Exon Skipping-Mediated Dystrophin Reading Frame Restoration for Small Mutations

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Tumor Necrosis Factor Receptor SF10A (TNFRSF10A) SNPs Correlate With Corticosteroid Response in Duchenne Muscular Dystrophy

Background Duchenne muscular dystrophy (DMD) is a rare and severe X-linked muscular dystrophy in which the standard of care with variable outcome, also due to different drug response, is chronic off-label treatment with corticosteroids (CS). In order to search for SNP biomarkers for corticosteroid responsiveness, we genotyped variants across 205 DMD-related genes in patients with differential response to steroid treatment. Methods and Findings We enrolled a total of 228 DMD patients with identified dystrophin mutations, 78 of these patients have been under corticosteroid treatment for at least 5 years. DMD patients were defined as high responders (HR) if they had maintained the ability to walk after 15 years of age and low responders (LR) for those who had lost ambulation before the age of 10 despite corticosteroid therapy. Based on interactome mapping, we prioritized 205 genes and sequenced them in 21 DMD patients (discovery cohort or DiC = 21). We identified 43 SNPs that discriminate between HR and LR. Discriminant Analysis of Principal Components (DAPC) prioritized 2 response-associated SNPs in theTNFRSF10Agene. Validation of this genotype was done in two additional larger cohorts composed of 46 DMD patients on corticosteroid therapy (validation cohorts or VaC1), and 150 non ambulant DMD patients and never treated with corticosteroids (VaC2). SNP analysis in all validation cohorts (N= 207) showed that the CT haplotype is significantly associated with HR DMDs confirming the discovery results. Conclusion We have shown that TNFRSF10A CT haplotype correlates with corticosteroid response in DMD patients and propose it as an exploratory CS response biomarker

Variabilité phénotypique et corrélations génotype phénotype des dystrophinopathies (contribution des banques de données.)

L'objectif de ce travail est de développer la partie clinique de la banque de données du gène DMD, afin d'étudier l'histoire naturelle des dystrophinopathies et les corrélations génotype phénotype, et de faciliter la sélection des patients pour les futurs essais thérapeutiques. La méthodologie créée pour le gène DMD peut être généralisée et utilisée pour d'autres banques de données dédiées à des maladies génétiques. La collecte de 70 000 données cliniques chez 600 patients avec un suivi longitudinal moyen de 12 ans permet de décrire l'histoire naturelle des dystrophies musculaires de Duchenne et de Becker et des formes symptomatiques chez les femmes. Nous avons pu préciser l'hétérogénéité phénotypique sur le plan moteur, orthopédique et respiratoire (forme sévère et forme intermédiaire de la dystrophie musculaire de Duchenne), sur le plan cardiaque (absence de corrélation entre les atteintes motrice et cardiaque, variabilité de l'atteinte cardiaque), et sur le plan cérébral (atteinte intellectuelle chez les patients avec dystrophie musculaire de Becker, troubles psychologiques des dystrophinopathies). L'utilisation de cet outil par les cliniciens et les généticiens devrait faciliter le travail de recherche clinique et la réalisation des futurs essais cliniques. Ceci nécessite maintenant de développer l'accessibilité de la banque de données et d'envisager sa pérennisation.The objective of this work is to develop the clinical part of the French dystrophinopathy data-base, in order to study the natural history and the genotype-phenotype correlations, and to facilitate the selection of the patients for the future therapeutic trials. The methodology developed for the DMD gene can be generalized and used for the other databases dedicated to genetic diseases. The collection of 70 000 clinical data for 600 patients with an average lon-gitudinal follow-up of 12 years allows to clarify the natural history of the muscular dystrophies of Duchenne and Becker and in symptomatic females. We were able to specify the pheno-typic heterogeneity of the motor, orthopaedic and respiratory involvements (severe form and intermediary form of the Duchenne muscular dystrophy), of the cardiac disorder (absence of correlation between motor and cardiac involvements, variability of the cardiomyopathy), and of the brain function (mental deficiency in the patients with Becker muscular dystrophy, psychological disorders in dystrophinopathies). The use of this tool by the clinicians and the ge-neticists should facilitate their clinical research work and the realization of the future clinical trials. This requires now to develop the accessibility of the database and to ensure its continued existence.MONTPELLIER-BU Médecine UPM (341722108) / SudocSudocFranceF

