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2.
Clin Genet ; 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31573083

RESUMO

Children with Neurofibromatosis Type 1 (NF1) may exhibit an incomplete clinical presentation, making difficult to reach a clinical diagnosis. A phenotypic overlap may exist with children with other RASopathies or with other genetic conditions if only multiple café-au-lait macules (CALMs) are present. The syndromes that can converge in these inconclusive phenotypes have different clinical courses. In this context, an early genetic testing has been proposed to be clinically useful to manage these patients. We present the validation and implementation into diagnostics of a custom NGS panel (I2HCP) for testing patients with a clinical suspicion of a RASopathy (n=48) and children presenting multiple CALMs (n=102). We describe the mutational spectrum and the detection rates identified in these two groups of individuals. We identified pathogenic variants in 21 out of 48 patients with clinical suspicion of RASopathy, with mutations in NF1 accounting for 10% of cases. Furthermore, we identified pathogenic mutations mainly in the NF1 gene, but also in SPRED1, in more than 50% of children with multiple CALMs, exhibiting a NF1 mutational spectrum different from a group of clinically diagnosed NF1 patients (n=80). An NGS panel strategy for the genetic testing of these two phenotype-defined groups outperforms previous strategies. This article is protected by copyright. All rights reserved.

3.
J Inherit Metab Dis ; 2019 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-30942483

RESUMO

There are many metabolic disorders that present with bone phenotypes. In some cases, the pathological bone symptoms are the main features of the disease whereas in others they are a secondary characteristic. In general, the generation of the bone problems in these disorders is not well understood and the therapeutic options for them are scarce. Bone development occurs in the early stages of embryonic development where the bone formation, or osteogenesis, takes place. This osteogenesis can be produced through the direct transformation of the pre-existing mesenchymal cells into bone tissue (intramembranous ossification) or by the replacement of the cartilage by bone (endochondral ossification). In contrast, bone remodeling takes place during the bone's growth, after the bone development, and continues throughout the whole life. The remodeling involves the removal of mineralized bone by osteoclasts followed by the formation of bone matrix by the osteoblasts, which subsequently becomes mineralized. In some metabolic diseases, bone pathological features are associated with bone development problems but in others they are associated with bone remodeling. Here, we describe three examples of impaired bone development or remodeling in metabolic diseases, including work by others and the results from our research. In particular, we will focus on hereditary multiple exostosis (or osteochondromatosis), Gaucher disease, and the susceptibility to atypical femoral fracture in patients treated with bisphosphonates for several years.

4.
Sci Rep ; 8(1): 694, 2018 01 12.
Artigo em Inglês | MEDLINE | ID: mdl-29330474

RESUMO

De novo FOXP1 mutations have been associated with intellectual disability (ID), motor delay, autistic features and a wide spectrum of speech difficulties. C syndrome (Opitz C trigonocephaly syndrome) is a rare and genetically heterogeneous condition, characterized by trigonocephaly, craniofacial anomalies and ID. Several different chromosome deletions and and point mutations in distinct genes have been associated with the disease in patients originally diagnosed as Opitz C. By whole exome sequencing we identified a de novo splicing mutation in FOXP1 in a patient, initially diagnosed as C syndrome, who suffers from syndromic intellectual disability with trigonocephaly. The mutation (c.1428 + 1 G > A) promotes the skipping of exon 16, a frameshift and a premature STOP codon (p.Ala450GLyfs*13), as assessed by a minigene strategy. The patient reported here shares speech difficulties, intellectual disability and autistic features with other FOXP1 syndrome patients, and thus the diagnosis for this patient should be changed. Finally, since trigonocephaly has not been previously reported in FOXP1 syndrome, it remains to be proved whether it may be associated with the FOXP1 mutation.


