Purpose: Most BRCA1/2 mutations are of unknown clinical relevance. An increasing amount of evidence indicates that there can be deleterious effects through the disruption of the splicing process. We have investigated the effect of aberrant splicing of BRCA1/2 on hereditary breast/ovarian cancer (HBOC).Experimental Design: DNA variants were analyzed with splicing prediction programs to select putative splicing mutations. Splicing assays of 57 genetic variants were done by lymphocyte reverse transcription-PCR and/or hybrid minigenes in HeLa and nontumor breast epithelial cells.Results: Twenty-four BRCA1/2 variants of Spanish HBOC patients were bioinformatically preselected. Functional assays showed that 12 variants induced anomalous splicing patterns, 6 of which accounted for 58.5% of BRCA1 families. To further evaluate the defective splicing of BRCA1/2, we analyzed 31 Breast Cancer Information Core Database (BIC) and two artificial variants that were generated by mutagenesis. Sixteen variants induced different degrees of aberrant splicing. Altogether, anomalous splicing was caused by 28 BRCA1/2 variants of all types, indicating that any DNA change can disrupt pre-mRNA processing. We show that a wide range of regulatory elements can be involved, including the canonical and cryptic splice sites, the polypyrimidine tract, and splicing enhancers/silencers. Twenty mutations were predicted to truncate the BRCA proteins and/or to delete essential domains, thus supporting a role in HBOC.Conclusions: An important fraction of DNA variants of BRCA1/2 presents splicing aberrations that may represent a relevant disease-causing mechanism in HBOC. The identification of splicing disruptions by functional assays is a valuable tool to discriminate between benign polymorphisms and pathogenic mutations. Clin Cancer Res; 16(6); 1957-67. ©2010 AACR.Inactivating mutations in BRCA1 (MIM 113705) and BRCA2 (MIM 600185) confer a high risk of developing breast and ovarian cancers (1, 2). Both genes are responsible for ∼16% of the familial breast cancer risk (3). Genetic testing for BRCA1 and BRCA2 provides valuable information in determining the clinical management of breast/ ovarian cancer patients. However, the data it provides are also difficult to interpret due to the identification of many DNA variants of unknown physiologic significance, or unclassified variants (UV) that hamper genetic counseling in hereditary breast/ovarian cancer (HBOC). The critical issue is to identify whether a given nucleotide change results in a benign polymorphism or a disease-causing mutation. In fact, approximately half of the 3,499 different sequence variations of the Breast Cancer Information Core Database (BIC) 5 are UVs and determining their biological effect remains a challenge (4, 5).The analysis of the deleterious effect of genetic variants in disease genes is usually focused on the predicted effect on protein structure and function. However, precise removal of introns from precursor mRNA or splicing is an essential step in eukaryotic gene ...