PRECLINICAL STUDY Investigating the effect of 28 BRCA1 and BRCA2 mutations on their related transcribed mRNA Francisco Quiles 1 • Mireia Mene ´ndez 1 • Eva Tornero 1 • Jesu ´s del Valle 1 • A ` lex Teule ´ 1 • Sarai Palanca 2 • Angel Izquierdo 1 • Carolina Go ´mez 1 • Olga Campos 1 • Rau ¨ l Santamaria 3 • Joan Brunet 1 • Gabriel Capella ´ 1 • Lı ´dia Feliubadalo ´ 1 • Conxi La ´zaro 1 Received: 8 October 2015 / Accepted: 31 December 2015 Ó Springer Science+Business Media New York 2016 Abstract Germline inactivating mutations in the BRCA1 and BRCA2 genes are responsible for hereditary breast and ovarian cancer syndrome (HBOCS). Genetic testing of these genes identifies a significant proportion of variants of uncertain significance (VUS). Elucidation of the clinical impact of these variants is an important challenge in genetic diagnostics and counseling. In this study, we assess the RNA effect of 28 BRCA1 and BRCA2 VUS identified in our set of HBOCS families with the aim of gaining insight into their clinical relevance. mRNA was extracted from VUS carriers and controls lymphocytes cultured for 5–6 days and treated with puromycin. RNA was reverse transcribed to perform transcriptional analysis for the study of splicing aberrations. In silico prediction tools were used to select those variants most likely to affect the RNA splicing process. Six out of the 28 variants analyzed showed an aberrant splicing pattern and could therefore be classified as probably pathogenic mutations. Reclassifica- tion of VUS improves the genetic counseling and clinical surveillance of carriers of these mutations and highlights the importance of RNA studies in routine diagnostic laboratories. Keywords BRCA1 Á BRCA2 Á VUS Á Splicing Á RNA Introduction Germline mutations in the BRCA1 and BRCA2 genes sig- nificantly increase the lifetime risk of breast and ovarian cancer, among other neoplasias [1–4]. Inactivating germ- line mutations in these genes account for around 20–25 % of familial cases [5]. However, 2–15 % of patients tested for BRCA mutations are carriers of variants of unknown significance (VUS); the exact figure changes constantly and depends on the expertise and experience of the intervening laboratory as well as on the population studied [6, 7]. The clinical uncertainty generated by VUS greatly hinders the clinical management of carriers. In addition, the clinical ambiguity can be difficult for carriers to interpret and may impact on their psychosocial outcomes [8–10]. Thus, the reclassification of VUS is paramount for a better clinical management of carriers. The efforts made in recent years to gather more information about VUS have allowed the use of multifactorial models and led to the classifications of some variants as deleterious mutations or benign poly- morphisms [11–13]. As consequence, the number of VUS in these genes is falling and large private laboratories with databases of thousands of mutations achieved a drastic decline in their VUS rate. For instance, Myriad laboratories claimed a drop in VUS from 12.8 % in 2002 to 2.7 % in 2012 [14, 15]. Unfortunately, in many cases there is still not enough data for these variants to be classified or the existing data are not available because not necessarily all laboratories share their information. In such cases, Electronic supplementary material The online version of this article (doi:10.1007/s10549-015-3676-9) contains supplementary material, which is available to authorized users. & Conxi La ´zaro conxi.lazaro@gmail.com 1 Hereditary Cancer Program, Molecular Diagnostics Unit, Catalan Institute of Oncology (ICO-IDIBELL), Hospital Duran i Reynals, Gran Via 199, L’Hospitalet de Llobregat, 08908 Barcelona, Spain 2 Molecular Biology Laboratory, Service of Clinical Analysis, University and Polytechnic Hospital La Fe, Valencia, Spain 3 Gene `tica Molecular, Laboratorio Echevarne, Barcelona, Spain 123 Breast Cancer Res Treat DOI 10.1007/s10549-015-3676-9