Assuntos
Craniossinostoses/diagnóstico , Fatores de Transcrição Forkhead/genética , Deficiência Intelectual/diagnóstico , Proteínas Repressoras/genética , Transtorno Autístico/complicações , Transtorno Autístico/diagnóstico , Craniossinostoses/genética , Éxons , Mutação da Fase de Leitura , Humanos , Deficiência Intelectual/genética , Masculino , Processamento de RNA , Distúrbios da Fala/complicações , Distúrbios da Fala/diagnóstico , Sequenciamento Completo do Exoma , Adulto Jovem
5.
Org Biomol Chem ; 15(37): 7977, 2017 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-28905961

RESUMO

Correction for 'Stereodivergent synthesis of right- and left-handed iminoxylitol heterodimers and monomers. Study of their impact on ß-glucocerebrosidase activity' by Fabien Stauffert et al., Org. Biomol. Chem., 2017, 15, 3681-3705.

6.
Org Biomol Chem ; 15(17): 3681-3705, 2017 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-28401966

RESUMO

A library of dimers and heterodimers of both enantiomers of 2-O-alkylated iminoxylitol derivatives has been synthesised and evaluated on ß-glucocerebrosidase (GCase), the enzyme responsible for Gaucher disease (GD). Although the objective was to target simultaneously the active site and a secondary binding site of the glucosidase, the (-)-2-iminoxylitol moiety seemed detrimental for imiglucerase inhibition and no significant enhancement was obtained in G202R, N370S and L444P fibroblasts. However, all compounds having at least one (+)-2-O-alkyl iminoxylitol are GCase inhibitors in the nano molar range and are significant GCase activity enhancers in G202R fibroblats, as confirmed by a decrease of glucosylceramide levels and by co-localization studies.


Assuntos
Dimerização , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Glucosilceramidase/antagonistas & inibidores , Xilitol/síntese química , Xilitol/farmacologia , Domínio Catalítico , Técnicas de Química Sintética , Inibidores Enzimáticos/química , Fibroblastos/efeitos dos fármacos , Fibroblastos/enzimologia , Doença de Gaucher/enzimologia , Glucosilceramidase/química , Glucosilceramidase/metabolismo , Humanos , Transporte Proteico , Estereoisomerismo , Xilitol/química
7.
Sci Rep ; 7: 44138, 2017 03 10.
Artigo em Inglês | MEDLINE | ID: mdl-28281571

RESUMO

Opitz trigonocephaly C syndrome (OTCS) is a rare genetic disorder characterized by craniofacial anomalies, variable intellectual and psychomotor disability, and variable cardiac defects with a high mortality rate. Different patterns of inheritance and genetic heterogeneity are known in this syndrome. Whole exome and genome sequencing of a 19-year-old girl (P7), initially diagnosed with OTCS, revealed a de novo nonsense mutation, p.Q638*, in the MAGEL2 gene. MAGEL2 is an imprinted, maternally silenced, gene located at 15q11-13, within the Prader-Willi region. Patient P7 carried the mutation in the paternal chromosome. Recently, mutations in MAGEL2 have been described in Schaaf-Yang syndrome (SHFYNG) and in severe arthrogryposis. Patient P7 bears resemblances with SHFYNG cases but has other findings not described in this syndrome and common in OTCS. We sequenced MAGEL2 in nine additional OTCS patients and no mutations were found. This study provides the first clear molecular genetic basis for an OTCS case, indicates that there is overlap between OTCS and SHFYNG syndromes, and confirms that OTCS is genetically heterogeneous. Genes encoding MAGEL2 partners, either in the retrograde transport or in the ubiquitination-deubiquitination complexes, are promising candidates as OTCS disease-causing genes.


Assuntos
Craniossinostoses , Deficiência Intelectual , Mutação de Sentido Incorreto , Proteínas , Adulto , Craniossinostoses/genética , Craniossinostoses/metabolismo , Feminino , Humanos , Deficiência Intelectual/genética , Deficiência Intelectual/metabolismo , Proteínas/genética , Proteínas/metabolismo
8.
Sci Rep ; 7: 41931, 2017 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-28167839

