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1.
Int J Mol Sci ; 23(7)2022 Mar 23.
Artigo em Inglês | MEDLINE | ID: mdl-35408837

RESUMO

Among the trinucleotide repeat disorders, myotonic dystrophy type 1 (DM1) is one of the most complex neuromuscular diseases caused by an unstable CTG repeat expansion in the DMPK gene. DM1 patients exhibit high variability in the dynamics of CTG repeat instability and in the manifestations and progression of the disease. The largest expanded alleles are generally associated with the earliest and most severe clinical form. However, CTG repeat length alone is not sufficient to predict disease severity and progression, suggesting the involvement of other factors. Several data support the role of epigenetic alterations in clinical and genetic variability. By highlighting epigenetic alterations in DM1, this review provides a new avenue on how these changes can serve as biomarkers to predict clinical features and the mutation behavior.


Assuntos
Distrofia Miotônica , Alelos , Biomarcadores , Epigênese Genética , Humanos , Distrofia Miotônica/genética , Expansão das Repetições de Trinucleotídeos
2.
Int J Mol Sci ; 22(5)2021 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-33807660

RESUMO

Myotonic dystrophy type 1 (DM1) is the most complex and variable trinucleotide repeat disorder caused by an unstable CTG repeat expansion, reaching up to 4000 CTG in the most severe cases. The genetic and clinical variability of DM1 depend on the sex and age of the transmitting parent, but also on the CTG repeat number, presence of repeat interruptions and/or on the degree of somatic instability. Currently, it is difficult to simultaneously and accurately determine these contributing factors in DM1 patients due to the limitations of gold standard methods used in molecular diagnostics and research laboratories. Our study showed the efficiency of the latest PacBio long-read sequencing technology to sequence large CTG trinucleotides, detect multiple and single repeat interruptions and estimate the levels of somatic mosaicism in DM1 patients carrying complex CTG repeat expansions inaccessible to most methods. Using this innovative approach, we revealed the existence of de novo CCG interruptions associated with CTG stabilization/contraction across generations in a new DM1 family. We also demonstrated that our method is suitable to sequence the DM1 locus and measure somatic mosaicism in DM1 families carrying more than 1000 pure CTG repeats. Better characterization of expanded alleles in DM1 patients can significantly improve prognosis and genetic counseling, not only in DM1 but also for other tandem DNA repeat disorders.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala , Mosaicismo , Distrofia Miotônica/genética , Expansão das Repetições de Trinucleotídeos , Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade
3.
Am J Hum Genet ; 100(3): 488-505, 2017 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-28257691

RESUMO

CTG repeat expansions in DMPK cause myotonic dystrophy (DM1) with a continuum of severity and ages of onset. Congenital DM1 (CDM1), the most severe form, presents distinct clinical features, large expansions, and almost exclusive maternal transmission. The correlation between CDM1 and expansion size is not absolute, suggesting contributions of other factors. We determined CpG methylation flanking the CTG repeat in 79 blood samples from 20 CDM1-affected individuals; 21, 27, and 11 individuals with DM1 but not CDM1 (henceforth non-CDM1) with maternal, paternal, and unknown inheritance; and collections of maternally and paternally derived chorionic villus samples (7 CVSs) and human embryonic stem cells (4 hESCs). All but two CDM1-affected individuals showed high levels of methylation upstream and downstream of the repeat, greater than non-CDM1 individuals (p = 7.04958 × 10-12). Most non-CDM1 individuals were devoid of methylation, where one in six showed downstream methylation. Only two non-CDM1 individuals showed upstream methylation, and these were maternally derived childhood onset, suggesting a continuum of methylation with age of onset. Only maternally derived hESCs and CVSs showed upstream methylation. In contrast, paternally derived samples (27 blood samples, 3 CVSs, and 2 hESCs) never showed upstream methylation. CTG tract length did not strictly correlate with CDM1 or methylation. Thus, methylation patterns flanking the CTG repeat are stronger indicators of CDM1 than repeat size. Spermatogonia with upstream methylation may not survive due to methylation-induced reduced expression of the adjacent SIX5, thereby protecting DM1-affected fathers from having CDM1-affected children. Thus, DMPK methylation may account for the maternal bias for CDM1 transmission, larger maternal CTG expansions, age of onset, and clinical continuum, and may serve as a diagnostic indicator.


