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1.
Nat Med ; 25(6): 911-919, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31160820

RESUMO

It is estimated that 350 million individuals worldwide suffer from rare diseases, which are predominantly caused by mutation in a single gene1. The current molecular diagnostic rate is estimated at 50%, with whole-exome sequencing (WES) among the most successful approaches2-5. For patients in whom WES is uninformative, RNA sequencing (RNA-seq) has shown diagnostic utility in specific tissues and diseases6-8. This includes muscle biopsies from patients with undiagnosed rare muscle disorders6,9, and cultured fibroblasts from patients with mitochondrial disorders7. However, for many individuals, biopsies are not performed for clinical care, and tissues are difficult to access. We sought to assess the utility of RNA-seq from blood as a diagnostic tool for rare diseases of different pathophysiologies. We generated whole-blood RNA-seq from 94 individuals with undiagnosed rare diseases spanning 16 diverse disease categories. We developed a robust approach to compare data from these individuals with large sets of RNA-seq data for controls (n = 1,594 unrelated controls and n = 49 family members) and demonstrated the impacts of expression, splicing, gene and variant filtering strategies on disease gene identification. Across our cohort, we observed that RNA-seq yields a 7.5% diagnostic rate, and an additional 16.7% with improved candidate gene resolution.


Assuntos
Doenças Raras/genética , Ceramidase Ácida/genética , Estudos de Casos e Controles , Criança , Pré-Escolar , Estudos de Coortes , Feminino , Variação Genética , Humanos , Masculino , Modelos Genéticos , Mutação , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/genética , Canais de Potássio/genética , RNA/sangue , RNA/genética , Processamento de RNA/genética , Doenças Raras/sangue , Análise de Sequência de RNA , Sequenciamento Completo do Exoma
2.
Arch Pharm Res ; 42(3): 232-243, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30661200

RESUMO

Sphingolipid metabolism plays an important role in determining the fate of a cell. Among several sphingolipid metabolites, ceramide is a key player in intracellular signal transduction. Ceramide is usually converted to various metabolites such as sphingomyelin, sphingosine, ceramide-1-phosphate, and glucosylceramide. If ceramide is accumulated in the cell, it induces apoptosis. On the other hand, its metabolite sphingosine is converted to sphingosine-1-phosphate (S1P), which promotes angiogenesis via G protein coupled receptor signaling. Therefore, the equilibrium in ceramide and S1P levels in cells plays an important role in angiogenesis as well as cell death. Acid ceramidase (AC) is a promising target protein in the development of multi-targeted anticancer drugs as its inhibition can simultaneously inhibit angiogenesis via the Akt and ERK 1/2 pathway and limit cancer growth through ceramide-induced apoptosis. Although some inhibitors of AC have been reported, they have not been proven effective for human therapy. Recent advancement in the elucidation of AC structure will facilitate the development of better inhibitors for treating human diseases.


Assuntos
Ceramidase Ácida/antagonistas & inibidores , Antineoplásicos/farmacologia , Inibidores Enzimáticos/farmacologia , Neoplasias/tratamento farmacológico , Neovascularização Patológica/tratamento farmacológico , Ceramidase Ácida/genética , Ceramidase Ácida/metabolismo , Antineoplásicos/química , Inibidores Enzimáticos/química , Humanos , Modelos Moleculares , Conformação Molecular , Neoplasias/metabolismo , Neoplasias/patologia , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia
3.
J Med Chem ; 62(2): 987-992, 2019 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-30525581

RESUMO

Human acid ceramidase (AC) is a lysosomal cysteine amidase, which has received a great deal of interest in recent years as a potential target for the development of new therapeutics against melanoma and glioblastoma tumors. Despite the strong interest in obtaining structural information, only the structures of the apo-AC enzyme in its zymogen and activated conformations are available. In this work, the crystal structure of AC in complex with the covalent carmofur inhibitor is presented. Carmofur is an antineoplastic drug containing an electrophilic carbonyl reactive group that targets the catalytic cysteine. This novel structural data explains the basis of the AC inhibition, provides insights into the enzymatic properties of the protein, and is a great aid toward the structure-based drug design of potent inhibitors for AC, providing the detailed mechanism, which has eluded the scientific community for more than 30 years, of carmofur's mysterious 5-fluorouracil-independent antitumor activity.


