Splicing Defects in the Ataxia-Telangiectasia Gene, ATM: Underlying Mutations and Consequences

Abstract

Mutations resulting in defective splicing constitute a significant proportion (30/62 [48%]) of a new series of mutations in the ATM gene in patients with ataxia-telangiectasia (AT) that were detected by the protein-truncation assay followed by sequence analysis of genomic DNA. Fewer than half of the splicing mutations involved the canonical AG splice-acceptor site or GT splice-donor site. A higher percentage of mutations occurred at less stringently conserved sites, including silent mutations of the last nucleotide of exons, mutations in nucleotides other than the conserved AG and GT in the consensus splice sites, and creation of splice-acceptor or splice-donor sites in either introns or exons. These splicing mutations led to a variety of consequences, including exon skipping and, to a lesser degree, intron retention, activation of cryptic splice sites, or creation of new splice sites. In addition, 5 of 12 nonsense mutations and 1 missense mutation were associated with deletion in the cDNA of the exons in which the mutations occurred. No ATM protein was detected by western blotting in any AT cell line in which splicing mutations were identified. Several cases of exon skipping in both normal controls and patients for whom no underlying defect could be found in genomic DNA were also observed, suggesting caution in the interpretation of exon deletions observed in ATM cDNA when there is no accompanying identification of genomic mutations.

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].…”

“…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].…”

Deep-intronic ATM mutation detected by genomic resequencing and corrected in vitro by antisense morpholino oligonucleotide (AMO)

“…10,11 This strategy has already found a proof of principle in the ATM gene where approximately half of unique mutations are splicing mutations. 12,13 In particular, some of these mutations have been demonstrated to occur in deep intronic regions that are associated with the retention of intronic sequences in the mRNAs and AONs have been already been designed to successfully abrogate these ATM mutations. 5,14 In the current study, we studied a new deep-intronic mutation detected in an A-T patient using a NGS strategy to resequence the entire 160-kb ATM genomic region, after standard mutation detection techniques (DHPLC, MLPA and cDNA sequencing), failed to identify the second mutation.…”

Six novel ATM mutations in Italian patients with classical ataxia-telangiectasia

“…One pathological allele showed an abnormally spliced cDNA fragment: in patient AT80RM the skipping of exon 63 appeared to be caused by a transversion of the last nucleotide of the same exon (8850G>T) which most probably interferes with the nearby donor site as in previously reported ATM mutations (Teraoka et al, 1999). This substitution was absent in 50 normal subjects.…”