title: Whole USH2A Gene Sequencing Identifies Several New Deep Intronic Mutations.
abstract: Deep intronic mutations leading to pseudoexon (PE) insertions are underestimated and most of these splicing alterations have been identified by transcript analysis, for instance, the first deep intronic mutation in USH2A, the gene most frequently involved in Usher syndrome type II (USH2). Unfortunately, analyzing USH2A transcripts is challenging and for 1.8%-19% of USH2 individuals carrying a single USH2A recessive mutation, a second mutation is yet to be identified. We have developed and validated a DNA next-generation sequencing approach to identify deep intronic variants in USH2A and evaluated their consequences on splicing. Three distinct novel deep intronic mutations have been identified. All were predicted to affect splicing and resulted in the insertion of PEs, as shown by minigene assays. We present a new and attractive strategy to identify deep intronic mutations, when RNA analyses are not possible. Moreover, the bioinformatics pipeline developed is independent of the gene size, implying the possible application of this approach to any disease-linked gene. Finally, an antisense morpholino oligonucleotide tested in vitro for its ability to restore splicing caused by the c.9959-4159A>G mutation provided high inhibition rates, which are indicative of its potential for molecular therapy.
authors.affiliation: Laboratoire de Génétique de Maladies Rares EA 7402, Université de Montpellier, Montpellier, France.
authors.forename: Alessandro
authors.initials: A
authors.lastname: Liquori
authors.affiliation: Laboratoire de Génétique de Maladies Rares EA 7402, Université de Montpellier, Montpellier, France.|Laboratoire de Génétique Moléculaire, CHRU Montpellier, Montpellier, France.
authors.forename: Christel
authors.initials: C
authors.lastname: Vaché
authors.affiliation: Laboratoire de Génétique de Maladies Rares EA 7402, Université de Montpellier, Montpellier, France.|Laboratoire de Génétique Moléculaire, CHRU Montpellier, Montpellier, France.
authors.forename: David
authors.initials: D
authors.lastname: Baux
authors.affiliation: Service ORL, CHRU Montpellier, Montpellier, France.|CHU Montpellier, Centre National de Référence Maladies Rares, "Affections Sensorielles Génétiques, France.
authors.forename: Catherine
authors.initials: C
authors.lastname: Blanchet
authors.affiliation: CHU Montpellier, Centre National de Référence Maladies Rares, "Affections Sensorielles Génétiques, France.
authors.forename: Christian
authors.initials: C
authors.lastname: Hamel
authors.affiliation: Genetics and Genomic Medicine Programme, Institute of Child Health, UCL, London, UK.
authors.forename: Sue
authors.initials: S
authors.lastname: Malcolm
authors.affiliation: Laboratoire de Génétique de Maladies Rares EA 7402, Université de Montpellier, Montpellier, France.|Laboratoire de Génétique Moléculaire, CHRU Montpellier, Montpellier, France.
authors.forename: Michel
authors.initials: M
authors.lastname: Koenig
authors.affiliation: Laboratoire de Génétique de Maladies Rares EA 7402, Université de Montpellier, Montpellier, France.|Laboratoire de Génétique Moléculaire, CHRU Montpellier, Montpellier, France.
authors.forename: Mireille
authors.initials: M
authors.lastname: Claustres
authors.affiliation: Laboratoire de Génétique de Maladies Rares EA 7402, Université de Montpellier, Montpellier, France.|Laboratoire de Génétique Moléculaire, CHRU Montpellier, Montpellier, France.
authors.forename: Anne-Françoise
authors.initials: AF
authors.lastname: Roux
mesh_terms: D001483:Base Sequence; D019295:Computational Biology; D004252:DNA Mutational Analysis; D005091:Exons; D016326:Extracellular Matrix Proteins / Q000235:genetics* / Q000378:metabolism; D005260:Female; D015870:Gene Expression; D006367:HeLa Cells; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D007438:Introns; D008297:Male; D008969:Molecular Sequence Data; D060172:Morpholinos / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D016376:Oligonucleotides, Antisense / Q000235:genetics / Q000378:metabolism; D010375:Pedigree; D012326:RNA Splicing; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism; D052245:Usher Syndromes / Q000235:genetics* / Q000378:metabolism / Q000473:pathology
chemical_list: D016326:Extracellular Matrix Proteins; D060172:Morpholinos; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; C112735:USH2A protein, human
keywords: NGS; USH2A; deep intronic mutation; minigene assays; pseudoexon; sequence capture; whole gene sequencing