RESUMO

Niemann-Pick disease type C (NPC) is a rare neurovisceral disease caused mainly by mutations in the NPC1 gene. This autosomal recessive lysosomal disorder is characterised by the defective lysosomal secretion of cholesterol and sphingolipids. No effective therapy exists for the disease. We previously described a deep intronic point mutation (c.1554-1009 G > A) in NPC1 that generated a pseudoexon, which could be corrected at the cellular level using antisense oligonucleotides. Here, we describe the generation of two mouse models bearing this mutation, one in homozygosity and the other in compound heterozygosity with the c.1920delG mutation. Both the homozygotes for the c.1554-1009 G > A mutation and the compound heterozygotes recapitulated the hallmarks of NPC. Lipid analysis revealed accumulation of cholesterol in the liver and sphingolipids in the brain, with both types of transgenic mice displaying tremor and ataxia at 7-8 weeks of age. Behavioural tests showed motor impairment, hyperactivity, reduced anxiety-like behaviour and impaired learning and memory performances, features consistent with those reported previously in NPC animal models and human patients. These mutant mice, the first NPC models bearing a pseudoexon-generating mutation, could be suitable for assessing the efficacy of specific splicing-targeted therapeutic strategies against NPC.


Assuntos
Comportamento Animal , Proteínas de Transporte/genética , Modelos Animais de Doenças , Éxons , Glicoproteínas de Membrana/genética , Mutação , Doença de Niemann-Pick Tipo C/fisiopatologia , Proteínas/fisiologia , Animais , Proteínas de Transporte/metabolismo , Feminino , Regulação da Expressão Gênica , Humanos , Masculino , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Transgênicos , Testes Neuropsicológicos , Doença de Niemann-Pick Tipo C/psicologia
9.
JIMD Rep ; 36: 41-48, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28105569

RESUMO

Niemann-Pick type C disease (NPC) is a neurovisceral lysosomal storage disease caused by mutations in either the NPC1 or the NPC2 gene. It is a cellular lipid trafficking disorder characterized by the accumulation of unesterified cholesterol and various sphingolipids in the lysosomes and late endosomes, and it exhibits a broad clinical spectrum. Today, over 420 disease-causing mutations have been identified in the NPC1 and the NPC2 genes. We present the clinical, biochemical, and molecular findings in 14 cases diagnosed in Greece during the last 28 years. Age at diagnosis ranged from 2.5 months to 48 years. Systemic manifestations were present in 7/14 patients. All developed neurological manifestations (age of onset 5 months to 42 years). Six patients are still alive (age: 5-50 years). Classical filipin staining pattern was observed in all but four patients (3 NPC1, 1 NPC2). The rate of LDL-induced cholesteryl ester formation was severely reduced in 4/7 and significantly reduced in 3/7 patients studied. Increased chitotriosidase activity was observed in 9/12 patients. Mutation analysis in 11 unrelated patients identified 12 different mutations in the NPC1 gene: eight previously described p.E1089K (c.3265G>A), p.F284Lfs*26 (c.852delT), p.A1132P(c.3394G>C), del promoter region and exons 1-10, p.R1186H (c.3557G>A), p.P1007A (c.3019C>G), p.Q92R(c.275A>G),p.S940L (c.2819C>T), and four novel ones: (p.N701K fs*13 (c.2102-2103insA), p.K1057R (c.3170A>G), IVS23+3insT(c.3591+3insT), p.C1119*(c.3357T>C); and the previously described IVS2+5G>A(c.190+5G>A) mutation in the NPC2 gene. All patients were of Greek origin. Assuming a birth rate of 100,000/year, a rough incidence estimate for NPC disease in Greece would be 0.5/100,000 births.

10.
Curr Protein Pept Sci ; 18(7): 758-764, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-26965692

RESUMO

Gaucher disease is an autosomal recessive lysosomal storage disorder, caused by mutations in the GBA gene. The frequency of Gaucher disease patients and heterozygote carriers that developed Parkinson disease has been found to be above that of the control population. This fact suggests that mutations in the GBA gene can be involved in Parkison's etiology. Analysis of large cohorts of patients with Parkinson disease has shown that there are significantly more cases bearing GBA mutations than those found among healthy individuals. Functional studies have proven an interaction between α-synuclein and GBA, the levels of which presented an inverse correlation. Mutant GBA proteins cause increases in α-synuclein levels, while an inhibition of GBA by α-synuclein has been also demonstrated. Saposin C, a coactivator of GBA, has been shown to protect GBA from this inhibition. Among the GBA variants associated with Parkinson disease, E326K seems to be one of the most prevalent. Interestingly, it is involved in Gaucher disease only when it forms part of a double-mutant allele, usually with the L444P mutation. Structural analyses have revealed that both residues (E326 and L444) interact with Saposin C and, probably, also with α-synuclein. This could explain the antagonistic role of these two proteins in relation to GBA.


