scispace - formally typeset
Search or ask a question
Journal ArticleDOI

Extensive in silico analysis of NF1 splicing defects uncovers determinants for splicing outcome upon 5′ splice‐site disruption

TL;DR: 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.
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.
Citations
More filters
Journal ArticleDOI
TL;DR: This article summarizes the current knowledge about the “splicing mutations” and methods that help to identify such changes in clinical diagnosis and recommends bioinformatic algorithms as a tool to assess the possible effect of the identified changes.
Abstract: Precise pre-mRNA splicing, essential for appropriate protein translation, depends on the presence of consensus “cis” sequences that define exon-intron boundaries and regulatory sequences recognized by splicing machinery. Point mutations at these consensus sequences can cause improper exon and intron recognition and may result in the formation of an aberrant transcript of the mutated gene. The splicing mutation may occur in both introns and exons and disrupt existing splice sites or splicing regulatory sequences (intronic and exonic splicing silencers and enhancers), create new ones, or activate the cryptic ones. Usually such mutations result in errors during the splicing process and may lead to improper intron removal and thus cause alterations of the open reading frame. Recent research has underlined the abundance and importance of splicing mutations in the etiology of inherited diseases. The application of modern techniques allowed to identify synonymous and nonsynonymous variants as well as deep intronic mutations that affected pre-mRNA splicing. The bioinformatic algorithms can be applied as a tool to assess the possible effect of the identified changes. However, it should be underlined that the results of such tests are only predictive, and the exact effect of the specific mutation should be verified in functional studies. This article summarizes the current knowledge about the “splicing mutations” and methods that help to identify such changes in clinical diagnosis.

383 citations


Cites background from "Extensive in silico analysis of NF1..."

  • ...According to Wimmer et al. (2007), splicing mutations can be briefly divided into five categories: (1) splicing mutations within the canonical splice sites leading to whole exon skipping, (2) deep intronic variants creating new splice sites resulting in the inclusion of cryptic exons, (3) exonic…...

    [...]

  • ...According to Wimmer et al. (2007), splicing mutations can be briefly divided into five categories that are further briefly discussed (Fig....

    [...]

  • ...Second, the presence of exonic changes that cause the disruption of exonic splicing enhancers may also lead to the entire exon skipping (so-called type V splicing mutation) (Wimmer et al. 2007)....

    [...]

Journal ArticleDOI
01 Oct 2007-RNA
TL;DR: The applicability of exon skipping for Duchenne muscular dystrophy and other diseases is described and retrospective analysis resulted in guidelines for AON design for DMD and most likely other genes as well.
Abstract: Antisense-mediated modulation of splicing is one of the few fields where antisense oligonucleotides (AONs) have been able to live up to their expectations. In this approach, AONs are implemented to restore cryptic splicing, to change levels of alternatively spliced genes, or, in case of Duchenne muscular dystrophy (DMD), to skip an exon in order to restore a disrupted reading frame. The latter allows the generation of internally deleted, but largely functional, dystrophin proteins and would convert a severe DMD into a milder Becker muscular dystrophy phenotype. In fact, exon skipping is currently one of the most promising therapeutic tools for DMD, and a successful first-in-man trial has recently been completed. In this review the applicability of exon skipping for DMD and other diseases is described. For DMD AONs have been designed for numerous exons, which has given us insight into their mode of action, splicing in general, and splicing of the DMD gene in particular. In addition, retrospective analysis resulted in guidelines for AON design for DMD and most likely other genes as well. This knowledge allows us to optimize therapeutic exon skipping, but also opens up a range of other applications for the exon skipping approach.

284 citations


Cites background from "Extensive in silico analysis of NF1..."

  • ...Famous examples are the neurofibromatosis type 1 gene and the ataxia telangiectasia mutated gene, where a significant number of mutations lead to exon skipping (Teraoka et al. 1999; Ars et al. 2000; Wimmer et al. 2007)....

    [...]

Journal ArticleDOI
TL;DR: This study embarked upon the largest BRCA1 and BRCa2 splice study to date by testing 272 VUSs within the BRC a splice network of Unicancer, and defined guidelines for transcript analysis along with a tentative classification of splice variants.
Abstract: Assessing the impact of variants of unknown significance (VUS) on splicing is a key issue in molecular diagnosis. This impact can be predicted by in silico tools, but proper evaluation and user guidelines are lacking. To fill this gap, we embarked upon the largest BRCA1 and BRCA2 splice study to date by testing 272 VUSs (327 analyses) within the BRCA splice network of Unicancer. All these VUSs were analyzed by using six tools (splice site prediction by neural network, splice site finder (SSF), MaxEntScan (MES), ESE finder, relative enhancer and silencer classification by unanimous enrichment, and human splicing finder) and the predictions obtained were compared with transcript analysis results. Combining MES and SSF gave 96% sensitivity and 83% specificity for VUSs occurring in the vicinity of consensus splice sites, that is, the surrounding 11 and 14 bases for the 5' and 3' sites, respectively. This study was also an opportunity to define guidelines for transcript analysis along with a tentative classification of splice variants. The guidelines drawn from this large series should be useful for the whole community, particularly in the context of growing sequencing capacities that require robust pipelines for variant interpretation.

