Germ-line mutations in the breast cancer susceptibility genes BRCA1 and BRCA2 account for a large proportion of hereditary breast/ovarian cancer families. A large number of disease-causing germ-line mutations and variants of unknown pathological significance have been identified in both genes. The majority of these variants have been studied only in genomic DNA and their effects at the mRNA level have not been reported. Our aim was to ascertain the pathological effect of six BRCA1 and two BRCA2 sequence unclassified variants by RNA analysis. Three of the BRCA1 variants are novel: IVS18+5G>A, IVS20-6_IVS20-4del and IVS22-2A>G. Three BRCA1 mutations showed aberrant splicing: Ala1693del, IVS18+5G>A and IVS22-2A>G. The variants G1706A, S1715N and IVS20-6_IVS20-4del in BRCA1, and T2515I and IVS25+9A>C in BRCA2 led to normal transcripts. We compared these RNA results with those obtained from two theoretical splicing prediction methods. The consensus values for the splice sequences (Shapiro and Senapathy 1987) involved in three of the BRCA1 splicing site variants agreed with the RNA results, lending support to the validity of this model. Moreover, we used previously established exonic splicing enhancer (ESE) sequences to ascertain whether the four exonic variants studied fell within predicted ESE motifs and whether they would disrupt ESE functions. Our results suggest that the splicing predictions based on this method are not definitive and should be considered with caution. This work highlights the importance of studying mutations at DNA and RNA levels in order to clarify their pathological effect. This information is essential for providing efficient counseling for breast/ovarian cancer families.
Copyright 2003 Wiley-Liss, Inc.