Assuntos
Ilhas de CpG , Metilação de DNA , Distrofia Miotônica/genética , Miotonina Proteína Quinase/genética , Adolescente , Adulto , Sequência de Bases , Linhagem Celular , Criança , Feminino , Células-Tronco Embrionárias Humanas/química , Humanos , Modelos Lineares , Masculino , Linhagem , Gravidez , Regiões Promotoras Genéticas , Análise de Sequência de DNA , Adulto Jovem
4.
Mol Ther ; 27(8): 1372-1388, 2019 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-31253581

RESUMO

Myotonic dystrophy type 1 (DM1) is caused by a CTG repeat expansion located in the 3' UTR of the DMPK gene. Expanded DMPK transcripts aggregate into nuclear foci and alter the function of RNA-binding proteins, leading to defects in the alternative splicing of numerous pre-mRNAs. To date, there is no curative treatment for DM1. Here we investigated a gene-editing strategy using the CRISPR-Cas9 system from Staphylococcus aureus (Sa) to delete the CTG repeats in the human DMPK locus. Co-expression of SaCas9 and selected pairs of single-guide RNAs (sgRNAs) in cultured DM1 patient-derived muscle line cells carrying 2,600 CTG repeats resulted in targeted DNA deletion, ribonucleoprotein foci disappearance, and correction of splicing abnormalities in various transcripts. Furthermore, a single intramuscular injection of recombinant AAV vectors expressing CRISPR-SaCas9 components in the tibialis anterior muscle of DMSXL (myotonic dystrophy mouse line carrying the human DMPK gene with >1,000 CTG repeats) mice decreased the number of pathological RNA foci in myonuclei. These results establish the proof of concept that genome editing of a large trinucleotide expansion is feasible in muscle and may represent a useful strategy to be further developed for the treatment of myotonic dystrophy.


Assuntos
Edição de Genes , Miotonina Proteína Quinase/genética , RNA Nuclear , Expansão das Repetições de Trinucleotídeos , Processamento Alternativo , Animais , Sequência de Bases , Sistemas CRISPR-Cas , Núcleo Celular , Modelos Animais de Doenças , Imunofluorescência , Expressão Gênica , Marcação de Genes , Vetores Genéticos/genética , Humanos , Camundongos , Camundongos Knockout , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Distrofia Miotônica/genética , Distrofia Miotônica/terapia , RNA Guia de Cinetoplastídeos , Transdução Genética
5.
Int J Mol Sci ; 21(2)2020 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-31936870

RESUMO

Myotonic dystrophy type 1 (DM1) is a complex neuromuscular disease caused by an unstable cardiotocography (CTG) repeat expansion in the DMPK gene. This disease is characterized by high clinical and genetic variability, leading to some difficulties in the diagnosis and prognosis of DM1. Better understanding the origin of this variability is important for developing new challenging therapies and, in particular, for progressing on the path of personalized treatments. Here, we reviewed CTG triplet repeat instability and its modifiers as an important source of phenotypic variability in patients with DM1.


Assuntos
Variação Biológica da População , Distrofia Miotônica/metabolismo , Repetições de Trinucleotídeos/fisiologia , Animais , Cardiotocografia , Humanos , Distrofia Miotônica/genética , Miotonina Proteína Quinase/genética , Miotonina Proteína Quinase/metabolismo , Fenótipo , Repetições de Trinucleotídeos/genética
6.
Hum Mutat ; 39(7): 970-982, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29664219

RESUMO

Myotonic dystrophy type 1 (DM1) is a dominant multisystemic disorder associated with high variability of symptoms and anticipation. DM1 is caused by an unstable CTG repeat expansion that usually increases in successive generations and tissues. DM1 family pedigrees have shown that ∼90% and 10% of transmissions result in expansions and contractions of the CTG repeat, respectively. To date, the mechanisms of CTG repeat contraction remain poorly documented in DM1. In this report, we identified two new DM1 families with apparent contractions and no worsening of DM1 symptoms in two and three successive maternal transmissions. A new and unique CAG interruption was found in 5' of the CTG expansion in one family, whereas multiple 5' CCG interruptions were detected in the second family. We showed that these interruptions are associated with maternal intergenerational contractions and low somatic mosaicism in blood. By specific triplet-prime PCR, we observed that CTG repeat changes (contractions/expansions) occur preferentially in 3' of the interruptions for both families.


