Adulto Jovem , Povo Asiático/genética , População do Leste AsiáticoRESUMO
OBJECTIVES: To investigate genotype-phenotype characteristics and long-term prognosis of neonatal carbamoyl phosphate synthetase 1 (CPS1) deficiency among children through newborn screening in Zhejiang province. METHODS: The clinical and follow-up data of children with CPS1 deficiency detected through neonatal screening and confirmed by tandem mass spectrometry and genetic testing in Zhejiang Province Newborn Disease Screening Center from September 2013 to August 2023 were retrospectively analyzed. RESULTS: A total of 4 056 755 newborns were screened and 6 cases of CPS1 deficiency were diagnosed through phenotypic and genetic testing. Ten different variations of CPS1 genewere identified in genetic testing, including 2 known pathogenic variations (c.2359C>T and c.1549+1G>T) and 8 unreported variations (c.3405-1G>T, c.2372C>T, c.1436C>T, c.2228T>C, c.2441G>A, c.3031G>A, c.3075T>C and c.390-403del). All patients had decreased citrulline levels (2.72-6.21 µmol/L), and varying degrees of elevated blood ammonia. The patients received restricted natural protein intake (special formula), arginine and supportive therapy after diagnosis, and were followed-up for a period ranging from 9 months to 10 years. Three patients experienced hyperammonemia, and one patient each had attention deficit hyperactivity disorder, transient facial twitching and increased muscle tone. One patient died, while the other five surviving patients had normal scores of the Ages & Stages Questionnaires (ASQ) and Griffiths Development Scales up to the present time; 4 cases had combined height or weight lag and one case was normal in height and weight. CONCLUSIONS: Low citrulline levels and hyperammonemia are common in CPS1 deficiency patients in Zhejiang. Most gene variants identified were specific to individual families, and no hotspot mutations were found. Early diagnosis through newborn screening and following standardized treatment can significantly improve the prognosis of the patients.
Assuntos
Doença da Deficiência da Carbamoil-Fosfato Sintase I , Hiperamonemia , Criança , Humanos , Recém-Nascido , Doença da Deficiência da Carbamoil-Fosfato Sintase I/diagnóstico , Doença da Deficiência da Carbamoil-Fosfato Sintase I/genética , Doença da Deficiência da Carbamoil-Fosfato Sintase I/terapia , Triagem Neonatal , Seguimentos , Citrulina/genética , Estudos Retrospectivos , MutaçãoRESUMO
BACKGROUND: Carbamoyl phosphate synthetase I defect (CPS1D) is a rare disease with clinical case reports mainly in early neonates or adults, with few reports of first onset in late neonatal to childhood. We studied the clinical and genotypic characteristics of children with childhood onset CPS1D caused by two loci mutations (one of these is a rarely reported non-frame shift mutation) in the CPS1. CASE PRESENTATION: We present a rare case of adolescent-onset CPS1D that had been misdiagnosed due to atypical clinical features, and further investigations revealed severe hyperammonemia (287µmol/L; reference range 11.2 ~ 48.2umol/L). MRI of the brain showed diffuse white matter lesions. Blood genetic metabolic screening showed elevated blood alanine (757.06umol/L; reference range 148.8 ~ 739.74umol/L) and decreased blood citrulline (4.26umol/L; reference range 5.45 ~ 36.77umol/L). Urine metabolic screening showed normal whey acids and uracil. Whole-exome sequencing revealed compound heterozygous mutations in the CPS1, a missense mutation (c.1145 C > T) and an unreported de novo non-frame shift mutation (c.4080_c.4091delAGGCATCCTGAT), respectively, which provided a clinical diagnosis. CONCLUSION: A comprehensive description of the clinical and genetic features of this patient, who has a rare age of onset and a relatively atypical clinical presentation, will facilitate the early diagnosis and management of this type of late onset CPS1D and reduce misdiagnosis, thus helping to reduce mortality and improve prognosis. It also provides a preliminary understanding of the relationship between genotype and phenotype, based on a summary of previous studies, which reminds us that it may help to explore the pathogenesis of the disease and contribute to genetic counselling and prenatal diagnosis.
