RESUMO
BACKGROUND: Titinopathies are caused by mutations in the titin gene (TTN). Titin is the largest known human protein; its gene has the longest coding phase with 364 exons. Titinopathies are very complex neuromuscular pathologies due to the variable age of onset of symptoms, the great diversity of pathological and muscular impairment patterns (cardiac, skeletal muscle or mixed) and both autosomal dominant and recessive modes of transmission. Until now, only few CNVs in TTN have been reported without clear genotype-phenotype associations. METHODS: Our study includes eight families with dominant titinopathies. We performed next-generation sequencing or comparative genomic hybridisation array analyses and found CNVs in the TTN gene. We characterised these CNVs by RNA sequencing (RNAseq) analyses in six patients' muscles and performed genotype-phenotype inheritance association study by combining the clinical and biological data of these eight families. RESULTS: Seven deletion-type CNVs in the TTN gene were identified among these families. Genotype and RNAseq results showed that five deletions do not alter the reading frame and one is out-of-reading frame. The main phenotype identified was distal myopathy associated with contractures. The analysis of morphological, clinical and genetic data and imaging let us draw new genotype-phenotype associations of titinopathies. CONCLUSION: Identifying TTN CNVs will further increase diagnostic sensitivity in these complex neuromuscular pathologies. Our cohort of patients enabled us to identify new deletion-type CNVs in the TTN gene, with unexpected autosomal dominant transmission. This is valuable in establishing new genotype-phenotype associations of titinopathies, mainly distal myopathy in most of the patients.
Assuntos
Miopatias Distais , Humanos , Conectina/genética , Miopatias Distais/genética , Variações do Número de Cópias de DNA/genética , Músculo Esquelético/patologia , Mutação/genética , FenótipoRESUMO
SpliceAI is an open-source deep learning splicing prediction algorithm that has demonstrated in the past few years its high ability to predict splicing defects caused by DNA variations. However, its outputs present several drawbacks: (1) although the numerical values are very convenient for batch filtering, their precise interpretation can be difficult, (2) the outputs are delta scores which can sometimes mask a severe consequence, and (3) complex delins are most often not handled. We present here SpliceAI-visual, a free online tool based on the SpliceAI algorithm, and show how it complements the traditional SpliceAI analysis. First, SpliceAI-visual manipulates raw scores and not delta scores, as the latter can be misleading in certain circumstances. Second, the outcome of SpliceAI-visual is user-friendly thanks to the graphical presentation. Third, SpliceAI-visual is currently one of the only SpliceAI-derived implementations able to annotate complex variants (e.g., complex delins). We report here the benefits of using SpliceAI-visual and demonstrate its relevance in the assessment/modulation of the PVS1 classification criteria. We also show how SpliceAI-visual can elucidate several complex splicing defects taken from the literature but also from unpublished cases. SpliceAI-visual is available as a Google Colab notebook and has also been fully integrated in a free online variant interpretation tool, MobiDetails ( https://mobidetails.iurc.montp.inserm.fr/MD ).
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Algoritmos , Splicing de RNA , Humanos , Splicing de RNA/genéticaRESUMO
The implementation of high-throughput diagnostic sequencing has led to the generation of large amounts of mutational data, making their interpretation more complex and responsible for long delays. It has been important to prioritize certain analyses, particularly those of "actionable" genes in diagnostic situations, involving specific treatment and/or management. In our project, we carried out an objective assessment of the clinical actionability of genes involved in myopathies, for which only few data obtained methodologically exist to date. Using the ClinGen Actionability criteria, we scored the clinical actionability of all 199 genes implicated in myopathies published by FILNEMUS for the "National French consensus on gene Lists for the diagnosis of myopathies using next generation sequencing". We objectified that 63 myopathy genes were actionable with the currently available data. Among the 36 myopathy genes with the highest actionability scores, only 8 had been scored to date by ClinGen. The data obtained through these methodological tools are an important resource for strategic choices in diagnostic approaches and the management of genetic myopathies. The clinical actionability of genes has to be considered as an evolving concept, in relation to progresses in disease knowledge and therapeutic approaches.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala , Doenças Musculares , Consenso , Humanos , Doenças Musculares/diagnóstico , Doenças Musculares/genética , Doenças Musculares/terapia , Mutação , Assistência ao PacienteRESUMO
We present the experimental realization of a long-lived superfluid flow of a quantum gas rotating in an anharmonic potential, sustained by its own angular momentum. The gas is set into motion by rotating an elliptical deformation of the trap. An evaporation selective in angular momentum yields an acceleration of rotation until the density vanishes at the trap center, resulting in a dynamical ring with ≃350â angular momentum per particle. The density profile of the ring corresponds to the one of a quasi two-dimensional superfluid, with a linear velocity reaching Mach 18 and a rotation lasting more than a minute.
