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1.
Gene ; 767: 145174, 2021 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-33007370

RESUMO

The expansion of trinucleotide CGG repeats in the promoter of fragile X mental retardation 1 (FMR1) gene is associated with fragile X and fragile X associated tremor/ataxia syndromes. While the expansion of CGG repeats has been associated with such neuro/psychiatric diseases, the contraction of CGG repeats has been recently suggested as an indication of ovarian dysfunction. This study aimed to evaluate a possible association of the short CGG repeats with poor ovarian responders (POR) and to test for a possible correlation between the CGG size and different known markers of the ovarian reserve, namely FSH, AMH, and the number of retrieved oocytes from Jordanian females. We found a significant difference between the CGG median allele size between the cases and the controls (p < 0.001), where poor ovarian responders had shorter CGG repeats compared to the healthy controls. Also, females with alleles <26 had twice the odds to be presented in the POR compared to the controls. However, we did not find a significant correlation between CGG sizes and the markers of ovarian reserve. We conclude that although low CGG repeats appear to be linked to POR, the clinical utility of FMR1 for predicting ovarian response needs further investigation.


Assuntos
Proteína do X Frágil de Retardo Mental/genética , Reserva Ovariana/genética , Repetições de Trinucleotídeos/genética , Adulto , Alelos , Hormônio Antimülleriano/genética , Ataxia , Feminino , Hormônio Foliculoestimulante/genética , Proteína do X Frágil de Retardo Mental/metabolismo , Síndrome do Cromossomo X Frágil , Frequência do Gene/genética , Humanos , Jordânia/epidemiologia , Reserva Ovariana/fisiologia , Ovário/metabolismo , Insuficiência Ovariana Primária/genética , Insuficiência Ovariana Primária/fisiopatologia , Regiões Promotoras Genéticas/genética , Tremor , Expansão das Repetições de Trinucleotídeos
2.
Am J Hum Genet ; 107(4): 654-669, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-32937144

RESUMO

There is growing recognition that epivariations, most often recognized as promoter hypermethylation events that lead to gene silencing, are associated with a number of human diseases. However, little information exists on the prevalence and distribution of rare epigenetic variation in the human population. In order to address this, we performed a survey of methylation profiles from 23,116 individuals using the Illumina 450k array. Using a robust outlier approach, we identified 4,452 unique autosomal epivariations, including potentially inactivating promoter methylation events at 384 genes linked to human disease. For example, we observed promoter hypermethylation of BRCA1 and LDLR at population frequencies of ∼1 in 3,000 and ∼1 in 6,000, respectively, suggesting that epivariations may underlie a fraction of human disease which would be missed by purely sequence-based approaches. Using expression data, we confirmed that many epivariations are associated with outlier gene expression. Analysis of variation data and monozygous twin pairs suggests that approximately two-thirds of epivariations segregate in the population secondary to underlying sequence mutations, while one-third are likely sporadic events that occur post-zygotically. We identified 25 loci where rare hypermethylation coincided with the presence of an unstable CGG tandem repeat, validated the presence of CGG expansions at several loci, and identified the putative molecular defect underlying most of the known folate-sensitive fragile sites in the genome. Our study provides a catalog of rare epigenetic changes in the human genome, gives insight into the underlying origins and consequences of epivariations, and identifies many hypermethylated CGG repeat expansions.


Assuntos
Proteína BRCA1/genética , Epigênese Genética , Doenças Genéticas Inatas/genética , Genoma Humano , Receptores de LDL/genética , Expansão das Repetições de Trinucleotídeos , Proteína BRCA1/metabolismo , Metilação de DNA , Feminino , Ácido Fólico/metabolismo , Inativação Gênica , Doenças Genéticas Inatas/diagnóstico , Doenças Genéticas Inatas/patologia , Loci Gênicos , Variação Genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Regiões Promotoras Genéticas , Receptores de LDL/metabolismo , Gêmeos Monozigóticos
3.
Proc Natl Acad Sci U S A ; 117(38): 23597-23605, 2020 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-32900932