Assuntos
Doença da Deficiência da Carbamoil-Fosfato Sintase I , Carbamoil-Fosfato , Humanos , Glicogênio Sintase/genética , Doença da Deficiência da Carbamoil-Fosfato Sintase I/genética , Doença da Deficiência da Carbamoil-Fosfato Sintase I/diagnóstico , Doença da Deficiência da Carbamoil-Fosfato Sintase I/patologia , Mutação , Carbamoil-Fosfato Sintase (Amônia)/genética , Carbamoil-Fosfato Sintase (Amônia)/metabolismoRESUMO
PURPOSE: Tumor radiation resistance is the main obstacle to effective radiation therapy for patients with hepatocellular carcinoma (HCC). We identified the role of urea cycle key enzyme carbamoyl phosphate synthetase 1 (CPS1) in radioresistance of HCC and explored its mechanism, aiming to provide a novel radiosensitization strategy for the CPS1-deficiency HCC subtype. METHODS AND MATERIALS: The expression of CPS1 was measured by western blot and immunohistochemistry. Cell growth assay, EdU assay, cell apoptosis assay, cell cycle assay, clone formation assay, and subcutaneous tumor assay were performed to explore the relationship between CPS1 and radioresistance of HCC cells. Lipid metabonomic analysis was used for investigating the effects of CPS1 on lipid synthesis of HCC cells. RNA sequencing and coimmunoprecipitation assay were carried out to reveal the mechanism of CPS1 participating in the regulation of HCC radiation therapy resistance. Furthermore, 10074-G5, the specific inhibitor of c-Myc, was administered to HCC cells to investigate the role of c-Myc in CPS1-deficiency HCC cells. RESULTS: We found that urea cycle key enzyme CPS1 was frequently lower in human HCC samples and positively associated with the patient's prognosis. Functionally, the present study proved that CPS1 depletion could accelerate the development of HCC and induce radiation resistance of HCC in vitro and in vivo, and deficiency of CPS1 promoted the synthesis of some lipid molecules. Regarding the mechanism, we uncovered that inhibition of CPS1 upregulated CyclinA2 and CyclinD1 by stabilizing oncoprotein c-Myc at the posttranscriptional level and generated radioresistance of HCC cells. Moreover, inactivation of c-Myc using 10074-G5, a specific c-Myc inhibitor, could partially attenuate the proliferation and radioresistance induced by depletion of CPS1. CONCLUSIONS: Our results recapitulated that silencing CPS1 could promote HCC progression and radioresistance via c-Myc stability mediated by the ubiquitin-proteasome system, suggesting that targeting c-Myc in CPS1-deficiency HCC subtype may be a valuable radiosensitization strategy in the treatment of HCC.
Assuntos
Doença da Deficiência da Carbamoil-Fosfato Sintase I , Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Carcinoma Hepatocelular/radioterapia , Carcinoma Hepatocelular/metabolismo , Carbamoil-Fosfato , Neoplasias Hepáticas/radioterapia , Neoplasias Hepáticas/metabolismo , Carbamoil-Fosfato Sintase (Amônia)/química , Carbamoil-Fosfato Sintase (Amônia)/genética , Carbamoil-Fosfato Sintase (Amônia)/metabolismo , Doença da Deficiência da Carbamoil-Fosfato Sintase I/genética , Doença da Deficiência da Carbamoil-Fosfato Sintase I/metabolismo , Doença da Deficiência da Carbamoil-Fosfato Sintase I/patologia , Ureia , Lipídeos , Linhagem Celular TumoralRESUMO
PURPOSE: Carbamoyl phosphate synthetase 1 (CPS1) deficiency affects the first step of urea cycle and is a severe form of urea cycle disorder (UCD). The severity of hyperammonemic encephalopathy determines the clinical course of UCDs. Here, we describe the genetic and clinical characteristics of CPS1 deficiency in Korea. PATIENT AND METHODS: This study included seven patients with CPS1 deficiency genetically confirmed from January 1992 to September 2020. The peak ammonia level during the first crisis, the half time of peak ammonia level, the initial plasma amino acid levels, and neurological outcomes were compared between CPS1 deficiency and two common UCDs (i.e., 17 patients with argininosuccinate synthetase 1 deficiency and 24 patients with ornithine transcarbamylase deficiency). RESULT: Eleven CPS1 mutations were identified, including 10 novel mutations. Eight mutations were missense. Six patients with CPS1 deficiency had neonatal type. The peak ammonia level, initial glutamate level, and accompanying rate of irreversible neurological damages were highest in patients with CPS1 deficiency. The patient with late-onset CPS1 deficiency responded dramatically to N-carbamylglutamate treatment. CONCLUSION: The clinical manifestations of CPS1 deficiency were the most severe among UCDs. Considering the high proportion of missense mutations, responsiveness to N-carbamylglutamate would be evaluated in a future study.
