RESUMO
Maternal inactivation of genes encoding components of the subcortical maternal complex (SCMC) and its associated member, PADI6, generally results in early embryo lethality. In humans, SCMC gene variants were found in the healthy mothers of children affected by multilocus imprinting disturbances (MLID). However, how the SCMC controls the DNA methylation required to regulate imprinting remains poorly defined. We generated a mouse line carrying a Padi6 missense variant that was identified in a family with Beckwith-Wiedemann syndrome and MLID. If homozygous in female mice, this variant resulted in interruption of embryo development at the two-cell stage. Single-cell multiomic analyses demonstrated defective maturation of Padi6 mutant oocytes and incomplete DNA demethylation, down-regulation of zygotic genome activation (ZGA) genes, up-regulation of maternal decay genes, and developmental delay in two-cell embryos developing from Padi6 mutant oocytes but little effect on genomic imprinting. Western blotting and immunofluorescence analyses showed reduced levels of UHRF1 in oocytes and abnormal localization of DNMT1 and UHRF1 in both oocytes and zygotes. Treatment with 5-azacytidine reverted DNA hypermethylation but did not rescue the developmental arrest of mutant embryos. Taken together, this study demonstrates that PADI6 controls both nuclear and cytoplasmic oocyte processes that are necessary for preimplantation epigenetic reprogramming and ZGA.
Assuntos
Oócitos , Zigoto , Animais , Criança , Feminino , Humanos , Camundongos , Proteínas Estimuladoras de Ligação a CCAAT/genética , Citoplasma/genética , Citoplasma/metabolismo , Metilação de DNA/genética , Desenvolvimento Embrionário/genética , Impressão Genômica/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Rare Diseases (RD) do not have an exact definition since local authorities define the criteria in different ways, from fewer than 5 people in 10,000, according to the European Union, to the standard world average of 40 cases per 100,000 people [...].
Assuntos
Medicina de Precisão , Doenças Raras , Humanos , Doenças Raras/diagnóstico , Doenças Raras/genética , Doenças Raras/terapiaRESUMO
Fabry disease is a lysosomal storage disease caused by mutations in the GLA gene that encodes alpha-galactosidase (AGAL). The disease causes abnormal globotriaosylceramide (Gb3) storage in the lysosomes. Variants responsible for the genotypic spectrum of Fabry disease include mutations that abolish enzymatic activity and those that cause protein instability. The latter can be successfully treated with small molecules that either bind and stabilize AGAL or indirectly improve its cellular activity. This paper describes the first attempt to reposition curcumin, a nutraceutical, to treat Fabry disease. We tested the efficacy of curcumin in a cell model and found an improvement in AGAL activity for 80% of the tested mutant genotypes (four out of five tested). The fold-increase was dependent on the mutant and ranged from 1.4 to 2.2. We produced evidence that supports a co-chaperone role for curcumin when administered with AGAL pharmacological chaperones (1-deoxygalactonojirimycin and galactose). The combined treatment with curcumin and either pharmacological chaperone was beneficial for four out of five tested mutants and showed fold-increases ranging from 1.1 to 2.3 for DGJ and from 1.1 to 2.8 for galactose. Finally, we tested a long-term treatment on one mutant (L300F) and detected an improvement in Gb3 clearance and lysosomal markers (LAMP-1 and GAA). Altogether, our findings confirmed the necessity of personalized therapies for Fabry patients and paved the way to further studies and trials of treatments for Fabry disease.
