Next Generation Sequencing Analysis of HIV-1 Group O Reverse Transcriptase Residue 181C Prevalence and Evolution over Time, With or Without Antiretroviral Selection Pressure

International audienc

Modélisation de la régulation de l'épissage alternatif au site A7 de HIV-1

Stage de DESS. Rapport de stage.L'épissage alternatif est un processus biologique qui intervient dans la régulation post-transcriptionelle. A travers l'élimination de certains introns, l'épissage alter- natif peut générer di érents ARNm. De récentes études biologiques sur HIV-1 mon- trent le pouvoir de la protéine Tat. Le site accepteur d'épissage A7 est requis pour la production des ARNm tat. La production de l'activateur transcriptionel Tat est hautement contrôlée à cause de ses propriétés apoptotiques. Notre objectif est de modéliser cette régulation dans le but de mieux comprendre la dynamique du virus. La site d'épissage A7 contient des éléments activateurs et des éléments inhibiteurs qui peuvent être xés par des protéines de régulation. Nous formalisons la régulation par des équations di érentielles, développant ainsi un modèle qualitatif pour le site A7 d'épissage de HIV-1. Le comportement qualitatif est dépendant des valeurs des paramètres des réactions cinétiques. Les résultats expérimentaux nous permettent de valider ce modèle à l'équilibre, confortant ainsi in silico les hypothèses formulées ex- périmentalement. Cette démarche joue ainsi un rôle essentiel dans la compréhension de la régulation de l'épissage alternatif

Implication des altérations de l'épissage de l'ARN dans les cancers héréditaires et dans l'amyotrophie spinale infantile

Cette thèse traite des altérations constitutionnelles affectant l épissage de l ARN dans deux pathologies : les prédispositions génétiques aux cancers et l amyotrophie spinale infantile (ASI). Nous avons mis en œuvre un test fonctionnel d épissage utilisant des constructions basées sur des minigènes afin de détecter les conséquences sur l épissage de toute variation nucléotidique de signification inconnue. Appliqué à l interprétation des mutations identifiées lors du diagnostic génétique de prédispositions aux cancers du colon, du sein et de l ovaire, ce test permet de reclasser près de 20% des variations de signification inconnue en mutations d épissage. Certains de ces variants sont situés à distance des sites consensus d épissage et peuvent altérer un motif régulateur d épissage. Nous avons développé un ensemble d outils d analyse permettant leur cartographie et leur analyse fonctionnelle. Mes travaux sur l ASI s inscrivent dans la perspective d essais cliniques, basés sur la correction de l épissage du transcrit SMN2-D7. Afin de proposer des biomarqueurs indicateurs de l efficacité du traitement, nous avons développé et validé une méthode sensible et robuste de dosage des ARNm issus de l expression du gène SMN2. La dernière partie de mes travaux décrit l étude d un patient portant seulement deux copies du gène SMN2 avec un phénotype exceptionnellement modéré d une ASI de type III. Nous avons montré qu une mutation rare, sur les deux copies du gène SMN2 de ce patient, induit l inclusion de l exon 7 dans l ARNm de SMN2. L investigation sur le plan biochimique de ce variant a permis d accéder à certains aspects de la régulation de l épissage de l exon 7 des gènes SMN qui n avaient pas encore été décrits.This thesis deals with constitutional changes affecting RNA splicing in in two groups of genetics diseases: genetic predisposition to cancer and the neuromuscular disease spinal muscular atrophy (SMA). We developed minigene-based constructs functional assay to detect the effect on splicing of nucleotide variations of unknown significance. We applied this assay to the interpretation of mutations identified in the molecular diagnosis of genetic predisposition to colon cancer and to breast and ovarian cancer, and found that nearly 20% of the variation of unknown significance could be reclassified into splicing mutations. Some of these variants are located at a distance of consensus splice sites and can affect a splicing regulatory element. We developed a set of analytical tools to map these regulatory elements and to analyze them functionally. My work on SMA is in the context of clinical trials based on the correction of the defective splicing of SMN2 transcripts. We developed and validated a sensitive and robust method to determine mRNA expression from the SMN2 gene. The last part of my work describes the study of a patient with only two copies of the SMN2 gene and an unexpectedly moderate clinical phenotype (type III SMA). We showed that a rare mutation on both copies of the SMN2 gene of this patient enhanced inclusion of exon 7 into the mRNA of SMN2. The biochemical investigation of this variant has highlighted previously unrecognized aspects of the regulation of splicing of exon 7 of SMN genes.ROUEN-BU Sciences (764512102) / SudocSudocFranceF