Assuntos
Carbamoil-Fosfato Sintase (Amônia) , Doença da Deficiência da Carbamoil-Fosfato Sintase I , Distúrbios Congênitos do Ciclo da Ureia , Carbamoil-Fosfato Sintase (Amônia)/deficiência , Carbamoil-Fosfato Sintase (Amônia)/genética , Doença da Deficiência da Carbamoil-Fosfato Sintase I/diagnóstico , Doença da Deficiência da Carbamoil-Fosfato Sintase I/genética , Carbamoil-Fosfato , Humanos , Recém-Nascido , Mutação , Distúrbios Congênitos do Ciclo da Ureia/diagnóstico , Distúrbios Congênitos do Ciclo da Ureia/genéticaRESUMO
A 36-year-old man experienced severely impaired consciousness twice after drinking because of hyperammonemia. No abnormal blood tests were found other than ammonia levels. However, magnetic resonance imaging (MRI) showed atrophy of the brain parenchyma. One the second occasion, the patient suffered severe impairment of consciousness, and because of seizures and glossoptosis, mechanical ventilation was started. Urea cycle disorders (UCDs) were assumed to be involved. Genetic testing revealed a monoallelic mutation of the carbamoyl phosphate synthase 1 (CPS1) gene. When transient hyperammonemia of unknown cause occurs repeatedly in adults, an active investigation for UCDs should be conducted.
Assuntos
Doença da Deficiência da Carbamoil-Fosfato Sintase I , Hiperamonemia , Distúrbios Congênitos do Ciclo da Ureia , Adulto , Carbamoil-Fosfato Sintase (Amônia)/genética , Doença da Deficiência da Carbamoil-Fosfato Sintase I/complicações , Doença da Deficiência da Carbamoil-Fosfato Sintase I/genética , Carbamoil-Fosfato , Estado de Consciência , Humanos , Hiperamonemia/complicações , Hiperamonemia/diagnóstico , Hiperamonemia/genética , Masculino , Mutação/genética , Distúrbios Congênitos do Ciclo da Ureia/complicaçõesRESUMO
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.
Assuntos
Carbamoil-Fosfato Sintase (Amônia)/genética , Doença da Deficiência da Carbamoil-Fosfato Sintase I/genética , Distúrbios Congênitos do Ciclo da Ureia/genética , Ureia/metabolismo , Amônia/metabolismo , Carbamoil-Fosfato Sintase (Amônia)/metabolismo , Doença da Deficiência da Carbamoil-Fosfato Sintase I/metabolismo , Doença da Deficiência da Carbamoil-Fosfato Sintase I/patologia , Homeostase/genética , Humanos , Distúrbios Congênitos do Ciclo da Ureia/metabolismo , Distúrbios Congênitos do Ciclo da Ureia/patologiaRESUMO
The urea cycle enzyme carbamoyl phosphate synthetase 1 (CPS1) catalyzes the initial step of the urea cycle; bi-allelic mutations typically present with hyperammonemia, vomiting, ataxia, lethargy progressing into coma, and death due to brain edema if ineffectively treated. The enzyme deficiency is particularly difficult to treat; early recognition is essential to minimize injury to the brain. Even under optimal conditions, therapeutic interventions are of limited scope and efficacy, with most patients developing long-term neurologic sequelae. One significant encumberment to gene therapeutic development is the size of the CPS1 cDNA, which, at 4.5 kb, nears the packaging capacity of adeno-associated virus (AAV). Herein we developed a split AAV (sAAV)-based approach, packaging the large transgene and its regulatory cassette into two separate vectors, thereby delivering therapeutic CPS1 by a dual vector system with testing in a murine model of the disorder. Cps1-deficient mice treated with sAAVs survive long-term with markedly improved ammonia levels, diminished dysregulation of circulating amino acids, and increased hepatic CPS1 expression and activity. In response to acute ammonia challenging, sAAV-treated female mice rapidly incorporated nitrogen into urea. This study demonstrates the first proof-of-principle that sAAV-mediated therapy is a viable, potentially clinically translatable approach to CPS1 deficiency, a devastating urea cycle disorder.
