title: Extensive in silico analysis of NF1 splicing defects uncovers determinants for splicing outcome upon 5' splice-site disruption.
abstract: We describe 94 pathogenic NF1 gene alterations in a cohort of 97 Austrian neurofibromatosis type 1 patients meeting the NIH criteria. All mutations were fully characterized at the genomic and mRNA levels. Over half of the patients carried novel mutations, and only a quarter carried recurrent minor-lesion mutations at 16 mutational warm spots. The remaining patients carried NF1 microdeletions (7%) and rare recurring mutations. Thirty-six of the mutations (38%) altered pre-mRNA splicing, and fall into five groups: exon skipping resulting from mutations at authentic splice sites (type I), cryptic exon inclusion caused by deep intronic mutations (type II), creation of de novo splice sites causing loss of exonic sequences (type III), activation of cryptic splice sites upon authentic splice-site disruption (type IV), and exonic sequence alterations causing exon skipping (type V). Extensive in silico analyses of 37 NF1 exons and surrounding intronic sequences suggested that the availability of a cryptic splice site combined with a strong natural upstream 3' splice site (3'ss)is the main determinant of cryptic splice-site activation upon 5' splice-site disruption. Furthermore, the exonic sequences downstream of exonic cryptic 5' splice sites (5'ss) resemble intronic more than exonic sequences with respect to exonic splicing enhancer and silencer density, helping to distinguish between exonic cryptic and pseudo 5'ss. This study provides valuable predictors for the splicing pathway used upon 5'ss mutation, and underscores the importance of using RNA-based techniques, together with methods to identify microdeletions and intragenic copy-number changes, for effective and reliable NF1 mutation detection.
authors.affiliation: Department of Medical Genetics, Medical University of Vienna, Vienna, Austria. katharina.wimmer@meduniwien.ac.at
authors.forename: K
authors.initials: K
authors.lastname: Wimmer
authors.forename: X
authors.initials: X
authors.lastname: Roca
authors.forename: H
authors.initials: H
authors.lastname: Beiglböck
authors.forename: T
authors.initials: T
authors.lastname: Callens
authors.forename: J
authors.initials: J
authors.lastname: Etzler
authors.forename: A R
authors.initials: AR
authors.lastname: Rao
authors.forename: A R
authors.initials: AR
authors.lastname: Krainer
authors.forename: C
authors.initials: C
authors.lastname: Fonatsch
authors.forename: L
authors.initials: L
authors.lastname: Messiaen
mesh_terms: D000328:Adult; D017398:Alternative Splicing / Q000235:genetics*; D001317:Austria; D002648:Child; D015331:Cohort Studies; D003198:Computer Simulation; D004252:DNA Mutational Analysis; D005091:Exons; D016514:Genes, Neurofibromatosis 1; D006801:Humans; D009154:Mutation; D009456:Neurofibromatosis 1 / Q000175:diagnosis / Q000235:genetics*; D022821:RNA Splice Sites / Q000235:genetics*; D012680:Sensitivity and Specificity; D017384:Sequence Deletion
chemical_list: D022821:RNA Splice Sites