Mutations affecting mRNA splicing are the most common molecular defects in patients with neurofibromatosis type 1

Abstract

Neurofibromatosis type 1 (NF1) is one of the most common inherited disorders in humans and is caused by mutations in the NF1 gene. To date, the majority of the reported NF1 mutations are predicted to result in protein truncation, but very few studies have correlated the causative NF1 mutation with its effect at the mRNA level. We have applied a whole NF1 cDNA screening methodology to the study of 80 unrelated NF1 patients and have identified 44 different mutations, 32 being novel, in 52 of these patients. Mutations were detected in 87% of the familial cases, but in 51% of the sporadic ones. At least 15 of the 80 NF1 patients (19%) had recurrent mutations. The study shows that in 50% of the patients in whom the mutations were identified, these resulted in splicing alterations. Most of the splicing mutations did not involve the conserved AG/GT dinucleotides of the splice sites. One frameshift, two nonsense and two missense mutations were also responsible for alterations in mRNA splicing. The location and type of mutation within the NF1 gene, and its putative effect at the protein level, do not indicate any relationship to any specific clinical feature of NF1. The high proportion of aberrant spliced transcripts detected in NF1 patients stresses the importance of studying mutations at both the genomic and RNA level. It is possible that part of the clinical variability in NF1 could be due to mutations affecting mRNA splicing, which is the most common molecular defect in NF1.

Aberrant splicing is a common mutational mechanism inMKS1, a key player in Meckel-Gruber syndrome

“…The only two non-splicing mutations were categorized as frameshifting changes and were not analysed at mRNA level given their clear pathogenic character. However, it is conceivable that these mutations may result in aberrant splicing of the MKS1 gene too, comparable to findings that have been reported for the ATM and NF1 genes [Teraoka et al, 1999;Ars et al, 2000;Messiaen et al, 2000]. Nevertheless, irrespective if these two mutations have a direct or indirect effect on splicing or not, we would suggest that splicing defects represent a main mechanism in MKS1 gene dysfunction.…”

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

“…As described in previous studies (Ars et al, 2000(Ars et al, , 2003, the cDNA-SSCP/HD approach was used to detect mutations in mRNA. In summary, 1-2.5μg of total RNA was reverse transcribed using SuperScript™ II reverse transcriptase (Invitrogen).…”

“…Fragments showing abnormal cDNA-SSCP/HD patterns were sequenced using automated sequencers (ABI PRISM™ 377 and 3100). DNA from the corresponding region was sequenced to determine the NF1 disease-causing mutation (Ars et al, 2000(Ars et al, , 2003. Of the 282 different mutations identified in our laboratory, 142 were reported in our previous papers (Ars et al, 2000(Ars et al, , 2003 and 140 are new.…”

“…DNA from the corresponding region was sequenced to determine the NF1 disease-causing mutation (Ars et al, 2000(Ars et al, , 2003. Of the 282 different mutations identified in our laboratory, 142 were reported in our previous papers (Ars et al, 2000(Ars et al, , 2003 and 140 are new. In the present paper, all mutations and their putative effect at the protein level have been renamed according to the Human Genome Variation Society guidelines (http//:www.hgvs.org) and sequence variations checked by Mutalyzer -sequence variant nomenclature check V1.0.1.…”

“…exon or multiexon deletions/duplications) (Wimmer et al, 2006), and the remaining patients present intragenic minor lesions including single nucleotide changes and small deletions and insertions. Although the existence of several recurrent NF1 mutations has been reported (Hoffmeyer et al, 1998;Ars et al, 2000Ars et al, , 2003Fahsold et al, 2000;Messiaen et al, 2000), each one accounts for only a small proportion of patients, which suggests that complete analysis of the entire coding region should be a mandatory procedure in any screening strategy. Exhaustive double DNA/RNA studies in early 2000 revealed that a significant proportion of mutations affected the correct splicing of the gene, even in cases where canonical splicing sequences were not affected (Ars et al, 2000(Ars et al, , 2003Fahsold et al, 2000;Messiaen, et al, 2000).…”