Assuntos
Predisposição Genética para Doença , Mosaicismo , Distrofia Miotônica/genética , Expansão das Repetições de Trinucleotídeos/genética , Alelos , Feminino , Humanos , Masculino , Distrofia Miotônica/fisiopatologia , Miotonina Proteína Quinase/genética , Linhagem
7.
Hum Mol Genet ; 23(6): 1606-18, 2014 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-24191263

RESUMO

Expansion of CAG/CTG trinucleotide repeats causes numerous inherited neurological disorders, including Huntington's disease (HD), several spinocerebellar ataxias and myotonic dystrophy type 1. Expanded repeats are genetically unstable with a propensity to further expand when transmitted from parents to offspring. For many alleles with expanded repeats, extensive somatic mosaicism has been documented. For CAG repeat diseases, dramatic instability has been documented in the striatum, with larger expansions noted with advancing age. In contrast, only modest instability occurs in the cerebellum. Using microarray expression analysis, we sought to identify the genetic basis of these regional instability differences by comparing gene expression in the striatum and cerebellum of aged wild-type C57BL/6J mice. We identified eight candidate genes enriched in cerebellum, and validated four--Pcna, Rpa1, Msh6 and Fen1--along with a highly associated interactor, Lig1. We also explored whether expression levels of mismatch repair (MMR) proteins are altered in a line of HD transgenic mice, R6/2, that is known to show pronounced regional repeat instability. Compared with wild-type littermates, MMR expression levels were not significantly altered in R6/2 mice regardless of age. Interestingly, expression levels of these candidates were significantly increased in the cerebellum of control and HD human samples in comparison to striatum. Together, our data suggest that elevated expression levels of DNA replication and repair proteins in cerebellum may act as a safeguard against repeat instability, and may account for the dramatically reduced somatic instability present in this brain region, compared with the marked instability observed in the striatum.


Assuntos
Cerebelo/metabolismo , Corpo Estriado/metabolismo , Reparo de Erro de Pareamento de DNA , Doença de Huntington/genética , Fatores Etários , Animais , DNA Ligase Dependente de ATP , DNA Ligases/genética , Proteínas de Ligação a DNA/genética , Feminino , Endonucleases Flap/genética , Regulação da Expressão Gênica , Humanos , Doença de Huntington/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Instabilidade de Microssatélites , Antígeno Nuclear de Célula em Proliferação/genética , Proteína de Replicação A/genética , Repetições de Trinucleotídeos
8.
PLoS Genet ; 9(2): e1003280, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23468640

RESUMO

Expansions of trinucleotide CAG/CTG repeats in somatic tissues are thought to contribute to ongoing disease progression through an affected individual's life with Huntington's disease or myotonic dystrophy. Broad ranges of repeat instability arise between individuals with expanded repeats, suggesting the existence of modifiers of repeat instability. Mice with expanded CAG/CTG repeats show variable levels of instability depending upon mouse strain. However, to date the genetic modifiers underlying these differences have not been identified. We show that in liver and striatum the R6/1 Huntington's disease (HD) (CAG)∼100 transgene, when present in a congenic C57BL/6J (B6) background, incurred expansion-biased repeat mutations, whereas the repeat was stable in a congenic BALB/cByJ (CBy) background. Reciprocal congenic mice revealed the Msh3 gene as the determinant for the differences in repeat instability. Expansion bias was observed in congenic mice homozygous for the B6 Msh3 gene on a CBy background, while the CAG tract was stabilized in congenics homozygous for the CBy Msh3 gene on a B6 background. The CAG stabilization was as dramatic as genetic deficiency of Msh2. The B6 and CBy Msh3 genes had identical promoters but differed in coding regions and showed strikingly different protein levels. B6 MSH3 variant protein is highly expressed and associated with CAG expansions, while the CBy MSH3 variant protein is expressed at barely detectable levels, associating with CAG stability. The DHFR protein, which is divergently transcribed from a promoter shared by the Msh3 gene, did not show varied levels between mouse strains. Thus, naturally occurring MSH3 protein polymorphisms are modifiers of CAG repeat instability, likely through variable MSH3 protein stability. Since evidence supports that somatic CAG instability is a modifier and predictor of disease, our data are consistent with the hypothesis that variable levels of CAG instability associated with polymorphisms of DNA repair genes may have prognostic implications for various repeat-associated diseases.