RESUMO

Trinucleotide repeat (TNR) expansions cause nearly 20 severe human neurological diseases which are currently untreatable. For some of these diseases, ongoing somatic expansions accelerate disease progression and may influence age of onset. This new knowledge emphasizes the importance of understanding the protein factors that drive expansions. Recent genetic evidence indicates that the mismatch repair factor MutSß (Msh2-Msh3 complex) and the histone deacetylase HDAC3 function in the same pathway to drive triplet repeat expansions. Here we tested the hypothesis that HDAC3 deacetylates MutSß and thereby activates it to drive expansions. The HDAC3-selective inhibitor RGFP966 was used to examine its biological and biochemical consequences in human tissue culture cells. HDAC3 inhibition efficiently suppresses repeat expansion without impeding canonical mismatch repair activity. Five key lysine residues in Msh3 are direct targets of HDAC3 deacetylation. In cells expressing Msh3 in which these lysine residues are mutated to arginine, the inhibitory effect of RGFP966 on expansions is largely bypassed, consistent with the direct deacetylation hypothesis. RGFP966 treatment does not alter MutSß subunit abundance or complex formation but does partially control its subcellular localization. Deacetylation sites in Msh3 overlap a nuclear localization signal, and we show that localization of MutSß is partially dependent on HDAC3 activity. Together, these results indicate that MutSß is a key target of HDAC3 deacetylation and provide insights into an innovative regulatory mechanism for triplet repeat expansions. The results suggest expansion activity may be druggable and support HDAC3-selective inhibition as an attractive therapy in some triplet repeat expansion diseases.


Assuntos
Reparo de Erro de Pareamento de DNA/genética , Histona Desacetilases , Expansão das Repetições de Trinucleotídeos/genética , Acetilação/efeitos dos fármacos , Acrilamidas/farmacologia , Linhagem Celular , Células Cultivadas , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Humanos , Fenilenodiaminas/farmacologia
4.
PLoS One ; 15(8): e0233820, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32804976

RESUMO

Molecular markers derived from cerebrospinal fluid (CSF) represent an accessible means of exploring the pathobiology of Huntington's disease (HD) in vivo. The endo-lysosomal/autophagy system is dysfunctional in HD, potentially contributing to disease pathogenesis and representing a potential target for therapeutic intervention. Several endo-lysosomal proteins have shown promise as biomarkers in other neurodegenerative diseases; however, they have yet to be fully explored in HD. We performed parallel reaction monitoring mass spectrometry analysis (PRM-MS) of multiple endo-lysosomal proteins in the CSF of 60 HD mutation carriers and 20 healthy controls. Using generalised linear models controlling for age and CAG, none of the 18 proteins measured displayed significant differences in concentration between HD patients and controls. This was affirmed by principal component analysis, in which no significant difference across disease stage was found in any of the three components representing lysosomal hydrolases, binding/transfer proteins and innate immune system/peripheral proteins. However, several proteins were associated with measures of disease severity and cognition: most notably amyloid precursor protein, which displayed strong correlations with composite Unified Huntington's Disease Rating Scale, UHDRS Total Functional Capacity, UHDRS Total Motor Score, Symbol Digit Modalities Test and Stroop Word Reading. We conclude that although endo-lysosomal proteins are unlikely to have value as disease state CSF biomarkers for Huntington's disease, several proteins demonstrate associations with clinical severity, thus warranting further, targeted exploration and validation in larger, longitudinal samples.