Assuntos
Ceramidase Ácida/antagonistas & inibidores , Antineoplásicos/química , Fluoruracila/análogos & derivados , Simulação de Dinâmica Molecular , Ceramidase Ácida/genética , Ceramidase Ácida/metabolismo , Antineoplásicos/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Fluoruracila/química , Fluoruracila/metabolismo , Humanos , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação
4.
Brain ; 140(12): 3191-3203, 2017 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-29140481

RESUMO

Mutations in the glucocerebrosidase gene (GBA), which cause Gaucher disease, are also potent risk factors for Parkinson's disease. We examined whether a genetic burden of variants in other lysosomal storage disorder genes is more broadly associated with Parkinson's disease susceptibility. The sequence kernel association test was used to interrogate variant burden among 54 lysosomal storage disorder genes, leveraging whole exome sequencing data from 1156 Parkinson's disease cases and 1679 control subjects. We discovered a significant burden of rare, likely damaging lysosomal storage disorder gene variants in association with Parkinson's disease risk. The association signal was robust to the exclusion of GBA, and consistent results were obtained in two independent replication cohorts, including 436 cases and 169 controls with whole exome sequencing and an additional 6713 cases and 5964 controls with exome-wide genotyping. In secondary analyses designed to highlight the specific genes driving the aggregate signal, we confirmed associations at the GBA and SMPD1 loci and newly implicate CTSD, SLC17A5, and ASAH1 as candidate Parkinson's disease susceptibility genes. In our discovery cohort, the majority of Parkinson's disease cases (56%) have at least one putative damaging variant in a lysosomal storage disorder gene, and 21% carry multiple alleles. Our results highlight several promising new susceptibility loci and reinforce the importance of lysosomal mechanisms in Parkinson's disease pathogenesis. We suggest that multiple genetic hits may act in combination to degrade lysosomal function, enhancing Parkinson's disease susceptibility.


Assuntos
Ceramidase Ácida/genética , Catepsina D/genética , Glucosilceramidase/genética , Transportadores de Ânions Orgânicos/genética , Doença de Parkinson/genética , Esfingomielina Fosfodiesterase/genética , Simportadores/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Estudos de Coortes , Exoma , Feminino , Predisposição Genética para Doença , Genótipo , Humanos , Doenças por Armazenamento dos Lisossomos/genética , Masculino , Pessoa de Meia-Idade , Mutação
5.
Eur J Hum Genet ; 25(10): 1155-1161, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28905881

RESUMO

Keloids result from abnormal proliferative scar formation with scar tissue expanding beyond the margin of the original wound and are mostly found in individuals of sub-Saharan African descent. The etiology of keloids has not been resolved but previous studies suggest that keloids are a genetically heterogeneous disorder. Although possible candidate genes have been suggested by genome-wide association studies using common variants, by upregulation in keloids or their involvement in syndromes that include keloid formation, rare coding variants that contribute to susceptibility in non-syndromic keloid formation have not been previously identified. Through analysis of whole-genome data we mapped a locus to chromosome 8p23.3-p21.3 with a statistically significant maximum multipoint LOD score of 4.48. This finding was followed up using exome sequencing and led to the identification of a c.1202T>C (p.(Leu401Pro)) variant in the N-acylsphingosine amidohydrolase (ASAH1) gene that co-segregates with the keloid phenotype in a large Yoruba family. ASAH1 is an acid ceramidase known to be involved in tumor formation by controlling the ratio of ceramide and sphingosine. ASAH1 is also involved in cell proliferation and inflammation, and may affect the development of keloids via multiple mechanisms. Functional studies need to clarify the role of the ASAH1 variant in wound healing.