RESUMO
Titin protein is responsible for muscle elasticity. The TTN gene, composed of 364 exons, is subjected to extensive alternative splicing and leads to different isoforms expressed in skeletal and cardiac muscle. Variants in TTN are responsible for myopathies with a wide phenotypic spectrum and autosomal dominant or recessive transmission. The I-band coding domain, highly subject to alternative splicing, contains a three-zone block of repeated sequences with 99% homology. Sequencing and localization of variants in these areas are complex when using short-reads sequencing, a second-generation sequencing technique. We have implemented a protocol based on the third-generation sequencing technology (long-reads sequencing). This new method allows us to localize variants in these repeated areas to improve the diagnosis of TTN-related myopathies and offer the analysis of relatives in postnatal or in prenatal screening.
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Doenças Musculares , Processamento Alternativo/genética , Conectina/genética , Éxons/genética , Humanos , Doenças Musculares/genética , Isoformas de Proteínas/genéticaRESUMO
Next generation sequencing (NGS) is strategically used for genetic diagnosis in patients with Charcot-Marie-Tooth disease (CMT) and related disorders called non-syndromic inherited peripheral neuropathies (NSIPN) in this paper. With over 100 different CMT-associated genes involved and ongoing discoveries, an important interlaboratory diversity of gene panels exists at national and international levels. Here, we present the work of the French National Network for Rare Neuromuscular Diseases (FILNEMUS) genetic diagnosis section which coordinates the seven French diagnosis laboratories using NGS for peripheral neuropathies. This work aimed to establish a unique, simple and accurate gene classification based on literature evidence. In NSIPN, three subgroups were usually distinguished: (1) HMSN, Hereditary Motor Sensory Neuropathy, (2) dHMN, distal Hereditary Motor Neuropathy, and (3) HSAN, Hereditary Sensory Autonomic Neuropathy. First, we reported ClinGen evaluation, and second, for the genes not evaluated yet by ClinGen, we classified them as "definitive" if reported in at least two clinical publications and associated with one report of functional evidence, or "limited" otherwise. In total, we report a unique consensus gene list for NSIPN including the three subgroups with 93 genes definitive and 34 limited, which is a good rate for our gene's panel for molecular diagnostic use.
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Doença de Charcot-Marie-Tooth , Neuropatias Hereditárias Sensoriais e Autônomas , Doença de Charcot-Marie-Tooth/diagnóstico , Doença de Charcot-Marie-Tooth/genética , Consenso , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Patologia MolecularRESUMO
The aim of this study was to analyze patients from two distinct families with a novel distal titinopathy phenotype associated with exactly the same CNV in the TTN gene. We used an integrated strategy combining deep phenotyping and complete molecular analyses in patients. The CNV is the most proximal out-of-frame TTN variant reported and leads to aberrant splicing transcripts leading to a frameshift. In this case, the dominant effect would be due to dominant-negative and/or haploinsufficiency. Few CNV in TTN have been reported to date. Our data represent a novel phenotype-genotype association and provides hypotheses for its dominant effects.