Assuntos
Proteínas do Líquido Cefalorraquidiano/metabolismo , Doença de Huntington/líquido cefalorraquidiano , Adulto , Idoso , Precursor de Proteína beta-Amiloide/líquido cefalorraquidiano , Biomarcadores/líquido cefalorraquidiano , Estudos de Casos e Controles , Cognição , Estudos Transversais , Progressão da Doença , Endossomos/metabolismo , Feminino , Proteína Ativadora de G(M2)/líquido cefalorraquidiano , Humanos , Proteína Huntingtina/genética , Doença de Huntington/genética , Doença de Huntington/psicologia , Modelos Lineares , Estudos Longitudinais , Proteína 2 de Membrana Associada ao Lisossomo/líquido cefalorraquidiano , Glicoproteínas de Membrana Associadas ao Lisossomo/líquido cefalorraquidiano , Masculino , Espectrometria de Massas/métodos , Pessoa de Meia-Idade , Análise de Componente Principal , Estudos Prospectivos , Proteínas/metabolismo , Expansão das Repetições de Trinucleotídeos
5.
Nat Commun ; 11(1): 3343, 2020 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-32620905

RESUMO

The expanded polyglutamine (polyQ) tract form of ataxin-1 drives disease progression in spinocerebellar ataxia type 1 (SCA1). Although known to form distinctive intranuclear bodies, the cellular pathways and processes that polyQ-ataxin-1 influences remain poorly understood. Here we identify the direct and proximal partners constituting the interactome of ataxin-1[85Q] in Neuro-2a cells, pathways analyses indicating a significant enrichment of essential nuclear transporters, pointing to disruptions in nuclear transport processes in the presence of elevated levels of ataxin-1. Our direct assessments of nuclear transporters and their cargoes confirm these observations, revealing disrupted trafficking often with relocalisation of transporters and/or cargoes to ataxin-1[85Q] nuclear bodies. Analogous changes in importin-ß1, nucleoporin 98 and nucleoporin 62 nuclear rim staining are observed in Purkinje cells of ATXN1[82Q] mice. The results highlight a disruption of multiple essential nuclear protein trafficking pathways by polyQ-ataxin-1, a key contribution to furthering understanding of pathogenic mechanisms initiated by polyQ tract proteins.


Assuntos
Ataxina-1/metabolismo , Núcleo Celular/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Células de Purkinje/metabolismo , Transporte Ativo do Núcleo Celular/genética , Animais , Ataxina-1/genética , Linhagem Celular Tumoral , Modelos Animais de Doenças , Células HeLa , Humanos , Camundongos , Mutação , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas de Transporte Nucleocitoplasmático/genética , Peptídeos/genética , Ligação Proteica , Ataxias Espinocerebelares/genética , Ataxias Espinocerebelares/metabolismo , Expansão das Repetições de Trinucleotídeos/genética
6.
Nucleic Acids Res ; 48(14): 7856-7863, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32619224

RESUMO

The Fragile X-related disorders (FXDs) are Repeat Expansion Diseases, genetic disorders that result from the expansion of a disease-specific microsatellite. In those Repeat Expansion Disease models where it has been examined, expansion is dependent on functional mismatch repair (MMR) factors, including MutLγ, a heterodimer of MLH1/MLH3, one of the three MutL complexes found in mammals and a minor player in MMR. In contrast, MutLα, a much more abundant MutL complex that is the major contributor to MMR, is either not required for expansion or plays a limited role in expansion in many model systems. How MutLγ acts to generate expansions is unclear given its normal role in protecting against microsatellite instability and while MLH3 does have an associated endonuclease activity, whether that contributes to repeat expansion is uncertain. We show here, using a gene-editing approach, that a point mutation that eliminates the endonuclease activity of MLH3 eliminates expansions in an FXD mouse embryonic stem cell model. This restricts the number of possible models for repeat expansion and supports the idea that MutLγ may be a useful druggable target to reduce somatic expansion in those disorders where it contributes to disease pathology.


