Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 3.991
Filtrar
1.
PLoS Genet ; 16(7): e1008924, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32673314

RESUMO

Microsatellites are short tandem repeats, ubiquitous in all eukaryotes and represent ~2% of the human genome. Among them, trinucleotide repeats are responsible for more than two dozen neurological and developmental disorders. Targeting microsatellites with dedicated DNA endonucleases could become a viable option for patients affected with dramatic neurodegenerative disorders. Here, we used the Streptococcus pyogenes Cas9 to induce a double-strand break within the expanded CTG repeat involved in myotonic dystrophy type 1, integrated in a yeast chromosome. Repair of this double-strand break generated unexpected large chromosomal deletions around the repeat tract. These deletions depended on RAD50, RAD52, DNL4 and SAE2, and both non-homologous end-joining and single-strand annealing pathways were involved. Resection and repair of the double-strand break (DSB) were totally abolished in a rad50Δ strain, whereas they were impaired in a sae2Δ mutant, only on the DSB end containing most of the repeat tract. This observation demonstrates that Sae2 plays significant different roles in resecting a DSB end containing a repeated and structured sequence as compared to a non-repeated DSB end. In addition, we also discovered that gene conversion was less efficient when the DSB could be repaired using a homologous template, suggesting that the trinucleotide repeat may interfere with gene conversion too. Altogether, these data show that SpCas9 may not be the best choice when inducing a double-strand break at or near a microsatellite, especially in mammalian genomes that contain many more dispersed repeated elements than the yeast genome.


Assuntos
Quebras de DNA de Cadeia Dupla , Distrofia Miotônica/genética , Recombinação Genética , Repetições de Trinucleotídeos/genética , Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas , Deleção Cromossômica , Cromossomos Fúngicos/genética , Reparo do DNA por Junção de Extremidades/genética , DNA Ligase Dependente de ATP/genética , Reparo do DNA/genética , Proteínas de Ligação a DNA/genética , Endonucleases/genética , Conversão Gênica/genética , Genoma Humano/genética , Humanos , Distrofia Miotônica/patologia , Proteína Rad52 de Recombinação e Reparo de DNA/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Expansão das Repetições de Trinucleotídeos/genética
2.
Nucleic Acids Res ; 48(12): 6740-6758, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32463444

RESUMO

How genetic defects trigger the molecular changes that cause late-onset disease is important for understanding disease progression and therapeutic development. Fuchs' endothelial corneal dystrophy (FECD) is an RNA-mediated disease caused by a trinucleotide CTG expansion in an intron within the TCF4 gene. The mutant intronic CUG RNA is present at one-two copies per cell, posing a challenge to understand how a rare RNA can cause disease. Late-onset FECD is a uniquely advantageous model for studying how RNA triggers disease because: (i) Affected tissue is routinely removed during surgery; (ii) The expanded CTG mutation is one of the most prevalent disease-causing mutations, making it possible to obtain pre-symptomatic tissue from eye bank donors to probe how gene expression changes precede disease; and (iii) The affected tissue is a homogeneous single cell monolayer, facilitating accurate transcriptome analysis. Here, we use RNA sequencing (RNAseq) to compare tissue from individuals who are pre-symptomatic (Pre_S) to tissue from patients with late stage FECD (FECD_REP). The abundance of mutant repeat intronic RNA in Pre_S and FECD_REP tissue is elevated due to increased half-life in a corneal cells. In Pre_S tissue, changes in splicing and extracellular matrix gene expression foreshadow the changes observed in advanced disease and predict the activation of the fibrosis pathway and immune system seen in late-stage patients. The absolute magnitude of splicing changes is similar in pre-symptomatic and late stage tissue. Our data identify gene candidates for early drivers of disease and biomarkers that may represent diagnostic and therapeutic targets for FECD. We conclude that changes in alternative splicing and gene expression are observable decades prior to the diagnosis of late-onset trinucleotide repeat disease.