Assuntos
Carbamoil-Fosfato Sintase (Amônia)/genética , Doença da Deficiência da Carbamoil-Fosfato Sintase I/terapia , Dependovirus/genética , Ureia/metabolismo , Amônia/metabolismo , Animais , Doença da Deficiência da Carbamoil-Fosfato Sintase I/genética , Doença da Deficiência da Carbamoil-Fosfato Sintase I/metabolismo , Empacotamento do DNA , Modelos Animais de Doenças , Feminino , Terapia Genética , Vetores Genéticos/administração & dosagem , Humanos , Camundongos , Estudo de Prova de ConceitoRESUMO
The enzyme carbamoyl phosphate synthetase 1 (CPS1; EC 6.3.4.16) forms carbamoyl phosphate from bicarbonate, ammonia, and adenosine triphosphate (ATP) and is activated allosterically by N-acetylglutamate. The neonatal presentation of bi-allelic mutations of CPS1 results in hyperammonemia with reduced citrulline and is reported as the most challenging nitrogen metabolism disorder to treat. As therapeutic interventions are limited, patients often develop neurological injury or die from hyperammonemia. Survivors remain vulnerable to nitrogen overload, being at risk for repetitive neurological injury. With transgenic technology, our lab developed a constitutive Cps1 mutant mouse and reports its characterization herein. Within 24 hours of birth, all Cps1 -/- mice developed hyperammonemia and expired. No CPS1 protein by Western blot or immunostaining was detected in livers nor was Cps1 mRNA present. CPS1 enzymatic activity was markedly decreased in knockout livers and reduced in Cps1+/- mice. Plasma analysis found markedly reduced citrulline and arginine and markedly increased glutamine and alanine, both intermolecular carriers of nitrogen, along with elevated ammonia, taurine, and lysine. Derangements in multiple other amino acids were also detected. While hepatic amino acids also demonstrated markedly reduced citrulline, arginine, while decreased, was not statistically significant; alanine and lysine were markedly increased while glutamine was trending towards significance. In conclusion we have determined that this constitutive neonatal mouse model of CPS1 deficiency replicates the neonatal human phenotype and demonstrates the key biochemical features of the disorder. These mice will be integral for addressing the challenges of developing new therapeutic approaches for this, at present, poorly treated disorder.
Assuntos
Carbamoil-Fosfato Sintase (Amônia)/genética , Doença da Deficiência da Carbamoil-Fosfato Sintase I/complicações , Doença da Deficiência da Carbamoil-Fosfato Sintase I/mortalidade , Glutamina/sangue , Hiperamonemia , Animais , Animais Recém-Nascidos , Carbamoil-Fosfato Sintase (Amônia)/deficiência , Doença da Deficiência da Carbamoil-Fosfato Sintase I/sangue , Doença da Deficiência da Carbamoil-Fosfato Sintase I/genética , Hiperamonemia/sangue , Hiperamonemia/complicações , Hiperamonemia/genética , Hiperamonemia/mortalidade , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , MutaçãoRESUMO
A liver-humanized mouse model for CPS1-deficiency was generated by the high-level repopulation of the mouse liver with CPS1-deficient human hepatocytes. When compared with mice that are highly repopulated with CPS1-proficient human hepatocytes, mice that are repopulated with CPS1-deficient human hepatocytes exhibited characteristic symptoms of human CPS1 deficiency including an 80% reduction in CPS1 metabolic activity, delayed clearance of an ammonium chloride infusion, elevated glutamine and glutamate levels, and impaired metabolism of [15 N]ammonium chloride into urea, with no other obvious phenotypic differences. Because most metabolic liver diseases result from mutations that alter critical pathways in hepatocytes, a model that incorporates actual disease-affected, mutant human hepatocytes is useful for the investigation of the molecular, biochemical, and phenotypic differences induced by that mutation. The model is also expected to be useful for investigations of modified RNA, gene, and cellular and small molecule therapies for CPS1-deficiency. Liver-humanized models for this and other monogenic liver diseases afford the ability to assess the therapy on actual disease-affected human hepatocytes, in vivo, for long periods of time and will provide data that are highly relevant for investigations of the safety and efficacy of gene-editing technologies directed to human hepatocytes and the translation of gene-editing technology to the clinic.
Assuntos
Carbamoil-Fosfato Sintase (Amônia)/genética , Doença da Deficiência da Carbamoil-Fosfato Sintase I/genética , Doença da Deficiência da Carbamoil-Fosfato Sintase I/patologia , Hepatócitos/transplante , Hidrolases/genética , Fígado/metabolismo , Animais , Carbamoil-Fosfato Sintase (Amônia)/metabolismo , Células Cultivadas , Criança , Modelos Animais de Doenças , Feminino , Hepatócitos/metabolismo , Humanos , Hidrolases/metabolismo , Lactente , Recém-Nascido , Fígado/patologia , Masculino , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Especificidade de Órgãos/genéticaRESUMO
The urea cycle and glutamine synthetase (GS) are the two main pathways for waste nitrogen removal and their deficiency results in hyperammonemia. Here, we investigated the efficacy of liver-specific GS overexpression for therapy of hyperammonemia. To achieve hepatic GS overexpression, we generated a helper-dependent adenoviral (HDAd) vector expressing the murine GS under the control of a liver-specific expression cassette (HDAd-GS). Compared to mice injected with a control vector expressing an unrelated reporter gene (HDAd-alpha-fetoprotein), wild-type mice with increased hepatic GS showed reduced blood ammonia levels and a concomitant increase of blood glutamine after intraperitoneal injections of ammonium chloride, whereas blood urea was unaffected. Moreover, injection of HDAd-GS reduced blood ammonia levels at baseline and protected against acute hyperammonemia following ammonia challenge in a mouse model with conditional hepatic deficiency of carbamoyl phosphate synthetase 1 (Cps1), the initial and rate-limiting step of ureagenesis. In summary, we found that upregulation of hepatic GS reduced hyperammonemia in wild-type and Cps1-deficient mice, thus confirming a key role of GS in ammonia detoxification. These results suggest that hepatic GS augmentation therapy has potential for treatment of both primary and secondary forms of hyperammonemia.