Assuntos
Síndrome do Cromossomo X Frágil/genética , Proteínas MutL/genética , Expansão das Repetições de Trinucleotídeos , Alelos , Animais , Linhagem Celular , Modelos Animais de Doenças , Masculino , Camundongos , Mutação Puntual , Domínios Proteicos/genética , Células-Tronco
7.
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
8.
Hum Genet ; 139(12): 1531-1539, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32533363

RESUMO

The fragile X premutation is defined by the expansion of the CGG trinucleotide repeat at the 5' UTR of the FMR1 gene to between 55 and 200 repeats, while repeat tracks longer than 200 are defined as full mutations. Men carrying a premutation are at increased risk for fragile X-associated tremor/ataxia syndrome (FXTAS); those with > 200 repeats have fragile X syndrome, a common genetic form of intellectual disabilities. In our study, we tested the hypothesis that men carrying a fragile X premutation or full mutation are "biologically older", as suggested by the associated age-related disorder in the presence of the fragile X premutation or the altered cellular pathology that affects both the fragile X premutation and full mutation carriers. Thus, we predicted that both groups would have shorter telomeres than men carrying the normal size repeat allele. Using linear regression models, we found that, on average, premutation carriers had shorter telomeres compared with non-carriers (n = 69 vs n = 36; p = 0.02) and that there was no difference in telomere length between full mutation carriers and non-carriers (n = 37 vs n = 29; p > 0.10). Among premutation carriers only, we also asked whether telomere length was shorter among men with vs without symptoms of FXTAS (n = 28 vs n = 38 and n = 27 vs n = 41, depending on criteria) and found no evidence for a difference (p > 0.10). Previous studies have shown that the premutation is transcribed whereas the full mutation is not, and the expanded repeat track in FMR1 transcript is thought to lead to the risk for premutation-associated disorders. Thus, our data suggest that the observed premutation-only telomere shortening may be a consequence of the toxic effect of the premutation transcript and suggest that premutation carriers are "biologically older" than men carrying the normal size allele in the same age group.


Assuntos
Ataxia/genética , Proteína do X Frágil de Retardo Mental/genética , Síndrome do Cromossomo X Frágil/genética , Telômero/genética , Tremor/genética , Regiões 5' não Traduzidas/genética , Adulto , Idoso , Alelos , Ataxia/patologia , Síndrome do Cromossomo X Frágil/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Mutação/genética , Telômero/patologia , Telômero/ultraestrutura , Homeostase do Telômero/genética , Tremor/patologia , Expansão das Repetições de Trinucleotídeos/genética , Adulto Jovem
9.
PLoS Genet ; 16(6): e1008902, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32589669

RESUMO

Expansion of a CGG-repeat tract in the 5' untranslated region of the FMR1 gene causes the fragile X-related disorders (FXDs; aka the FMR1 disorders). The expansion mechanism is likely shared by the 35+ other diseases resulting from expansion of a disease-specific microsatellite, but many steps in this process are unknown. We have shown previously that expansion is dependent upon functional mismatch repair proteins, including an absolute requirement for MutLγ, one of the three MutL heterodimeric complexes found in mammalian cells. We demonstrate here that both MutLα and MutLß, the two other MutL complexes present in mammalian cells, are also required for most, if not all, expansions in a mouse embryonic stem cell model of the FXDs. A role for MutLα and MutLß is consistent with human GWA studies implicating these complexes as modifiers of expansion risk in other Repeat Expansion Diseases. The requirement for all three complexes suggests a novel model in which these complexes co-operate to generate expansions. It also suggests that the PMS1 subunit of MutLß may be a reasonable therapeutic target in those diseases in which somatic expansion is an important disease modifier.


Assuntos
Síndrome do Cromossomo X Frágil/genética , Endonuclease PMS2 de Reparo de Erro de Pareamento/genética , Proteínas MutL/genética , Expansão das Repetições de Trinucleotídeos/genética , Animais , Linhagem Celular , Reparo de Erro de Pareamento de DNA , Modelos Animais de Doenças , Células-Tronco Embrionárias , Técnicas de Inativação de Genes , Humanos , Camundongos , Endonuclease PMS2 de Reparo de Erro de Pareamento/metabolismo , Proteínas MutL/metabolismo
10.
Am J Hum Genet ; 106(6): 793-804, 2020 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-32413282