Assuntos
Distrofia Endotelial de Fuchs/genética , Fator de Transcrição 4/genética , Expansão das Repetições de Trinucleotídeos/genética , Repetições de Trinucleotídeos/genética , Adulto , Idoso , Biomarcadores/metabolismo , Córnea/metabolismo , Córnea/patologia , Feminino , Distrofia Endotelial de Fuchs/patologia , Distrofia Endotelial de Fuchs/terapia , Regulação da Expressão Gênica/genética , Predisposição Genética para Doença , Humanos , Íntrons/genética , Masculino , Pessoa de Meia-Idade , Mutação/genética , Especificidade de Órgãos/genética , Análise de Sequência de RNA
6.
BMC Bioinformatics ; 21(1): 75, 2020 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-32093602

RESUMO

BACKGROUND: MicroRNA (miRNA) regulation is associated with several diseases, including neurodegenerative diseases. Several approaches can be used for modeling miRNA regulation. However, their precision may be limited for analyzing multidimensional data. Here, we addressed this question by integrating shape analysis and feature selection into miRAMINT, a methodology that we used for analyzing multidimensional RNA-seq and proteomic data from a knock-in mouse model (Hdh mice) of Huntington's disease (HD), a disease caused by CAG repeat expansion in huntingtin (htt). This dataset covers 6 CAG repeat alleles and 3 age points in the striatum and cortex of Hdh mice. RESULTS: Remarkably, compared to previous analyzes of this multidimensional dataset, the miRAMINT approach retained only 31 explanatory striatal miRNA-mRNA pairs that are precisely associated with the shape of CAG repeat dependence over time, among which 5 pairs with a strong change of target expression levels. Several of these pairs were previously associated with neuronal homeostasis or HD pathogenesis, or both. Such miRNA-mRNA pairs were not detected in cortex. CONCLUSIONS: These data suggest that miRNA regulation has a limited global role in HD while providing accurately-selected miRNA-target pairs to study how the brain may compute molecular responses to HD over time. These data also provide a methodological framework for researchers to explore how shape analysis can enhance multidimensional data analytics in biology and disease.


Assuntos
Doença de Huntington/genética , Aprendizado de Máquina , MicroRNAs/metabolismo , Animais , Encéfalo/metabolismo , Modelos Animais de Doenças , Regulação da Expressão Gênica , Técnicas de Introdução de Genes , Humanos , Proteína Huntingtina/genética , Doença de Huntington/metabolismo , Camundongos , Neurônios/metabolismo , Proteômica , RNA Mensageiro/metabolismo , RNA-Seq , Repetições de Trinucleotídeos
7.
Nat Neurosci ; 23(3): 386-397, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32066985

RESUMO

Repeat-associated non-AUG-initiated translation of expanded CGG repeats (CGG RAN) from the FMR1 5'-leader produces toxic proteins that contribute to neurodegeneration in fragile X-associated tremor/ataxia syndrome. Here we describe how unexpanded CGG repeats and their translation play conserved roles in regulating fragile X protein (FMRP) synthesis. In neurons, CGG RAN acts as an inhibitory upstream open reading frame to suppress basal FMRP production. Activation of mGluR5 receptors enhances FMRP synthesis. This enhancement requires both the CGG repeat and CGG RAN initiation sites. Using non-cleaving antisense oligonucleotides (ASOs), we selectively blocked CGG RAN. This ASO blockade enhanced endogenous FMRP expression in human neurons. In human and rodent neurons, CGG RAN-blocking ASOs suppressed repeat toxicity and prolonged survival. These findings delineate a native function for CGG repeats and RAN translation in regulating basal and activity-dependent FMRP synthesis, and they demonstrate the therapeutic potential of modulating CGG RAN translation in fragile X-associated disorders.


Assuntos
Expansão das Repetições de DNA/genética , Proteína do X Frágil de Retardo Mental/genética , Síndrome do Cromossomo X Frágil/genética , Repetições de Trinucleotídeos/genética , Animais , Linhagem Celular , Sobrevivência Celular/genética , Feminino , Proteína do X Frágil de Retardo Mental/biossíntese , Células-Tronco Pluripotentes Induzidas , Masculino , Camundongos , Neurônios/metabolismo , Oligonucleotídeos Antissenso/farmacologia , Biossíntese de Proteínas , Ratos , Ratos Long-Evans , Ratos Sprague-Dawley , Receptor de Glutamato Metabotrópico 5/biossíntese , Receptor de Glutamato Metabotrópico 5/genética
8.
Proc Natl Acad Sci U S A ; 117(8): 4411-4417, 2020 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-32029588