Assuntos
Doença de Huntington/genética , Proteínas/genética , Expansão das Repetições de Trinucleotídeos/genética , Repetições de Trinucleotídeos/genética , Animais , Corpo Estriado/metabolismo , Modelos Animais de Doenças , Instabilidade Genômica , Humanos , Camundongos , Proteína 3 Homóloga a MutS , Distrofia Miotônica/genética , Distrofia Miotônica/metabolismo , Neostriado/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Polimorfismo Genético , Estabilidade Proteica
9.
Hum Mol Genet ; 20(1): 1-15, 2011 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-21044947

RESUMO

Myotonic dystrophy (DM1) affects multiple organs, shows age-dependent progression and is caused by CTG expansions at the DM1 locus. We determined the DM1 CpG methylation profile and CTG length in tissues from DM1 foetuses, DM1 adults, non-affected individuals and transgenic DM1 mice. Analysis included CTCF binding sites upstream and downstream of the CTG tract, as methylation-sensitive CTCF binding affects chromatinization and transcription of the DM1 locus. In humans, in a given foetus, expansions were largest in heart and smallest in liver, differing by 40-400 repeats; in adults, the largest expansions were in heart and cerebral cortex and smallest in cerebellum, differing by up to 5770 repeats in the same individual. Abnormal methylation was specific to the mutant allele. In DM1 adults, heart, liver and cortex showed high-to-moderate methylation levels, whereas cerebellum, kidney and skeletal muscle were devoid of methylation. Methylation decreased between foetuses and adults. Contrary to previous findings, methylation was not restricted to individuals with congenital DM1. The expanded repeat demarcates an abrupt boundary of methylation. Upstream sequences, including the CTCF site, were methylated, whereas the repeat itself and downstream sequences were not. In DM1 mice, expansion-, tissue- and age-specific methylation patterns were similar but not identical to those in DM1 individuals; notably in mice, methylation was present up- and downstream of the repeat, but greater upstream. Thus, in humans, the CpG-free expanded CTG repeat appears to maintain a highly polarized pattern of CpG methylation at the DM1 locus, which varies markedly with age and tissues.


Assuntos
Distrofia Miotônica/genética , Proteínas Serina-Treonina Quinases/genética , Expansão das Repetições de Trinucleotídeos/genética , Adolescente , Adulto , Idoso de 80 Anos ou mais , Alelos , Animais , Sequência de Bases , Fator de Ligação a CCCTC , Metilação de DNA , Feminino , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Instabilidade de Microssatélites , Pessoa de Meia-Idade , Dados de Sequência Molecular , Miotonina Proteína Quinase , Especificidade de Órgãos/genética , Proteínas Serina-Treonina Quinases/química , Proteínas Repressoras/genética , Adulto Jovem
10.
Hum Mol Genet ; 20(11): 2131-43, 2011 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-21378394