228 citations


Cites background or methods from "Extensive in silico analysis of NF1..."

  • ...One million cells were suspended in 10 ml, treated with puromycin (10 mg/ml) for 5 h the following day [Dehainault et al., 2007; Wimmer et al., 2007]; (3) RNA was extracted from PAXgene blood RNA tubes (allowing RNA stabilization at collection) using the dedicated kit and according to the manufacturer’s protocols (Preanalytix, Qiagen, Hombreachtikon, Switzerland); and (4) RNA was extracted from stimulated T lymphocytes (see supplementary data) and RNA was analyzed as described in the supplementary section....

    [...]

  • ...This absence of interpretation guidelines can be explained by the small-scale series published to date [Holla et al., 2009; Houdayer et al., 2008; Thery et al., 2011; Vreeswijk et al., 2009; Whiley et al., 2011; Wimmer et al., 2007]....

    [...]

  • ...One million cells were suspended in 10 ml, treated with puromycin (10 mg/ml) for 5 h the following day [Dehainault et al., 2007; Wimmer et al., 2007]; (3) RNA was extracted from PAXgene blood RNA tubes (allowing RNA stabilization at collection) using the dedicated kit and according to the…...

    [...]

Journal ArticleDOI
TL;DR: The history of research on 5' Splice sites selection is reviewed, highlighting the difficulties of establishing how base-pairing strength determines splicing outcomes and proposed that protein complexes propagate along the exon, thereby changing its physical behavior so as to affect 5'ss selection.
Abstract: Splice site selection is fundamental to pre-mRNA splicing and the expansion of genomic coding potential. 5' Splice sites (5'ss) are the critical elements at the 5' end of introns and are extremely diverse, as thousands of different sequences act as bona fide 5'ss in the human transcriptome. Most 5'ss are recognized by base-pairing with the 5' end of the U1 small nuclear RNA (snRNA). Here we review the history of research on 5'ss selection, highlighting the difficulties of establishing how base-pairing strength determines splicing outcomes. We also discuss recent work demonstrating that U1 snRNA:5'ss helices can accommodate noncanonical registers such as bulged duplexes. In addition, we describe the mechanisms by which other snRNAs, regulatory proteins, splicing enhancers, and the relative positions of alternative 5'ss contribute to selection. Moreover, we discuss mechanisms by which the recognition of numerous candidate 5'ss might lead to selection of a single 5'ss and propose that protein complexes propagate along the exon, thereby changing its physical behavior so as to affect 5'ss selection.

184 citations


Cites result from "Extensive in silico analysis of NF1..."

  • ...Predicting the precise consequence of the mutation is far more difficult, although recent analyses have shown some progress (Wimmer et al. 2007; Divina et al. 2009)....

    [...]

Journal ArticleDOI
TL;DR: A combination of complementary in silico tools is necessary to guide molecular geneticists (balance between the time and cost required by RNA analysis and the risk of missing a deleterious mutation) because the weaknesses of one in silICO tool may be overcome by the results of another tool.
Abstract: It appears that all types of genomic nucleotide variations can be deleterious by affecting normal pre-mRNA splicing via disruption/creation of splice site consensus sequences. As it is neither pertinent nor realistic to perform functional testing for all of these variants, it is important to identify those that could lead to a splice defect in order to restrict transcript analyses to the most appropriate cases. Web-based tools designed to provide such predictions are available. We evaluated the performance of six of these tools (Splice Site Prediction by Neural Network [NNSplice], Splice-Site Finder [SSF], MaxEntScan [MES], Automated Splice-Site Analyses [ASSA], Exonic Splicing Enhancer [ESE] Finder, and Relative Enhancer and Silencer Classification by Unanimous Enrichment [RESCUE]-ESE) using 39 unrelated retinoblastoma patients carrying different RB1 variants (31 intronic and eight exonic). These 39 patients were screened for abnormal splicing using puromycin-treated cell lines and the results were compared to the predictions. As expected, 17 variants impacting canonical AG/GT splice sites were correctly predicted as deleterious. A total of 22 variations occurring at loosely defined positions (±60 nucleotides from an AG/GT site) led to a splice defect in 19 cases and 16 of them were classified as deleterious by at least one tool (84% sensitivity). In other words, three variants escaped in silico detection and the remaining three were correctly predicted as neutral. Overall our results suggest that a combination of complementary in silico tools is necessary to guide molecular geneticists (balance between the time and cost required by RNA analysis and the risk of missing a deleterious mutation) because the weaknesses of one in silico tool may be overcome by the results of another tool. Hum Mutat 29(7), 975–982, 2008. © 2008 Wiley-Liss, Inc.

