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PURPOSE: Clinical next-generation sequencing is an effective approach for identifying pathogenic sequence variants that are medically actionable for participants and families but are not associated with the participant's primary diagnosis. These variants are called secondary findings (SFs). According to the literature, there is no report of the types and frequencies of SFs in a large pediatric cohort which includes substantial African-American participants. We sought to investigate the types (including American College of Medical Genetics and Genomics [ACMG] and non-ACMG recommended gene lists), frequencies, and rates of SFs, as well as the effects of SF disclosure on the participants and families of a large pediatric cohort at the Center for Applied Genomics at The Children's Hospital of Philadelphia (CHOP). METHODS: We systematically identified pathogenic (P) and likely pathogenic (LP) variants in established disease-causing genes, adhering to ACMG v3.2 secondary finding guidelines and beyond. For non-ACMG secondary findings, akin to incidental findings in clinical settings, we utilized a set of criteria focusing on pediatric onset, high penetrance, moderate to severe phenotypes, and the clinical actionability of the variants. This criteria-based approach was applied rather than using a fixed gene list to ensure that the variants identified are likely to impact participant health significantly. To identify and categorize these variants, we employed a clinical-grade variant classification standard per ACMG/AMP recommendations; additionally, we conducted a detailed literature search to ensure a comprehensive exploration of potential secondary findings relevant to pediatric participants. RESULTS: We report a distinctive distribution of 1,464 P/LP SF variants in 16,713 participants. There were 427 unique variants in ACMG genes and 265 in non-ACMG genes. The most frequently mutated genes among the ACMG and non-ACMG gene lists were TTR (41.6%) and CHEK2 (7.16%), respectively. Overall, variants of possible medical importance were found in 8.76% of participants in both ACMG (5.81%) and non-ACMG (2.95%) genes.
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Urea cycle disorders (UCDs) are a group of rare inherited metabolic diseases caused by a deficiency of one of the enzymes or transporters that constitute the urea cycle. Defects in these enzymes lead to acute accumulation (hyperammonemic crises, HAC) or chronically elevated levels (hyperammonemia) of ammonia in the blood and/or various tissues including the brain, which can cause persistent neurological deficits, irreversible brain damage, coma, and death. Ongoing treatment of UCDs include the use of nitrogen-scavenging agents, such as sodium phenylbutyrate (salt of 4-phenylbutyric acid; NaPBA) or glycerol phenylbutyrate (GPB). These treatments provide an alternative pathway for nitrogen disposal through the urinary excretion of phenylacetylglutamine. ACER-001 is a novel formulation of NaPBA with polymer coated pellets in suspension, which is designed to briefly mask the unpleasant bitter taste of NaPBA and is being developed as a treatment option for patients with UCDs. Four Phase 1 studies were conducted to characterize the bioavailability (BA) and/or bioequivalence (BE) of ACER-001 (in healthy volunteers) and taste assessment relative to NaPBA powder (in taste panelists). ACER-001 was shown to be bioequivalent to NaPBA powder under both fed and fasting conditions. Lower systemic exposure of phenylacetate (PAA) and phenylbutyrate (PBA) was observed when ACER-001 was administered with a high-fat meal relative to a fasting state suggesting that the lower doses of PBA administered under fasting conditions may yield similar efficacy with potentially fewer dose dependent adverse effects relative to higher doses with a meal. ACER-001 appeared to be adequately taste-masked, staying below the aversive taste threshold for the first 3 min after the formulation was prepared and remaining palatable when taken within 5 min.
Assuntos
Hiperamonemia , Distúrbios Congênitos do Ciclo da Ureia , Humanos , Fenilbutiratos , Paladar , Pós/uso terapêutico , Hiperamonemia/tratamento farmacológico , Nitrogênio , Doenças Raras/tratamento farmacológico , UreiaRESUMO
Arginase 1 Deficiency (ARG1-D) is a rare urea cycle disorder that results in persistent hyperargininemia and a distinct, progressive neurologic phenotype involving developmental delay, intellectual disability, and spasticity, predominantly affecting the lower limbs and leading to mobility impairment. Unlike the typical presentation of other urea cycle disorders, individuals with ARG1-D usually appear healthy at birth and hyperammonemia is comparatively less severe and less common. Clinical manifestations typically begin to develop in early childhood in association with high plasma arginine levels, with hyperargininemia (and not hyperammonemia) considered to be the primary driver of disease sequelae. Nearly five decades of clinical experience with ARG1-D and empirical studies in genetically manipulated models have generated a large body of evidence that, when considered in aggregate, implicates arginine directly in disease pathophysiology. Severe dietary protein restriction to minimize arginine intake and diversion of ammonia from the urea cycle are the mainstay of care. Although this approach does reduce plasma arginine and improve patients' cognitive and motor/mobility manifestations, it is inadequate to achieve and maintain sufficiently low arginine levels and prevent progression in the long term. This review presents a comprehensive discussion of the clinical and scientific literature, the effects and limitations of the current standard of care, and the authors' perspectives regarding the past, current, and future management of ARG1-D.
