Three different premature stop codons lead to skipping of exon 7 in neurofibromatosis type I patients

Wimmer, Katharina; Eckart, Markus; Stadler, Peter F.; Rehder, Helga; Fonatsch, Christa

doi:10.1002/1098-1004(200007)16:1<90::aid-humu20>3.0.co;2-j

Cited by 15 publications

(12 citation statements)

References 18 publications

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“…The same has been shown in another study of exon 7 skipping in which the RNA structure overall was not affected by mutations, suggesting that the skipping of exon 7 can be easily induced or that the mutations disrupt exon enhancer sites as an alternative mechanism (Wimmer et al 2000a). Wimmer et al (2000a) have also found 11 different RT-PCR aberrations within exons 1-10a by using aged blood of a normal individual, showing that this can mimic results from alterations of exon splicing caused by mutations in NF1 patients (Wimmer et al 2000b). However, other than the exon 7 observation, we have not clearly detected any of their other reported abnormalities suggesting that laboratory-specific methods and factors may also play a role in the precision of these results.…”

Section: Discussionsupporting

confidence: 68%

“…Stress conditions, such as cold shock, could lead to alterations in the secondary structure of RNA in certain regions of the molecule and might be responsible for the exon skipping in aged blood samples. The same has been shown in another study of exon 7 skipping in which the RNA structure overall was not affected by mutations, suggesting that the skipping of exon 7 can be easily induced or that the mutations disrupt exon enhancer sites as an alternative mechanism (Wimmer et al 2000a). Wimmer et al (2000a) have also found 11 different RT-PCR aberrations within exons 1-10a by using aged blood of a normal individual, showing that this can mimic results from alterations of exon splicing caused by mutations in NF1 patients (Wimmer et al 2000b).…”

Section: Discussionsupporting

confidence: 64%

“…Related to alternate/aberrant splicing, some nonsense NF1 mutations have been found to cause the skipping of their corresponding exons, viz., 7 and 37 (Hoffmeyer et al 1998;Messiaen et al 1997;Wimmer et al 2000a). Since these exon skips are in-frame (the coding sequence length is a multiple of three, and no stop codons are formed at the splice joint), the resulting protein could theoretically retain some function, missing only the residues encoded by that exon.…”

Section: Introductionmentioning

confidence: 99%

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RT-PCR splicing analysis of the NF1 open reading frame

Thomson

Wallace

2002

Hum Genet

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Neurofibromatosis 1 (NF1) is an autosomal dominant condition whose molecular diagnosis is challenging because of the large size of the gene and the vast number of unique NF1 gene mutations. Some splicing and nonsense mutations have been shown to cause exon skipping. Recently, temperature-induced abnormal splicing has been found in NF1 in ex-vivo tissues. This prompted us to investigate the entire NF1 transcript for such aberrant splicing. We found several novel exon skips that appeared de novo or were present initially and increased in aged/cooled blood: exon 20, exons 20 and 21 combined, exon 33, exon 34, exon 37, exon 40, exon 45, exons 43 and 45 combined, part of exon 43, and the first codon of exon 12b. Some aberrant splice forms were undetectable when blood was drawn into Qiagen PAXgene tubes, rather than EDTA vacutainers, and we demonstrate how these aberrant splicing events are a potential pitfall for RNA-based NF1 mutation characterization. The same reverse transcription/polymerase chain reaction strategy was used to screen for novel NF1 alternative splicing in Schwann cells and seven other tissues. Even though no Schwann-specific alternative exons were identified, we found minor novel splicing isoforms differentially expressed such as skips of exon 37 and exon 40. Skipping of exon 43, part of exon 43, and the first codon of exon 12b were found in all tissues analyzed. These forms suggest greater tissue-based variability in the NF1 message than was previously thought and may indicate minor amounts of heterogeneity at the protein level.

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Section: Discussionsupporting

confidence: 68%

Section: Discussionsupporting

confidence: 64%

Section: Introductionmentioning

confidence: 99%

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RT-PCR splicing analysis of the NF1 open reading frame

Thomson

Wallace

2002

Hum Genet

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“…A total of 124 (44%) mutations of various types affected splicing (173/374 patients). We and others have shown that several of these mutations generate a NF1-transcriptional profile rather than a single mutated mRNA, where correctly spliced mRNAs containing the original DNA mutation coexist with mRNAs exhibiting a splicing alteration (Messiaen et al, 2000;Wimmer et al, 2000b;Zatkova et al, 2004;Pros et al, 2006). For example, we have already shown that mutations c.910C>T, c.5546G>A, c.6791dupA and c.6792C>A generate different proportions of mutated transcripts for each NF1 mutation (Pros et al, 2006) and that these proportions were similar among patients with the same mutations.…”