Altérations de l'épissage et pathologies humaines

MONTPELLIER-BU Médecine UPM (341722108) / SudocPARIS-BIUP (751062107) / SudocMONTPELLIER-BU Médecine (341722104) / SudocSudocFranceF

Mutations des éléments cis-régulateurs de l'épissage (rôle en pathologie humaine)

L'épissage des ARN pre-messagers joue un rôle fondamental dans l'expression des gènes chez les eucaryotes supérieurs. La régulation de ce processus extrêmement complexe, qui permet l'excision des introns, repose sur la reconnaissance de multiples éléments, incluant les séquences consensus (sites accepteurs et donneurs) et cis-régulatrices auxiliaires, dont la combinaison définit un "code de l'épissage" propre à chaque exon. Les exemples de mutation associées à des anomalies d'épissage dans les maladies génétiques humaines sont de plus en plus nombreux, néanmoins leur contribution reste très probablement sous estimée. En effet, un nombre important de variants non classés (Unknown Variants, UVs) introniques ou exoniques est identifié par séquençage des gènes, dont une partie est susceptible d'altérer l'épissage. Quand les transcrits spécifiques des patients ne sont pas accessibles, l'utilisation combinée d'outils bio-informatiques et de tests fonctionnels basés sur l'utilisation de minigènes rapporteurs d'épissage permet d'évaluer l'impact de ces UVs sur l'épissage. Cette stratégie, appliquée aux variants identifiés dans les gènes responsables du syndrome de Usher, a montré que 76% des UVs localisés à des positions peu conservées des sites consensus avaient des conséquences majeures sur l'épissage. Au cours de ces travaux, nous avons confirmé l'importance de l'interdépendance des positions nucléotidiques dans la définition des sites d'épissage, et avons pu établir que les positions -1 et +4 du site donneur d'épissage permettaient de compenser un mésappariement en position +3 avec la protéine U1 snRNP. Les évènements d'épissage alternatifs jouent également un rôle important dans la modulation de sévérité du phénotype en pathologie humaine. On observe ainsi pour le gène DMD (Duchenne muscular Dystrophy) que des mutations non-sens (introduisant un codon stop prématuré) sont retrouvées dans des formes modérées de la maladie (Myopathie de Becker) lorsqu'elles sont à l'origine d'un épissage alternatif en phase de l'exon muté. L'identification des éléments cis-régulateurs de l'épissage impliqués (ou SREs, splicing regulatory elements), notamment les séquences activatrices de type ESE (Exonic Splicing Enhancer), représente un enjeu majeur pour la mise en place des stratégies thérapeutiques par saut d'exon dans la myopathie de DuchenneSplicing of pre-messenger RNAs to mature transcripts is a crucial step in eukaryotic gene expression. This highly regulated mechanism involves multiple signals, including the core splice site motifs (donor and acceptor splice sites) and auxiliary ci-regulatory elements, which are part of integrated "splicing code". More and more examples of splicing mutations resulting in genetic diseases are described in the literature, however their true contribution is probably underestimated. Indeed, sequence-based approach to study disease-causing genes in diagnostic procedures leads to the identification of an increasing number of variants of unknown significance (UVs). The biological consequences of the so-called UVs and notably their purative impact of splicing ar unknown. When specific analysis is not achievable in patients, in silico predictions associated with minigene studies are performed to determine the impact of these UVs on splicing. This strategy was applied to a set of UVs located in poorly conserved positions of acceptor and donor splice sites of Usher genes, and 76% of them were found to cause aberrant splicing. From our results, we further confirmed the importance of the overall nucleotidic environment in splice sites selection. In particular, we could establish that positions -1 and +4 in donor splice sites can compensate a mismatch at position +3 with the U1 snRNP and contribute to the correct recognition of such donor splice sites. Alernative splicing events play also a major role in modulation of disease severity. In the DMD gene, nonsense mutations introducing a premature stop codon are found in patients with a milder phenotype (Becker-like) than expected (Duchenne-like), and usually result from the elimination of the truncating mutation from dystrophin mRNA by skipping of an in-frame exon. This kind of disease-causing mutations can contribute to shed light on new cis-regulatory elements, in particular Exonic Splicing Enhancer (ESE) motifs, essential for splicing in the DMD gene, which may be important for the exon skipping therapeuric strategyMONTPELLIER-BU Médecine UPM (341722108) / SudocMONTPELLIER-BU Médecine (341722104) / SudocSudocFranceF