Next Generation Sequencing Analysis of HIV-1 Group O Reverse Transcriptase Residue 181C Prevalence and Evolution over Time, With or Without Antiretroviral Selection Pressure

International audienc

Pontocerebellar hypoplasia with rhombencephalosynapsis and microlissencephaly expands the spectrum of PCH type 1B

International audienc

A leaky splicing mutation affecting SMN1 exon 7 inclusion explains an unexpected mild case of spinal muscular atrophy

International audienceSpinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder resulting, in most cases, from homozygous deletions of the SMN1 gene or, in rare cases, from SMN1 intragenic mutations. Here we describe the identification and characterization of c.835-3C>T, a novel SMA-causing mutation detected in the intron 6 of the single SMN1 allele of a type IV SMA patient. We demonstrate both ex vivo and in vivo that c.835-3C>T is a deleterious splicing mutation that induces a modest but unequivocal exclusion of exon 7 from the SMN1 transcripts, its "leakiness" explaining the exceptionally mild phenotype of this patient. This mutation creates a putative high-affinity binding site for the splicing repressor protein hnRNP A1 overlapping the splice acceptor site of exon 7 (UAG|GGU). Our findings support the current therapeutic strategies aiming at correcting exon 7 splicing in SMA patients, and bring clues about the level of exon 7 inclusion required to achieve a therapeutic effect

Neuropathological hallmarks of fetal hydrocephalus linked to CCDC88C pathogenic variants

Abstract The prevalence of congenital hydrocephalus has been estimated at 1.1 per 1000 infants when including cases diagnosed before 1 year of age after exclusion of neural tube defects. Classification criteria are based either on CSF dynamics, pathophysiological mechanisms or associated lesions. Whereas inherited syndromic hydrocephalus has been associated with more than 100 disease-causing genes, only four genes are currently known to be linked to congenital hydrocephalus either isolated or as a major clinical feature: L1CAM, AP1S2, MPDZ and CCDC88C. In the past 10 years, pathogenic variants in CCDC88C have been documented but the neuropathology remains virtually unknown. We report the neuropathology of two foetuses from one family harbouring two novel compound heterozygous pathogenic variants in the CCDC88C gene: a maternally inherited indel in exon 22, c.3807_3809delinsACCT;p.(Gly1270Profs*53) and a paternally inherited deletion of exon 23, c.3967-?_c.4112-?;p.(Leu1323Argfs*10). Medical termination of pregnancy was performed at 18 and 23 weeks of gestation for severe bilateral ventriculomegaly. In both fetuses, brain lesions consisted of multifocal atresia-forking along the aqueduct of Sylvius and the central canal of the medulla, periventricular neuronal heterotopias and choroid plexus hydrops. The second fetus also presented lumbar myelomeningocele, left diaphragmatic hernia and bilateral renal agenesis. CCDC88C encodes the protein DAPLE which contributes to ependymal cell planar polarity by inhibiting the non-canonical Wnt signaling pathway and interacts with MPDZ and PARD3. Interestingly, heterozygous variants in PARD3 result in neural tube defects by defective tight junction formation and polarization process of the neuroepithelium. Besides, during organ formation Wnt signalling is a prerequisite for planar cell polarity pathway activation, and mutations in planar cell polarity genes lead to heart, lung and kidney malformations. Hence, candidate variants in CCDC88C should be carefully considered whether brain lesions are isolated or associated with malformations suspected to result from disorders of planar cell polarity