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Hiperamonemia , Hiperargininemia , Distúrbios Congênitos do Ciclo da Ureia , Pré-Escolar , Humanos , Arginase/genética , Arginina , Hiperamonemia/metabolismoRESUMO
Arginase deficiency is caused by biallelic mutations in arginase 1 (ARG1), the final step of the urea cycle, and results biochemically in hyperargininemia and the presence of guanidino compounds, while it is clinically notable for developmental delays, spastic diplegia, psychomotor function loss, and (uncommonly) death. There is currently no completely effective medical treatment available. While preclinical strategies have been demonstrated, disadvantages with viral-based episomal-expressing gene therapy vectors include the risk of insertional mutagenesis and limited efficacy due to hepatocellular division. Recent advances in messenger RNA (mRNA) codon optimization, synthesis, and encapsulation within biodegradable liver-targeted lipid nanoparticles (LNPs) have potentially enabled a new generation of safer, albeit temporary, treatments to restore liver metabolic function in patients with urea cycle disorders, including ARG1 deficiency. In this study, we applied such technologies to successfully treat an ARG1-deficient murine model. Mice were administered LNPs encapsulating human codon-optimized ARG1 mRNA every 3 d. Mice demonstrated 100% survival with no signs of hyperammonemia or weight loss to beyond 11 wk, compared with controls that perished by day 22. Plasma ammonia, arginine, and glutamine demonstrated good control without elevation of guanidinoacetic acid, a guanidino compound. Evidence of urea cycle activity restoration was demonstrated by the ability to fully metabolize an ammonium challenge and by achieving near-normal ureagenesis; liver arginase activity achieved 54% of wild type. Biochemical and microscopic data showed no evidence of hepatotoxicity. These results suggest that delivery of ARG1 mRNA by liver-targeted nanoparticles may be a viable gene-based therapeutic for the treatment of arginase deficiency.
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Hiperargininemia/tratamento farmacológico , Lipídeos/farmacologia , Hepatopatias/tratamento farmacológico , Fígado/efeitos dos fármacos , Nanopartículas/administração & dosagem , RNA Mensageiro/metabolismo , Amônia/metabolismo , Animais , Arginase/metabolismo , Arginina/metabolismo , Códon/metabolismo , Modelos Animais de Doenças , Glutamina/metabolismo , Hiperamonemia/tratamento farmacológico , Hiperamonemia/metabolismo , Hiperargininemia/metabolismo , Fígado/metabolismo , Hepatopatias/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ureia/metabolismoRESUMO
The transplantation, engraftment, and expansion of primary hepatocytes have the potential to be an effective therapy for metabolic disorders of the liver including those of nitrogen metabolism. To date, such methods for the treatment of urea cycle disorders in murine models has only been minimally explored. Arginase deficiency, an inherited disorder of nitrogen metabolism that presents in the first two years of life, has the potential to be treated by such methods. To explore the potential of this approach, we mated the conditional arginase deficient mouse with a mouse model deficient in fumarylacetoacetate hydrolase (FAH) and with Rag2 and IL2-Rγ mutations to give a selective advantage to transplanted (normal) human hepatocytes. On day -1, a uroplasminogen-expressing adenoviral vector was administered intravenously followed the next day with the transplantation of 1â¯×â¯106 human hepatocytes (or vehicle alone) by intrasplenic injection. As the initial number of administered hepatocytes would be too low to prevent hepatotoxicity-induced mortality, NTBC cycling was performed to allow for hepatocyte expansion and repopulation. While all control mice died, all except one human hepatocyte transplanted mice survived. Four months after hepatocyte transplantation, 2â¯×â¯1011 genome copies of AAV-TBG-Cre recombinase was administered IV to disrupt endogenous hepatic arginase expression. While all control mice died within the first month, human hepatocyte transplanted mice did well. Ammonia and amino acids, analyzed in both groups before and after disruption of endogenous arginase expression, while well-controlled in the transplanted group, were markedly abnormal in the controls. Ammonium challenging further demonstrated the durability and functionality of the human repopulated liver. In conclusion, these studies demonstrate that human hepatocyte repopulation in the murine liver can result in effective treatment of arginase deficiency.
