miércoles, 31 de marzo de 2010

A High Proportion of DNA Variants of BRCA1 and BRCA2 Is Associated with Aberrant Splicing in Breast/Ovarian Cancer Patients

A High Proportion of DNA Variants of BRCA1 and BRCA2 Is Associated with Aberrant Splicing in Breast/Ovarian Cancer Patients
David J. Sanz1, Alberto Acedo1, Mar Infante1, Mercedes Durán1, Lucía Pérez-Cabornero1, Eva Esteban-Cardeñosa2, Enrique Lastra3, Franco Pagani4, Cristina Miner1 and Eladio A. Velasco1

+ Author Affiliations

Authors' Affiliations:1Grupo de Genetica del Cancer, Instituto de Biologia y Genetica Molecular, Consejo Superior de Investigaciones Cientificas-Universidad de Valladolid, Valladolid, Spain; 2Laboratorio de Biologia Molecular, Servicio de Biopatologia Clinica, Hospital La Fe, Valencia, Spain; 3Servicio de Oncologia, Complejo Hospitalario de Burgos, Burgos, Spain; and 4International Centre of Genetic Engineering and Biotechnology, Trieste, Italy

Corresponding Author:
Eladio A. Velasco, Grupo de Genética del Cáncer (B-7), Instituto de Biología y Genética Molecular, CSIC-UVa, Sanz y Forés s/n, 47003 Valladolid, Spain. Phone: 34-98318480; Fax: 34-983184800; E-mail: eavelsam@ibgm.uva.es.
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
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