Impact of BRCA1 and BRCA2 variants on splicing: clues from an allelic imbalance study

Abstract

Nearly one-half of BRCA1 and BRCA2 sequence variations are variants of uncertain significance (VUSs) and are candidates for splice alterations for example, by disrupting/creating splice sites. As out-of-frame splicing defects lead to a marked reduction of the level of the mutant mRNA cleared through nonsense-mediated mRNA decay, a cDNA-based test was developed to show the resulting allelic imbalance (AI). Fifty-four VUSs identified in 53 hereditary breast/ovarian cancer (HBOC) patients without BRCA1/2 mutation were included in the study. Two frequent exonic single-nucleotide polymorphisms on both BRCA1 and BRCA2 were investigated by using a semiquantitative single-nucleotide primer extension approach and the cDNA allelic ratios obtained were corrected using genomic DNA ratios from the same sample. A total of five samples showed AI. Subsequent transcript analyses ruled out the implication of VUS on AI and identified a deletion encompassing BRCA2 exons 12 and 13 in one sample. No sequence abnormality was found in the remaining four samples, suggesting implication of cis-or trans-acting factors in allelic expression regulation that might be disease causative in these HBOC patients. Overall, this study showed that AI screening is a simple way to detect deleterious splicing defects and that a major role for VUSs and deep intronic mutations in splicing anomalies is unlikely in BRCA1/2 genes. Methods to analyze gene expression and identify regulatory elements in BRCA1/2 are now needed to complement standard approaches to mutational analysis.

A High Proportion of DNA Variants ofBRCA1andBRCA2Is Associated with Aberrant Splicing in Breast/Ovarian Cancer Patients

“…For example, the GTAA deletion of intron 20 of the ATM gene promotes the exonization of a cryptic exon (46). However, other authors have suggested a minor role for such mutations in the molecular spectrum of BRCA1/2 (47).…”

Alternative splicing and breast cancer

“…However, when BRCA mRNAs from tumors bearing such unclassified variants are analyzed, they often show splicing alterations. [76][77][78][79][80][81][82] Further mechanistic analyses have revealed that such unclassified variants are directly responsible for splicing alterations. For example, several unclassified variants in BRCA1 exon 18 induce exon skipping by altering the binding of splicing factors that enhance (SF2/ASF) or inhibit (hnRNP A1 and hnRNP H/F) exon 18 recognition.…”

EMMA, a cost- and time-effective diagnostic method for simultaneous detection of point mutations and large-scale genomic rearrangements: application to BRCA1 and BRCA2 in 1,525 patients

“…The simultaneous detection of point mutations and large rearrangements is another advantage. However promoter regions are not covered and this must be taken into account, since deletions of the promoter regions are a probably rare but significant cause of HBOC (Brown, et al, 2002;Caux-Moncoutier, et al, 2009). Although technical performance was acceptable, the complexity of the currently available Emmalys interface prevents routine use of large rearrangement detection.…”