RESUMO

Identification of repeat-associated non-AUG (RAN) translation in trinucleotide (CAG) repeat diseases has led to the emerging concept that CAG repeat diseases are caused by nonpolyglutamine products. Nonetheless, the in vivo contribution of RAN translation to the pathogenesis of CAG repeat diseases remains elusive. Via CRISPR/Cas9-mediated genome editing, we established knock-in mouse models that harbor expanded CAG repeats in the mouse huntingtin gene to express RAN-translated products with or without polyglutamine peptides. We found that RAN translation is not detected in the knock-in mouse models when expanded CAG repeats are expressed at the endogenous level. Consistently, the expanded CAG repeats that cannot be translated into polyglutamine repeats do not yield the neuropathological and behavioral phenotypes that were found in knock-in mice expressing expanded polyglutamine repeats. Our findings suggest that RAN-translated products do not play a major role in the pathogenesis of CAG repeat diseases and underscore the importance in targeting polyglutamine repeats for therapeutics.


Assuntos
Doença de Huntington/genética , RNA/genética , Animais , Modelos Animais de Doenças , Feminino , Técnicas de Introdução de Genes , Humanos , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Doença de Huntington/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Biossíntese de Proteínas , RNA/metabolismo , Expansão das Repetições de Trinucleotídeos , Repetições de Trinucleotídeos
9.
Proc Natl Acad Sci U S A ; 117(3): 1628-1637, 2020 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-31911468

RESUMO

Friedreich's ataxia (FRDA) is a human hereditary disease caused by the presence of expanded (GAA)n repeats in the first intron of the FXN gene [V. Campuzano et al., Science 271, 1423-1427 (1996)]. In somatic tissues of FRDA patients, (GAA)n repeat tracts are highly unstable, with contractions more common than expansions [R. Sharma et al., Hum. Mol. Genet. 11, 2175-2187 (2002)]. Here we describe an experimental system to characterize GAA repeat contractions in yeast and to conduct a genetic analysis of this process. We found that large-scale contraction is a one-step process, resulting in a median loss of ∼60 triplet repeats. Our genetic analysis revealed that contractions occur during DNA replication, rather than by various DNA repair pathways. Repeats contract in the course of lagging-strand synthesis: The processivity subunit of DNA polymerase δ, Pol32, and the catalytic domain of Rev1, a translesion polymerase, act together in the same pathway to counteract contractions. Accumulation of single-stranded DNA (ssDNA) in the lagging-strand template greatly increases the probability that (GAA)n repeats contract, which in turn promotes repeat instability in rfa1, rad27, and dna2 mutants. Finally, by comparing contraction rates for homopurine-homopyrimidine repeats differing in their mirror symmetry, we found that contractions depend on a repeat's triplex-forming ability. We propose that accumulation of ssDNA in the lagging-strand template fosters the formation of a triplex between the nascent and fold-back template strands of the repeat. Occasional jumps of DNA polymerase through this triplex hurdle, result in repeat contractions in the nascent lagging strand.


Assuntos
Replicação do DNA , Ataxia de Friedreich/genética , Saccharomyces cerevisiae/genética , Repetições de Trinucleotídeos , DNA Polimerase III , Reparo do DNA , DNA de Cadeia Simples , DNA Polimerase Dirigida por DNA , Endonucleases Flap , Humanos , Mutação , Nucleotidiltransferases/genética , Proteína de Replicação A , Proteínas de Saccharomyces cerevisiae
10.
Nucleic Acids Res ; 48(5): 2232-2245, 2020 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-31974547

RESUMO

DNA trinucleotide repeats (TRs) can exhibit dynamic expansions by integer numbers of trinucleotides that lead to neurodegenerative disorders. Strand slipped hairpins during DNA replication, repair and/or recombination may contribute to TR expansion. Here, we combine single-molecule FRET experiments and molecular dynamics studies to elucidate slipping dynamics and conformations of (CAG)n TR hairpins. We directly resolve slipping by predominantly two CAG units. The slipping kinetics depends on the even/odd repeat parity. The populated states suggest greater stability for 5'-AGCA-3' tetraloops, compared with alternative 5'-CAG-3' triloops. To accommodate the tetraloop, even(odd)-numbered repeats have an even(odd) number of hanging bases in the hairpin stem. In particular, a paired-end tetraloop (no hanging TR) is stable in (CAG)n = even, but such situation cannot occur in (CAG)n = odd, where the hairpin is "frustrated'' and slips back and forth between states with one TR hanging at the 5' or 3' end. Trinucleotide interrupts in the repeating CAG pattern associated with altered disease phenotypes select for specific conformers with favorable loop sequences. Molecular dynamics provide atomic-level insight into the loop configurations. Reducing strand slipping in TR hairpins by sequence interruptions at the loop suggests disease-associated variations impact expansion mechanisms at the level of slipped hairpins.