Assuntos
Curcumina , Doença de Fabry , Humanos , Doença de Fabry/tratamento farmacológico , Doença de Fabry/genética , alfa-Galactosidase/metabolismo , Curcumina/farmacologia , Curcumina/uso terapêutico , Curcumina/metabolismo , Galactose/metabolismo , Mutação , Lisossomos/metabolismo , 1-Desoxinojirimicina/farmacologia , 1-Desoxinojirimicina/uso terapêuticoRESUMO
BACKGROUND: Triploidy is one of the most common chromosome abnormalities affecting human gestation and accounts for an important fraction of first-trimester miscarriages. Triploidy has been demonstrated in a few cases of recurrent pregnancy loss (RPL) but its molecular mechanisms are unknown. This study aims to identify the genetic cause of RPL associated with fetus triploidy. METHODS: We investigated genomic imprinting, genotyped sequence-tagged site (STS) markers and performed exome sequencing in a family including two sisters with RPL. Moreover, we evaluated oocyte maturation in vivo and in vitro and effect of the candidate protein variant in silico. RESULTS: While features of hydatidiform mole were excluded, the presence of triploidy of maternal origin was demonstrated in the fetuses. Oocyte maturation was deficient and all the maternally inherited pericentromeric STS alleles were homozygous in the fetuses. A deleterious missense variant (p.V1251D) of the cyclin B3 gene (CCNB3) affecting a residue conserved in placental mammals and located in a region that can interact with the cyclin-dependent kinase 1 or cyclin-dependent kinase 2 cosegregated in homozygosity with RPL. CONCLUSION: Here, we report a family in which a damaging variant in cyclin B3 is associated with the failure of oocyte meiosis II and recurrent fetus triploidy, implicating a rationale for CCNB3 testing in RPL.
Assuntos
Aborto Habitual/genética , Ciclina B/genética , Triploidia , Ciclina B/química , Feminino , Humanos , Meiose/genética , Oócitos/fisiologia , Gravidez , Sequenciamento do ExomaRESUMO
Ambroxol (ABX) is a mucolytic agent used for the treatment of respiratory diseases. Bioactivity has been demonstrated as an enhancement effect on lysosomal acid ß-glucosidase (ß-Glu) activity in Gaucher disease (GD). The positive effects observed have been attributed to a mechanism of action similar to pharmacological chaperones (PCs), but an exact mechanistic description is still pending. The current study uses cell culture and in vitro assays to study the effects of ABX on ß-Glu activity, processing, and stability upon ligand binding. Structural analogues bromohexine, 4-hydroxybromohexine, and norbromohexine were screened for chaperone efficacy, and in silico docking was performed. The sugar mimetic isofagomine (IFG) strongly inhibits ß-Glu, while ABX exerts its inhibitory effect in the micromolar range. In GD patient fibroblasts, IFG and ABX increase mutant ß-Glu activity to identical levels. However, the characteristics of the banding patterns of Endoglycosidase-H (Endo-H)-digested enzyme and a substantially lower half-life of ABX-treated ß-Glu suggest different intracellular processing. In line with this observation, IFG efficiently stabilizes recombinant ß-Glu against thermal denaturation in vitro, whereas ABX exerts no significant effect. Additional ß-Glu enzyme activity testing using Bromohexine (BHX) and two related structures unexpectedly revealed that ABX alone can refunctionalize ß-Glu in cellula. Taken together, our data indicate that ABX has little in vitro ability to act as PC, so the mode of action requires further clarification.
Assuntos
Ambroxol , Doença de Gaucher , Ambroxol/farmacologia , Ambroxol/uso terapêutico , Doença de Gaucher/tratamento farmacológico , Glucosilceramidase/metabolismo , Humanos , Chaperonas Moleculares/metabolismo , beta-Glucosidase/químicaRESUMO
Fabry disease is caused by a deficiency of lysosomal alpha galactosidase and has a very large genotypic and phenotypic spectrum. Some patients who carry hypomorphic mutations can benefit from oral therapy with a pharmacological chaperone. The drug requires a very precise regimen because it is a reversible inhibitor of alpha-galactosidase. We looked for molecules that can potentiate this pharmacological chaperone, among drugs that have already been approved for other diseases. We tested candidate molecules in fibroblasts derived from a patient carrying a large deletion in the gene GLA, which were stably transfected with a plasmid expressing hypomorphic mutants. In our cell model, three drugs were able to potentiate the action of the pharmacological chaperone. We focused our attention on one of them, acetylsalicylic acid. We expect that acetylsalicylic acid can be used in synergy with the Fabry disease pharmacological chaperone and prolong its stabilizing effect on alpha-galactosidase.