RESUMO

Oculopharyngodistal myopathy (OPDM) is an adult-onset inherited neuromuscular disorder characterized by progressive ptosis, external ophthalmoplegia, and weakness of the masseter, facial, pharyngeal, and distal limb muscles. The myopathological features are presence of rimmed vacuoles (RVs) in the muscle fibers and myopathic changes of differing severity. Inheritance is variable, with either putative autosomal-dominant or autosomal-recessive pattern. Here, using a comprehensive strategy combining whole-genome sequencing (WGS), long-read whole-genome sequencing (LRS), linkage analysis, repeat-primed polymerase chain reaction (RP-PCR), and fluorescence amplicon length analysis polymerase chain reaction (AL-PCR), we identified an abnormal GGC repeat expansion in the 5' UTR of GIPC1 in one out of four families and three sporadic case subjects from a Chinese OPDM cohort. Expanded GGC repeats were further confirmed as the cause of OPDM in an additional 2 out of 4 families and 6 out of 13 sporadic Chinese individuals with OPDM, as well as 7 out of 194 unrelated Japanese individuals with OPDM. Methylation, qRT-PCR, and western blot analysis indicated that GIPC1 mRNA levels were increased while protein levels were unaltered in OPDM-affected individuals. RNA sequencing indicated p53 signaling, vascular smooth muscle contraction, ubiquitin-mediated proteolysis, and ribosome pathways were involved in the pathogenic mechanisms of OPDM-affected individuals with GGC repeat expansion in GIPC1. This study provides further evidence that OPDM is associated with GGC repeat expansions in distinct genes and highly suggests that expanded GGC repeat units are essential in the pathogenesis of OPDM, regardless of the genes in which the expanded repeats are located.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Distrofias Musculares/genética , Adolescente , Adulto , Grupo com Ancestrais do Continente Asiático/genética , Cromossomos Humanos Par 19/genética , Metilação de DNA , Feminino , Humanos , Escore Lod , Masculino , Músculo Esquelético/diagnóstico por imagem , Músculo Esquelético/patologia , Distrofias Musculares/patologia , Distrofias Musculares/fisiopatologia , Linhagem , RNA-Seq , Expansão das Repetições de Trinucleotídeos/genética , Proteína Supressora de Tumor p53/metabolismo , Adulto Jovem
11.
Gene ; 753: 144793, 2020 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-32446918

RESUMO

BACKGROUND: Fragile X syndrome (FXS) is a monogenic disorder and a common cause of intellectual disability (ID). Up to now, very few pathological variants other than the typical CGG-repeat expansion have been reported in the FMR1 gene. METHODS: A panel of 56 intellectual disability (ID) genes including the FMR1 gene was sequenced in a cohort of 300 patients with unexplained ID. To determine the effect of a new FMR1 variant, total RNA from peripheral blood cells was reverse transcribed, amplified by polymerase chain reaction and sequenced. RESULTS: We report a novel G to A point variant (c.801G > A) located at the last nucleotide of exon 8 in the FMR1 gene in one patient with ID. Direct sequencing of the RT-PCR products revealed that the transcript from the allele with G to A variant skips exon 8 entirely, resulting in a joining of exons 7 and 9. Skipping of exon 8 may result in an abnormal FMR1 protein (FMRP), which removes the highly conserved region that encoding the KH1 domain of FMRP. CONCLUSIONS: This report describes for the first time that a synonymous variant in the FMR1 gene is associated with an error in mRNA processing, leading preferentially to the production of an aberrant transcript without exon 8. This splice variant was associated with an unspecific clinical presentation, suggesting the need for more detailed investigation of silent variants in ID patients with a large spectrum of phenotypes.