Assuntos
Amônia/metabolismo , Terapia Genética/métodos , Glutamato-Amônia Ligase/genética , Hiperamonemia/genética , Hiperamonemia/terapia , Fígado/metabolismo , Amônia/toxicidade , Animais , Carbamoil-Fosfato Sintase (Amônia)/genética , Carbamoil-Fosfato Sintase (Amônia)/metabolismo , Doença da Deficiência da Carbamoil-Fosfato Sintase I/genética , Doença da Deficiência da Carbamoil-Fosfato Sintase I/metabolismo , Doença da Deficiência da Carbamoil-Fosfato Sintase I/terapia , Modelos Animais de Doenças , Feminino , Técnicas de Transferência de Genes , Glutamato-Amônia Ligase/metabolismo , Hiperamonemia/metabolismo , Hiperamonemia/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Especificidade de Órgãos/genéticaRESUMO
OBJECTIVE: To explore the genetic basis for a neonate featuring hyperammonemia. METHODS: The patient was examined and tested by tandem mass spectrometry and next generation sequencing (NGS). Suspected mutations were confirmed by Sanger sequencing of the proband and her parents. Potential impact of the mutation was predicted with SIFT, PolyPhen-2 and MutationTaste software. RESULTS: Plasma ammonia and alanine were significantly increased in the proband, while serum citrulline was decreased. The neonate was found to harbor compound heterozygous mutations of the CPS1 gene [c.1631C>T(p.T544M) and c.1981G>T(p.G661C)], which were respectively inherited from her father and mother. CONCLUSION: The carbamoyl phosphate synthetase I deficiency of the proband can probably be attributed to the mutations of the CPS1 gene. Above finding has expanded the spectrum of CPS1 mutations in association with carbamoyl phosphate synthetase I deficiency.
Assuntos
Carbamoil-Fosfato Sintase (Amônia)/genética , Doença da Deficiência da Carbamoil-Fosfato Sintase I/genética , Hiperamonemia/diagnóstico , Hiperamonemia/genética , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Recém-Nascido , MutaçãoRESUMO
Carbamoyl phosphate synthetase 1 (CPS1) is a urea cycle enzyme that forms carbamoyl phosphate from bicarbonate, ammonia and ATP. Bi-allelic mutations of the CPS1 gene result in a urea cycle disorder presenting with hyperammonemia, often with reduced citrulline, and without orotic aciduria. CPS1 deficiency is particularly challenging to treat and lack of early recognition typically results in early neonatal death. Therapeutic interventions have limited efficacy and most patients develop long-term neurologic sequelae. Using transgenic techniques, we generated a conditional Cps1 knockout mouse. By loxP/Cre recombinase technology, deletion of the Cps1 locus was achieved in adult transgenic animals using a Cre recombinase-expressing adeno-associated viral vector. Within four weeks from vector injection, all animals developed hyperammonemia without orotic aciduria and died. Minimal CPS1 protein was detectable in livers. To investigate the efficacy of gene therapy for CPS deficiency following knock-down of hepatic endogenous CPS1 expression, we injected these mice with a helper-dependent adenoviral vector (HDAd) expressing the large murine CPS1 cDNA under control of the phosphoenolpyruvate carboxykinase promoter. Liver-directed HDAd-mediated gene therapy resulted in survival, normalization of plasma ammonia and glutamine, and 13% of normal Cps1 expression. A gender difference in survival suggests that female mice may require higher hepatic CPS1 expression. We conclude that this conditional murine model recapitulates the clinical and biochemical phenotype detected in human patients with CPS1 deficiency and will be useful to investigate ammonia-mediated neurotoxicity and for the development of cell- and gene-based therapeutic approaches.