Retrotransposon insertion as a novel mutational cause of spinal muscular atrophy

International audienceSpinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder resulting from biallelic alterations of the SMN1 gene: deletion, gene conversion or, in rare cases, intragenic variants. The disease severity is mainly influenced by the copy number of SMN2, a nearly identical gene, which produces only low amounts of full-length (FL) mRNA. Here we describe the first example of retrotransposon insertion as a pathogenic SMN1 mutational event. The 50-year-old patient is clinically affected by SMA type III with a diagnostic odyssey spanning nearly 30 years. Despite a mild disease course, he carries a single SMN2 copy. Using Exome Sequencing and Sanger sequencing, we characterized a SINE-VNTR-Alu (SVA) type F retrotransposon inserted in SMN1 intron 7. Using RT-PCR and RNASeq experiments on lymphoblastoid cell lines, we documented the dramatic decrease of FL transcript production in the patient compared to subjects with the same SMN1 and SMN2 copy number, thus validating the pathogenicity of this SVA insertion. We described the mutant FL-SMN1-SVA transcript characterized by exon extension and showed that it is subject to degradation by nonsense-mediated mRNA decay. The stability of the SMN-SVA protein may explain the mild course of the disease. This observation exemplifies the role of retrotransposons in human genetic disorders

A large fraction of unclassified variants of the mismatch repair genes MLH1 and MSH2 is associated with splicing defects

International audienceNumerous unclassified variants (UVs) have been found in the mismatch repair genes MLH1 and MSH2 involved in hereditary nonpolyposis colorectal cancer (HNPCC or Lynch syndrome). Some of these variants may have an effect on pre‐mRNA splicing, either by altering degenerate positions of splice site sequences or by affecting intronic or exonic splicing regulatory sequences such as exonic splicing enhancers (ESEs). In order to determine the consequences of UVs on splicing, we used a functional assay of exon inclusion. For each variant, mutant and wild‐type exons to be tested were PCR‐amplified from patient genomic DNA together with ∼150 bp of flanking sequences and were inserted into a splicing reporter minigene. After transfection into HeLa cells, the effects on splicing were evaluated by RT‐PCR analysis and systematic sequencing. A total of 22 UVs out of 85 different variant alleles examined in 82 families affected splicing, including four exonic variants that affected putative splicing regulatory elements. We analyzed short stretches spanning the latter variants by cloning them into the ESE‐dependent central exon of a three‐exon splicing minigene and we showed in cell transfection experiments that the wild‐type sequences indeed contain functional ESEs. We then used this construct to query for ESE elements in the MLH1 or MSH2 regions affected by 14 previously reported exonic splicing mutations and showed that they also contain functional ESEs. These splicing assays represent a valuable tool for the interpretation of UVs and should contribute to the optimization of the molecular diagnosis of the Lynch syndrome and of other genetic diseases

Retrotransposon insertion as a novel mutational cause of spinal muscular atrophy

International audienceSpinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder resulting from biallelic alterations of the SMN1 gene: deletion, gene conversion or, in rare cases, intragenic variants. The disease severity is mainly influenced by the copy number of SMN2, a nearly identical gene, which produces only low amounts of full-length (FL) mRNA. Here we describe the first example of retrotransposon insertion as a pathogenic SMN1 mutational event. The 50-year-old patient is clinically affected by SMA type III with a diagnostic odyssey spanning nearly 30 years. Despite a mild disease course, he carries a single SMN2 copy. Using Exome Sequencing and Sanger sequencing, we characterized a SINE-VNTR-Alu (SVA) type F retrotransposon inserted in SMN1 intron 7. Using RT-PCR and RNASeq experiments on lymphoblastoid cell lines, we documented the dramatic decrease of FL transcript production in the patient compared to subjects with the same SMN1 and SMN2 copy number, thus validating the pathogenicity of this SVA insertion. We described the mutant FL-SMN1-SVA transcript characterized by exon extension and showed that it is subject to degradation by nonsense-mediated mRNA decay. The stability of the SMN-SVA protein may explain the mild course of the disease. This observation exemplifies the role of retrotransposons in human genetic disorders