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Arginase/fisiologia , Predisposição Genética para Doença , Hepatócitos/transplante , Hepatopatias/terapia , Doenças Metabólicas/terapia , Animais , Células Cultivadas , Modelos Animais de Doenças , Feminino , Hepatócitos/citologia , Humanos , Hepatopatias/enzimologia , Hepatopatias/patologia , Masculino , Doenças Metabólicas/enzimologia , Doenças Metabólicas/patologia , Camundongos , Camundongos KnockoutRESUMO
UNLABELLED: Arginase 1 deficiency is a urea cycle disorder associated with hyperargininemia, spastic diplegia, loss of ambulation, intellectual disability, and seizures. To gain insight on how loss of arginase expression affects the excitability and synaptic connectivity of the cortical neurons in the developing brain, we used anatomical, ultrastructural, and electrophysiological techniques to determine how single-copy and double-copy arginase deletion affects cortical circuits in mice. We find that the loss of arginase 1 expression results in decreased dendritic complexity, decreased excitatory and inhibitory synapse numbers, decreased intrinsic excitability, and altered synaptic transmission in layer 5 motor cortical neurons. Hepatic arginase 1 gene therapy using adeno-associated virus rescued nearly all these abnormalities when administered to neonatal homozygous knock-out animals. Therefore, gene therapeutic strategies can reverse physiological and anatomical markers of arginase 1 deficiency and therefore may be of therapeutic benefit for the neurological disabilities in this syndrome. SIGNIFICANCE STATEMENT: These studies are one of the few investigations to try to understand the underlying neurological dysfunction that occurs in urea cycle disorders and the only to examine arginase deficiency. We have demonstrated by multiple modalities that, in murine layer 5 cortical neurons, a gradation of abnormalities exists based on the functional copy number of arginase: intrinsic excitability is altered, there is decreased density in asymmetrical and perisomatic synapses, and analysis of the dendritic complexity is lowest in the homozygous knock-out. With neonatal administration of adeno-associated virus expressing arginase, there is near-total recovery of the abnormalities in neurons and cortical circuits, supporting the concept that neonatal gene therapy may prevent the functional abnormalities that occur in arginase deficiency.
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Arginase/uso terapêutico , Terapia Genética , Hiperargininemia/patologia , Hiperargininemia/terapia , Córtex Motor/fisiologia , Recuperação de Função Fisiológica/fisiologia , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Amônia/sangue , Animais , Animais Recém-Nascidos , Arginase/genética , Arginase/metabolismo , Modelos Animais de Doenças , Hiperargininemia/sangue , Técnicas In Vitro , Camundongos , Camundongos Transgênicos , Córtex Motor/citologia , Córtex Motor/ultraestrutura , Rede Nervosa/patologia , Rede Nervosa/fisiologia , Rede Nervosa/ultraestrutura , Neurônios/fisiologia , Neurônios/ultraestrutura , Picrotoxina/farmacologia , Bloqueadores dos Canais de Sódio/farmacologia , Sinapses/ultraestrutura , Tetrodotoxina/farmacologiaRESUMO
Hyperargininemia caused by Arginase 1 deficiency is a rare disorder of the urea cycle that can be diagnosed by elevation of arginine in newborn screening blood spots when analyzed by tandem mass spectrometry. Hyperargininemia is currently included as a secondary target on the U.S. Recommended Uniform Screening Panel, which directly influences state-based newborn screening. Because of the apparent low disease frequency and lack of case detection and treatment data, detailed attention has not been given to a model newborn screening algorithm including appropriate analytical cutoff values for disease indicators. In this paper we assess the frequency of hyperargininemia in the U.S. identified by newborn screening to date and document the current status and variability of hyperargininemia newborn screening across U.S. newborn screening programs. We also review other data that support improved screening efficacy by utilizing the arginine/ornithine ratio and other amino acid ratios as discriminators in the screening algorithm. Analysis of archived California screening data showed that an arginine cutoff of 50µM combined with an arginine/ornithine ratio of 1.4 would have resulted in a recall rate of 0.01%. Using an arginine cutoff of 60µM and an arginine/(phenylalanine x leucine) ratio of 1.4, reportedly used in one screening program, or the R4S Tool Runner, would have resulted in a recall rate of <0.005%. All 9 diagnosed patients would have been found for either protocol. Thus, use of appropriate ratios as part of the screening algorithm has the potential to increase both screening sensitivity and specificity. Improved newborn screening effectiveness should lead to better case detection and more rapid treatment to lower plasma arginine levels hence improving long term outcome of individuals with hyperargininemia.
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Arginase/genética , Hiperargininemia/diagnóstico , Triagem Neonatal , Algoritmos , Arginina/sangue , California , Feminino , Ensaios de Triagem em Larga Escala , Humanos , Hiperargininemia/sangue , Hiperargininemia/epidemiologia , Incidência , Recém-Nascido , Masculino , Sensibilidade e Especificidade , Estados Unidos/epidemiologiaRESUMO
Human arginase deficiency is characterized by hyperargininemia and infrequent episodes of hyperammonemia that cause neurological impairment and growth retardation. We previously developed a neonatal mouse adeno-associated viral vector (AAV) rh10-mediated therapeutic approach with arginase expressed by a chicken ß-actin promoter that controlled plasma ammonia and arginine, but hepatic arginase declined rapidly. This study tested a codon-optimized arginase cDNA and compared the chicken ß-actin promoter to liver- and muscle-specific promoters. ARG1(-/-) mice treated with AAVrh10 carrying the liver-specific promoter also exhibited long-term survival and declining hepatic arginase accompanied by the loss of AAV episomes during subsequent liver growth. Although arginase expression in striated muscle was not expected to counteract hyperammonemia, due to muscle's lack of other urea cycle enzymes, we hypothesized that the postmitotic phenotype in muscle would allow vector genomes to persist, and hence contribute to decreased plasma arginine. As anticipated, ARG1(-/-) neonatal mice treated with AAVrh10 carrying a modified creatine kinase-based muscle-specific promoter did not survive longer than controls; however, their plasma arginine levels remained normal when animals were hyperammonemic. These data imply that plasma arginine can be controlled in arginase deficiency by muscle-specific expression, thus suggesting an alternative approach to utilizing the liver for treating hyperargininemia.
