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 GenéticoRESUMO
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 TrinucleotídeosRESUMO
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/genéticaRESUMO
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.