Identification of three novel OA1 gene mutations identified in three families misdiagnosed with congenital nystagmus and carrier status determination by real-time quantitative PCR assay

<p>Abstract</p> <p>Background</p> <p>X-linked ocular albinism type 1 (OA1) is caused by mutations in <it>OA1 </it>gene, which encodes a membrane glycoprotein localised to melanosomes. OA1 mainly affects pigment production in the eye, resulting in optic changes associated with albinism including hypopigmentation of the retina, nystagmus, strabismus, foveal hypoplasia, abnormal crossing of the optic fibers and reduced visual acuity. Affected Caucasian males usually appear to have normal skin and hair pigment.</p> <p>Results</p> <p>We identified three previously undescribed mutations consisting of two intragenic deletions (one encompassing exon 6, the other encompassing exons 7–8), and a point mutation (310delG) in exon 2. We report the development of a new method for diagnosis of heterozygous deletions in <it>OA1 </it>gene based on measurement of gene copy number using real-time quantitative PCR from genomic DNA.</p> <p>Conclusion</p> <p>The identification of <it>OA1 </it>mutations in families earlier reported as families with hereditary nystagmus indicate that ocular albinism type 1 is probably underdiagnosed. Our method of real-time quantitative PCR of <it>OA1 </it>exons with <it>DMD exon </it>as external standard performed on the LightCycler™ allows quick and accurate carrier-status assessment for at-risk females.</p

Normal and altered pre-mRNA processing in the DMD gene

International audienceSplicing of pre-mRNA is a crucial regulatory stage in the pathway of gene expression controlled by multiple post- and co-transcriptional mechanisms. The large Duchenne muscular dystrophy gene encoding the protein dystrophin provides a striking example of the complexity of human pre-mRNAs. In this review, we summarize the current state of knowledge about canonical and non-canonical splicing in the DMD pre-mRNA, with a focus on mechanisms that take place in the full-length transcript isoform expressed in human skeletal muscle. In particular, we highlight recent work demonstrating that multi-step events are required for long DMD intron removal. The role of temporary intron retention in the occurrence of alternative splicing events is also discussed. Even though the proportion of splicing mutations is lower than reported in other genes, a great diversity of splicing defects linked to point mutations, but also large genomic rearrangements are observed in the DMD gene. We provide an overview of the molecular mechanisms underlying aberrant splicing in patients with Duchenne or Becker muscular dystrophy, and we also detail how alternative splicing can serve as a disease modifier in patients by changing the outcome of the primary defect

Primary dystonia: In search of new genes...

Primary dystonia are movement disorders, genetically heterogeneous. The only gene identified for primary dystonia is the DYT1 gene (or TOR1A) implicated in generalized forms. Three loci have been implicated in focal dystonia but no genes have been identified. Many families excluded these known loci suggesting larger heterogeneity and existence of other genes. In this article, we will review the strategies useful for identifying disease gene in primary dystonia

Identification of Splicing Factors Involved in DMD Exon Skipping Events Using an In Vitro RNA Binding Assay

International audienceMutation-induced exon skipping in the DMD gene can modulate the severity of the phenotype in patients with Duchenne or Becker Muscular Dystrophy. These alternative splicing events are most likely the result of changes in recruitment of splicing factors at cis-acting elements in the mutated DMD pre-mRNA. The identification of proteins involved can be achieved by an affinity purification procedure. Here, we provide a detailed protocol for the in vitro RNA binding assay that we routinely apply to explore molecular mechanisms underlying splicing defects in the DMD gene. In vitro transcribed RNA probes containing either the wild type or mutated sequence are oxidized and bound to adipic acid dihydrazide-agarose beads. Incubation with a nuclear extract allows the binding of nuclear proteins to the RNA probes. The unbound proteins are washed off and then the specifically RNA-bound proteins are released from the beads by an RNase treatment. After separation by SDS-PAGE, proteins that display differential binding affinities for the wild type and mutant RNA probes are identified by mass spectrometry