Assuntos
Ceramidase Ácida/genética , Queloide/genética , Mutação de Sentido Incorreto , Adulto , Feminino , Humanos , Queloide/diagnóstico , Masculino , Linhagem
6.
Hum Mutat ; 38(6): 611-614, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28251733

RESUMO

At least 15% of the disease-causing mutations affect mRNA splicing. Many splicing mutations are missed in a clinical setting due to limitations of in silico prediction algorithms or their location in noncoding regions. Whole-transcriptome sequencing is a promising new tool to identify these mutations; however, it will be a challenge to obtain disease-relevant tissue for RNA. Here, we describe an individual with a sporadic atypical spinal muscular atrophy, in whom clinical DNA sequencing reported one pathogenic ASAH1 mutation (c.458A>G;p.Tyr153Cys). Transcriptome sequencing on patient leukocytes identified a highly significant and atypical ASAH1 isoform not explained by c.458A>G(p<10-16 ). Subsequent Sanger-sequencing identified the splice mutation responsible for the isoform (c.504A>C;p.Lys168Asn) and provided a molecular diagnosis of autosomal-recessive spinal muscular atrophy with progressive myoclonic epilepsy. Our findings demonstrate the utility of RNA sequencing from blood to identify splice-impacting disease mutations for nonhematological conditions, providing a diagnosis for these otherwise unsolved patients.


Assuntos
Ceramidase Ácida/genética , Atrofia Muscular Espinal/sangue , Epilepsias Mioclônicas Progressivas/sangue , Processamento de RNA/genética , Ceramidase Ácida/sangue , Pré-Escolar , Humanos , Masculino , Atrofia Muscular Espinal/complicações , Atrofia Muscular Espinal/genética , Mutação , Epilepsias Mioclônicas Progressivas/complicações , Epilepsias Mioclônicas Progressivas/genética , Patologia Molecular , Análise de Sequência de DNA , Transcriptoma/genética
7.
Adv Biol Regul ; 63: 122-131, 2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-27771292

RESUMO

Over the past three decades, extensive research has been able to determine the biologic functions for the main bioactive sphingolipids, namely ceramide, sphingosine, and sphingosine 1-phosphate (S1P) (Hannun, 1996; Hannun et al., 1986; Okazaki et al., 1989). These studies have managed to define the metabolism, regulation, and function of these bioactive sphingolipids. This emerging body of literature has also implicated bioactive sphingolipids, particularly S1P and ceramide, as key regulators of cellular homeostasis. Ceramidases have the important role of cleaving fatty acid from ceramide and producing sphingosine, thereby controlling the interconversion of these two lipids. Thus far, five human ceramidases encoded by five different genes have been identified: acid ceramidase (AC), neutral ceramidase (NC), alkaline ceramidase 1 (ACER1), alkaline ceramidase 2 (ACER2), and alkaline ceramidase 3 (ACER3). These ceramidases are classified according to their optimal pH for catalytic activity. AC, which is localized to the lysosomal compartment, has been associated with Farber's disease and is involved in the regulation of cell viability. Neutral ceramidase, which is localized to the plasma membrane and primarily expressed in the small intestine and colon, is involved in digestion, and has been implicated in colon carcinogenesis. ACER1 which can be found in the endoplasmic reticulum and is highly expressed in the skin, plays an important role in keratinocyte differentiation. ACER2, localized to the Golgi complex and highly expressed in the placenta, is involved in programed cell death in response to DNA damage. ACER3, also localized to the endoplasmic reticulum and the Golgi complex, is ubiquitously expressed, and is involved in motor coordination-associated Purkinje cell degeneration. This review seeks to consolidate the current knowledge regarding these key cellular players.


Assuntos
Ceramidase Ácida/metabolismo , Ceramidase Alcalina/metabolismo , Ceramidase Neutra/metabolismo , Esfingolipídeos/metabolismo , Ceramidase Ácida/genética , Ceramidase Alcalina/genética , Animais , Lipogranulomatose de Farber/enzimologia , Lipogranulomatose de Farber/genética , Lipogranulomatose de Farber/patologia , Expressão Gênica , Humanos , Concentração de Íons de Hidrogênio , Inflamação , Cinética , Neoplasias/enzimologia , Neoplasias/genética , Neoplasias/patologia , Doenças Neurodegenerativas/enzimologia , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/patologia , Ceramidase Neutra/genética , Transdução de Sinais , Especificidade por Substrato
8.
Oncotarget ; 7(50): 83208-83222, 2016 Dec 13.
Artigo em Inglês | MEDLINE | ID: mdl-27825124