Assuntos
Proteína do X Frágil de Retardo Mental/genética , Síndrome do Cromossomo X Frágil/genética , Deficiência Intelectual/genética , Adolescente , Adulto , Alelos , Estudos de Coortes , Éxons , Humanos , Masculino , Mutação , Fenótipo , Polimorfismo de Nucleotídeo Único , Domínios Proteicos , Processamento de RNA , Análise de Sequência de RNA/métodos , Mutação Silenciosa , Expansão das Repetições de Trinucleotídeos
12.
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
13.
PLoS One ; 15(4): e0227464, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32240172

RESUMO

Expanded CAG nucleotide repeats are the underlying genetic cause of at least 14 incurable diseases, including Huntington's disease (HD). The toxicity associated with many CAG repeat expansions is thought to be due to the translation of the CAG repeat to create a polyQ protein, which forms toxic oligomers and aggregates. However, recent studies show that HD CAG repeats undergo a non-canonical form of translation called Repeat-associated non-AUG dependent (RAN) translation. RAN translation of the CAG sense and CUG anti-sense RNAs produces six distinct repeat peptides: polyalanine (polyAla, from both CAG and CUG repeats), polyserine (polySer), polyleucine (polyLeu), polycysteine (polyCys), and polyglutamine (polyGln). The toxic potential of individual CAG-derived RAN polypeptides is not well understood. We developed pure C. elegans protein models for each CAG RAN polypeptide using codon-varied expression constructs that preserve RAN protein sequence but eliminate repetitive CAG/CUG RNA. While all RAN polypeptides formed aggregates, only polyLeu was consistently toxic across multiple cell types. In GABAergic neurons, which exhibit significant neurodegeneration in HD patients, codon-varied (Leu)38, but not (Gln)38, caused substantial neurodegeneration and motility defects. Our studies provide the first in vivo evaluation of CAG-derived RAN polypeptides in a multicellular model organism and suggest that polyQ-independent mechanisms, such as RAN-translated polyLeu peptides, may have a significant pathological role in CAG repeat expansion disorders.


Assuntos
Doença de Huntington/genética , Neurônios Motores/metabolismo , Agregados Proteicos/genética , Expansão das Repetições de Trinucleotídeos/genética , Animais , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Modelos Animais de Doenças , Humanos , Doença de Huntington/patologia , Neurônios Motores/patologia , Peptídeos/genética , Peptídeos/metabolismo , Biossíntese de Proteínas , RNA Antissenso/genética , Sequências Repetitivas de Aminoácidos/genética
14.
PLoS One ; 15(4): e0231000, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32287265

RESUMO

Myotonic dystrophy type 1 (DM1) is a rare genetic disorder, characterised by muscular dystrophy, myotonia, and other symptoms. DM1 is caused by the expansion of a CTG repeat in the 3'-untranslated region of DMPK. Longer CTG expansions are associated with greater symptom severity and earlier age at onset. The primary mechanism of pathogenesis is thought to be mediated by a gain of function of the CUG-containing RNA, that leads to trans-dysregulation of RNA metabolism of many other genes. Specifically, the alternative splicing (AS) and alternative polyadenylation (APA) of many genes is known to be disrupted. In the context of clinical trials of emerging DM1 treatments, it is important to be able to objectively quantify treatment efficacy at the level of molecular biomarkers. We show how previously described candidate mRNA biomarkers can be used to model an effective reduction in CTG length, using modern high-dimensional statistics (machine learning), and a blood and muscle mRNA microarray dataset. We show how this model could be used to detect treatment effects in the context of a clinical trial.


Assuntos
Distrofia Miotônica/genética , Distrofia Miotônica/terapia , RNA Mensageiro/genética , Processamento Alternativo , Bioestatística , Ensaios Clínicos como Assunto/métodos , Ensaios Clínicos como Assunto/estatística & dados numéricos , Bases de Dados de Ácidos Nucleicos/estatística & dados numéricos , Marcadores Genéticos , Humanos , Análise dos Mínimos Quadrados , Aprendizado de Máquina , Modelos Genéticos , Músculos/metabolismo , Distrofia Miotônica/metabolismo , Miotonina Proteína Quinase/genética , Análise de Sequência com Séries de Oligonucleotídeos/estatística & dados numéricos , Poliadenilação , RNA Mensageiro/metabolismo , Resultado do Tratamento , Expansão das Repetições de Trinucleotídeos
15.
Mol Cell ; 78(2): 346-358.e9, 2020 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-32268123