Assuntos
Doença de Gaucher/genética , Glucosilceramidase/genética , Mutação , Doença de Parkinson/genética , alfa-Sinucleína/genética , Alelos , Doença de Gaucher/complicações , Doença de Gaucher/metabolismo , Doença de Gaucher/patologia , Expressão Gênica , Frequência do Gene , Glucosilceramidase/metabolismo , Humanos , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Lisossomos/patologia , Doença de Parkinson/complicações , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Saposinas/farmacologia , alfa-Sinucleína/metabolismo
11.
Am J Med Genet A ; 170A(1): 24-31, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26768331

RESUMO

Opitz C trigonocephaly (or Opitz C syndrome, OTCS) and Bohring-Opitz syndrome (BOS or C-like syndrome) are two rare genetic disorders with phenotypic overlap. The genetic causes of these diseases are not understood. However, two genes have been associated with OTCS or BOS with dominantly inherited de novo mutations. Whereas CD96 has been related to OTCS (one case) and to BOS (one case), ASXL1 has been related to BOS only (several cases). In this study we analyze CD96 and ASXL1 in a group of 11 affected individuals, including 2 sibs, 10 of them were diagnosed with OTCS, and one had a BOS phenotype. Exome sequences were available on six patients with OTCS and three parent pairs. Thus, we could analyze the CD96 and ASXL1 sequences in these patients bioinformatically. Sanger sequencing of all exons of CD96 and ASXL1 was carried out in the remaining patients. Detailed scrutiny of the sequences and assessment of variants allowed us to exclude putative pathogenic and private mutations in all but one of the patients. In this patient (with BOS) we identified a de novo mutation in ASXL1 (c.2100dupT). By nature and location within the gene, this mutation resembles those previously described in other BOS patients and we conclude that it may be responsible for the condition. Our results indicate that in 10 of 11, the disease (OTCS or BOS) cannot be explained by small changes in CD96 or ASXL1. However, the cohort is too small to make generalizations about the genetic etiology of these diseases.


Assuntos
Antígenos CD/genética , Craniossinostoses/genética , Deficiência Intelectual/genética , Mutação/genética , Proteínas Repressoras/genética , Adolescente , Criança , Pré-Escolar , Craniossinostoses/patologia , Exoma/genética , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Lactente , Deficiência Intelectual/patologia , Masculino , Linhagem , Fenótipo , Prognóstico
12.
JIMD Rep ; 25: 57-64, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26108647

RESUMO

Krabbe disease is an autosomal recessive neurodegenerative lysosomal storage disease caused by the deficiency of ß-galactocerebrosidase. This deficiency results in the impaired degradation of ß-galactocerebroside, a major myelin lipid, and of galactosylsphingosine. Based on the age of onset of neurological symptoms, an infantile form (90% patients) and late-onset forms (10% patients) of the disease are recognized. Over 130 disease-causing mutations have been identified in the ß-galactocerebrosidase gene. We present the biochemical and molecular findings in 19 cases of Krabbe disease, 17 of them unrelated, diagnosed in Greece over the last 30 years. ß-Galactocerebrosidase activity assayed in leukocyte homogenates using either the tritium-labeled or the fluorescent substrate was diagnostic for all. Increased plasma chitotriosidase activity was found in 11/15 patients.Mutational analysis, carried out in 11 unrelated cases, identified seven different mutations, four previously described (p.I250T, c.1161+6532_polyA+9kbdel, p.K139del, p.D187V) and three novel mutations (p.D610A, c.583-1 G>C, p.W132X), and seven distinct genotypes. The most prevalent mutation was mutation p.I250T, first described in a patient of Greek origin. It accounted for 36.4% (8/22) of the mutant alleles. The second most frequent mutation was c.1161+6532_polyA+9kbdel that accounted for 22.7% (5/22) of the mutant alleles. The observed frequency was lower than that described in Northern European countries and closer to that described in Italian patients.