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Arginase/genética , Regulação da Expressão Gênica , Hiperamonemia/genética , Hiperargininemia/genética , Células Musculares/metabolismo , Animais , Arginase/metabolismo , Linhagem Celular , Códon , Dependovirus/genética , Modelos Animais de Doenças , Feminino , Vetores Genéticos/genética , Hepatócitos/metabolismo , Humanos , Hiperamonemia/metabolismo , Hiperargininemia/metabolismo , Hiperargininemia/mortalidade , Masculino , Camundongos , Camundongos Knockout , Fibras Musculares Esqueléticas/metabolismo , Mioblastos Cardíacos/metabolismo , Especificidade de Órgãos/genética , Regiões Promotoras GenéticasRESUMO
BACKGROUND AND METHODS: There are considerable uncertainty and debate regarding all aspects of newborn screen-positive cases of 3-methylcrotonyl-CoA carboxylase deficiency (3-MCCD), including diagnostic criteria, clinical spectrum, morbidity, prognosis, and appropriate management. To address some of these questions, we queried data from the California Newborn Screening Program's Screening Information System (SIS) and available scanned laboratory reports on cases of 3-MCCD reported by 15 state contracted metabolic specialty care centers born between July 2005 and December 2010. We evaluated the completeness and utility of the database as a tool for clinical disease characterization. RESULTS: During the study period, 2,959,108 infants were screened and 71 infants were diagnosed with 3-MCCD for an overall incidence of 1:41,676. The availability of diagnostic biochemical laboratory data varied significantly from subject to subject. Using a new case classification based on biochemical severity, we found that 8 of the cases met our criteria for biochemically severe (category 1), 19 cases met our criteria for biochemically mild (category 2) that we suspect to possibly be hypomorphic variants or heterozygote carriers, and 44 cases could not be classified (category 3) as mild or severe based on the data available in SIS. Documentation of the treatment regimens also varied significantly with 49% receiving dietary modification and 44% receiving carnitine. 15% of cases were documented to have experienced at least one of the following symptoms: lethargy, vomiting, irritability, ketosis, poor feeding, or poor tone. The majority of the subjects were completely developmentally age appropriate at their last assessment. CONCLUSIONS: The results suggest that a significant portion of the 3-MCCD "confirmed" cases have a mild biochemical phenotype. Moreover the majority of cases had insufficient data entered to allow for adequate clinical characterization of the cases. These findings raise the concern that a significant number of individuals receiving treatment for 3-MCCD may not have a clinically significant condition. Additionally, the utility of this data system could be improved if centers provided complete confirmatory test results and more specific documentation of clinical outcomes and health/developmental status. Further studies, including a clinical chart review, are necessary to validate the data and further characterize this cohort.
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Carbono-Carbono Ligases/deficiência , Triagem Neonatal , Distúrbios Congênitos do Ciclo da Ureia/genética , Acetonitrilas , California , Carbono-Carbono Ligases/genética , Carnitina , Humanos , Lactente , Recém-Nascido , Distúrbios Congênitos do Ciclo da Ureia/epidemiologia , Distúrbios Congênitos do Ciclo da Ureia/patologiaRESUMO
Human arginase deficiency is characterized by hyperargininemia and infrequent episodes of hyperammonemia, which lead to neurological impairment with spasticity, loss of ambulation, seizures, and severe mental and growth retardation; uncommonly, patients suffer early death from this disorder. In a murine targeted knockout model, onset of the phenotypic abnormality is heralded by weight loss at around day 15, and death occurs typically by postnatal day 17 with hyperargininemia and markedly elevated ammonia. This discrepancy between the more attenuated juvenile-onset human disease and the lethal neonatal murine model has remained suboptimal for studying and developing therapy for the more common presentation of arginase deficiency. These investigations aimed to address this issue by creating an adult conditional knockout mouse to determine whether later onset of arginase deficiency also resulted in lethality. Animal survival and ammonia levels, body weight, circulating amino acids, and tissue arginase levels were examined as outcome parameters after widespread Cre-recombinase activation in a conditional knockout model of arginase 1 deficiency. One hundred percent of adult female and 70% of adult male mice died an average of 21.0 and 21.6 days, respectively, after the initiation of tamoxifen administration. Animals demonstrated elevated circulating ammonia and arginine at the onset of phenotypic abnormalities. In addition, brain and liver amino acids demonstrated abnormalities. These studies demonstrate that (a) the absence of arginase in adult animals results in a disease profile (leading to death) similar to that of the targeted knockout and (b) the phenotypic abnormalities seen in the juvenile-onset model are not exclusive to the age of the animal but instead to the biochemistry of the disorder. This adult model will be useful for developing gene- and cell-based therapies for this disorder that will not be limited by the small animal size of neonatal therapy and for developing a better understanding of the characteristics of hyperargininemia.
