HUMAN MUTATION 27(10), 1024^1029, 2006 RESEARCH ARTICLE The 185delAG Mutation (c.68_69delAG) in the BRCA1 Gene Triggers Translation Reinitiation at a Downstream AUG Codon Monique Buisson, Olga Anczuko ´w, Almoutassem B. Zetoune, Mark D. Ware, and Sylvie Mazoyer à Laboratoire de Ge ´ne ´tique Mole ´culaire, Signalisation et Cancer Unite ´ Mixte de Recherche 5201 Centre National de la Recherche Scientifique, Universite ´ Claude Bernard Lyon I, Lyon, France Communicated by David N. Cooper The 185delAG mutation (c.68_69delAG; ter39) in the BRCA1 gene is a founder Jewish Ashkenazi mutation that is carried by 1% of this population and has been identified in thousands of breast or ovarian cancer patients. We have previously described that transcripts bearing this mutation, as well as transcripts bearing the 188del11 mutation (c.71_81del; ter36), are not degraded by nonsense-mediated mRNA decay (NMD), contrary to our observations of other truncating mutations that introduce premature termination codons (PTCs) farther downstream in the coding sequence [Perrin-Vidoz et al., 2002]. To test the hypothesis that these two mutations fail to trigger NMD because of translation reinitiation, we have constructed BRCA1 minigenes and studied their protein expression after transient expression in HeLa cells. We show here that in the presence of a PTC at position 36 or 39, translation reinitiation occurs in the BRCA1 minigenes at position 128. Hum Mutat 27(10), 1024–1029, 2006. r r 2006 Wiley-Liss, Inc. KEY WORDS: BRCA1; nonsense-mediated mRNA decay; translation reinitiation INTRODUCTION Germ-line mutations in the BRCA1 gene (MIM] 113705) predispose women to breast and ovarian cancer. The BRCA1 protein is 1863 amino-acid long and possesses two well conserved structural domains: a zing finger of the RING type at its N-terminus, and a BRCT (BRCA1 C-terminus) domain at its C terminus. BRCA1 plays a central role in DNA repair, but also in cell-cycle-checkpoint control, protein ubiquitinylation, and chromatin remodeling [Billack and Monteiro, 2005]. Germ-line mutations are scattered throughout the 81 kb-long gene that encompasses the 22 coding exons of BRCA1 [Smith et al., 1996]. The majority of these mutations introduce premature termination codons (PTCs) into the 5.7-kb open reading frame, thereby making them targets of nonsense-mediated mRNA decay (NMD), an evolutionarily conserved mechanism that prevents the synthesis of potentially harmful truncated protein products [Conti and Izaurralde, 2005; Lejeune and Maquat, 2005]. We have previously observed the effect of NMD on mutated BRCA1 transcripts by measuring relative allelic abundance of BRCA1 transcripts expressed in lymphoblastoid cells from mutation carriers [Perrin-Vidoz et al., 2002]. About one third of the documented BRCA1 mutations have been identified in more than one family. The most frequent mutations, 185delAG (c.68_69delAG) and 5385insC (c.5266dupC), are present in 1% and 0.15%, respectively, of the Jewish Ashkenazim population due to a founder effect [Roa et al., 1996]. (Please see Table 1 for mutation nomenclature definitions.) Thousands of carriers of 185delAG, which introduces a PTC very early in the coding sequence at amino acid 39, have been identified worldwide. This mutation is often described as leading to the expression of a short peptide, as NMD is rarely taken into account when one describes potential repercussions of BRCA1 mutations. However, NMD rules suggest that the 185delAG mutation should trigger decay of the corresponding transcript, as the introduced PTC is followed by numerous exon–exon junctions (EEJs) that allow the NMD mechanism to discriminate between a normal termination codon (usually located in the last exon) and a premature one. However, contrary to expectations, we observed that 185delAG, and the similar 188del11 mutation (c.71_81del; see Table 1) were not associated with a reduction in the steady- state level of mutant transcripts in the carriers that we tested [Perrin-Vidoz et al., 2002]. Mutations 185delAG and 188del11 change the frame in the same way and the resulting truncated open reading frame of 39 and 36 codons, respectively, use the same stop codon in exon 3. To explain why 185delAG and 188del11 failed to trigger NMD, we hypothesized that the transcripts carrying these mutations were subjected to translation reinitiation, as the presence of a PTC shortly after the first methionine codon Published online 28 August 2006 in Wiley InterScience (www. interscience.wiley.com). DOI 10.1002/humu.20384 Received 14 March 2006; accepted revised manuscript 19 May 2006. Grant sponsor: Association pour la Recherche sur le Cancer; Grant sponsor: Comite L De L partemental de la Loire de la Ligue Nationale contre le Cancer; Grant sponsor: Ministe Ø re de la Recherche and the Association pour la Recherche sur le Cancer (M.D.W.); Grant sponsor: Comite L De L partemental de la Sao Œ ne-et-Loire de la Ligue Nationale contre le Cancer (O.A.). à Correspondence to: Sylvie Mazoyer, Laboratoire de Ge L ne L tique Mole L culaire, Signalisation et Cancer UMR5201 CNRS, Faculte L de Me L decine,8 avenue Rockefeller,69373 Lyon Cedex 08, France. E-mail: sylvie.mazoyer@recherche.univ-lyon1.fr r r 2006 WILEY-LISS, INC.