Assuntos
DNA/química , Sequências Repetidas Invertidas , Repetições de Trinucleotídeos , Pareamento de Bases , Transferência Ressonante de Energia de Fluorescência , Cinética , Simulação de Dinâmica Molecular , Conformação de Ácido Nucleico , Imagem Individual de Molécula , Termodinâmica
11.
Nucleic Acids Res ; 48(1): 36-54, 2020 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-31745548

RESUMO

Huntington disease (HD) is a fatal neurodegenerative disease caused by a pathogenic expansion of a CAG repeat in the huntingtin (HTT) gene. There are no disease-modifying therapies for HD. Artificial microRNAs targeting HTT transcripts for degradation have shown preclinical promise and will soon enter human clinical trials. Here, we examine the tolerability and efficacy of non-selective HTT lowering with an AAV5 encoded miRNA targeting human HTT (AAV5-miHTT) in the humanized Hu128/21 mouse model of HD. We show that intrastriatal administration of AAV5-miHTT results in potent and sustained HTT suppression for at least 7 months post-injection. Importantly, non-selective suppression of huntingtin was generally tolerated, however high dose AAV5-miHTT did induce astrogliosis. We observed an improvement of select behavioural and modest neuropathological HD-like phenotypes in Hu128/21 mice, suggesting a potential therapeutic benefit of miRNA-mediated non-selective HTT lowering. Finally, we also observed that potent reduction of wild type HTT (wtHTT) in Hu21 control mice was tolerated up to 7 months post-injection but may induce impairment of motor coordination and striatal atrophy. Taken together, our data suggests that in the context of HD, the therapeutic benefits of mHTT reduction may outweigh the potentially detrimental effects of wtHTT loss following non-selective HTT lowering.


Assuntos
Proteína Huntingtina/genética , Doença de Huntington/terapia , MicroRNAs/genética , Terapia de Alvo Molecular/métodos , Parvovirinae/genética , RNA Mensageiro/genética , Animais , Animais Geneticamente Modificados , Astrócitos/metabolismo , Astrócitos/patologia , Sequência de Bases , Corpo Estriado/metabolismo , Corpo Estriado/patologia , Modelos Animais de Doenças , Dosagem de Genes , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Proteína Huntingtina/antagonistas & inibidores , Proteína Huntingtina/metabolismo , Doença de Huntington/genética , Doença de Huntington/metabolismo , Doença de Huntington/patologia , Camundongos , MicroRNAs/administração & dosagem , MicroRNAs/metabolismo , Neuroglia/metabolismo , Neuroglia/patologia , Neurônios/metabolismo , Neurônios/patologia , Parvovirinae/metabolismo , Desempenho Psicomotor , Estabilidade de RNA , RNA Mensageiro/antagonistas & inibidores , RNA Mensageiro/metabolismo , Repetições de Trinucleotídeos
12.
Gene ; 730: 144257, 2020 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-31759983

RESUMO

Genetic sequence analysis, classification of genome sequence and evolutionary relationship between species using their biological sequences, are the emerging research domain in Bioinformatics. Several methods have already been applied to DNA sequence comparison under tri-nucleotide representation. In this paper, a new form of tri-nucleotide representation is proposed for sequence comparison. The comparison does not depend on the alignment of the sequences. In this representation, the bio-chemical properties of the nucleotides are considered. The novelty of this method is that the sequences of unequal lengths are represented by vectors of the same length and each of the tri-nucleotide formed out of the given sequence has its unique representation. To validate the proposed method, it is verified on several data sets related to mammalians, viruses and bacteria. The results of this method are further compared with those obtained by methods such as probabilistic method, natural vector method, Fourier power spectrum method, multiple encoding vector method, and feature frequency profiles method. Moreover, this method produces accurate phylogeny in all the cases. It is also proved that the time complexity of the present method is less.