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Genes Letais , Hiperargininemia/genética , Hiperargininemia/metabolismo , Fenótipo , Aminoácidos/sangue , Aminoácidos/metabolismo , Animais , Arginase/genética , Modelos Animais de Doenças , Feminino , Deleção de Genes , Genótipo , Hiperamonemia/genética , Hiperamonemia/metabolismo , Hiperargininemia/tratamento farmacológico , Hiperargininemia/mortalidade , Rim/efeitos dos fármacos , Rim/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Camundongos , Camundongos Knockout , Tamoxifeno/administração & dosagem , Tamoxifeno/farmacologia , Redução de PesoRESUMO
Arginase deficiency is characterized by hyperargininemia and infrequent episodes of hyperammonemia. Human patients suffer from neurological impairment with spasticity, loss of ambulation, seizures, and severe mental and growth retardation. In a murine model, onset of the phenotypic abnormality is heralded by weight loss beginning around day 15 with death occurring typically by postnatal day 17 with hyperargininemia and markedly elevated ammonia. The goal of this study was to address the development of a gene therapy approach for arginase deficiency beginning in the neonatal period. Lifespan extension, body weight, circulating amino acids and ammonia levels were examined as outcome parameters after gene therapy with an adeno-associated viral vector expressing arginase was administered to mice on the second day of life (DOL). One-hundred percent of untreated arginase-deficient mice died by DOL 24, whereas 89% of the adeno-associated virus (AAV)-treated arginase deficient mice have survived for >8 months. While animals at 8 months demonstrate elevated glutamine levels, ammonia is less than three times that of controls and arginine levels are normal. These studies are the first to demonstrate that AAV-based therapy for arginase deficiency is effective and supports the development of gene therapy for this and the other urea cycle disorders.
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Dependovirus/genética , Terapia Genética/métodos , Vetores Genéticos/genética , Hiperargininemia/terapia , Aminoácidos/sangue , Amônia/sangue , Animais , Arginase/genética , Arginase/metabolismo , Western Blotting , Feminino , Fertilidade/genética , Regulação da Expressão Gênica , Técnicas de Transferência de Genes , Genótipo , Glutamina/sangue , Hiperargininemia/genética , Imuno-Histoquímica , Masculino , Camundongos , Camundongos KnockoutRESUMO
Antibiotics-induced release of the pore-forming virulence factor pneumolysin (PLY) in patients with pneumococcal pneumonia results in its presence days after lungs are sterile and is a major factor responsible for the induction of permeability edema. Here we sought to identify major mechanisms mediating PLY-induced endothelial dysfunction. We evaluated PLY-induced endothelial hyperpermeability in human lung microvascular endothelial cells (HL-MVECs) and human lung pulmonary artery endothelial cells in vitro and in mice instilled intratracheally with PLY. PLY increases permeability in endothelial monolayers by reducing stable and dynamic microtubule content and modulating VE-cadherin expression. These events, dependent upon an increased calcium influx, are preceded by protein kinase C (PKC)-α activation, perturbation of the RhoA/Rac1 balance, and an increase in myosin light chain phosphorylation. At later time points, PLY treatment increases the expression and activity of arginase in HL-MVECs. Arginase inhibition abrogates and suppresses PLY-induced endothelial barrier dysfunction by restoring NO generation. Consequently, a specific PKC-α inhibitor and the TNF-derived tonoplast intrinsic protein peptide, which blunts PLY-induced PKC-α activation, are able to prevent activation of arginase in HL-MVECs and to reduce PLY-induced endothelial hyperpermeability in mice. Arginase I (AI)(+/-)/arginase II (AII)(-/-) C57BL/6 mice, displaying a significantly reduced arginase I expression in the lungs, are significantly less sensitive to PLY-induced capillary leak than their wild-type or AI(+/+)/AII(-/-) counterparts, indicating an important role for arginase I in PLY-induced endothelial hyperpermeability. These results identify PKC-α and arginase I as potential upstream and downstream therapeutic targets in PLY-induced pulmonary endothelial dysfunction.