RESUMO

CAG-repeat expansions in at least eight different genes cause neurodegeneration. The length of the extended polyglutamine stretches in the corresponding proteins is proportionally related to their aggregation propensity. Although these proteins are ubiquitously expressed, they predominantly cause toxicity to neurons. To understand this neuronal hypersensitivity, we generated induced pluripotent stem cell (iPSC) lines of spinocerebellar ataxia type 3 and Huntington's disease patients. iPSC generation and neuronal differentiation are unaffected by polyglutamine proteins and show no spontaneous aggregate formation. However, upon glutamate treatment, aggregates form in neurons but not in patient-derived neural progenitors. During differentiation, the chaperone network is drastically rewired, including loss of expression of the anti-amyloidogenic chaperone DNAJB6. Upregulation of DNAJB6 in neurons antagonizes glutamate-induced aggregation, while knockdown of DNAJB6 in progenitors results in spontaneous polyglutamine aggregation. Loss of DNAJB6 expression upon differentiation is confirmed in vivo, explaining why stem cells are intrinsically protected against amyloidogenesis and protein aggregates are dominantly present in neurons.


Assuntos
Proteínas Amiloidogênicas/genética , Diferenciação Celular/genética , Proteínas de Choque Térmico HSP40/genética , Chaperonas Moleculares/genética , Proteínas do Tecido Nervoso/genética , Células-Tronco Neurais/metabolismo , Regulação da Expressão Gênica/genética , Técnicas de Inativação de Genes , Ácido Glutâmico/metabolismo , Humanos , Doença de Huntington/genética , Doença de Huntington/metabolismo , Doença de Huntington/patologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/patologia , Doença de Machado-Joseph/genética , Doença de Machado-Joseph/metabolismo , Doença de Machado-Joseph/patologia , Células-Tronco Neurais/patologia , Neurônios/metabolismo , Neurônios/patologia , Agregados Proteicos/genética , Expansão das Repetições de Trinucleotídeos/genética
17.
Muscle Nerve ; 62(2): 201-207, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32270505

RESUMO

INTRODUCTION: In this study we aimed to document the prevalence and age of onset of motor impairments and other key symptoms in oculopharyngeal muscular dystrophy (OPMD). METHODS: Retrospective chart review of patients followed at the Saguenay Neuromuscular Clinic (Quebec, Canada). RESULTS: A total of 333 participants with the (GCN)13 mutation were included. Before the age of 75 years, 27% of them had walking limitations, 14% could not climb stairs independently, and 14% used a wheelchair for long distances or daily living. The median age of onset was 54 years for ptosis and dysphagia and 58 years for lower limb proximal weakness. Other frequent symptoms included fatigue, pharyngeal pooling of thickened secretions, and dysphonia. The median age at death was 77 years and the main cause was respiratory disease. DISCUSSION: This study provides important information to help anticipatory guidance for affected people and for the development of therapeutic trials in OPMD.


Assuntos
Atividades Cotidianas , Blefaroptose/fisiopatologia , Transtornos de Deglutição/fisiopatologia , Disfonia/fisiopatologia , Fadiga/fisiopatologia , Limitação da Mobilidade , Debilidade Muscular/fisiopatologia , Distrofia Muscular Oculofaríngea/fisiopatologia , Adulto , Idade de Início , Idoso , Idoso de 80 Anos ou mais , Causas de Morte , Creatina Quinase/sangue , Progressão da Doença , Eletromiografia , Feminino , Humanos , Extremidade Inferior , Masculino , Pessoa de Meia-Idade , Distrofia Muscular Oculofaríngea/sangue , Distrofia Muscular Oculofaríngea/genética , Proteína I de Ligação a Poli(A)/genética , Estudos Retrospectivos , Taxa de Sobrevida , Expansão das Repetições de Trinucleotídeos , Cadeiras de Rodas
19.
J Clin Neurosci ; 74: 242-244, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32063450