Assuntos
Carbamoil-Fosfato Sintase (Amônia)/genética , Doença da Deficiência da Carbamoil-Fosfato Sintase I/terapia , Terapia Genética , Hiperamonemia/terapia , Amônia/metabolismo , Animais , Carbamoil-Fosfato Sintase (Amônia)/uso terapêutico , Doença da Deficiência da Carbamoil-Fosfato Sintase I/genética , Doença da Deficiência da Carbamoil-Fosfato Sintase I/metabolismo , Doença da Deficiência da Carbamoil-Fosfato Sintase I/patologia , Carbamoil-Fosfato/metabolismo , Feminino , Regulação Enzimológica da Expressão Gênica , Glutamina/metabolismo , Humanos , Hiperamonemia/genética , Hiperamonemia/metabolismo , Hiperamonemia/patologia , Fígado/enzimologia , Fígado/patologia , Masculino , Camundongos , Camundongos Knockout , Mutação , Orotato Fosforribosiltransferase/deficiência , Orotato Fosforribosiltransferase/genética , Orotidina-5'-Fosfato Descarboxilase/deficiência , Orotidina-5'-Fosfato Descarboxilase/genética , Erros Inatos do Metabolismo da Purina-Pirimidina/genética , Erros Inatos do Metabolismo da Purina-Pirimidina/patologiaRESUMO
BACKGROUND: Carbamoyl phosphate synthetase 1 deficiency (CPS1D) is a rare autosomal recessive disorder of the urea cycle, mostly characterized by hyperammonemia and the concomitant leukodystrophy. The onset of CPS1D can be at any age, and the clinical manifestations are variable and atypical. Genetic tests are indispensable for accurate diagnosis of CPS1D on the basis of biochemical tests. METHODS: Blood tandem mass spectrometric analysis and urea organic acidemia screening were performed on a Chinese neonatal patient with low activity, recurrent seizures, and hyperammonemia. Next-generation sequencing and Sanger sequencing were followed up for making a definite diagnosis. Bioinformatics tools were used for the conservation analysis and pathogenicity predictions of the identified mutations. RESULTS: Increased lactate in urea and decreased citrulline in blood were detected in the patient. Two novel mutations (c.173G>T, p.G58V in exon 2 and c.796G>A, p.G266R in exon 8) in CPS1 identified in the neonatal patient were found through coseparation verification. Both of the two mutations were predicted to be deleterious, and the two relevant amino acids exerted highly evolutionarily conserved. The final diagnosis of the patient was compound heterozygous CPS1D. CONCLUSION: This study described the specific clinical characteristics and the variations of physiological and biochemical indices in a Chinese neonatal patient with CPS1D, which facilitated the diagnosis and mechanism research of the disease. Two novel causative missense mutations were identified, which enriched the mutation spectrum of CPS1D in China and worldwide. Advice of prenatal diagnosis was given to the family for a new pregnancy.
Assuntos
Carbamoil-Fosfato Sintase (Amônia)/genética , Doença da Deficiência da Carbamoil-Fosfato Sintase I/complicações , Doença da Deficiência da Carbamoil-Fosfato Sintase I/genética , Hiperamonemia/etiologia , Doenças por Armazenamento dos Lisossomos do Sistema Nervoso/etiologia , Mutação/genética , Carbamoil-Fosfato Sintase (Amônia)/metabolismo , Análise Mutacional de DNA , Saúde da Família , Feminino , Genótipo , Humanos , Masculino , Modelos Moleculares , Espectrometria de Massas em TandemRESUMO
BACKGROUND: Carbamoyl Phosphate Synthetase 1 deficiency (CPS1D) is a rare autosomal recessive inborn metabolic disease characterized mainly by hyperammonemia. The fatal nature of CPS1D and its similar symptoms with other urea cycle disorders (UCDs) make its diagnosis difficult, and the molecular diagnosis is hindered due to the large size of the causative gene CPS1. Therefore, the objective of the present study was to investigate the clinical applicability of exome sequencing in molecular diagnosis of CPS1D in Chinese population. METHODS: We described two Chinese neonates presented with unconsciousness and drowsiness due to deepening encephalopathy with hyperammonemia. Whole exome sequencing was performed. Candidate mutations were validated by Sanger sequencing. In-silicon analysis was processed for the pathogenicity predictions of the identified mutations. RESULTS: Two compound heterozygous mutations in the gene carbamoyl phosphate synthetase 1(CPS1) were identified. One is in Case 1 with two novel missense mutations (c.2537C>T, p. Pro846Leu and c.3443T>A, p.Met1148Lys), and the other one is in Case 2 with a novel missense mutation (c.1799G>A, p.Cys600Tyr) and a previously reported 12-bp deletion (c.4088_4099del, p.Leu 1363_Ile1366del). Bioinformatics deleterious predictions indicated pathogenicity of the missense mutations. Conversation analysis and homology modeling showed that the substituted amino acids were highly evolutionary conserved and necessary for enzyme stability or function. CONCLUSION: The present study initially and successfully applied whole exome sequencing to the molecular diagnosis of CPS1D in Chinese neonates, indicating its applicability in cost-effective molecular diagnosis of CPS1D. Three novel pathogenic missense mutations were identified, expanded the mutational spectrum of the CPS1 gene.