Assuntos
Nucleotídeos/química , Análise de Sequência de DNA/métodos , Repetições de Trinucleotídeos/genética , Algoritmos , Animais , Bactérias/genética , Sequência de Bases , Mapeamento Cromossômico/métodos , Análise por Conglomerados , Biologia Computacional/métodos , Genômica/métodos , Humanos , Mamíferos/genética , Nucleotídeos/genética , Filogenia , Alinhamento de Sequência , Vírus/genética
13.
PLoS One ; 14(12): e0226865, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31860647

RESUMO

Siraitia grosvenorii fruit, known as Luo-Han-Guo, has been used as a traditional Chinese medicine for many years, and mogrosides are its primary active ingredients. Unfortunately, Siraitia siamensis, its wild relative, might be misused due to its indistinguishable appearance, not only threatening the reliability of the medication but also partly exacerbating wild resource scarcity. Therefore, high-resolution genetic markers must be developed to discriminate between these species. Here, the complete chloroplast genomes of S. grosvenorii and S. siamensis were assembled and analyzed for the first time; they were 158,757 and 159,190 bp in length, respectively, and possessed conserved quadripartite circular structures. Both contained 134 annotated genes, including 8 rRNA, 37 tRNA and 89 protein-coding genes. Twenty divergences (Pi > 0.03) were found in the intergenic regions. Nine protein-coding genes, accD, atpA, atpE, atpF, clpP, ndhF, psbH, rbcL, and rpoC2, underwent selection within Cucurbitaceae. Phylogenetic relationship analysis indicated that these two species originated from the same ancestor. Finally, four pairs of molecular markers were developed to distinguish the two species. The results of this study will be beneficial for taxonomic research, identification and conservation of Siraitia Merrill wild resources in the future.


Assuntos
Cloroplastos/genética , Cucurbitaceae/genética , Genes de Plantas , Marcadores Genéticos/genética , Genoma de Cloroplastos , Códon/genética , Cucurbitaceae/classificação , Frutas/genética , Medicina Tradicional Chinesa , Anotação de Sequência Molecular , Filogenia , Repetições de Trinucleotídeos/genética , Sequenciamento Completo do Genoma
14.
Int J Mol Sci ; 20(22)2019 Nov 13.
Artigo em Inglês | MEDLINE | ID: mdl-31766224

RESUMO

The congenital form of myotonic dystrophy type 1 (cDM) is caused by the large-scale expansion of a (CTG•CAG)n repeat in DMPK and DM1-AS. The production of toxic transcripts with long trinucleotide tracts from these genes results in impairment of the myogenic differentiation capacity as cDM's most prominent morpho-phenotypic hallmark. In the current in vitro study, we compared the early differentiation programs of isogenic cDM myoblasts with and without a (CTG)2600 repeat obtained by gene editing. We found that excision of the repeat restored the ability of cDM myoblasts to engage in myogenic fusion, preventing the ensuing myotubes from remaining immature. Although the cDM-typical epigenetic status of the DM1 locus and the expression of genes therein were not altered upon removal of the repeat, analyses at the transcriptome and proteome level revealed that early abnormalities in the temporal expression of differentiation regulators, myogenic progression markers, and alternative splicing patterns before and immediately after the onset of differentiation became normalized. Our observation that molecular and cellular features of cDM are reversible in vitro and can be corrected by repeat-directed genome editing in muscle progenitors, when already committed and poised for myogenic differentiation, is important information for the future development of gene therapy for different forms of myotonic dystrophy type 1 (DM1).


Assuntos
Mioblastos/patologia , Distrofia Miotônica/genética , Repetições de Trinucleotídeos , Linhagem Celular , Epigênese Genética , Edição de Genes , Terapia Genética , Humanos , Desenvolvimento Muscular , Mioblastos/citologia , Mioblastos/metabolismo , Distrofia Miotônica/patologia , Distrofia Miotônica/terapia , Miotonina Proteína Quinase/genética
15.
Int J Mol Sci ; 20(21)2019 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-31683630