RESUMO

The instability of (CTG)•(CAG) repeats can cause >15 diseases including myotonic dystrophy, DM1. Instability can arise during DNA replication, repair or recombination, where sealing of nicks by DNA ligase I (LIGI) is a final step. The role of LIGI in CTG/CAG instability was determined using in vitro and in vivo approaches. Cell extracts from a human (46BR) harbouring a deficient LIGI (∼3% normal activity) were used to replicate CTG/CAG repeats; and DM1 mice with >300 CTG repeats were crossed with mice harbouring the 46BR LigI. In mice, the defective LigI reduced the frequency of CTG expansions and increased CTG contraction frequencies on female transmissions. Neither male transmissions nor somatic CTG instability was affected by the 46BR LigI - indicating a post-female germline segregation event. Replication-mediated instability was affected by the 46BR LIGI in a manner that depended upon the location of Okazaki fragment initiation relative to the repeat tract; on certain templates, the expansion bias was unaltered by the mutant LIGI, similar to paternal transmissions and somatic tissues; however, a replication fork-shift reduced expansions and increased contractions, similar to maternal transmissions. The presence of contractions in oocytes suggests that the DM1 replication profile specific to pre-meiotic oogenesis replication of maternal alleles is distinct from that occurring in other tissues and, when mediated by the mutant LigI, is predisposed to CTG contractions. Thus, unlike other DNA metabolizing enzymes studied to date, LigI has a highly specific role in CTG repeat maintenance in the maternal germline, involved in mediating CTG expansions and in the avoidance of maternal CTG contractions.


Assuntos
DNA Ligases/metabolismo , Replicação do DNA , Instabilidade Genômica , Expansão das Repetições de Trinucleotídeos/genética , Alelos , Animais , Linhagem Celular , DNA/genética , DNA Ligase Dependente de ATP , DNA Ligases/genética , Análise Mutacional de DNA , Feminino , Homozigoto , Humanos , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Distrofia Miotônica/genética , Oócitos/metabolismo , Fosforilação
11.
PLoS Genet ; 5(5): e1000482, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-19436705

RESUMO

Myotonic dystrophy type 1 (DM1) is associated with one of the most highly unstable CTG*CAG repeat expansions. The formation of further repeat expansions in transgenic mice carrying expanded CTG*CAG tracts requires the mismatch repair (MMR) proteins MSH2 and MSH3, forming the MutSbeta complex. It has been proposed that binding of MutSbeta to CAG hairpins blocks its ATPase activity compromising hairpin repair, thereby causing expansions. This would suggest that binding, but not ATP hydrolysis, by MutSbeta is critical for trinucleotide expansions. However, it is unknown if the MSH2 ATPase activity is dispensible for instability. To get insight into the mechanism by which MSH2 generates trinucleotide expansions, we crossed DM1 transgenic mice carrying a highly unstable >(CTG)(300) repeat tract with mice carrying the G674A mutation in the MSH2 ATPase domain. This mutation impairs MSH2 ATPase activity and ablates base-base MMR, but does not affect the ability of MSH2 (associated with MSH6) to bind DNA mismatches. We found that the ATPase domain mutation of MSH2 strongly affects the formation of CTG expansions and leads instead to transmitted contractions, similar to a Msh2-null or Msh3-null deficiency. While a decrease in MSH2 protein level was observed in tissues from Msh2(G674) mice, the dramatic reduction of expansions suggests that the expansion-biased trinucleotide repeat instability requires a functional MSH2 ATPase domain and probably a functional MMR system.


Assuntos
Instabilidade Genômica , Proteína 2 Homóloga a MutS/química , Proteína 2 Homóloga a MutS/genética , Mutação Puntual , Expansão das Repetições de Trinucleotídeos , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Animais , Sequência de Bases , Encéfalo/metabolismo , Reparo de Erro de Pareamento de DNA/genética , Primers do DNA/genética , Proteínas de Ligação a DNA/metabolismo , Feminino , Humanos , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Proteína 2 Homóloga a MutS/deficiência , Proteína 2 Homóloga a MutS/metabolismo , Proteína 3 Homóloga a MutS , Distrofia Miotônica/genética , Distrofia Miotônica/metabolismo , Miotonina Proteína Quinase , Ovário/metabolismo , Proteínas Serina-Treonina Quinases/genética , Estrutura Terciária de Proteína , Proteínas/genética , Proteínas/metabolismo , Testículo/metabolismo
12.
J Mol Diagn ; 24(11): 1143-1154, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-36084803