13.
Stem Cell Reports ; 5(4): 546-57, 2015 Oct 13.
Artigo em Inglês | MEDLINE | ID: mdl-26411903

RESUMO

Induced pluripotent stem cell (iPSC) technology has been successfully used to recapitulate phenotypic traits of several human diseases in vitro. Patient-specific iPSC-based disease models are also expected to reveal early functional phenotypes, although this remains to be proved. Here, we generated iPSC lines from two patients with Sanfilippo type C syndrome, a lysosomal storage disorder with inheritable progressive neurodegeneration. Mature neurons obtained from patient-specific iPSC lines recapitulated the main known phenotypes of the disease, not present in genetically corrected patient-specific iPSC-derived cultures. Moreover, neuronal networks organized in vitro from mature patient-derived neurons showed early defects in neuronal activity, network-wide degradation, and altered effective connectivity. Our findings establish the importance of iPSC-based technology to identify early functional phenotypes, which can in turn shed light on the pathological mechanisms occurring in Sanfilippo syndrome. This technology also has the potential to provide valuable readouts to screen compounds, which can prevent the onset of neurodegeneration.


Assuntos
Células-Tronco Pluripotentes Induzidas/patologia , Mucopolissacaridose III/patologia , Rede Nervosa/patologia , Neurônios/patologia , Técnicas de Cultura de Células , Células Cultivadas , Humanos , Neurogênese
14.
Sci Rep ; 5: 13654, 2015 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-26347037

RESUMO

Sanfilippo syndrome is a rare lysosomal storage disorder caused by an impaired degradation of heparan sulfate (HS). It presents severe and progressive neurodegeneration and currently there is no effective treatment. Substrate reduction therapy (SRT) may be a useful option for neurological disorders of this kind, and several approaches have been tested to date. Here we use different siRNAs targeting EXTL2 and EXTL3 genes, which are important for HS synthesis, as SRT in Sanfilippo C patients' fibroblasts in order to decrease glycosaminoglycan (GAG) storage inside the lysosomes. The results show a high inhibition of the EXTL gene mRNAs (around 90%), a decrease in GAG synthesis after three days (30-60%) and a decrease in GAG storage after 14 days (up to 24%). Moreover, immunocytochemistry analyses showed a clear reversion of the phenotype after treatment. The in vitro inhibition of HS synthesis genes using siRNAs shown here is a first step in the development of a future therapeutic option for Sanfilippo C syndrome.


Assuntos
Proteínas de Membrana/genética , Mucopolissacaridose III/genética , N-Acetilglucosaminiltransferases/genética , Interferência de RNA , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Fibroblastos/metabolismo , Expressão Gênica , Glicosaminoglicanos/metabolismo , Heparitina Sulfato/metabolismo , Humanos , Imuno-Histoquímica , Proteínas de Membrana/metabolismo , Mucopolissacaridose III/metabolismo , Mucopolissacaridose III/terapia , N-Acetilglucosaminiltransferases/metabolismo , Transfecção
15.
PLoS One ; 10(8): e0135873, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26287674