RESUMO

There is an urgent unmet need for new therapeutics in acute myeloid leukemia (AML) as standard therapy has not changed in the past three decades and outcome remains poor for most patients. Sphingolipid dysregulation through decreased ceramide levels and elevated sphingosine 1-phosphate (S1P) promotes cancer cell growth and survival. Acid ceramidase (AC) catalyzes ceramide breakdown to sphingosine, the precursor for S1P. We report for the first time that AC is required for AML blast survival. Transcriptome analysis and enzymatic assay show that primary AML cells have high levels of AC expression and activity. Treatment of patient samples and cell lines with AC inhibitor LCL204 reduced viability and induced apoptosis. AC overexpression increased the expression of anti-apoptotic Mcl-1, significantly increased S1P and decreased ceramide. Conversely, LCL204 induced ceramide accumulation and decreased Mcl-1 through post-translational mechanisms. LCL204 treatment significantly increased overall survival of C57BL/6 mice engrafted with leukemic C1498 cells and significantly decreased leukemic burden in NSG mice engrafted with primary human AML cells. Collectively, these studies demonstrate that AC plays a critical role in AML survival through regulation of both sphingolipid levels and Mcl-1. We propose that AC warrants further exploration as a novel therapeutic target in AML.


Assuntos
Ceramidase Ácida/antagonistas & inibidores , Antineoplásicos/farmacologia , Biomarcadores Tumorais/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Leucemia Mieloide Aguda/tratamento farmacológico , Ceramidase Ácida/genética , Ceramidase Ácida/metabolismo , Animais , Apoptose/efeitos dos fármacos , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Ceramidas/metabolismo , Relação Dose-Resposta a Droga , Feminino , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Células HL-60 , Humanos , Leucemia Mieloide Aguda/enzimologia , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Lisofosfolipídeos/metabolismo , Camundongos Endogâmicos C57BL , Terapia de Alvo Molecular , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Interferência de RNA , Transdução de Sinais/efeitos dos fármacos , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Fatores de Tempo , Transfecção , Células Tumorais Cultivadas , Regulação para Cima , Ensaios Antitumorais Modelo de Xenoenxerto
10.
Pediatrics ; 138(4)2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27650050

RESUMO

Survival of motor neuron 1-------negative spinal muscular atrophy (SMA) is heterogeneous and remains a diagnostic challenge. The clinical spectrum continues to expand and ∼33 genes have been identified to date. The present report describes a 9-year-old girl with novel clinical phenotype of a patient with polyarticular arthritis followed by symptoms of SMA due to acid ceramidase deficiency. Whole exome sequencing identified compound heterozygous pathogenic mutation in the N-acylsphingosine amidohydrolase 1 gene. Functional assay with leukocyte acid ceramidase activity showed a decreased level in the proband confirming pathogenicity of the mutations. Mutations of N-acylsphingosine amidohydrolase 1 are known to separately cause Farber disease (arthritis, subcutaneous nodules, and dysphonia) or SMA with progressive myoclonic epilepsy. The present combined phenotype is novel, bringing together SMA with progressive myoclonic epilepsy and Farber disease and establishing a phenotypic spectrum. Acid ceramidase deficiency is an important consideration in patients presenting with polyarticular arthritis and motor neuron disease.


Assuntos
Ceramidase Ácida/genética , Artrite Juvenil/genética , Lipogranulomatose de Farber/genética , Atrofia Muscular Espinal/genética , Criança , Lipogranulomatose de Farber/complicações , Evolução Fatal , Feminino , Humanos , Mutação , Fenótipo , Insuficiência Respiratória/etiologia
11.
Epileptic Disord ; 18(S2): 128-134, 2016 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-27647482