Assuntos
Conectina/genética , Distrofias Musculares/genética , Distrofias Musculares/patologia , Distrofias Musculares/fisiopatologia , Idoso , Idoso de 80 Anos ou mais , Variações do Número de Cópias de DNA , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Linhagem , FenótipoRESUMO
Diagnosis of myopathies is challenged by the high genetic heterogeneity and clinical overlap of the various etiologies. We previously reported a Next-Generation Sequencing strategy to identify genetic etiology in patients with undiagnosed Limb-Girdle Muscular Dystrophies, Congenital Myopathies, Congenital Muscular Dystrophies, Distal Myopathies, Myofibrillar Myopathies, and hyperCKemia or effort intolerance, using a large gene panel including genes classically associated with other entry diagnostic categories. In this study, we report the comprehensive clinical-biological strategy used to interpret NGS data in a cohort of 156 pediatric and adult patients, that included Copy Number Variants search, variants filtering and interpretation according to ACMG guidelines, segregation studies, deep phenotyping of patients and relatives, transcripts and protein studies, and multidisciplinary meetings. Genetic etiology was identified in 74 patients, a diagnostic yield (47.4%) similar to previous studies. We identified 18 patients (10%) with causative variants in different genes (ACTA1, RYR1, NEB, TTN, TRIP4, CACNA1S, FLNC, TNNT1, and PAPBN1) that resulted in milder and/or atypical phenotypes, with high intrafamilial variability in some cases. Mild phenotypes could mostly be explained by a less deleterious effect of variants on the protein. Detection of inter-individual variability and atypical phenotype-genotype associations is essential for precision medicine, patient care, and to progress in the understanding of the molecular mechanisms of myopathies.
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Genótipo , Doenças Musculares/patologia , Fenótipo , Adulto , Criança , Estudos de Coortes , Feminino , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Masculino , Doenças Musculares/diagnóstico , Doenças Musculares/genéticaRESUMO
Congenital titinopathies are myopathies with variable phenotypes and inheritance modes. Here, we fully characterized, using an integrated approach (deep phenotyping, muscle morphology, mRNA and protein evaluation in muscle biopsies), two siblings with congenital multicore myopathy harboring three TTN variants predicted to affect titin stability and titin-myosin interactions. Muscle biopsies showed multicores, type 1 fiber uniformity and sarcomeric structure disruption with some thick filament loss. Immunohistochemistry and Western blotting revealed a marked reduction of fast myosin heavy chain isoforms. This is the first observation of a titinopathy suggesting that titin defect leads to secondary loss of fast myosin heavy chain isoforms.
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Conectina/genética , Doenças Musculares , Cadeias Pesadas de Miosina/deficiência , Adolescente , Criança , Músculo Deltoide/patologia , Feminino , Humanos , Masculino , Doenças Musculares/congênito , Doenças Musculares/genética , Doenças Musculares/metabolismo , Doenças Musculares/patologia , Linhagem , IrmãosRESUMO
Next generation sequencing (NGS) has allowed the titin gene (TTN) to be identified as a major contributor to neuromuscular disorders, with high clinical heterogeneity. The mechanisms underlying the phenotypic variability and the dominant or recessive pattern of inheritance are unclear. Titin is involved in the formation and stability of the sarcomeres. The effects of the different TTN variants can be harmless or pathogenic (recessive or dominant) but the interpretation is tricky because the current bioinformatics tools can not predict their functional impact effectively. Moreover, TTN variants are very frequent in the general population. The combination of deep phenotyping associated with RNA molecular analyses, western blot (WB) and functional studies is often essential for the interpretation of genetic variants in patients suspected of titinopathy. In line with the current guidelines and suggestions, we implemented for patients with skeletal myopathy and with potentially disease causing TTN variant(s) an integrated genotype-transcripts-protein-phenotype approach, associated with phenotype and variants segregation studies in relatives and confrontation with published data on titinopathies to evaluate pathogenic effects of TTN variants (even truncating ones) on titin transcripts, amount, size and functionality. We illustrate this integrated approach in four patients with recessive congenital myopathy.