RESUMO

Nonsense mutations are quite prevalent in inherited diseases. Readthrough drugs could provide a therapeutic option for any disease caused by this type of mutation. Geneticin (G418) and gentamicin were among the first to be described. Novel compounds have been generated, but only a few have shown improved results. PTC124 is the only compound to have reached clinical trials. Here we first investigated the readthrough effects of gentamicin on fibroblasts from one patient with Sanfilippo B, one with Sanfilippo C, and one with Maroteaux-Lamy. We found that ARSB activity (Maroteaux-Lamy case) resulted in an increase of 2-3 folds and that the amount of this enzyme within the lysosomes was also increased, after treatment. Since the other two cases (Sanfilippo B and Sanfilippo C) did not respond to gentamicin, the treatments were extended with the use of geneticin and five non-aminoglycoside (PTC124, RTC13, RTC14, BZ6 and BZ16) readthrough compounds (RTCs). No recovery was observed at the enzyme activity level. However, mRNA recovery was observed in both cases, nearly a two-fold increase for Sanfilippo B fibroblasts with G418 and around 1.5 fold increase for Sanfilippo C cells with RTC14 and PTC124. Afterwards, some of the products were assessed through in vitro analyses for seven mutations in genes responsible for those diseases and, also, for Niemann-Pick A/B. Using the coupled transcription/translation system (TNT), the best results were obtained for SMPD1 mutations with G418, reaching a 35% recovery at 0.25 µg/ml, for the p.W168X mutation. The use of COS cells transfected with mutant cDNAs gave positive results for most of the mutations with some of the drugs, although to a different extent. The higher enzyme activity recovery, of around two-fold increase, was found for gentamicin on the ARSB p.W146X mutation. Our results are promising and consistent with those of other groups. Further studies of novel compounds are necessary to find those with more consistent efficacy and fewer toxic effects.


Assuntos
Códon de Terminação/genética , Gentamicinas/uso terapêutico , Mucopolissacaridose III/genética , Mucopolissacaridose VI/genética , Animais , Células COS , Linhagem Celular , Cercopithecus aethiops , Códon sem Sentido/efeitos dos fármacos , Códon sem Sentido/genética , Códon de Terminação/efeitos dos fármacos , Fibroblastos/citologia , Humanos , Lisossomos/metabolismo , Mucopolissacaridose III/tratamento farmacológico , Mucopolissacaridose VI/tratamento farmacológico , Doença de Niemann-Pick Tipo A/tratamento farmacológico , Doença de Niemann-Pick Tipo A/genética , Doença de Niemann-Pick Tipo B/tratamento farmacológico , Doença de Niemann-Pick Tipo B/genética , RNA Mensageiro/genética
16.
Orphanet J Rare Dis ; 9: 180, 2014 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-25491247

RESUMO

BACKGROUND: Mutations affecting RNA splicing represent more than 20% of the mutant alleles in Sanfilippo syndrome type C, a rare lysosomal storage disorder that causes severe neurodegeneration. Many of these mutations are localized in the conserved donor or acceptor splice sites, while few are found in the nearby nucleotides. METHODS: In this study we tested several therapeutic approaches specifically designed for different splicing mutations depending on how the mutations affect mRNA processing. For three mutations that affect the donor site (c.234 + 1G > A, c.633 + 1G > A and c.1542 + 4dupA), different modified U1 snRNAs recognizing the mutated donor sites, have been developed in an attempt to rescue the normal splicing process. For another mutation that affects an acceptor splice site (c.372-2A > G) and gives rise to a protein lacking four amino acids, a competitive inhibitor of the HGSNAT protein, glucosamine, was tested as a pharmacological chaperone to correct the aberrant folding and to restore the normal trafficking of the protein to the lysosome. RESULTS: Partial correction of c.234 + 1G > A mutation was achieved with a modified U1 snRNA that completely matches the splice donor site suggesting that these molecules may have a therapeutic potential for some splicing mutations. Furthermore, the importance of the splice site sequence context is highlighted as a key factor in the success of this type of therapy. Additionally, glucosamine treatment resulted in an increase in the enzymatic activity, indicating a partial recovery of the correct folding. CONCLUSIONS: We have assayed two therapeutic strategies for different splicing mutations with promising results for the future applications.