RESUMO

Spinocerebellar ataxia type 3 (SCA3) is a devastating neurodegenerative disease for which there is currently no cure, nor effective treatment strategy. One of nine polyglutamine disorders known to date, SCA3 is clinically heterogeneous and the main feature is progressive ataxia, which in turn affects speech, balance and gait of the affected individual. SCA3 is caused by an expanded polyglutamine tract in the ataxin-3 protein, resulting in conformational changes that lead to toxic gain of function. The expanded glutamine tract is located at the 5' end of the penultimate exon (exon 10) of ATXN3 gene transcript. Other studies reported removal of the expanded glutamine tract using splice switching antisense oligonucleotides. Here, we describe improved efficiency in the removal of the toxic polyglutamine tract of ataxin-3 in vitro using phosphorodiamidate morpholino oligomers, when compared to antisense oligonucleotides composed of 2'-O-methyl modified bases on a phosphorothioate backbone. Significant downregulation of both the expanded and non-expanded protein was induced by the morpholino antisense oligomer, with a greater proportion of ataxin-3 protein missing the polyglutamine tract. With growing concerns over toxicity associated with long-term administration of phosphorothioate oligonucleotides, the use of a phosphorodiamidate morpholino oligomer may be preferable for clinical application. These results suggest that morpholino oligomers may provide greater therapeutic benefit for the treatment of spinocerebellar ataxia type 3, without toxic effects.


Assuntos
Ataxina-3/genética , Peptídeos/genética , Precursores de RNA/genética , Repetições de Trinucleotídeos/genética , Animais , Ataxina-3/metabolismo , Sequência de Bases , Células Cultivadas , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Doença de Machado-Joseph/genética , Doença de Machado-Joseph/metabolismo , Doença de Machado-Joseph/patologia , Modelos Genéticos , Morfolinos/genética , Morfolinos/metabolismo , Precursores de RNA/metabolismo
16.
J Assist Reprod Genet ; 36(11): 2245-2250, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31625034

RESUMO

INTRODUCTION: Recurrent spontaneous abortion is a multifactorial disorder and till date, various factors have been attributed in its pathogenesis. Still, approximately 50% of RSA cases remain unexplained. Premutation (PM) expanded allele of fragile-X mental retardation 1 (FMR1) gene is known to contribute to ovarian dysfunction in 20% of the cases. Recently, the link between expanded FMR1 allele and recurrent miscarriages has been reported. METHOD: In the present prospective case-control study, we have investigated the status of CGG repeat size at 5'UTR of the FMR1 gene in women with unexplained RSA in comparison to age-matched healthy control women (n = 100 each). The genomic DNA from these samples was subjected to molecular analysis for characterization of CGG repeat size and composition at FMR1 gene RESULTS: As compared to the control women, the RSA women cohort had a higher frequency of carriers with alleles in gray zone (GZ) and expanded PM range, i.e., 2% (2/100) versus 5% (5/100), respectively. Also, the RSA cohort had a significantly higher number of normal alleles with ≥ 35 CGG repeats (24 out of 200 alleles) as compared to control cohort (8 out of 200 alleles). The number of larger FMR1 alleles with pure CGG repeat tract was found to be significantly higher (P = 0.0063) in the RSA cohort (15 out of 200 alleles) as compared to that in control cohort (3 out of 200 alleles). CONCLUSION: Henceforth, the CGG expanded uninterrupted FMR1 allele might be associated with recurrent abortions and may help to explain many of these unexplained cases.


Assuntos
Aborto Habitual/genética , Proteína do X Frágil de Retardo Mental/genética , Adulto , Alelos , Estudos de Casos e Controles , Feminino , Síndrome do Cromossomo X Frágil/genética , Heterozigoto , Humanos , Mutação/genética , Gravidez , Insuficiência Ovariana Primária/genética , Estudos Prospectivos , Expansão das Repetições de Trinucleotídeos/genética , Repetições de Trinucleotídeos/genética
17.
Cell ; 178(5): 1159-1175.e17, 2019 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-31442405

RESUMO

Expansion of CAG trinucleotide repeats in ATXN1 causes spinocerebellar ataxia type 1 (SCA1), a neurodegenerative disease that impairs coordination and cognition. While ATXN1 is associated with increased Alzheimer's disease (AD) risk, CAG repeat number in AD patients is not changed. Here, we investigated the consequences of ataxin-1 loss of function and discovered that knockout of Atxn1 reduced CIC-ETV4/5-mediated inhibition of Bace1 transcription, leading to increased BACE1 levels and enhanced amyloidogenic cleavage of APP, selectively in AD-vulnerable brain regions. Elevated BACE1 expression exacerbated Aß deposition and gliosis in AD mouse models and impaired hippocampal neurogenesis and olfactory axonal targeting. In SCA1 mice, polyglutamine-expanded mutant ataxin-1 led to the increase of BACE1 post-transcriptionally, both in cerebrum and cerebellum, and caused axonal-targeting deficit and neurodegeneration in the hippocampal CA2 region. These findings suggest that loss of ataxin-1 elevates BACE1 expression and Aß pathology, rendering it a potential contributor to AD risk and pathogenesis.