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Arginase/metabolismo , Permeabilidade Capilar , Células Endoteliais/metabolismo , Pulmão/patologia , Proteína Quinase C-alfa/metabolismo , Estreptolisinas/farmacologia , Animais , Antígenos CD/metabolismo , Arginase/antagonistas & inibidores , Proteínas de Bactérias/farmacologia , Caderinas/metabolismo , Sinalização do Cálcio , Células Cultivadas , Células Endoteliais/enzimologia , Inibidores Enzimáticos/farmacologia , Humanos , Pulmão/irrigação sanguínea , Pulmão/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microtúbulos/metabolismo , Microvasos/patologia , Pneumonia/enzimologia , Pneumonia/imunologia , Pneumonia/patologia , Proteína Quinase C-alfa/antagonistas & inibidores , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
Arginase can cause vascular dysfunction by competing with nitric oxide synthase for l-arginine and by increasing cell proliferation and collagen formation, which promote vascular fibrosis/stiffening. We have shown that increased arginase expression/activity contribute to vascular endothelial cell (EC) dysfunction. Here, we examined the roles of the two arginase isoforms, arginase I and II (AI and AII, respectively), in this process. Experiments were performed using streptozotocin-induced diabetic mice: wild-type (WT) mice and knockout mice lacking the AII isoform alone (AI(+/+)AII(-/-)) or in combination with partial deletion of AI (AI(+/-)AII (-/-)). EC-dependent vasorelaxation of aortic rings and arterial fibrosis and stiffness were assessed in relation to arginase activity and expression. Diabetes reduced mean EC-dependent vasorelaxation markedly in diabetic WT and AI(+/+)AII(-/-) aortas (53% and 44% vs. controls, respectively) compared with a 27% decrease in AI(+/-)AII (-/-) vessels. Coronary fibrosis was also increased in diabetic WT and AI(+/+)AII(-/-) mice (1.9- and 1.7-fold vs. controls, respectively) but was not altered in AI(+/-)AII (-/-) diabetic mice. Carotid stiffness was increased by 142% in WT diabetic mice compared with 51% in AI(+/+)AII(-/-) mice and 19% in AI(+/-)AII (-/-) mice. In diabetic WT and AI(+/+)AII(-/-) mice, aortic arginase activity and AI expression were significantly increased compared with control mice, but neither parameter was altered in AI(+/-)AII (-/-) mice. In summary, AI(+/-)AII (-/-) mice exhibit better EC-dependent vasodilation and less vascular stiffness and coronary fibrosis compared with diabetic WT and AI(+/+)AII(-/-) mice. These data indicate a major involvement of AI in diabetes-induced vascular dysfunction.
Assuntos
Arginase/metabolismo , Artérias/enzimologia , Diabetes Mellitus Experimental/complicações , Angiopatias Diabéticas/etiologia , Vasodilatação , Animais , Aorta/enzimologia , Aorta/fisiopatologia , Arginase/genética , Artérias/efeitos dos fármacos , Artérias/patologia , Artérias/fisiopatologia , Artérias Carótidas/enzimologia , Artérias Carótidas/fisiopatologia , Complacência (Medida de Distensibilidade) , Vasos Coronários/enzimologia , Vasos Coronários/fisiopatologia , Diabetes Mellitus Experimental/enzimologia , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Experimental/fisiopatologia , Angiopatias Diabéticas/enzimologia , Angiopatias Diabéticas/genética , Angiopatias Diabéticas/patologia , Angiopatias Diabéticas/fisiopatologia , Relação Dose-Resposta a Droga , Fibrose , Peróxido de Hidrogênio/metabolismo , Hidroxiprolina/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Superóxidos/metabolismo , Vasoconstrição , Vasoconstritores/farmacologia , Vasodilatadores/farmacologiaRESUMO
BACKGROUND: Short-chain acyl-CoA dehydrogenase deficiency (SCADD) is an autosomal recessive inborn error of mitochondrial fatty acid oxidation with highly variable biochemical, genetic, and clinical characteristics. SCADD has been associated with accumulation of butyryl-CoA byproducts, including butyrylcarnitine (C4), butyrylglycine, ethylmalonic acid (EMA), and methylsuccinic acid (MS) in body fluid and tissues. Differences in genotype frequencies have been shown between patients diagnosed clinically versus those diagnosed by newborn screening. Moreover, while patients diagnosed clinically have a variable clinical presentation including developmental delay, ketotic hypoglycemia, epilepsy and behavioral disorders, studies suggest patients diagnosed by newborn screening are largely asymptomatic. Scant information is published about the biochemical, genetic and clinical outcome of SCADD patients diagnosed by newborn screening. METHODS: We collected California newborn screening, follow-up biochemical levels, and ACADS mutation data from September, 2005 through April, 2010. We retrospectively reviewed available data on SCADD cases diagnosed by newborn screening for clinical outcomes. RESULTS: During the study period, 2,632,058 newborns were screened and 76 confirmed SCADD cases were identified. No correlations between initial C4 value and follow-up biochemical markers (C4, EMA or MS levels) were found in the 76 cases studied. We found significant correlation between urine EMA versus MS, and correlation between follow-up C4 versus urine EMA. Of 22 cases where ACADS gene sequencing was performed: 7 had two or more deleterious mutations; 8 were compound heterozygotes for a deleterious mutation and common variant; 7 were homozygous for the common variant c.625G>A; and 1 was heterozygous for c.625G>A. Significant increases in mean urine EMA and MS levels were noted in patients with two or more deleterious mutations versus mutation heterozygotes or common polymorphism homozygotes. Clinical outcome data was available in 31 patients with follow-up extending from 0.5 to 60 months. None developed epilepsy or behavioral disorders, and three patients had isolated speech delay. Hypoglycemia occurred in two patients, both in the neonatal period. The first patient had concomitant meconium aspiration; the other presented with central apnea, poor feeding, and hypotonia. The latter, a c.625G>A homozygote, has had persistent elevations in both short- and medium-chain acylcarnitines; diagnostic workup in this case is extensive and ongoing. CONCLUSIONS: This study examines the largest series to date of SCADD patients identified by newborn screening. Our results suggest that confirmatory tests may be useful to differentiate patients with common variants from those with deleterious mutations. This study also provides evidence to suggest that, even when associated with deleterious mutations, SCADD diagnosed by newborn screening presents largely as a benign condition.