Assuntos
Chaperonas Moleculares/genética , Mucopolissacaridose III/genética , Mucopolissacaridose III/terapia , Mutação/genética , Processamento de RNA/genética , RNA Nuclear Pequeno/genética , Animais , Células COS , Células Cultivadas , Cercopithecus aethiops , Terapia Genética/métodos , Humanos , Mucopolissacaridose III/diagnóstico
17.
Int J Biochem Cell Biol ; 54: 245-54, 2014 09.
Artigo em Inglês | MEDLINE | ID: mdl-25084554

RESUMO

Gaucher disease is an autosomal recessive lysosomal disorder characterized by the accumulation of glucosylceramide as a result of a deficiency of the enzyme glucocerebrosidase. Several competitive glucocerebrosidase inhibitors are able to act as pharmacological chaperones for an efficient rescue of the mutated, misfolded forms of the enzyme. Along this line, we report in this work on the ability of several aminocyclitols to increase the residual glucocerebrosidase activity in patient fibroblasts with different genotypes. Some of the compounds were slightly active on fibroblasts bearing some mutations, including the highly prevalent N370S mutation. All compounds were highly active as enzyme activity enhancers on fibroblasts from Gaucher disease patients containing the G202R mutation. Moreover, using the novel tagged sphingolipid ω-azidosphingosine, a reduction in the tagged glucosylceramide accumulation was also observed for selected aminocyclitols. Attempts to explain the activity impairment observed in glucocerebrosidase bearing the G202R mutation by comparative molecular dynamic studies on wild type and the G202R mutated proteins (free and isofagomine-bound, in both cases) were unsuccessful. Under the simulation conditions used, no clear effect of the G202R mutation neither over the global structure of the protein nor on the loops that constitute the glucocerebrosidase active site was observed. Since the G202R residue is located on the protein surface, altered protein-membrane or protein-protein interactions could account for the observed differences. In conclusion, we have tested novel compounds that have shown some chaperone effect on particular glucocerebrosidase mutant enzymes, supporting the enhancement therapy as an alternative approach for Gaucher disease.


Assuntos
Fibroblastos/efeitos dos fármacos , Doença de Gaucher/tratamento farmacológico , Glucosilceramidase/genética , Imino Piranoses/farmacologia , Chaperonas Moleculares/farmacologia , Mutação/genética , Pele/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Células Cultivadas , Cristalografia por Raios X , Inibidores Enzimáticos/farmacologia , Fibroblastos/enzimologia , Fibroblastos/patologia , Imunofluorescência , Doença de Gaucher/enzimologia , Doença de Gaucher/genética , Glucosilceramidase/química , Glucosilceramidase/metabolismo , Humanos , Lipídeos , Microscopia Confocal , Simulação de Dinâmica Molecular , Conformação Proteica , Dobramento de Proteína , Pele/enzimologia , Pele/patologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Esfingolipídeos
18.
ChemMedChem ; 9(8): 1744-54, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24976039

RESUMO

A series of hybrid analogues was designed by combination of the iminoxylitol scaffold of parent 1C9-DIX with triazolylalkyl side chains. The resulting compounds were considered potential pharmacological chaperones in Gaucher disease. The DIX analogues reported here were synthesized by CuAAC click chemistry from scaffold 1 (α-1-C-propargyl-1,5-dideoxy-1,5-imino-D-xylitol) and screened as imiglucerase inhibitors. A set of selected compounds were tested as ß-glucocerebrosidase (GBA1) enhancers in fibroblasts from Gaucher patients bearing different genotypes. A number of these DIX compounds were revealed as potent GBA1 enhancers in genotypes containing the G202R mutation, particularly compound DIX-28 (α-1-C-[(1-(3-trimethylsilyl)propyl)-1H-1,2,3-triazol-4-yl)methyl]-1,5-dideoxy-1,5-imino-D-xylitol), bearing the 3-trimethylsilylpropyl group as a new surrogate of a long alkyl chain, with approximately threefold activity enhancement at 10 nM. Despite their structural similarities with isofagomine and with our previously reported aminocyclitols, the present DIX compounds behaved as non-competitive inhibitors, with the exception of the mixed-type inhibitor DIX-28.