RESUMO

A rare syndrome characterized by lower motor neuron disease associated with progressive myoclonic epilepsy, referred to as "spinal muscular atrophy associated with progressive myoclonic epilepsy" (SMA-PME), has been described in childhood and is inherited as an autosomal recessive trait. SMA-PME is caused by mutation in the ASAH1 gene encoding acid ceramidase. Ceramide and the metabolites participate in various cellular events as lipid mediators. The catabolism of ceramide in mammals occurs in lysosomes through the activity of ceramidase. Three different ceramidases (acid, neutral and alkaline) have been identified and appear to play distinct roles in sphingolipid metabolism. The enzymatic activity of acid ceramidase is deficient in two rare inherited disorders; Farber disease and SMA-PME. Farber disease is a very rare and severe autosomal recessive condition with a distinct clinical phenotype. The marked difference in disease manifestations may explain why Farber and SMA-PME diseases were not previously suspected to be allelic conditions. The precise molecular mechanism underlying the phenotypic differences remains to be clarified. Recently, a condition with mutation in CERS1, the gene encoding ceramide synthase 1, has been identified as a novel form of PME. This finding underlies the essential role of enzymes regulating either the synthesis (CERS1) or degradation (ASAH1) of ceramide, and the link between defects in ceramide metabolism and PME.


Assuntos
Ceramidase Ácida/genética , Lipogranulomatose de Farber/genética , Atrofia Muscular Espinal/genética , Epilepsias Mioclônicas Progressivas/genética , Animais , Humanos , Atrofia Muscular Espinal/fisiopatologia , Epilepsias Mioclônicas Progressivas/fisiopatologia , Síndrome
12.
Am J Med Genet A ; 170(11): 3023-3027, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27411168

RESUMO

Farber disease is a very rare autosomal recessive disease caused by mutation of ASAH1 that results in the accumulation of ceramide in various tissues. Clinical symptoms of classic Farber disease comprise painful joint deformity, hoarseness of voice, and subcutaneous nodules. Here, we describe a patient with Farber disease with atypical presentation of early onset hypotonia, sacral mass, congenital heart disease, and dysmorphic face since birth. Severe cognitive disability, failure to gain motor skills, failure to thrive, and joint contractures developed. Using whole-exome sequencing, we identified the compound heterozygote missense mutations of ASAH1 (p.R333C and p.G235R). Because of the diagnostic delay, she underwent sacral mass excision, which revealed enlarged lysosomes and zebra bodies. We report an atypical presentation of Farber disease with her pathology and associated genetic defect. This case expands the phenotypic spectrum of Farber disease to include novel mutations of ASAH1, which pose a diagnostic challenge. We also discuss the clinical utility of whole-exome sequencing for diagnosis of ultra-rare diseases. © 2016 Wiley Periodicals, Inc.


Assuntos
Ceramidase Ácida/genética , Lipogranulomatose de Farber/diagnóstico , Lipogranulomatose de Farber/genética , Mutação , Fenótipo , Idade de Início , Alelos , Sequência de Aminoácidos , Substituição de Aminoácidos , Encéfalo/patologia , Análise Mutacional de DNA , Exoma , Feminino , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Lactente , Recém-Nascido , Imagem por Ressonância Magnética , Linhagem
14.
Arthritis Rheumatol ; 68(9): 2323-7, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-26945816

RESUMO

OBJECTIVE: To establish a diagnosis and provide counseling and treatment for 3 adult patients from one family presenting with peripheral osteolysis. METHODS: Following clinical and radiographic assessment, exome sequencing, targeted gene resequencing, and determination of enzyme activity in cultured fibroblasts were performed. RESULTS: The proband (age 40 years) had a history of episodic fever and pain in childhood that subsided around puberty. He and 2 of his older sisters (ages 58 and 60 years, respectively) showed adult-onset progressive shortening of fingers and toes with redundancy of the overlying skin. Radiographs showed severe osteolysis of the distal radius and ulna, carpal bones, metacarpal bones, and phalanges. Sequencing of the known genes for recessively inherited osteolysis, MMP2 and MMP14, failed to show pathogenic mutations. Exome sequencing revealed compound heterozygosity for mutations c.505T>C (p.Trp169Arg) and c.760A>G (p.Arg254Gly) in ASAH1, the gene coding for acid ceramidase. Sanger sequencing confirmed correct segregation in the family, and enzyme activity in fibroblast cultures from the patients was reduced to ∼8% of that in controls, confirming a diagnosis of Farber's disease. CONCLUSION: Our findings indicate that hypomorphic mutations in ASAH1 may result in an osteoarticular phenotype with a juvenile phase resembling rheumatoid arthritis that evolves to osteolysis as the final stage in the absence of neurologic signs. This observation delineates a novel type of recessively inherited peripheral osteolysis and illustrates the long-term skeletal manifestations of acid ceramidase deficiency (Farber's disease) in what appear to be the oldest affected individuals known so far.