Assuntos
Carbamoil-Fosfato Sintase (Amônia)/genética , Doença da Deficiência da Carbamoil-Fosfato Sintase I/diagnóstico , Doença da Deficiência da Carbamoil-Fosfato Sintase I/genética , Sequenciamento do Exoma/métodos , Técnicas de Diagnóstico Molecular/métodos , Feminino , Humanos , Recém-Nascido , Masculino , Modelos Moleculares , Mutação/genéticaRESUMO
RATIONALE: The carbamoyl phosphate synthetase I deficiency (CPS1D) was rare and hard to diagnose due to its atypical symptoms. Brain magnetic resonance imaging (MRI) was typically unavailable in other reports because most patients died before diagnosis was confirmed. Furthermore, it was found a new mutation that had not been described previously. PATIENT CONCERNS: This is a case of neonatal-onset CPS1D with nonspecific clinical manifestations and deteriorating rapidly. Poor feeding, low activity, and tachypnoea were observed, with rapid progression on day 2 after birth. Severe systematic infection was considered first. However, blood culture and cerebrospinal fluid examination were negative. Symptoms were relief temporarily. Then seizure and tachypnoea reappeared as intravenous amino acids were provided. Further examination indicated severe hyperammonemia (serum ammonia level >500mmol/L). Brain MRI showed diffused white matter lesions. DIAGNOSES: Genetic analysis revealed 2 heterozygous mutations in the CPS1 gene: c.2407C>G (p.803, R>G) in exon 20 and C.323G>A (p.108, G>E) in exon 4. The diagnosis of CSP1D was confirmed. INTERVENTIONS: Fasting, the withdrawal of amino acids and plans to treat hyperammonemia were immediately implemented. OUTCOMES: The parents decided to discontinue medical care. LESSONS: Many CPS1D patients died before the diagnoses are confirmed due to its sudden onset, rapid deterioration, atypical symptoms, and low morbidity. Once hyperammonemia is confirmed, blood and urea amino acid analysis in combination with genetic examinations should be performed as early as possible, this approach would help establish diagnoses at an early stage and thus contribute to reducing mortality and improving prognosis.
Assuntos
Doença da Deficiência da Carbamoil-Fosfato Sintase I/diagnóstico , Encéfalo/diagnóstico por imagem , Carbamoil-Fosfato Sintase (Amônia)/genética , Doença da Deficiência da Carbamoil-Fosfato Sintase I/genética , Doença da Deficiência da Carbamoil-Fosfato Sintase I/terapia , Diagnóstico Precoce , Feminino , Humanos , Recém-Nascido , Recusa do Paciente ao TratamentoRESUMO
The urea cycle disorder carbamoyl phosphate synthetase I deficiency is an important differential diagnosis in the encephalopathic neonate. This intoxication type inborn error of metabolism often leads to neonatal death or severe and irreversible damage of the central nervous system, even despite appropriate treatment. Timely diagnosis is crucial, but can be difficult on routine metabolite level. Here, we report ten neonates from eight families (finally) diagnosed with CPS1 deficiency at three tertiary metabolic centres. In seven of them the laboratory findings were dominated by significantly elevated urinary 3-methylglutaconic acid levels which complicated the diagnostic process. Our findings are both important for the differential diagnosis of patients with urea cycle disorders and also broaden the differential diagnosis of hyperammonemia associated with 3-methylglutaconic aciduria, which was earlier only reported in TMEM70 and SERAC1 defect.
Assuntos
Doença da Deficiência da Carbamoil-Fosfato Sintase I/urina , Glutaratos/urina , Doença da Deficiência da Carbamoil-Fosfato Sintase I/diagnóstico , Doença da Deficiência da Carbamoil-Fosfato Sintase I/genética , Feminino , Humanos , Recém-Nascido , Masculino , Mutação , LinhagemRESUMO
INTRODUCTION: Carbamoyl phosphate synthetase 1 (CPS1) deficiency (CPS1D) is a rare autosomal recessive urea cycle disorder (UCD), which can lead to life-threatening hyperammonemia. Unless promptly treated, it can result in encephalopathy, coma and death, or intellectual disability in surviving patients. Over recent decades, therapies for CPS1D have barely improved leaving the management of these patients largely unchanged. Additionally, in many cases, current management (protein-restriction and supplementation with citrulline and/or arginine and ammonia scavengers) is insufficient for achieving metabolic stability, highlighting the importance of developing alternative therapeutic approaches. Areas covered: After describing UCDs and CPS1D, we give an overview of the structure- function of CPS1. We then describe current management and potential novel treatments including N-carbamoyl-L-glutamate (NCG), pharmacological chaperones, and gene therapy to treat hyperammonemia. Expert opinion: Probably, the first novel CPS1D therapies to reach the clinics will be the already commercial substance NCG, which is the standard treatment for N-acetylglutamate synthase deficiency and has been proven to rescue specific CPS1D mutations. Pharmacological chaperones and gene therapy are under development too, but these two technologies still have key challenges to be overcome. In addition, current experimental therapies will hopefully add further treatment options.