Assuntos
Inibidores Enzimáticos/química , Glucosilceramidase/antagonistas & inibidores , Xilitol/química , Células Cultivadas , Química Click , Fibroblastos/citologia , Doença de Gaucher/metabolismo , Doença de Gaucher/patologia , Genótipo , Glucosilceramidase/genética , Glucosilceramidase/metabolismo , Humanos , Imino Piranoses/síntese química , Imino Piranoses/química , Imino Piranoses/metabolismo , Mutação , Ligação Proteica , Xilitol/síntese química , Xilitol/metabolismo
19.
Cell Rep ; 7(3): 883-97, 2014 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-24746815

RESUMO

Inhibition of cholesterol export from late endosomes causes cellular cholesterol imbalance, including cholesterol depletion in the trans-Golgi network (TGN). Here, using Chinese hamster ovary (CHO) Niemann-Pick type C1 (NPC1) mutant cell lines and human NPC1 mutant fibroblasts, we show that altered cholesterol levels at the TGN/endosome boundaries trigger Syntaxin 6 (Stx6) accumulation into VAMP3, transferrin, and Rab11-positive recycling endosomes (REs). This increases Stx6/VAMP3 interaction and interferes with the recycling of αVß3 and α5ß1 integrins and cell migration, possibly in a Stx6-dependent manner. In NPC1 mutant cells, restoration of cholesterol levels in the TGN, but not inhibition of VAMP3, restores the steady-state localization of Stx6 in the TGN. Furthermore, elevation of RE cholesterol is associated with increased amounts of Stx6 in RE. Hence, the fine-tuning of cholesterol levels at the TGN-RE boundaries together with a subset of cholesterol-sensitive SNARE proteins may play a regulatory role in cell migration and invasion.


Assuntos
Colesterol/metabolismo , Endossomos/metabolismo , Proteínas Qa-SNARE/metabolismo , Rede trans-Golgi/metabolismo , Animais , Células CHO , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Movimento Celular , Cricetinae , Cricetulus , Humanos , Integrina alfa5beta1/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Ligação Proteica , Transporte Proteico , Proteínas Qa-SNARE/química , Receptores de Vitronectina/metabolismo , Proteínas SNARE/metabolismo , Proteína 3 Associada à Membrana da Vesícula/química , Proteína 3 Associada à Membrana da Vesícula/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo
20.
Nucleic Acid Ther ; 24(1): 48-56, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24506780

RESUMO

In the past few years, research in targeted mutation therapies has experienced significant advances, especially in the field of rare diseases. In particular, the efficacy of antisense therapy for suppression of normal, pathogenic, or cryptic splice sites has been demonstrated in cellular and animal models and has already reached the clinical trials phase for Duchenne muscular dystrophy. In different inherited metabolic diseases, splice switching oligonucleotides (SSOs) have been used with success in patients' cells to force pseudoexon skipping or to block cryptic splice sites, in both cases recovering normal transcript and protein and correcting the enzyme deficiency. However, future in vivo studies require individual approaches for delivery depending on the gene defect involved, given the different patterns of tissue and organ expression. Herein we review the state of the art of antisense therapy targeting RNA splicing in metabolic diseases, grouped according to their expression patterns-multisystemic, hepatic, or in central nervous system (CNS)-and summarize the recent progress achieved in the field of in vivo delivery of oligonucleotides to each organ or system. Successful body-wide distribution of SSOs and preferential distribution in the liver after systemic administration have been reported in murine models for different diseases, while for CNS limited data are available, although promising results with intratechal injections have been achieved.


Assuntos
Erros Inatos do Metabolismo/genética , Erros Inatos do Metabolismo/terapia , Oligonucleotídeos Antissenso/administração & dosagem , Oligonucleotídeos Antissenso/uso terapêutico , Processamento de RNA , Reparo Gênico Alvo-Dirigido/métodos , Animais , Sistema Nervoso Central/metabolismo , Sistemas de Liberação de Medicamentos , Éxons , Doenças Genéticas Inatas/genética , Doenças Genéticas Inatas/metabolismo , Doenças Genéticas Inatas/terapia , Humanos , Fígado/metabolismo , Masculino , Erros Inatos do Metabolismo/metabolismo , Camundongos , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/terapia , Oligonucleotídeos Antissenso/genética
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