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

Abstract

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.

Prediction of single‐nucleotide substitutions that result in exon skipping: identification of a splicing silencer inBRCA1exon 6

“…For example, BRCA2 substitutions deposited in BIC and screened by RT-PCR and minigene assays did not show a single example of aberrant splicing [Whiley et al, 2010]. On the other hand, more systematic studies [Sanz et al, 2010] confirm previous findings [Pros et al, 2008;Teraoka et al, 1999] that in large genes with many introns up to a half of all disease gene mutations affect splicing, although only a few were in exons. For example, 2 of four exonic unclassified variants in BRCA2 showed aberrant splicing [Bonnet et al, 2008].…”

126 novel mutations in Italian patients with neurofibromatosis type 1

“…2014) This difference could be due to the cohort of novel mutations or our DNA‐based protocol; RNA studies reveal that new missense and new silent mutations could alter correct splicing if they fall at consensus sites (Pros et al. 2008). Only 2 of the 15 novel missense alterations were identified by the RNA approach.…”

Genetic variant in the telomerase gene modifies cancer risk in Lynch syndrome

“…The strength of the pseudoexon splice sites was analyzed using three different prediction programs: www.fruitfly.org/seq_tools/splice.html, http://genes.mit.edu/burgelab/maxent/ Xmaxentscan_scoreseq.html and http://ast.bioinfo.tau.ac.il/SpliceSiteFrame.html, as previously described. 31 Putative binding sites for transcription factors were identified using PROMO 3.0, a web-based program that employs the TRANSFAC database version 8.3 to construct specific binding-site weight matrices for prediction of transcription factor-binding sites. 32,33 Relative telomere length assessment…”