Assuntos
Doença de Fabry , alfa-Galactosidase , 1-Desoxinojirimicina/farmacologia , 1-Desoxinojirimicina/uso terapêutico , Aspirina/farmacologia , Aspirina/uso terapêutico , Reposicionamento de Medicamentos , Doença de Fabry/tratamento farmacológico , Doença de Fabry/genética , Humanos , Lisossomos , Chaperonas Moleculares/genética , Mutação , alfa-Galactosidase/genética , alfa-Galactosidase/uso terapêuticoRESUMO
The lysosomal storage disorder Fabry disease is characterized by a deficiency of the lysosomal enzyme α-Galactosidase A. The observation that missense variants in the encoding GLA gene often lead to structural destabilization, endoplasmic reticulum retention and proteasomal degradation of the misfolded, but otherwise catalytically functional enzyme has resulted in the exploration of alternative therapeutic approaches. In this context, we have investigated proteostasis regulators (PRs) for their potential to increase cellular enzyme activity, and to reduce the disease-specific accumulation of the biomarker globotriaosylsphingosine in patient-derived cell culture. The PRs also acted synergistically with the clinically approved 1-deoxygalactonojirimycine, demonstrating the potential of combination treatment in a therapeutic application. Extensive characterization of the effective PRs revealed inhibition of the proteasome and elevation of GLA gene expression as paramount effects. Further analysis of transcriptional patterns of the PRs exposed a variety of genes involved in proteostasis as potential modulators. We propose that addressing proteostasis is an effective approach to discover new therapeutic targets for diseases involving folding and trafficking-deficient protein mutants.
Assuntos
Doença de Fabry/genética , Doenças por Armazenamento dos Lisossomos/genética , Proteostase/genética , alfa-Galactosidase/genética , 1-Desoxinojirimicina/análogos & derivados , 1-Desoxinojirimicina/uso terapêutico , Biomarcadores/metabolismo , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Doença de Fabry/tratamento farmacológico , Doença de Fabry/enzimologia , Doença de Fabry/patologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Doenças por Armazenamento dos Lisossomos/tratamento farmacológico , Doenças por Armazenamento dos Lisossomos/enzimologia , Doenças por Armazenamento dos Lisossomos/patologia , Lisossomos/enzimologia , Lisossomos/genética , Lisossomos/metabolismo , Mutação de Sentido Incorreto/genética , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Transporte Proteico/efeitos dos fármacos , Esfingosina/análogos & derivados , Esfingosina/metabolismoRESUMO
BACKGROUND: Bioinformatics has pervaded all fields of biology and has become an indispensable tool for almost all research projects. Although teaching bioinformatics has been incorporated in all traditional life science curricula, practical hands-on experiences in tight combination with wet-lab experiments are needed to motivate students. RESULTS: We present a tutorial that starts from a practical problem: finding novel enzymes from marine environments. First, we introduce the idea of metagenomics, a recent approach that extends biotechnology to non-culturable microbes. We presuppose that a probe for the screening of metagenomic cosmid library is needed. The students start from the chemical structure of the substrate that should be acted on by the novel enzyme and end with the sequence of the probe. To attain their goal, they discover databases such as BRENDA and programs such as BLAST and Clustal Omega. Students' answers to a satisfaction questionnaire show that a multistep tutorial integrated into a research wet-lab project is preferable to conventional lectures illustrating bioinformatics tools. CONCLUSION: Experimental biologists can better operate basic bioinformatics if a problem-solving approach is chosen.