Assuntos
Acil Coenzima A , Erros Inatos do Metabolismo Lipídico/diagnóstico , Erros Inatos do Metabolismo Lipídico/genética , Triagem Neonatal , Acil Coenzima A/sangue , Acil Coenzima A/genética , Acil Coenzima A/urina , Acil-CoA Desidrogenase/deficiência , Acil-CoA Desidrogenase/genética , California , Carnitina/análogos & derivados , Carnitina/sangue , Carnitina/urina , Feminino , Seguimentos , Humanos , Recém-Nascido , Masculino , Malonatos/sangue , Malonatos/urina , Deleção de Sequência , Succinatos/sangue , Succinatos/urinaRESUMO
Arginase deficiency is associated with prominent neuromotor features, including spastic diplegia, clonus, and hyperreflexia; intellectual disability and progressive neurological decline are other signs. In a constitutive murine model, we recently described leukodystrophy as a significant component of the central nervous system features of arginase deficiency. In the present studies, we sought to examine if the administration of a lipid nanoparticle carrying human ARG1 mRNA to constitutive knockout mice could prevent abnormalities in myelination associated with arginase deficiency. Imaging of the cingulum, striatum, and cervical segments of the corticospinal tract revealed a drastic reduction of myelinated axons; signs of degenerating axons were also present with thin myelin layers. Lipid nanoparticle/ARG1 mRNA administration resulted in both light and electron microscopic evidence of a dramatic recovery of myelin density compared with age-matched controls; oligodendrocytes were seen to be extending processes to wrap many axons. Abnormally thin myelin layers, when myelination was present, were resolved with intermittent mRNA administration, indicative of not only a greater density of myelinated axons but also an increase in the thickness of the myelin sheath. In conclusion, lipid nanoparticle/ARG1 mRNA administration in arginase deficiency prevents the associated leukodystrophy and restores normal oligodendrocyte function.
RESUMO
The mucopolysaccharidoses (MPS) are lysosomal storage disorders caused by defects in the enzymes involved in the degradation of glycosaminoglycans. Hurler syndrome (MPS I) and Sanfilippo syndrome (MPS III) are among the more common diseases in the group, each occurring with an incidence of approximately 1 in 100,000. We present a case of siblings, born of a consanguineous union, affected with both MPS I and MPS IIIa. The diagnoses were confirmed with fibroblast enzyme assays and sequence analysis of the genes, which identified homozygous mutations in IDUA and SGSH. We discuss their clinical features and course and examine the psychosocial aspects of their case, specifically, the decision-making process that the medical team and family faced regarding treatment with enzyme replacement therapy.
Assuntos
Terapia de Reposição de Enzimas , Iduronidase/uso terapêutico , Mucopolissacaridose III/diagnóstico , Mucopolissacaridose III/terapia , Mucopolissacaridose I/diagnóstico , Mucopolissacaridose I/terapia , Pré-Escolar , Feminino , Genótipo , Humanos , Hidrolases/genética , Lactente , Masculino , Mucopolissacaridose I/genética , Mucopolissacaridose III/genética , Mutação/genética , IrmãosRESUMO
We describe the outcome of two consecutive pregnancies with a clinical presentation of ornithine transcarbamylase (OTC) deficiency (OTCD) without a molecular diagnosis. A 119kb deletion on Xp11.4 including the OTC gene was detected in the mother. The same deletion was identified in the blood spots from deceased male newborns. In patients with a clinical and biochemical presentation of OTCD and negative OTC sequencing, whole genome or targeted chromosomal microarray analysis (CMA) with coverage of the OTC and neighboring genes should be performed as a reflex test.