Assuntos
Carbamoil-Fosfato Sintase (Amônia)/metabolismo , Doença da Deficiência da Carbamoil-Fosfato Sintase I/terapia , Glutamatos/uso terapêutico , Animais , Carbamoil-Fosfato Sintase (Amônia)/genética , Doença da Deficiência da Carbamoil-Fosfato Sintase I/genética , Doença da Deficiência da Carbamoil-Fosfato Sintase I/fisiopatologia , Terapia Genética/métodos , Humanos , Chaperonas Moleculares/farmacologia , Mutação , Distúrbios Congênitos do Ciclo da Ureia/fisiopatologia , Distúrbios Congênitos do Ciclo da Ureia/terapiaRESUMO
Diagnosis of the urea cycle disorder (USD) carbamoyl-phosphate synthetase 1 (CPS1) deficiency (CPS1D) based on only the measurements of biochemical intermediary metabolites is not sufficient to properly exclude other UCDs with similar symptoms. We report the first Korean CPS1D patient using whole exome sequencing (WES). A four-day-old female neonate presented with respiratory failure due to severe metabolic encephalopathy with hyperammonemia (1,690 µmol/L; reference range, 11.2-48.2 µmol/L). Plasma amino acid analysis revealed markedly elevated levels of alanine (2,923 µmol/L; reference range, 131-710 µmol/L) and glutamine (5,777 µmol/L; reference range, 376-709 µmol/L), whereas that of citrulline was decreased (2 µmol/L; reference range, 10-45 µmol/L). WES revealed compound heterozygous pathogenic variants in the CPS1 gene: one novel nonsense pathogenic variant of c.580C>T (p.Gln194*) and one known pathogenic frameshift pathogenic variant of c.1547delG (p.Gly516Alafs*5), which was previously reported in Japanese patients with CPS1D. We successfully applied WES to molecularly diagnose the first Korean patient with CPS1D in a clinical setting. This result supports the clinical applicability of WES for cost-effective molecular diagnosis of UCDs.
Assuntos
Carbamoil-Fosfato Sintase (Amônia)/genética , Doença da Deficiência da Carbamoil-Fosfato Sintase I/genética , Sequência de Bases , Carbamoil-Fosfato Sintase (Amônia)/química , Doença da Deficiência da Carbamoil-Fosfato Sintase I/diagnóstico , Códon sem Sentido , Éxons , Feminino , Mutação da Fase de Leitura , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Recém-Nascido , República da Coreia , Análise de Sequência de DNA , Distúrbios Congênitos do Ciclo da Ureia/diagnósticoRESUMO
UNLABELLED: Carbamoyl phosphate synthetase 1 (CPS1) deficiency is a rare autosomal recessive disorder of ureagenesis presenting as life-threatening hyperammonemia. In this study, we present the main clinical features and biochemical and molecular data of six Malaysian patients with CPS1 deficiency. All the patients have neonatal-onset symptoms, initially diagnosed as infections before hyperammonemia was recognized. They have typical biochemical findings of hyperglutaminemia, hypocitrullinemia, and low to normal urinary excretion of orotate. One neonate succumbed to the first hyperammonemic decompensation. Five neonatal survivors received long-term treatment consisting of dietary protein restriction and ammonia-scavenging drugs. They have delayed neurocognitive development of varying severity. Genetic analysis revealed eight mutations in CPS1 gene, five of which were not previously reported. Five mutations were missense changes while another three were predicted to create premature stop codons. In silico analyses showed that these new mutations affected different CPS1 enzyme domains and were predicted to interrupt interactions at enzyme active sites, disturb local enzyme conformation, and destabilize assembly of intact enzyme complex. CONCLUSION: All mutations are private except one mutation; p.Ile1254Phe was found in three unrelated families. Identification of a recurrent p.Ile1254Phe mutation suggests the presence of a common and unique mutation in our population. Our study also expands the mutational spectrum of the CPS1 gene.