RESUMO

Myotonic dystrophy type 1 (DM1) exhibits highly heterogeneous clinical manifestations caused by an unstable CTG repeat expansion reaching up to 4000 CTG. The clinical variability depends on CTG repeat number, CNG repeat interruptions, and somatic mosaicism. Currently, none of these factors are simultaneously and accurately determined due to the limitations of gold standard methods used in clinical and research laboratories. An amplicon method for targeting the DMPK locus using single-molecule real-time sequencing was recently developed to accurately analyze expanded alleles. However, amplicon-based sequencing still depends on PCR, and the inherent bias toward preferential amplification of smaller repeats can be problematic in DM1. Thus, an amplification-free long-read sequencing method was developed by using CRISPR/Cas9 technology in DM1. This method was used to sequence the DMPK locus in patients with CTG repeat expansion ranging from 130 to >1000 CTG. We showed that elimination of PCR amplification improves the accuracy of measurement of inherited repeat number and somatic repeat variations, two key factors in DM1 severity and age at onset. For the first time, an expansion composed of >85% CCG repeats was identified by using this innovative method in a DM1 family with an atypical clinical profile. No-amplification targeted sequencing represents a promising method that can overcome research and diagnosis shortcomings, with translational implications for clinical and genetic counseling in DM1.


Assuntos
Distrofia Miotônica , Humanos , Distrofia Miotônica/diagnóstico , Distrofia Miotônica/genética , Miotonina Proteína Quinase/genética , Alelos , Expansão das Repetições de Trinucleotídeos/genética , Aconselhamento Genético
13.
J Cell Biochem ; 112(6): 1612-21, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21344488

RESUMO

The mismatch repair protein, MSH3, together with MSH2, forms the MutSß heterodimer which recognizes and repairs base pair mismatches and larger insertion/deletion loops in DNA. Lack of specific antibodies against mouse MSH3 has hampered studies of its expression and localization. Mouse MSH3 is not immunogenic in normal mice. This problem was overcome by immunizing msh3-knockout mice and generating a panel of ten monoclonal antibodies, two of which localize MSH3 specifically in cultured mouse cells and bind to an epitope containing amino-acids 33-37. The panel also includes two antibodies that recognise both mouse and human MSH3 and bind to a conserved epitope containing amino-acids 187-194. The mouse MSH3-specific antibodies show that MSH3 is a nuclear protein with a finely-granular nucleoplasmic distribution, largely absent from areas of condensed heterochromatin. Specificity of the localization was demonstrated by absence of immunostaining in a cell line from the msh3-knockout mouse. Furthermore, we show for the first time that stress treatment of mouse cells with ethanol or hydrogen peroxide caused the re-distribution of MSH3 into nuclear bodies containing the proliferating cell nuclear antigen (PCNA), a known binding partner of MutSß.


Assuntos
Núcleo Celular/metabolismo , Proteínas/metabolismo , Animais , Anticorpos Monoclonais , Western Blotting , Linhagem Celular , Núcleo Celular/efeitos dos fármacos , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Mapeamento de Epitopos , Células HeLa , Humanos , Hibridomas , Peróxido de Hidrogênio/farmacologia , Camundongos , Camundongos Knockout , Proteína 3 Homóloga a MutS , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Antígeno Nuclear de Célula em Proliferação/metabolismo , Ligação Proteica/efeitos dos fármacos , Proteínas/genética
14.
Med Sci (Paris) ; 37 Hors série n° 1: 6-10, 2021 Nov.
Artigo em Francês | MEDLINE | ID: mdl-34878385

RESUMO

Myotonic dystrophy type 1 (DM1) is a multisystemic neuromuscular disease caused by an abnormal CTG repeat expansion in the 3'UTR region of the DMPK gene. In patients, the CTG repeat size varies from fifty to thousands CTG and usually increases across generations (intergenerational instability) and over time in tissues (somatic instability). Larger expansions are associated with more severe symptoms and a decreasing age of onset. Thus, the larger expansions are often associated with the most severe clinical form of DM1 (congenital form). Our PhD project is to identify new genetic and chemical factors reducing the number of repeats and to better understand the mechanisms underlying instability. To this end, genetic and pharmacological screenings are carried out in a HEK293 cell model allowing the rapid detection of expansions (increase in CTG repeat number) and contractions (decrease in CTG repeat number). The effects of different genes and chemical factors, selected during the screening, on the dynamics of the CTG repeat instability will be studied in a DM1 cell model. The results of our work will provide a better understanding of the mechanisms behind contractions. In addition, the identification of new pharmacological compounds promoting CTG contractions and thus reducing or even reversing the progression of disease will offer new therapeutic prospects for DM1 but also for other triplet repeat diseases.