Assuntos
Éxons , Doença da Deficiência de Ornitina Carbomoiltransferase/diagnóstico , Doença da Deficiência de Ornitina Carbomoiltransferase/genética , Ornitina Carbamoiltransferase/genética , Complicações na Gravidez/enzimologia , Complicações na Gravidez/genética , Adulto , Feminino , Humanos , Recém-Nascido , Masculino , Análise em Microsséries , Análise de Sequência com Séries de Oligonucleotídeos , Doença da Deficiência de Ornitina Carbomoiltransferase/enzimologia , Polimorfismo de Nucleotídeo Único , Gravidez , Diagnóstico Pré-Natal , Deleção de SequênciaRESUMO
The paucity of hyperammonemic crises together with spasticity, only seen in human arginase I deficient patients and not in patients with other urea cycle disorders, forces a search for candidates other than ammonia to associate with the pathophysiology and symptomatology. Therefore, we determined arginine together with some catabolites of arginine in blood and cerebrospinal fluid of these patients as well as in extremely rare post-mortem brain material of two patients with argininemia. The levels of alpha-keto-delta-guanidinovaleric acid, argininic acid and alpha-N-acetylarginine correlate with the arginine levels in blood and cerebrospinal fluid of patients with imposed or spontaneous protein restriction. The levels in blood are higher than the upper limit of normal in all studied patients. In addition to the highly increased levels of these same compounds in blood of a child with argininemia, the increase of guanidinoacetic acid, 24h before death, is remarkable. However, the manifest increases of these studied catabolites of arginine are not seen in post-mortem brain material of the same pediatric patient. Otherwise a clear increase of guanidinoacetic acid in post-mortem brain material of an adult patient was shown. A similar, comparable increase of homoarginine in both studied post-mortem brain materials is observed. Therefore the study of the pathobiochemistry of arginine in argininemia must be completed in the future by the determination of the end catabolites of the nitric oxide and agmatine biosynthesis pathways in the knockouts as well as in the patients to evaluate their role, together with the here studied catabolites, as candidates for association with pathophysiology and symptomatology.
Assuntos
Encéfalo/patologia , Guanidinas/sangue , Guanidinas/líquido cefalorraquidiano , Hiperargininemia/sangue , Hiperargininemia/líquido cefalorraquidiano , Adolescente , Adulto , Autopsia , Criança , Pré-Escolar , Seguimentos , Humanos , Hiperargininemia/patologia , Masculino , Insuficiência de Múltiplos Órgãos/sangue , Insuficiência de Múltiplos Órgãos/líquido cefalorraquidiano , Insuficiência de Múltiplos Órgãos/complicações , Fatores de Tempo , Ureia/sangue , Adulto JovemRESUMO
Neonatal gene therapy has the potential to ameliorate abnormalities before disease onset. Our gene knockout of arginase I (AI) deficiency is characterized by increasing hyperammonemia, neurological deterioration, and early death. We constructed a helper-dependent adenoviral vector (HDV) carrying AI and examined for correction of this defect. Neonates were administered 5 x 10(9) viral particles/g and analyzed for survival, arginase activity, and ammonia and amino acids levels. The life expectancy of arg(-/-) mice increased to 27 days while controls died at 14 days with hyperammonemia and in extremis. Death correlated with a decrease in viral DNA/RNA per cell as liver mass increased. Arginase assays demonstrated that vector-injected hepatocytes had ~20% activity of heterozygotes at 2 weeks of age. Hepatic arginine and ornithine in treated mice were similar to those of saline-injected heterozygotes at 2 weeks, whereas ammonia was normal. By 26 days, arginase activity in the treated arg(-/-) livers declined to <10%, and arginine and ornithine increased. Ammonia levels began increasing by day 25, suggesting the cause of death to be similar to that of uninjected arg(-/-) mice, albeit at a later time. These studies demonstrate that the AI deficient newborn mouse can be temporarily corrected and rescued using a HDV.
Assuntos
Adenoviridae/genética , Arginase/genética , Terapia Genética/métodos , Vetores Genéticos/genética , Hiperamonemia/terapia , Hiperargininemia/terapia , Aminoácidos/metabolismo , Amônia/metabolismo , Animais , Animais Recém-Nascidos , Arginase/metabolismo , Arginase/fisiologia , Camundongos , Camundongos KnockoutRESUMO
BACKGROUND: Clinical care teams providing presymptomatic genetic testing often employ advanced confidentiality practices for documentation and result storage. However, patient requests for increased confidentiality may be in conflict with the legal obligations of medical providers to document patient care activities in the electronic health record (EHR). Huntington disease presents a representative case study for investigating the ways centers currently balance the requirements of EHRs with the privacy demands of patients seeking presymptomatic genetic testing. METHODS: We surveyed 23 HD centers (53% response rate) regarding their use of the EHR for presymptomatic HD testing. RESULTS: Our survey revealed that clinical care teams and laboratories have each developed their own practices, which are cumbersome and often include EHR avoidance. We found that a majority of HD care teams record appointments in the EHR (91%), often using vague notes. Approximately half of the care teams (52%) keep presymptomatic results of out of the EHR. CONCLUSION: As genetic knowledge grows, linking more genes to late-onset conditions, institutions will benefit from having professional recommendations to guide development of policies for EHR documentation of presymptomatic genetic results. Policies must be sensitive to the ethical differences and patient demands for presymptomatic genetic testing compared to those undergoing confirmatory genetic testing.