Assuntos
Doença de Alzheimer/patologia , Secretases da Proteína Precursora do Amiloide/metabolismo , Ataxina-1/metabolismo , Encéfalo/metabolismo , Doença de Alzheimer/metabolismo , Secretases da Proteína Precursora do Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Ataxina-1/deficiência , Ataxina-1/genética , Encéfalo/patologia , Região CA2 Hipocampal/metabolismo , Região CA2 Hipocampal/patologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Modelos Animais de Doenças , Feminino , Frequência do Gene , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Neurogênese , Proteínas Proto-Oncogênicas c-ets/genética , Proteínas Proto-Oncogênicas c-ets/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição Genética , Repetições de Trinucleotídeos/genética , Regulação para Cima
18.
Biosens Bioelectron ; 142: 111537, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31376709

RESUMO

Electrochemical detection of specific nucleic acid sequence remains a hot topic in current bioanalytical research. Here, a novel ratiometric electrochemical biosensor based on Exo III-assisted recycling amplification and graphene-modified electrode was fabricated for quantitative detection of trinucleotide repeat sequence d(CAG)n. The double-signals used are the hairpin DNAs labeled with ferrocene and methylene blue respectively as report DNAs, which can hybridize to target DNA. The hybridized DNA was digested by Exo III, resulting in the release of target and report fragments. The graphene-modified electrode can selectively adsorb the released report fragments to generate double electrochemical signals. The signal ratio (F/M) of ferrocene and methylene blue was used to determine the repeat length accurately: a linear relationship was found between F/M and numbers of repeats (n), F/M = 0.061 n + 1.97, with a correlation coefficient of 0.992. Moreover, any electrochemical signal can be used to test repeat concentration with detection limit of 0.22 pM. Therefore, this novel ratiometric electrochemical biosensor provided a reliable and efficient method for the analysis of d(CAG)n trinucleotide repeat and a potential simplified clinical tool for neurodegenerative diseases.


Assuntos
Técnicas Biossensoriais/métodos , Sondas de DNA/química , Exodesoxirribonucleases/química , Técnicas de Amplificação de Ácido Nucleico/métodos , Repetições de Trinucleotídeos , Técnicas Eletroquímicas/métodos , Compostos Ferrosos/química , Grafite/química , Humanos , Limite de Detecção , Metalocenos/química , Azul de Metileno/química , Hibridização de Ácido Nucleico/métodos
20.
Nanotechnology ; 30(46): 465502, 2019 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-31426052

RESUMO

Huntington's disease is a chronic progressive neurodegeneration which is caused by CAG repeat sequences expanding in the huntingtin gene. There is currently no disease-modifying treatment for the disease, and its progression can only be slowed down before the onset of symptoms. A novel fluorescent platform which contains an RNA probe and graphene oxide for detection of the biomarker of Huntington's disease, CAG repeat sequences, was constructed in this investigation. In addition, RNase H was employed in the fluorescent system to enhance the sensitivity of the detection capability. The fluorescent signal was increased through the cyclic amplified reaction, which results from RNase H, specifically digestion of the RNA strand in the complement of the RNA-DNA duplex. The designed measurement method can detect CAG repeat sequences with a detection limit of 108 pM (R2 = 0.968) under which we optimized assay conditions. Furthermore, the detection limit is approximately 18 times lower than the traditional DNA and graphene oxide detection method without assistance of RNase H. Additionally, the probing platform also shows stronger ability to discriminate between the fluorescence of the target sequence and that of other non-target sequences. The results of our studies demonstrate that the RNase H amplified RNA probe and graphene oxide system exhibited excellent sensitivity and selectivity to the target of CAG repeats sequences.


Assuntos
Grafite/química , Doença de Huntington/diagnóstico , Sondas RNA/química , Ribonuclease H/metabolismo , Repetições de Trinucleotídeos , Diagnóstico Precoce , Marcadores Genéticos/genética , Humanos , Doença de Huntington/genética , Sensibilidade e Especificidade , Espectrometria de Fluorescência
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA