Contribution of bioinformatics predictions and functional splicing assays to the interpretation of unclassified variants of the BRCA genes

Abstract

A large fraction of sequence variants of unknown significance (VUS) of the breast and ovarian cancer susceptibility genes BRCA1 and BRCA2 may induce splicing defects. We analyzed 53 VUSs of BRCA1 or BRCA2, detected in consecutive molecular screenings, by using five splicing prediction programs, and we classified them into two groups according to the strength of the predictions. In parallel, we tested them by using functional splicing assays. A total of 10 VUSs were predicted by two or more programs to induce a significant reduction of splice site strength or activation of cryptic splice sites or generation of new splice sites. Minigene-based splicing assays confirmed four of these predictions. Five additional VUSs, all at internal exon positions, were not predicted to induce alterations of splice sites, but revealed variable levels of exon skipping, most likely induced by the modification of exonic splicing regulatory elements. We provide new data in favor of the pathogenic nature of the variants BRCA1 c.212+3A4G and BRCA1 c.5194À12G4A, which induced aberrant out-of-frame mRNA forms. Moreover, the novel variant BRCA2 c.7977À7C4G induced in frame inclusion of 6 nt from the 3¢ end of intron 17. The novel variants BRCA2 c.520C4T and BRCA2 c.7992T4A induced incomplete skipping of exons 7 and 18, respectively. This work highlights the contribution of splicing minigene assays to the assessment of pathogenicity, not only when patient RNA is not available, but also as a tool to improve the accuracy of bioinformatics predictions. Keywords: variants of unknown significance; splicing defects; splicing reporter minigene; breast and ovarian cancer; BRCA1 and BRCA2 INTRODUCTIONThe interpretation of variants of unknown significance (VUS) found in the molecular screenings of the breast and ovarian cancer susceptibility genes, BRCA1 and BRCA2, is essential for genetic counseling of patients and their families and for the implementation of new therapies targeted to carriers of BRCA mutations, such as those based on poly-(ADP-ribose) polymerase inhibitors. 1 The number of these variants already exceeds that of the reported pathogenic mutations, and is expected to increase rapidly with the use of new high throughput sequencing technologies that are based on massive parallel sequencing. 2 It is now widely accepted that RNA analyses should be used to improve the assessment of pathogenicity of sequence variation, because a large fraction of sequence variants, both intronic and exonic, may induce splicing defects. However, in many cases, patient RNA is either not available or it has been obtained in ways that do not ensure its stability. Moreover, it is sometimes difficult to detect the mRNA affected by a truncating mutation because of the activation of the nonsense-mediated mRNA decay pathway. 3 Functional assays of the effect of VUS on RNA splicing, using patient genomic DNA and a splicing reporter hybrid minigene,

Analysis of the effects of rare variants on splicing identifies alterations in GABAA receptor genes in autism spectrum disorder individuals

“…These programs have been extensively used by other groups and proved their usefulness for the predictions of splicing defects. 14,15,31 To make in vivo/in vitro assessments of a variant true impact on splicing, the analysis of patient RNA is ideal but the availability of disease relevant material is often limited, especially for neurological disorders like ASD and SCZ for which brain biopsies are rare. The minigene approach allows the rapid testing of any variant and it circumvents the problems associated with the difficult access to patient RNA (from patients' derived biopsies, immortalized cell lines or blood sample recollection several years after the original recruitment).…”

“…Furthermore, it also helps to overcome challenges like low level of expression in blood cells of genes involved in neurodevelopmental diseases, and the masking effect of preferential amplification of the wild-type allele. As an alternative, we and others showed that using monoallelic minigene is a powerful tool to assess the impact of sequence variants on splicing in vitro, 14,15,32 as high correlation is observed between minigene results and patient RNA analysis. Among the 14 variants predicted to affect splicing by different programs and thus tested through the functional splicing assay, 29% (4/14) had an actual effect on splicing.…”

“…In addition to substitutions that affect canonical splice sites, variants are analyzed for effects on splicing only in well-documented disease-causing genes such as BRCA1 (Breast cancer 1) 14,15 or ATM (Ataxia-telangiectasia mutated) 16 or when a silent or intronic mutation is identified by familial linkage analysis. 17,18 Cartegni et al 19 published an important list of silent mutations associated with altered splicing in different diseases, showing that it is a mechanism to take into account when potential effects of mutations are analyzed.…”

Multiple sequence variants ofBRCA2exon 7 alter splicing regulation

“…The criteria for diagnostic mutation screening of the BRCA2 gene in patients were applied according to the current French recommendations. Screening for BRCA2 mutations was performed as previously described 16. Informed consent was obtained from all patients.…”

“…We have previously shown that the BRCA2 c.520C>T variant induces exon 7 skipping 16. Here, we investigated the effects on splicing of seven additional VUS located in this exon and identified four new splicing mutations.…”