Abstract
Screening for pathogenic mutations in breast and ovarian cancer genes such as BRCA1/2, CHEK2 and RAD51C is common practice for individuals from high-risk families. However, test results may be ambiguous due to the presence of unclassified variants (UCV) in the concurrent absence of clearly cancer-predisposing mutations. Especially the presence of intronic or exonic variants within these genes that possibly affect proper pre-mRNA processing poses a challenge as their functional implications are not immediately apparent. Therefore, it appears necessary to characterize potential splicing UCV and to develop appropriate classification tools. We investigated 30 distinct BRCA1 variants, both intronic and exonic, regarding their spliceogenic potential by commonly used in silico prediction algorithms (HSF, MaxEntScan) along with in vitro transcript analyses. A total of 25 variants were identified spliceogenic, either causing/enhancing exon skipping or activation of cryptic splice sites, or both. Except from a single intronic variant causing minor effects on BRCA1 pre-mRNA processing in our analyses, 23 out of 24 intronic variants were correctly predicted by MaxEntScan, while HSF was less accurate in this cohort. Among the 6 exonic variants analyzed, 4 severely impair correct pre-mRNA processing, while the remaining two have partial effects. In contrast to the intronic alterations investigated, only half of the spliceogenic exonic variants were correctly predicted by HSF and/or MaxEntScan. These data support the idea that exonic splicing mutations are commonly disease-causing and concurrently prone to escape in silico prediction, hence necessitating experimental in vitro splicing analysis.
Highlights
Between 1997 and 2012, more than 13.000 families fulfilling the criteria for hereditary breast and ovarian cancer were tested for mutations affecting the major susceptibility genes BRCA1 and BRCA2 [1,2] by the German Consortium of Hereditary Breast and Ovarian Cancer (GC-HBOC)
BRCA1 mutations within invariant splice sites We analyzed a total of 12 BRCA1 variants located within invariant donor or acceptor dinucleotides, all of which are predicted to be damaging according to both, Human Splice Finder (HSF) and MaxEntScan analyses
While some variants have previously been described on genomic level (6/12, see below), the assessment of their functional consequences for BRCA1 pre-mRNA processing is pending in all cases
Summary
Between 1997 and 2012, more than 13.000 families fulfilling the criteria for hereditary breast and ovarian cancer were tested for mutations affecting the major susceptibility genes BRCA1 and BRCA2 [1,2] by the German Consortium of Hereditary Breast and Ovarian Cancer (GC-HBOC). Numerous BRCA1/2 splicing mutations have been identified by using either mRNA derived from mutation carriers or by employing BRCA1/2 minigene constructs [4,5,6,7,8,9,10]. The majority of these studies focuses on variants located within or in the close proximity of intronic splice sites only, suggesting that many mutations located deeper in the intron or exon that impair proper BRCA1/2 pre-mRNA processing remain elusive
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