TITLE: Identification de nouveaux facteurs entraînant des contractions CTG.CAG dans la dystrophie myotonique de type 1. ABSTRACT: La dystrophie myotonique de type 1 (DM1 ou maladie de Steinert) est une maladie neuromusculaire multi-systémique causée par une expansion anormale de triplets CTG instables dans la région 3'UTR du gène DMPK. Le nombre de répétitions augmente au cours des générations (instabilité intergénérationnelle) mais également avec l'âge du patient (instabilité somatique). Chez les patients, la taille des répétitions CTG est généralement corrélée à l'âge d'apparition et à la sévérité des symptômes. Ainsi, les expansions les plus grandes sont souvent associées à la forme clinique la plus grave de la DM1 (forme congénitale). Notre projet de thèse vise à identifier des nouveaux facteurs génétiques et chimiques capables de diminuer la taille des répétitions, et de mieux comprendre les mécanismes d'instabilité. Pour cela, un criblage génétique et pharmacologique est réalisé dans un modèle cellulaire HEK293 permettant de détecter rapidement les expansions (augmentation de la taille des triplets CTG) et les contractions (diminution de la taille des CTG). Les effets des différents gènes et facteurs chimiques, sélectionnés au cours du criblage, sur la dynamique de l'instabilité des triplets CTG seront étudiés dans un modèle cellulaire DM1. Les résultats de nos travaux permettront de mieux comprendre les mécanismes à l'origine des contractions. Par ailleurs, l'identification de nouveaux composés pharmacologiques susceptibles de favoriser les contractions CTG et ainsi réduire, voire inverser, la progression de la maladie, offrira de nouvelles perspectives thérapeutiques pour la DM1 mais aussi pour d'autres maladies à triplets répétés.


Assuntos
Distrofia Miotônica , Células HEK293 , Humanos , Distrofia Miotônica/genética , Expansão das Repetições de Trinucleotídeos/genética
15.
Methods Mol Biol ; 2056: 11-23, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31586339

RESUMO

Different interrupted repeat expansions have been found in several trinucleotide repeat (TNR) diseases such as fragile X syndrome (FXS), spinocerebellar ataxias (SCAs), and myotonic dystrophies (DMs). Their origins and roles remain poorly understood, especially in myotonic dystrophy type 1 (DM1). We present here the triplet repeat primed polymerase chain reaction (TP-PCR) and restriction enzyme-digested PCR to detect and identify interrupted triplet repeat alleles in DM1. TP-PCR consists of a PCR amplification using a fluoresceinated (FAM) primer flanking the repeat region and a primer pair in CTG.CAG repeats. A detailed analysis of interrupted triplet repeat tracts is essential to fully understand the role of interruptions in the pathogenesis and molecular mechanisms observed in TNR diseases.


Assuntos
Distrofia Miotônica/genética , Reação em Cadeia da Polimerase/métodos , Expansão das Repetições de Trinucleotídeos , Enzimas de Restrição do DNA/metabolismo , Fluoresceína/química , Predisposição Genética para Doença , Humanos , Oligonucleotídeos/química , Fatores de Tempo
16.
Med Sci (Paris) ; 39 Hors série n° 1: 64, 2023 11.
Artigo em Francês | MEDLINE | ID: mdl-37975773
17.
PLoS Curr ; 62014 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-24611127