161 citations

References
More filters
Journal ArticleDOI
TL;DR: In this paper, a different approach to problems of multiple significance testing is presented, which calls for controlling the expected proportion of falsely rejected hypotheses -the false discovery rate, which is equivalent to the FWER when all hypotheses are true but is smaller otherwise.
Abstract: SUMMARY The common approach to the multiplicity problem calls for controlling the familywise error rate (FWER). This approach, though, has faults, and we point out a few. A different approach to problems of multiple significance testing is presented. It calls for controlling the expected proportion of falsely rejected hypotheses -the false discovery rate. This error rate is equivalent to the FWER when all hypotheses are true but is smaller otherwise. Therefore, in problems where the control of the false discovery rate rather than that of the FWER is desired, there is potential for a gain in power. A simple sequential Bonferronitype procedure is proved to control the false discovery rate for independent test statistics, and a simulation study shows that the gain in power is substantial. The use of the new procedure and the appropriateness of the criterion are illustrated with examples.

83,420 citations


"Extensive in silico analysis of NF1..." refers methods in this paper

  • ...To detect putative false positives due to multiple testing, the false discovery rate (FDR) controlling procedure [Benjamini and Hochberg, 1995] was applied on the P-values obtained from three test groups, separately....

    [...]

Journal ArticleDOI
22 Apr 1993-Nature
TL;DR: The spontaneous decay of DNA is likely to be a major factor in mutagenesis, carcinogenesis and ageing, and also sets limits for the recovery of DNA fragments from fossils.
Abstract: Although DNA is the carrier of genetic information, it has limited chemical stability. Hydrolysis, oxidation and nonenzymatic methylation of DNA occur at significant rates in vivo, and are counteracted by specific DNA repair processes. The spontaneous decay of DNA is likely to be a major factor in mutagenesis, carcinogenesis and ageing, and also sets limits for the recovery of DNA fragments from fossils.

5,209 citations


"Extensive in silico analysis of NF1..." refers background in this paper

  • ..., 2005] and mutations 1466A4G and 57491332A4G, found so far 11 and five times, respectively, may result from spontaneous adenine deamination [Lindahl, 1993] that leads to the formation of hypoxanthine, which mispairs with cytosine, and hence represents a premutagenic lesion for A4G/T4C transitions....

    [...]

  • ...A possible explanation for the recurrence of these two A4G mutations is a high susceptibility to spontaneous adenosine deamination [Lindahl, 1993]....

    [...]

Journal ArticleDOI
TL;DR: A striking similarity among the rare splice junctions which do not contain AG at the 3' splice site or GT at the 5'splice site indicates the existence of special mechanisms to recognize them, and that these unique signals may be involved in crucial gene-regulation events and in differentiation.
Abstract: A systematic analysis of the RNA splice junction sequences of eukaryotic protein coding genes was carried out using the GENBANK databank. Nucleotide frequencies obtained for the highly conserved regions around the splice sites for different categories of organisms closely agree with each other. A striking similarity among the rare splice junctions which do not contain AG at the 3' splice site or GT at the 5' splice site indicates the existence of special mechanisms to recognize them, and that these unique signals may be involved in crucial gene-regulation events and in differentiation. A method was developed to predict potential exons in a bare sequence, using a scoring and ranking scheme based on nucleotide weight tables. This method was used to find a majority of the exons in selected known genes, and also predicted potential new exons which may be used in alternative splicing situations.

2,235 citations


"Extensive in silico analysis of NF1..." refers methods in this paper

  • ...The position-weight matrices developed by Shapiro and Senapathy (S&S) [Senapathy et al., 1990; Shapiro and Senapathy, 1987] evaluate concordance of a sequence to the splice-site consensus motifs....

    [...]

Journal ArticleDOI
TL;DR: The best models out-perform previous probabilistic models in the discrimination of human 5' and 3' splice sites from decoys and mechanistically motivated ways of comparing models are discussed.
Abstract: We propose a framework for modeling sequence motifs based on the maximum entropy principle (MEP). We recommend approximating short sequence motif distributions with the maximum entropy distribution (MED) consistent with low-order marginal constraints estimated from available data, which may include dependencies between nonadjacent as well as adjacent positions. Many maximum entropy models (MEMs) are specified by simply changing the set of constraints. Such models can be utilized to discriminate between signals and decoys. Classification performance using different MEMs gives insight into the relative importance of dependencies between different positions. We apply our framework to large datasets of RNA splicing signals. Our best models out-perform previous probabilistic models in the discrimination of human 5' (donor) and 3' (acceptor) splice sites from decoys. Finally, we discuss mechanistically motivated ways of comparing models.

1,667 citations