Assuntos
Biotecnologia/educação , Biologia Computacional/educação , Biologia Marinha/educação , Metagenômica , Sequência de Aminoácidos , Proteínas de Bactérias/química , Sequência de Bases , Bases de Dados Factuais , Bases de Dados de Proteínas , Objetivos , Humanos , Aprendizagem , Interface Usuário-ComputadorRESUMO
The term "pharmacological chaperone" was introduced 20 years ago. Since then the approach with this type of drug has been proposed for several diseases, lysosomal storage disorders representing the most popular targets. The hallmark of a pharmacological chaperone is its ability to bind a protein specifically and stabilize it. This property can be beneficial for curing diseases that are associated with protein mutants that are intrinsically active but unstable. The total activity of the affected proteins in the cell is lower than normal because they are cleared by the quality control system. Although most pharmacological chaperones are reversible competitive inhibitors or antagonists of their target proteins, the inhibitory activity is neither required nor desirable. This issue is well documented by specific examples among which those concerning Fabry disease. Direct specific binding is not the only mechanism by which small molecules can rescue mutant proteins in the cell. These drugs and the properly defined pharmacological chaperones can work together with different and possibly synergistic modes of action to revert a disease phenotype caused by an unstable protein.
Assuntos
Doença de Fabry , Chaperonas Moleculares/uso terapêutico , Mutação de Sentido Incorreto , alfa-Galactosidase , Doença de Fabry/tratamento farmacológico , Doença de Fabry/enzimologia , Doença de Fabry/genética , Humanos , alfa-Galactosidase/genética , alfa-Galactosidase/metabolismoRESUMO
Fabry disease is one of the most common lysosomal storage disorders caused by mutations in the gene encoding lysosomal α-galactosidase A (α-Gal A) and resultant accumulation of glycosphingolipids. The sugar mimetic 1-deoxygalactonojirimycin (DGJ), an orally available pharmacological chaperone, was clinically approved as an alternative to intravenous enzyme replacement therapy. The decision as to whether a patient should be treated with DGJ depends on the genetic variant within the α-galactosidase A encoding gene (GLA). A good laboratory practice (GLP)-validated cell culture-based assay to investigate the biochemical responsiveness of the variants is currently the only source available to obtain pivotal information about susceptibility to treatment. Herein, variants were defined amenable when an absolute increase in enzyme activity of ≥3% of wild type enzyme activity and a relative increase in enzyme activity of ≥1.2-fold was achieved following DGJ treatment. Efficacy testing was carried out for over 1000 identified GLA variants in cell culture. Recent data suggest that about one-third of the variants comply with the amenability criteria. A recent study highlighted the impact of inter-assay variability on DGJ amenability, thereby reducing the power of the assay to predict eligible patients. This prompted us to compare our own α-galactosidase A enzyme activity data in a very similar in-house developed assay with those from the GLP assay. In an essentially retrospective approach, we reviewed 148 GLA gene variants from our former studies for which enzyme data from the GLP study were available and added novel data for 30 variants. We also present data for 18 GLA gene variants for which no data from the GLP assay are currently available. We found that both differences in experimental biochemical data and the criteria for the classification of amenability cause inter-assay discrepancy. We conclude that low baseline activity, borderline biochemical responsiveness, and inter-assay discrepancy are alarm signals for misclassifying a variant that must not be ignored. Furthermore, there is no solid basis for setting a minimum response threshold on which a clinical indication with DGJ can be justified.