Assuntos
Ceramidase Ácida/genética , Lipogranulomatose de Farber/genética , Mutação , Osteólise/genética , Adulto , Lipogranulomatose de Farber/diagnóstico , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Linhagem , Fenótipo
15.
Eur J Hum Genet ; 24(11): 1578-1583, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27026573

RESUMO

ASAH1 gene encodes for acid ceramidase that is involved in the degradation of ceramide into sphingosine and free fatty acids within lysosomes. ASAH1 variants cause both the severe and early-onset Farber disease and rare cases of spinal muscular atrophy (SMA) with progressive myoclonic epilepsy (SMA-PME), phenotypically characterized by childhood onset of proximal muscle weakness and atrophy due to spinal motor neuron degeneration followed by occurrence of severe and intractable myoclonic seizures and death in the teenage years. We studied two subjects, a 30-year-old pregnant woman and her 17-year-old sister, affected with a very slowly progressive non-5q SMA since childhood. No history of seizures or myoclonus has been reported and EEG was unremarkable. The molecular study of ASAH1 gene showed the presence of the homozygote nucleotide variation c.124A>G (r.124a>g) that causes the amino acid substitution p.Thr42Ala. Biochemical evaluation of cultured fibroblasts showed both reduction in ceramidase activity and accumulation of ceramide compared with the normal control. This study describes for the first time the association between ASAH1 variants and an adult SMA phenotype with no myoclonic epilepsy nor death in early age, thus expanding the phenotypic spectrum of ASAH1-related SMA. ASAH1 molecular analysis should be considered in the diagnostic testing of non-5q adult SMA patients.


Assuntos
Ceramidase Ácida/genética , Epilepsias Mioclônicas/genética , Atrofia Muscular Espinal/genética , Mutação de Sentido Incorreto , Fenótipo , Ceramidase Ácida/metabolismo , Adolescente , Adulto , Células Cultivadas , Epilepsias Mioclônicas/diagnóstico , Feminino , Homozigoto , Humanos , Atrofia Muscular Espinal/diagnóstico , Gravidez , Irmãos
16.
FEBS Lett ; 590(6): 716-25, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26898341

RESUMO

Glycosphingoid bases are elevated in inherited lysosomal storage disorders with deficient activity of glycosphingolipid catabolizing glycosidases. We investigated the molecular basis of the formation of glucosylsphingosine and globotriaosylsphingosine during deficiency of glucocerebrosidase (Gaucher disease) and α-galactosidase A (Fabry disease). Independent genetic and pharmacological evidence is presented pointing to an active role of acid ceramidase in both processes through deacylation of lysosomal glycosphingolipids. The potential pathophysiological relevance of elevated glycosphingoid bases generated through this alternative metabolism in patients suffering from lysosomal glycosidase defects is discussed.


Assuntos
Ceramidase Ácida/metabolismo , Doença de Fabry/metabolismo , Doença de Gaucher/metabolismo , Glicoesfingolipídeos/metabolismo , Ceramidase Ácida/genética , Acilação , Animais , Modelos Animais de Doenças , Doença de Fabry/genética , Feminino , Doença de Gaucher/genética , Glucosilceramidase/genética , Glucosilceramidase/metabolismo , Glicoesfingolipídeos/química , Células HEK293 , Humanos , Lisossomos/metabolismo , Masculino , Camundongos , Camundongos Knockout , alfa-Galactosidase/genética , alfa-Galactosidase/metabolismo
17.
Eur J Cancer ; 52: 163-72, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26687835