RESUMO

Many human diseases are associated with the abnormal expansion of unstable trinucleotide repeat sequences. The mechanisms of trinucleotide repeat size mutation have not been fully dissected, and their understanding must be grounded on the detailed analysis of repeat size distributions in human tissues and animal models. Small-pool PCR (SP-PCR) is a robust, highly sensitive and efficient PCR-based approach to assess the levels of repeat size variation, providing both quantitative and qualitative data. The method relies on the amplification of a very low number of DNA molecules, through sucessive dilution of a stock genomic DNA solution. Radioactive Southern blot hybridization is sensitive enough to detect SP-PCR products derived from single template molecules, separated by agarose gel electrophoresis and transferred onto DNA membranes. We describe a variation of the detection method that uses digoxigenin-labelled locked nucleic acid probes. This protocol keeps the sensitivity of the original method, while eliminating the health risks associated with the manipulation of radiolabelled probes, and the burden associated with their regulation, manipulation and waste disposal.

18.
DNA Repair (Amst) ; 12(1): 46-52, 2013 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-23228367

RESUMO

Mismatch repair (MMR) proteins have critical roles in the maintenance of genomic stability, both class-switch recombination and somatic hypermutation of immunoglobulin genes and disease-associated trinucleotide repeat expansions. In the genetic absence of MMR, certain tissues are predisposed to mutations and cancer. MMR proteins are involved in various functions including protection from replication-associated and non-mitotic mutations, as well as driving programmed and deleterious mutations, including disease-causing trinucleotide repeat expansions. Here we have assessed the levels of MSH2, MSH3, and MSH6 expression in a large number of murine tissues by transcript analysis and simultaneous Western blotting. We observed that MMR expression patterns varied widely between 14 different tissue types, but did not vary with age (13-84 weeks). MMR protein expression is highest in testis, thymus and spleen and lowest in pancreas, quadriceps and heart, with intermediate levels in liver, kidney, intestine, colon, cortex, striatum and cerebellum. By equalizing antibody signal intensity to represent levels found in mMutSα and mMutSß purified proteins, we observed that mMSH3 protein levels are greater than mMSH6 levels in the multiple tissues analyzed, with more MSH6 in proliferating tissues. In the intestinal epithelium MSH3 and MSH6 are more highly expressed in the proliferative undifferentiated cells of the crypts than in the differentiated villi cells, as reported for MSH2. This finding correlates with the higher level of MMR expression in highly proliferative mouse tissues such as the spleen and thymus. The relative MMR protein expression levels may explain the functional and tissue-specific reliance upon the roles of each MMR protein.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas/metabolismo , Fatores Etários , Animais , Proliferação de Células , Reparo de Erro de Pareamento de DNA/genética , Proteínas de Ligação a DNA/genética , Mucosa Intestinal/metabolismo , Camundongos , Proteína 2 Homóloga a MutS/genética , Proteína 2 Homóloga a MutS/metabolismo , Proteína 3 Homóloga a MutS , Especificidade de Órgãos , Proteínas/genética , RNA Mensageiro/biossíntese , Transcrição Gênica
19.
Nat Struct Mol Biol ; 17(9): 1079-87, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20711191

RESUMO

Myotonic dystrophy, caused by DM1 CTG/CAG repeat expansions, shows varying instability levels between tissues and across ages within patients. We determined DNA replication profiles at the DM1 locus in patient fibroblasts and tissues from DM1 transgenic mice of various ages showing different instability. In patient cells, the repeat is flanked by two replication origins demarcated by CTCF sites, with replication diminished at the expansion. In mice, the expansion replicated from only the downstream origin (CAG as lagging template). In testes from mice of three different ages, replication toward the repeat paused at the earliest age and was relieved at later ages-coinciding with increased instability. Brain, pancreas and thymus replication varied with CpG methylation at DM1 CTCF sites. CTCF sites between progressing forks and repeats reduced replication depending on chromatin. Thus, varying replication progression may affect tissue- and age-specific repeat instability.


Assuntos
Envelhecimento , Replicação do DNA , DNA/metabolismo , Distrofia Miotônica/genética , Distrofia Miotônica/metabolismo , Animais , Sítios de Ligação , Fator de Ligação a CCCTC , Cromossomos Humanos Par 19 , Ilhas de CpG , Metilação de DNA , Loci Gênicos , Humanos , Camundongos , Camundongos Transgênicos , Especificidade de Órgãos , Proteínas Repressoras/metabolismo
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