Assuntos
Substituição de Aminoácidos , Doença de Fabry/genética , alfa-Galactosidase/metabolismo , 1-Desoxinojirimicina/análogos & derivados , 1-Desoxinojirimicina/uso terapêutico , Bioensaio , Doença de Fabry/tratamento farmacológico , Doença de Fabry/metabolismo , Células HEK293 , Humanos , Medicina de Precisão , Reprodutibilidade dos Testes , Estudos Retrospectivos , alfa-Galactosidase/genéticaRESUMO
The urokinase receptor (uPAR) stimulates cell proliferation by forming a macromolecular complex with αvß3 integrin and the epidermal growth factor receptor (EGFR, ErbB1 or HER1) that we name the uPAR proliferasome. uPAR transactivates EGFR, which in turn mediates uPAR-initiated mitogenic signal to the cell. EGFR activation and EGFR-dependent cell growth are blocked in the absence of uPAR expression or when uPAR activity is inhibited by antibodies against either uPAR or EGFR. The mitogenic sequence of uPAR corresponds to the D2A motif present in domain 2. NMR analysis revealed that D2A synthetic peptide has a particular three-dimensional structure, which is atypical for short peptides. D2A peptide is as effective as EGF in promoting EGFR phosphorylation and cell proliferation that were inhibited by AG1478, a specific inhibitor of the tyrosine kinase activity of EGFR. Both D2A and EGF failed to induce proliferation of NR6-EGFR-K721A cells expressing a kinase-defective mutant of EGFR. Moreover, D2A peptide and EGF phosphorylate ERK demonstrating the involvement of the MAP kinase signalling pathway. Altogether, this study reveals the importance of sequence D2A of uPAR, and the interdependence of uPAR and EGFR.
Assuntos
Proliferação de Células , Receptores ErbB/metabolismo , Integrina alfaVbeta3/metabolismo , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Sequência de Aminoácidos , Linhagem Celular Tumoral , Células HEK293 , Humanos , Sistema de Sinalização das MAP Quinases , Modelos Moleculares , Fosforilação , Receptores de Ativador de Plasminogênio Tipo Uroquinase/químicaRESUMO
A large number of mutations causing PMM2-CDG, which is the most frequent disorder of glycosylation, destabilize phosphomannomutase2. We looked for a pharmacological chaperone to cure PMM2-CDG, starting from the structure of a natural ligand of phosphomannomutase2, α-glucose-1,6-bisphosphate. The compound, ß-glucose-1,6-bisphosphate, was synthesized and characterized via 31P-NMR. ß-glucose-1,6-bisphosphate binds its target enzyme in silico. The binding induces a large conformational change that was predicted by the program PELE and validated in vitro by limited proteolysis. The ability of the compound to stabilize wild type phosphomannomutase2, as well as frequently encountered pathogenic mutants, was measured using thermal shift assay. ß-glucose-1,6-bisphosphate is relatively resistant to the enzyme that specifically hydrolyses natural esose-bisphosphates.
Assuntos
Defeitos Congênitos da Glicosilação/genética , Defeitos Congênitos da Glicosilação/metabolismo , Glucose-6-Fosfato/análogos & derivados , Mutação , Fosfotransferases (Fosfomutases)/deficiência , Defeitos Congênitos da Glicosilação/tratamento farmacológico , Glucose-6-Fosfato/química , Glucose-6-Fosfato/metabolismo , Glucose-6-Fosfato/farmacologia , Humanos , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Conformação Molecular , Fosfotransferases (Fosfomutases)/genética , Ligação ProteicaRESUMO
BACKGROUND: Severity gradation of missense mutations is a big challenge for exome annotation. Predictors of deleteriousness that are most frequently used to filter variants found by next generation sequencing, produce qualitative predictions, but also numerical scores. It has never been tested if these scores correlate with disease severity. RESULTS: wANNOVAR, a popular tool that can generate several different types of deleteriousness-prediction scores, was tested on Fabry disease. This pathology, which is caused by a deficit of lysosomal alpha-galactosidase, has a very large genotypic and phenotypic spectrum and offers the possibility of associating a quantitative measure of the damage caused by mutations to the functioning of the enzyme in the cells. Some predictors, and in particular VEST3 and PolyPhen2 provide scores that correlate with the severity of lysosomal alpha-galactosidase mutations in a statistically significant way. CONCLUSIONS: Sorting disease mutations by severity is possible and offers advantages over binary classification. Dataset for testing and training in silico predictors can be obtained by transient transfection and evaluation of residual activity of mutants in cell extracts. This approach consents to quantitative data for severe, mild and non pathological variants.
