Assessment of unclassified variants in the BRCA1 and BRCA2 genes identified by clinical genetic testing

R.B. van der Luijt1, W. Gaasbeek1, A.Y. Bronkhorst1, C.W.F. van Helvert1, H. van der Spaa1, M. Gijzen1, B.G. Smith1, R.P.M. Jansen1, P.H.A. van Zon1, M.G.E.M. Ausems2, B.P.M. van Nesselrooij2, J.G. Post2, C.J.M. van der Sijs-Bos 2, T.G.W. Letteboer2, H.Y. Kroes2, E. van Riel2, A.E. Schoemaker2, J. van Echtelt2, Y. Cuperus2, R.J. Sinke1, J.K. Ploos van Amstel JK1
Section of Genome Diagnostics1 and Clinical Genetics2, Department of Medical Genetics, Division of Biomedical Genetics, University Medical Center Utrecht

Clinical testing for BRCA1 and BRCA2 mutations and oncogenetic counselling has been performed by our department in approximately 1,750 families with suspected hereditary breast and/or ovarian cancer. In all of the families, mutation scanning of the entire coding region of both genes was performed using a combination of methods. For the detection of missense, nonsense, splice site mutations, small deletions and insertions in both genes, we used Denaturing Gradient Gel Electrophoresis and the Protein Truncation Test in combination with semi-automated DNA-sequencing or TaqMan-assays. Deletions and insertions of one or more BRCA1 exons were investigated by Multiplex Ligation-dependent Probe Amplification. In 244 of our families, a deleterious BRCA1 (n=164) or BRCA2 (n=80) mutation was identified. Also, 124 unclassified variants (UVs), including missense and synonymous changes, and intronic changes located outside the conserved splice donor and acceptor consensus sequences, were found in BRCA1 (n=51) and BRCA2 (n=73). Following the technological and logistic challenge of diagnostic BRCA1 and BRCA2 mutation screening, with an increasing need for rapid genetic testing for therapeutic purposes, the evaluation of UVs has become the next challenge for DNA laboratories offering BRCA analysis. It has become evident that the evaluation of UVs requires a multi-disciplinary approach. To allow risk assessent and clinical management in families carrying UVs, we evaluated the functional and clinical relevance of UVs by integrating both genetic (co-segregation, tumor studies) and in silico approaches (locus-specific databases, literature searches, multiple sequence alignments, web-based prediction programs). We will present an overview of the UVs detected by our department and, to illustrate the difficult process of UV assessment, we will focus on a particularly interesting BRCA1 UV detected in two of our families.