Synonymous mutations in CFTR exon 12 affect splicing and are not neutral in evolution

Abstract

It is well established that exonic sequences contain regulatory elements of splicing that overlap with coding capacity. However, the conflict between ensuring splicing efficiency and preserving the coding capacity for an optimal protein during evolution has not been specifically analyzed. In fact, studies on genomic variability in fields as diverse as clinical genetics and molecular evolution mainly focus on the effect of mutations on protein function. Synonymous variations, in particular, are assumed to be functionally neutral both in clinical diagnosis and when measuring evolutionary distances between species. Using the cystic fibrosis transmembrane conductance regulator (CFTR) exon 12 splicing as a model, we have established that about one quarter of synonymous variations result in exon skipping and, hence, in an inactive CFTR protein. Furthermore, comparative splicing evaluation of mammalian sequence divergences showed that artificial combinations of CFTR exon 12 synonymous and nonsynonymous substitutions are incompatible with normal RNA processing. In particular, the combination of the mouse synonymous with the human missense variations causes exon skipping. It follows that there are two sequential levels at which evolutionary selection of genomic variants take place: splicing control and protein function optimization.composite exonic regulatory elements of splicing ͉ exonic splicing regulatory sequences ͉ molecular evolution ͉ synonymous variations S ince the first original observations that nucleotide changes in the coding regions can affect normal and alternative cellular pre-mRNA processing (1, 2), extensive evidence has accumulated that exonic variants may affect pre-mRNA splicing (3-6). Mutations within exons are responsible of aberrant splicing profiles of pre-mRNA in several human disease genes, including ataxia telangiectasia mutated (ATM) (7), SMN2 (8-10), BRCA1 (11), neurofibromin 1 (NF-1) (12), cystic fibrosis transmembrane regulator (CFTR) (13-15) genes and in some viral systems such as HIV-1 (16). Nonsense, missense, and even synonymous mutations can induce aberrant skipping of the mutant exon producing nonfunctional proteins. Because direct analysis of mRNA is not routinely performed in the diagnostic field, it is possible that a high amount of exonic mutations may unexpectedly affect splicing, as reported in ATM (7), CFTR (13-15), and NF-1 (12) genes.In the pre-mRNA of higher eukaryotes, the correct identification of exonic sequences from the larger introns requires conserved discrete sequence elements located at the 5Ј and 3Ј splice sites and the branch point. In addition, exonic splicing elements that interact with several classes of positive and negative trans-acting splicing regulatory factors contribute significantly to constitutive and alternative splicing regulation (3, 17). The sequence composition of exonic splicing regulatory sequences is highly degenerated (3,(18)(19)(20)(21), and their effect on splicing may be significantly affected by the context (13,14) and͞or depend on the fo...

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

“…For example, 2 of four exonic unclassified variants in BRCA2 showed aberrant splicing [Bonnet et al, 2008]. Thus, although the overall fraction of pathogenic exonic substitutions that cause aberrant splicing is likely to be relatively low, they should always be considered, particularly in exons weakly included in mRNAs, such as CFTR exon 12, where almost a third of all point mutations induced exon skipping in a minigene system [Pagani et al, 2005] (Supplemental Table 5). …”

“…We also compared previously published exon inclusion data for 42 mutations in CFTR exon 12 that were determined ex vivo [Pagani et al, 2005;Pagani et al, 2003]. We found a significant correlation of exon 12 inclusion with the ESS/ESE ratio (r=-0.28, P=0.03) as well as for the total number of ESSs (-0.35), but not for ESEs or any of the remaining individual elements, except for trusted NI-ESSs that gave the highest correlation coefficient (-0.40, P=0.004).…”

“…the first and the last three exon positions. Set 2 consisted of exon 12 inclusion levels measured after transfection of the wild-type CFTR minigene and its 42 mutated versions, as published previously [Pagani et al, 2005;Pagani et al, 2003] (Supplementary Table 5). …”

“…Splicing defects resulting from exonic substitutions have been found in a growing number of disease genes, such as CFTR [Pagani et al, 2005;Raponi et al, 2007], MLH1 [Auclair et al, 2006;Stella et al, 2001;Tournier et al, 2008], ATM [Teraoka et al, 1999], NF1 [Ars et al, 2000], SMN2 [Lorson and Androphy, 2000] or BRCA1 . The latter gene is one of the two most important breast cancer susceptibility genes [King et al, 2003], encoding a 1863 amino-acid protein involved in DNA damage repair and transcription regulation [Gowen et al, 1998].…”

“…Missense (amino acid altering) and nonsense (stop codon creating) mutations form by far the largest group of mutations deposited in the Human Gene Mutation Database, accounting for ~56% of over 92,000 alterations recorded until May 2010 [Stenson et al, 2009]. However, nonsense, missense and also translationally silent mutations can inactivate genes by altering the inclusion of the mutant exon in mature transcripts Cooper and Mattox, 1997;Pagani et al, 2005;Valentine, 1998]. The most common outcome of these DNA alterations is exon skipping, which is associated with diminished expression of canonical mRNAs and is usually dramatic in severe phenotypes.…”