RESUMO

BACKGROUND: Acid ceramidase (AC), a key enzyme in ceramide metabolism, plays a role in cancer progression and resistance to therapy. However, the role of AC in head and neck cancer (HNC) has not been addressed. Here, we investigate the effect of AC inhibition on the response to cisplatin-based chemotherapy for HNC. METHODS: AC protein and messenger RNA (mRNA) expression were examined in primary tumours and paired normal tissues, and in HNC cell lines. The effects of genetic and pharmacological AC inhibition using small hairpin RNA (shRNA) and N-oleoyl-ethanolamine (NOE), alone and in combination with cisplatin, were assessed in human HNC cells by measuring cell viability, cell cycle progression, apoptosis, mRNA, and protein expression, and in preclinical tumour xenograft mouse models. FINDINGS: AC overexpression was observed in four of six primary tumour tissues and six of nine HNC cell lines. Cisplatin sensitivity was significantly decreased by AC overexpression and significantly increased by AC downregulation in HNC cells (P<0.01). NOE or AC shRNA-mediated AC inhibition enhanced cisplatin-induced HNC cell death by increasing ceramide production and activating pro-apoptotic proteins, and these effects were abrogated by PUMA small interfering RNA transfection. AC inhibition promoted cisplatin-induced apoptosis of HNC cells in vitro and in vivo. INTERPRETATIONS: AC overexpression is associated with cisplatin sensitivity, suggesting its potential role as a chemotherapeutic target for HNC. Genetic or pharmacological AC inhibition promotes cisplatin cytotoxicity in HNC cells.


Assuntos
Ceramidase Ácida/antagonistas & inibidores , Antineoplásicos/farmacologia , Carcinoma de Células Escamosas/tratamento farmacológico , Cisplatino/farmacologia , Inibidores Enzimáticos/farmacologia , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Terapêutica com RNAi , Ceramidase Ácida/genética , Ceramidase Ácida/metabolismo , Animais , Apoptose/efeitos dos fármacos , Carcinoma de Células Escamosas/enzimologia , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Ceramidas/metabolismo , Relação Dose-Resposta a Droga , Regulação Neoplásica da Expressão Gênica , Neoplasias de Cabeça e Pescoço/enzimologia , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , Interferência de RNA , RNA Mensageiro/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço , Fatores de Tempo , Transfecção , Ensaios Antitumorais Modelo de Xenoenxerto
18.
Neuromuscul Disord ; 25(12): 959-63, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26526000

RESUMO

Spinal muscular atrophy with progressive myoclonic epilepsy (SMA-PME) is an extremely rare disorder related to the lysosomal storage disease, Farber lipogranulomatosis. Both disorders are autosomal recessive conditions caused by mutations in the ASAH1 gene encoding acid ceramidase. Farber disease is associated with joint deformities, lipomatous skin nodules, and often is fatal by 2-3 years of age; while SMA-PME is characterized by childhood-onset motor neuron disease and progressive myoclonic epilepsy. We report a case of SMA-PME with a novel mutation in the ASAH1 gene encoding acid ceramidase. The proband presented with childhood-onset of diffuse muscle atrophy and hypotonia. He also had diffuse weakness with greater proximal than distal involvement. Tongue fasciculations were present and his reflexes were either diminished or absent. He ambulated with an unsteady and hesitant gait. He subsequently developed myoclonic epilepsy along with other associated features including tremor, polymyoclonus, and sensorineural hearing loss. Neurophysiological studies revealed a motor neuron disorder and generalized epilepsy. Exome sequencing analysis identified compound heterozygous variants and biochemical analysis indicated acid ceramidase activity was approximately 12 percent of normal controls. Our proband was phenotypically similar to other cases of SMA-PME, albeit with somewhat lesser severity, slower progression, and greater longevity. As lysosomal disorders are sometimes amendable to early interventions, it is important to make early diagnoses in these cases. The combination of motor neuron disease and progressive myoclonic epilepsy should prompt genetic evaluation of ASAH1.


Assuntos
Ceramidase Ácida/genética , Lipogranulomatose de Farber/genética , Atrofia Muscular Espinal/genética , Epilepsias Mioclônicas Progressivas/genética , Adulto , Atrofia , Encéfalo/fisiopatologia , Cerebelo/patologia , Lipogranulomatose de Farber/complicações , Lipogranulomatose de Farber/patologia , Lipogranulomatose de Farber/fisiopatologia , Humanos , Masculino , Músculo Esquelético/ultraestrutura , Atrofia Muscular Espinal/complicações , Atrofia Muscular Espinal/patologia , Atrofia Muscular Espinal/fisiopatologia , Mutação , Epilepsias Mioclônicas Progressivas/complicações , Epilepsias Mioclônicas Progressivas/patologia , Epilepsias Mioclônicas Progressivas/fisiopatologia
19.
Cell Metab ; 22(2): 266-278, 2015 Aug 04.
Artigo em Inglês | MEDLINE | ID: mdl-26190650