Assuntos
Lisossomos/enzimologia , Anotação de Sequência Molecular/métodos , Mutação de Sentido Incorreto/genética , alfa-Galactosidase/genética , Doença de Fabry/enzimologia , Doença de Fabry/genética , Humanos , FenótipoRESUMO
Type I disorders of glycosylation (CDG), the most frequent of which is phosphomannomutase 2 (PMM2-CDG), are a group of diseases causing the incomplete N-glycosylation of proteins. PMM2-CDG is an autosomal recessive disease with a large phenotypic spectrum, and is associated with mutations in the PMM2 gene. The biochemical analysis of mutants does not allow a precise genotypeâ»phenotype correlation for PMM2-CDG. PMM2 is very tolerant to missense and loss of function mutations, suggesting that a partial deficiency of activity might be beneficial under certain circumstances. The patient phenotype might be influenced by variants in other genes associated with the type I disorders of glycosylation in the general population.
Assuntos
Defeitos Congênitos da Glicosilação/diagnóstico , Defeitos Congênitos da Glicosilação/genética , Mutação , Fosfotransferases (Fosfomutases)/genética , Estudos de Associação Genética , Glicosilação , Humanos , Modelos Moleculares , Mutação de Sentido Incorreto , Fenótipo , Fosfotransferases (Fosfomutases)/química , Conformação ProteicaRESUMO
Fabry disease is caused by mutations in the GLA gene and is characterized by a large genotypic and phenotypic spectrum. Missense mutations pose a special problem for graduating diagnosis and choosing a cost-effective therapy. Some mutants retain enzymatic activity, but are less stable than the wild type protein. These mutants can be stabilized by small molecules which are defined as pharmacological chaperones. The first chaperone to reach clinical trial is 1-deoxygalactonojirimycin, but others have been tested in vitro. Residual activity of GLA mutants has been measured in the presence or absence of pharmacological chaperones by several authors. Data obtained from transfected cells correlate with those obtained in cells derived from patients, regardless of whether 1-deoxygalactonojirimycin was present or not. The extent to which missense mutations respond to 1-deoxygalactonojirimycin is variable and a reference table of the results obtained by independent groups that is provided with this paper can facilitate the choice of eligible patients. A review of other pharmacological chaperones is provided as well. Frequent mutations can have residual activity as low as one-fourth of normal enzyme in vitro. The reference table with residual activity of the mutants facilitates the identification of non-pathological variants.
Assuntos
1-Desoxinojirimicina/análogos & derivados , Doença de Fabry/tratamento farmacológico , Doença de Fabry/genética , alfa-Galactosidase/genética , 1-Desoxinojirimicina/uso terapêutico , Doença de Fabry/patologia , Humanos , Chaperonas Moleculares/genética , Mutação de Sentido Incorreto/genética , Medicina de Precisão , Bibliotecas de Moléculas Pequenas/uso terapêuticoRESUMO
The most common glycosylation disorder is caused by mutations in the gene encoding phosphomannomutase2, producing a disease still without a cure. Phosphomannomutase2, a homodimer in which each chain is composed of two domains, requires a bisphosphate sugar (either mannose or glucose) as activator, opening a possible drug design path for therapeutic purposes. The crystal structure of human phosphomannomutase2, however, lacks bound substrate and a key active site loop. To speed up drug discovery, we present here the first structural model of a bisphosphate substrate bound to human phosphomannomutase2. Taking advantage of recent developments in all-atom simulation techniques in combination with limited and site-directed proteolysis, we demonstrated that α-glucose 1,6-bisphosphate can adopt two low energy orientations as required for catalysis. Upon ligand binding, the two domains come close, making the protein more compact, in analogy to the enzyme in the crystals from Leishmania mexicana. Moreover, proteolysis was also carried out on two common mutants, R141H and F119L. It was an unexpected finding that the mutant most frequently found in patients, R141H, although inactive, does bind α-glucose 1,6-bisphosphate and changes conformation.