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Conectina/genética , Genótipo , Doenças Musculares/genética , Fenótipo , Adolescente , Criança , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Pessoa de Meia-Idade , Músculo Esquelético/patologia , MutaçãoRESUMO
BACKGROUND: Genomic imprinting is essential for mammalian development and provides a unique paradigm to explore intra-cellular differences in chromatin configuration. So far, the detailed allele-specific chromatin organization of imprinted gene domains has mostly been lacking. Here, we explored the chromatin structure of the two conserved imprinted domains controlled by paternal DNA methylation imprints-the Igf2-H19 and Dlk1-Dio3 domains-and assessed the involvement of the insulator protein CTCF in mouse cells. RESULTS: Both imprinted domains are located within overarching topologically associating domains (TADs) that are similar on both parental chromosomes. At each domain, a single differentially methylated region is bound by CTCF on the maternal chromosome only, in addition to multiple instances of bi-allelic CTCF binding. Combinations of allelic 4C-seq and DNA-FISH revealed that bi-allelic CTCF binding alone, on the paternal chromosome, correlates with a first level of sub-TAD structure. On the maternal chromosome, additional CTCF binding at the differentially methylated region adds a further layer of sub-TAD organization, which essentially hijacks the existing paternal-specific sub-TAD organization. Perturbation of maternal-specific CTCF binding site at the Dlk1-Dio3 locus, using genome editing, results in perturbed sub-TAD organization and bi-allelic Dlk1 activation during differentiation. CONCLUSIONS: Maternal allele-specific CTCF binding at the imprinted Igf2-H19 and the Dlk1-Dio3 domains adds an additional layer of sub-TAD organization, on top of an existing three-dimensional configuration and prior to imprinted activation of protein-coding genes. We speculate that this allele-specific sub-TAD organization provides an instructive or permissive context for imprinted gene activation during development.
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Fator de Ligação a CCCTC/metabolismo , Impressão Genômica , Animais , Proteínas de Ligação ao Cálcio/genética , Fator de Crescimento Insulin-Like II/genética , Iodeto Peroxidase/genética , Camundongos , RNA Longo não Codificante/genéticaRESUMO
Ubiquitin and ubiquitin-like chains are finely balanced by conjugating and de-conjugating enzymes. Alterations in this balance trigger the response to stress conditions and are often observed in pathologies. How such changes are detected is not well understood. We identify the HSP70 chaperone as a sensor of changes in the balance between mono- and poly-NEDDylation. Upon DNA damage, the induction of the de-NEDDylating enzyme NEDP1 restricts the formation of NEDD8 chains, mainly through lysines K11/K48. This promotes APAF1 oligomerization and apoptosis induction, a step that requires the HSP70 ATPase activity. HSP70 binds to NEDD8, and, in vitro, the conversion of NEDD8 chains into mono-NEDD8 stimulates HSP70 ATPase activity. This effect is independent of NEDD8 conjugation onto substrates. The study indicates that the NEDD8 cycle is a regulatory module of HSP70 function. These findings may be important in tumorigenesis, as we find decreased NEDP1 levels in hepatocellular carcinoma with concomitant accumulation of NEDD8 conjugates.
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Adenosina Trifosfatases/genética , Dano ao DNA/genética , Endopeptidases/genética , Proteínas de Choque Térmico HSP70/genética , Proteína NEDD8/genética , Sequência de Aminoácidos , Animais , Carcinogênese/genética , Linhagem Celular Tumoral , Feminino , Humanos , Lisina/genética , Células MCF-7 , Camundongos , Ubiquitina/genéticaRESUMO
Next-generation sequencing (NGS) gene-panel-based analyses constitute diagnosis strategies which are adapted to the genetic heterogeneity within the field of myopathies, including more than 200 implicated genes to date. Nonetheless, important inter-laboratory diversity of gene panels exists at national and international levels, complicating the exchange of data and the visibility of the diagnostic offers available for referring neurologists. To address this issue, we here describe the initiative of the genetic diagnosis section of the French National Network for Rare Neuromuscular Diseases (Filière Nationale des Maladies Rares Neuromusculaires, FILNEMUS), which led to set up a consensual nationwide diagnostic strategy among the nine French genetic diagnosis laboratories using NGS for myopathies. The strategy is based on the determination of 13 clinical and/or histological entry-diagnosis groups, and consists for each group either in a successive NGS analysis of a "core gene list" followed in case of a negative result by the analysis of an "exhaustive gene list", or in the NGS analysis of a "unique exhaustive gene list".