Section: Resultsmentioning

confidence: 81%

Nature and mRNA effect of 282 differentNF1point mutations: focus on splicing alterations

et al. 2008

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Neurofibromatosis type 1 (NF1) is a common autosomal dominant genetic disorder caused by mutations in the NF1 gene. In this paper we report our experience using the cDNA-SSCP/HD analysis as a mutational screening approach and the double characterization of all mutations at the DNA and RNA levels. Two hundred and eighty-two different mutations (in 374 independent patients) were identified, 140 of which were novel in our population. Most of these mutations are unique and distributed along the gene. However, we also detected 37 recurrent mutations. Our approach is limited with respect to the detection of single base substitutions, but it is highly effective in the detection of frameshift mutations and mutations that affect the correct splicing. Due to this bias we focus here in the characterization of these two types of mutations. Forty-seven percent of mutations found were frameshift mutations, with small deletions being 2.3 times more common than small insertions. At the mRNA level, 44% of mutations affected the correct splicing, 80% of them located in the consensus sequences, with the donor site being much more frequently involved. The remaining 20% consisted of mutations located in deep intronic sites and mutations located in the coding region. In general the latter group produces different types of mutated transcripts with specific proportions for each mutation. The double characterization of mutations at the DNA and RNA levels enables to detect a broader spectrum of mutations than any single level approach, and provides a greater understanding of their molecular pathogenesis.

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“…For this reason, although in silico predictions represent an invaluable tool for the researcher in this field, special care should be exercised when predicted premRNA structures are correlated with splicing behavior. As an example, in silico studies of NF-1 gene transcripts (50,58), which are implicated in the generation of human tumors, have been challenged by successive reports (99,106). In fact, these studies have shown that the analyses reporting correlations between in silico predicted changes in secondary structure and splicing in these systems are heavily dependent on the RNA window taken into consideration, making it very difficult to assign significance to the suggested correlations.…”

Section: Can Alterations In Pre-mrna Secondary Structure Be Involved mentioning

confidence: 99%

Influence of RNA Secondary Structure on the Pre-mRNA Splicing Process

Buratti

Baralle

2004

Molecular and Cellular Biology

380

302

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Pre-mRNA splicing in eukaryotes requires joining together the nucleotides of the various mRNA-coding regions (exons) after recognizing them from the normally vastly superior number of non-mRNA-coding sequences (introns). For three excellent reviews on general splicing and its regulation, refer to references 14, 62, and 70. In eukaryotes, the vast majority of splicing processes are catalyzed by the spliceosome, a very complex RNA-protein aggregate which has been estimated to contain several hundred different proteins in addition to five spliceosomal snRNAs (1,54,62,63,81,109). These factors are responsible for the accurate positioning of the spliceosome on the 5Ј and 3Ј splice site sequences. The reason why so many factors are needed reflects the observation that exon recognition can be affected by many pre-mRNA features such as exon length (5, 97), the presence of enhancer and silencer elements (8, 62), the strength of splicing signals (45), the promoter architecture (29,55), and the rate of RNA processivity (86). In addition, the general cellular environment also exerts an effect, as recent observations suggest the existence of extensive coupling between splicing and many other gene expression steps (69) and even its modification by external stimuli (96).In the midst of all this complexity, it has also been proposed that pre-mRNA secondary structures can potentially influence splicing activity. However, despite a steady increase of reports invoking their effects on splicing regulation, the last specific review on this subject is now more than 10 years old (3). Here, we propose to address again this specific issue in the current perspective of the general field. Before we do this, however, we have to answer a basic question. DO PRE-mRNAS PRESENT SECONDARY STRUCTURE IN VIVO?Two properties of RNA molecules cannot be denied: their natural tendency to form highly stable secondary and tertiary structures in vitro and in vivo (9, 27, 39) and the observation that alterations in these structures represent a well-known regulatory mechanism for many RNA cellular processes (60).In this particular respect, however, a question that still remains to be addressed conclusively regards the presence of secondary structures in pre-mRNAs in vivo. That this existence may not simply be taken for granted comes from early experimental evidence. In fact, it was suggested that in vitro evidence regarding the possible influence of RNA structure on splicing (94) could not be accurately reproduced in vivo (95). The reason why this should be so goes back to the classical concept that RNA is coated in vivo by proteins. In fact, heterogeneous ribonucleoprotein particles have been known since early studies and the major protein family involved, the hnRNP proteins, are very abundant in mammalian cells. These RNAprotein interactions may well prevent mRNAs from folding in stable secondary structures (34) (Fig. 1a). For this reason, it was hypothesized that, following transcription, pre-mRNA may be allowed only a very limited timespan to fold (36)....

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Three different premature stop codons lead to skipping of exon 7 in neurofibromatosis type I patients

Cited by 15 publications

References 18 publications

RT-PCR splicing analysis of the NF1 open reading frame

RT-PCR splicing analysis of the NF1 open reading frame

Nature and mRNA effect of 282 differentNF1point mutations: focus on splicing alterations

Influence of RNA Secondary Structure on the Pre-mRNA Splicing Process

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