Assuntos
Erros Inatos do Metabolismo/enzimologia , Fosfotransferases (Fosfomutases)/química , Fosfotransferases (Fosfomutases)/metabolismo , Sequência de Aminoácidos , Animais , Glucose-6-Fosfato/análogos & derivados , Glucose-6-Fosfato/metabolismo , Glicosilação , Humanos , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Peptídeo Hidrolases/metabolismo , Fosfotransferases (Fosfomutases)/genética , Ligação Proteica , Conformação Proteica , Desdobramento de Proteína , Proteólise , TemperaturaRESUMO
BACKGROUND: The majority of the disease-causing mutations affect protein stability, but not functional sites and are amenable, in principle, to be treated with pharmacological chaperones. These drugs enhance the thermodynamic stability of their targets. Fabry disease, a disorder caused by mutations in the gene encoding lysosomal alpha-galactosidase, represents an excellent model system to develop experimental protocols to test the efficiency of such drugs. METHODS: The stability of lysosomal alpha-galactosidase under different conditions was studied by urea-induced unfolding followed by limited proteolysis and Western blotting. RESULTS: We measured the concentration of urea needed to obtain half-maximal unfolding because this parameter represents an objective indicator of protein stability. CONCLUSIONS: Urea-induced unfolding is a versatile technique that can be adapted to cell extracts containing tiny amounts of wild-type or mutant proteins. It allows testing of protein stability as a function of pH, in the presence or in the absence of drugs. Results are not influenced by the method used to express the protein in transfected cells. GENERAL SIGNIFICANCE: Scarce and dispersed populations pose a problem for the clinical trial of drugs for rare diseases. This is particularly true for pharmacological chaperones that must be tested on each mutation associated with a given disease. Diverse in vitro tests are needed. We used a method based on chemically induced unfolding as a tool to assess whether a particular Fabry mutation is responsive to pharmacological chaperones, but, by no means is our protocol limited to this disease.
Assuntos
Doença de Fabry/genética , Chaperonas Moleculares/química , Dobramento de Proteína , Termodinâmica , alfa-Galactosidase/química , Animais , Células COS , Chlorocebus aethiops , Doença de Fabry/tratamento farmacológico , Células HEK293 , Humanos , Mutação , Estabilidade Proteica , Ureia/farmacologia , alfa-Galactosidase/genéticaRESUMO
At chromosome 11p15.5, the imprinting centre 1 (IC1) controls the parent of origin-specific expression of the IGF2 and H19 genes. The 5 kb IC1 region contains multiple target sites (CTS) for the zinc-finger protein CTCF, whose binding on the maternal chromosome prevents the activation of IGF2 and allows that of H19 by common enhancers. CTCF binding helps maintaining the maternal IC1 methylation-free, whereas on the paternal chromosome gamete-inherited DNA methylation inhibits CTCF interaction and enhancer-blocking activity resulting in IGF2 activation and H19 silencing. Maternally inherited 1.4-2.2 kb deletions are associated with methylation of the residual CTSs and Beckwith-Wiedemann syndrome, although with different penetrance and expressivity. We explored the relationship between IC1 microdeletions and phenotype by analysing a number of previously described and novel mutant alleles. We used a highly quantitative assay based on next generation sequencing to measure DNA methylation in affected families and analysed enhancer-blocking activity and CTCF binding in cultured cells. We demonstrate that the microdeletions mostly affect IC1 function and CTCF binding by changing CTS spacing. Thus, the extent of IC1 inactivation and the clinical phenotype are influenced by the arrangement of the residual CTSs. A CTS spacing similar to the wild-type allele results in moderate IC1 inactivation and is associated with stochastic DNA methylation of the maternal IC1 and incomplete penetrance. Microdeletions with different CTS spacing display severe IC1 inactivation and are associated with IC1 hypermethylation and complete penetrance. Careful characterization of the IC1 microdeletions is therefore needed to predict recurrence risks and phenotypical outcomes.