Assuntos
Consenso , Testes Genéticos/normas , Doenças Neuromusculares/genética , Guias de Prática Clínica como Assunto , Análise de Sequência de DNA/normas , França , Testes Genéticos/métodos , Humanos , Doenças Neuromusculares/diagnóstico , Análise de Sequência de DNA/métodos , Sociedades MédicasRESUMO
Although many long non-coding RNAs (lncRNAs) are imprinted, their roles often remain unknown. The Dlk1-Dio3 domain expresses the lncRNA Meg3 and multiple microRNAs and small nucleolar RNAs (snoRNAs) on the maternal chromosome and constitutes an epigenetic model for development. The domain's Dlk1 (Delta-like-1) gene encodes a ligand that inhibits Notch1 signaling and regulates diverse developmental processes. Using a hybrid embryonic stem cell (ESC) system, we find that Dlk1 becomes imprinted during neural differentiation and that this involves transcriptional upregulation on the paternal chromosome. The maternal Dlk1 gene remains poised. Its protection against activation is controlled in cis by Meg3 expression and also requires the H3-Lys-27 methyltransferase Ezh2. Maternal Meg3 expression additionally protects against de novo DNA methylation at its promoter. We find that Meg3 lncRNA is partially retained in cis and overlaps the maternal Dlk1 in embryonic cells. Combined, our data evoke an imprinting model in which allelic lncRNA expression prevents gene activation in cis.
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Impressão Genômica , RNA Longo não Codificante/metabolismo , Alelos , Animais , Sistemas CRISPR-Cas/genética , Proteínas de Ligação ao Cálcio , Diferenciação Celular , Linhagem Celular , Metilação de DNA , Células-Tronco Embrionárias , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Histonas/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/citologia , Neurônios/metabolismo , Proteínas do Grupo Polycomb/metabolismo , Regiões Promotoras Genéticas , RNA Longo não Codificante/genética , Ativação Transcricional , Regulação para CimaRESUMO
Spatial management of stress-induced protein aggregation is an integral part of the proteostasis network. Protein modification by the ubiquitin-like molecule NEDD8 increases upon proteotoxic stress and it is characterised by the formation of hybrid NEDD8/ubiquitin conjugates. However, the biological significance of this response is unclear. Combination of quantitative proteomics with biological analysis shows that, during proteotoxic stress, NEDDylation promotes nuclear protein aggregation, including ribosomal proteins as a major group. This correlates with protection of the nuclear Ubiquitin Proteasome System from stress-induced dysfunction. Correspondingly, we show that NEDD8 compromises ubiquitination and prevents targeting and processing of substrates by the proteasome. Moreover, we identify HUWE1 as a key E3-ligase that is specifically required for NEDDylation during proteotoxic stress. The study reveals a specific role for NEDD8 in nuclear protein aggregation upon stress and is consistent with the concept that transient aggregate formation is part of a defence mechanism against proteotoxicity.
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Proteína NEDD8/metabolismo , Proteínas Nucleares/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitinas/metabolismo , Western Blotting , Linhagem Celular Tumoral , Células HEK293 , Humanos , Microscopia de Fluorescência , Proteína NEDD8/genética , Proteínas Nucleares/genética , Proteômica , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/genética , Ubiquitinação/fisiologia , Ubiquitinas/genéticaRESUMO
How metazoan genomes are structured at the nanoscale in living cells and tissues remains unknown. Here, we adapted a quantitative FRET (Förster resonance energy transfer)-based fluorescence lifetime imaging microscopy (FLIM) approach to assay nanoscale chromatin compaction in living organisms. Caenorhabditis elegans was chosen as a model system. By measuring FRET between histone-tagged fluorescent proteins, we visualized distinct chromosomal regions and quantified the different levels of nanoscale compaction in meiotic cells. Using RNAi and repetitive extrachromosomal array approaches, we defined the heterochromatin state and showed that its architecture presents a nanoscale-compacted organization controlled by Heterochromatin Protein-1 (HP1) and SETDB1 H3-lysine-9 methyltransferase homologs in vivo. Next, we functionally explored condensin complexes. We found that condensin I and condensin II are essential for heterochromatin compaction and that condensin I additionally controls lowly compacted regions. Our data show that, in living animals, nanoscale chromatin compaction is controlled not only by histone modifiers and readers but also by condensin complexes.