RESUMO

Myotonic dystrophy type 1 (DM1) is an autosomal dominant inherited muscular dystrophy caused by an expanded CTG repeat in the dystrophia myotonica protein kinase (DMPK) gene. Cardiac involvements in DM1 are characterized by cardiac conduction delays and atrial or ventricular tachycardia, which increase the risk of sudden cardiac death when compared with general population. Only a few reports have investigated the association between DM1 and inherited arrhythmias, including Brugada syndrome and a splicing abnormality of the SCN5A gene, encodes the α-subunit of cardiac voltage-gated Na+ channels. Here we report a 24-year-old male patient with progressive grip myotonia and dysphagia, who was genetically diagnosed with idiopathic ventricular fibrillation (IVF) caused by a novel V1764fsX1786 frameshift mutation in the SCN5A gene at the age of 18 years. Family history was negative for arrhythmia, cardiac sudden death, and neuromuscular disorders. Genetic analysis using the Southern blot technique revealed 350 CTG repeats in the DMPK gene. This is the first case of DM1 with genetically confirmed overlapping CTG repeat expansion and a V1764fsX1786 frameshift mutation in the SCN5A gene. Our case suggests that a loss-of-function in the cardiac sodium channel may contribute to the cardiac complications in DM1 patients.


Assuntos
Distrofia Miotônica/complicações , Distrofia Miotônica/genética , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Fibrilação Ventricular/etiologia , Fibrilação Ventricular/genética , Adulto , Arritmias Cardíacas , Síndrome de Brugada , Mutação da Fase de Leitura , Humanos , Masculino , Processamento de RNA , Taquicardia Ventricular/complicações , Expansão das Repetições de Trinucleotídeos , Adulto Jovem
20.
Nat Commun ; 11(1): 1101, 2020 02 27.
Artigo em Inglês | MEDLINE | ID: mdl-32107387

RESUMO

Spinocerebellar ataxias 17 (SCA17) is caused by polyglutamine (polyQ) expansion in the TATA box-binding protein (TBP). The selective neurodegeneration in the cerebellum in SCA17 raises the question of why ubiquitously expressed polyQ proteins can cause neurodegeneration in distinct brain regions in different polyQ diseases. By expressing mutant TBP in different brain regions in adult wild-type mice via stereotaxic injection of adeno-associated virus, we found that adult cerebellar neurons are particularly vulnerable to mutant TBP. In SCA17 knock-in mice, mutant TBP inhibits SP1-mediated gene transcription to down-regulate INPP5A, a protein that is highly abundant in the cerebellum. CRISPR/Cas9-mediated deletion of Inpp5a in the cerebellum of wild-type mice leads to Purkinje cell degeneration, and Inpp5a overexpression decreases inositol 1,4,5-trisphosphate (IP3) levels and ameliorates Purkinje cell degeneration in SCA17 knock-in mice. Our findings demonstrate the important contribution of a tissue-specific protein to the polyQ protein-mediated selective neuropathology.


Assuntos
Inositol Polifosfato 5-Fosfatases/genética , Células de Purkinje/patologia , Ataxias Espinocerebelares/patologia , Proteína de Ligação a TATA-Box/genética , Animais , Modelos Animais de Doenças , Regulação para Baixo , Técnicas de Introdução de Genes , Células HEK293 , Humanos , Inositol 1,4,5-Trifosfato/metabolismo , Inositol Polifosfato 5-Fosfatases/metabolismo , Camundongos , Camundongos Transgênicos , Peptídeos/genética , Peptídeos/metabolismo , Células de Purkinje/metabolismo , Fator de Transcrição Sp1/metabolismo , Ataxias Espinocerebelares/genética , Proteína de Ligação a TATA-Box/metabolismo , Expansão das Repetições de Trinucleotídeos
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