RESUMO

Sphingolipids have garnered attention for their role in insulin resistance and lipotoxic cell death. We have developed transgenic mice inducibly expressing acid ceramidase that display a reduction in ceramides in adult mouse tissues. Hepatic overexpression of acid ceramidase prevents hepatic steatosis and prompts improvements in insulin action in liver and adipose tissue upon exposure to high-fat diet. Conversely, overexpression of acid ceramidase within adipose tissue also prevents hepatic steatosis and systemic insulin resistance. Induction of ceramidase activity in either tissue promotes a lowering of hepatic ceramides and reduced activation of the ceramide-activated protein kinase C isoform PKCζ, though the induction of ceramidase activity in the adipocyte prompts more rapid resolution of hepatic steatosis than overexpression of the enzyme directly in the liver. Collectively, our observations suggest the existence of a rapidly acting "cross-talk" between liver and adipose tissue sphingolipids, critically regulating glucose metabolism and hepatic lipid uptake.


Assuntos
Ceramidase Ácida/biossíntese , Ceramidas/metabolismo , Fígado Gorduroso/metabolismo , Fígado/metabolismo , Ceramidase Ácida/genética , Tecido Adiposo/metabolismo , Tecido Adiposo/patologia , Animais , Ceramidas/genética , Indução Enzimática , Fígado Gorduroso/genética , Fígado Gorduroso/patologia , Fígado/patologia , Camundongos , Camundongos Transgênicos , Proteína Quinase C-épsilon/genética , Proteína Quinase C-épsilon/metabolismo
20.
Epilepsia ; 56(5): 692-8, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25847462

RESUMO

OBJECTIVE: To present the clinical features and the results of laboratory investigations in three patients with spinal muscular atrophy associated with progressive myoclonic epilepsy (SMA-PME), a rare condition caused by mutations in the N-acylsphingosine amidohydrosilase 1 (ASAH1) gene. METHODS: The patients were submitted to clinical evaluation, neurophysiologic investigations (that included wakefulness and sleep electroencephalography [EEG], video-polygraphic recording with jerk-locked back-averaging, multimodal evoked potentials, and electromyography), brain magnetic resonance imaging (MRI), biochemical screening, muscle and skin biopsies, and molecular genetic analysis. RESULTS: The main clinical features were onset in childhood with proximal muscular weakness, generalized epilepsy with absences and myoclonic seizures, cognitive impairment of variable degree; the course was progressive with muscle wasting and uncontrolled epileptic seizures. In one patient, earlier onset before the age of 2 years was associated with a more complex clinical picture, with abnormal eye movements, progressive cognitive impairment, and a more rapid and severe course. EEG/polygraphic data were consistent with PME, demonstrating generalized spike-and-wave discharges, evidence of positive and negative myoclonia, and prominent photosensitivity. In one patient, transcranial magnetic stimulation showed a hyperexcitable motor cortex, whereas somatosensory evoked potentials were unaffected. Possible involvement of the central acoustic and visual pathways was suggested by abnormal auditory and visual evoked potentials. Muscle biopsies showed typical signs of neurogenic damage. Molecular genetic analysis showed mutations of the ASAH1 gene. SIGNIFICANCE: Our data indicate that SMA-PME associated with ASAH1 mutations is a genetically distinct condition with specific clinical and neurophysiologic features. Further studies are warranted to explore the role of the ASAH1 gene in muscle and brain function.


Assuntos
Ceramidase Ácida/genética , Atrofia Muscular Espinal/complicações , Atrofia Muscular Espinal/genética , Mutação/genética , Epilepsias Mioclônicas Progressivas/complicações , Epilepsias Mioclônicas Progressivas/genética , Adolescente , Criança , Pré-Escolar , Análise Mutacional de DNA , Eletroencefalografia , Potenciais Evocados/genética , Feminino , Humanos , Neuroimagem , Estimulação Transcraniana por Corrente Contínua
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