Assuntos
Deleção de Genes , Impressão Genômica , Fator de Crescimento Insulin-Like II/genética , Fenótipo , RNA Longo não Codificante/genética , Proteínas Repressoras/genética , Alelos , Sítios de Ligação/genética , Fator de Ligação a CCCTC , Células Cultivadas , Imunoprecipitação da Cromatina , Cromossomos Humanos Par 11/genética , Metilação de DNA , Regulação da Expressão Gênica , Inativação Gênica , Loci Gênicos , Humanos , Fator de Crescimento Insulin-Like II/metabolismo , Linhagem , RNA Longo não Codificante/metabolismo , Proteínas Repressoras/metabolismo , Análise de Sequência de DNARESUMO
Silver-Russell syndrome (SRS) is a heterogeneous disorder characterized by intrauterine and post-natal growth retardation, dysmorphic facial features and body asymmetry. About 50% of the patients carry (epi)genetic alterations involving chromosomes 7 or 11.The high proportion of patients with unidentified molecular etiology suggests the involvement of other genes. Interestingly, SRS patients share clinical features with the 12q14 microdeletion syndrome, characterized by several deletions with a 2.6 Mb region of overlap. Among the genes present in this interval, high mobility AT-hook 2 (HMGA2) appears to be the most likely cause of the growth deficiency, due to its described growth control function. To define the role of HMGA2 in SRS, we looked for 12q14 chromosome imbalances and HMGA2 mutations in a cohort of 45 patients with growth retardation and SRS-like phenotype but no 11p15 (epi)mutations or maternal uniparental disomy of chromosome 7 (matUPD7). We identified a novel 7 bp intronic deletion in HMGA2 present in heterozygosity in the proband and her mother both displaying the typical features of SRS. We demonstrated that the deletion affected normal splicing, indicating that it is a likely cause of HMGA2 deficiency. This study provides the first evidence that a loss-of-function mutation of HMGA2 can be associated with a familial form of SRS. We suggest that HMGA2 mutations leading to haploinsufficiency should be investigated in the SRS patients negative for the typical 11p15 (epi)mutations and matUPD7.
Assuntos
Proteína HMGA2/genética , Síndrome de Silver-Russell/genética , Sequência de Bases , Estudos de Casos e Controles , Pré-Escolar , Análise Mutacional de DNA , Feminino , Estudos de Associação Genética , Humanos , Linhagem , Fenótipo , Sítios de Splice de RNA , Deleção de SequênciaRESUMO
A cluster of imprinted genes at chromosome 11p15.5 is associated with the growth disorders, Silver-Russell syndrome (SRS) and Beckwith-Wiedemann syndrome (BWS). The cluster is divided into two domains with independent imprinting control regions (ICRs). We describe two maternal 11p15.5 microduplications with contrasting phenotypes. The first is an inverted and in cis duplication of the entire 11p15.5 cluster associated with the maintenance of genomic imprinting and with the SRS phenotype. The second is a 160 kb duplication also inverted and in cis, but resulting in the imprinting alteration of the centromeric domain. It includes the centromeric ICR (ICR2) and the most 5' 20 kb of the non-coding KCNQ1OT1 gene. Its maternal transmission is associated with ICR2 hypomethylation and the BWS phenotype. By excluding epigenetic mosaicism, cell clones analysis indicated that the two closely located ICR2 sequences resulting from the 160 kb duplication carried discordant DNA methylation on the maternal chromosome and supported the hypothesis that the ICR2 sequence is not sufficient for establishing imprinted methylation and some other property, possibly orientation-dependent, is needed. Furthermore, the 1.2 Mb duplication demonstrated that all features are present for correct imprinting at ICR2 when this is duplicated and inverted within the entire cluster. In the individuals maternally inheriting the 160 kb duplication, ICR2 hypomethylation led to the expression of a truncated KCNQ1OT1 transcript and to down-regulation of CDKN1C. We demonstrated by chromatin RNA immunopurification that the KCNQ1OT1 RNA interacts with chromatin through its most 5' 20 kb sequence, providing a mechanism likely mediating the silencing activity of this long non-coding RNA.