Pathogenicity Evaluation of BRCA1 and BRCA2 Unclassified Variants Identified in Portuguese Breast/Ovarian Cancer Families Catarina Santos, Ana Peixoto,

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Pathogenicity Evaluation of BRCA1 and BRCA2 Unclassified Variants Identified in Portuguese Breast/Ovarian Cancer Families Catarina Santos, Ana Peixoto, Patrícia Rocha, Pedro Pinto, Susana Bizarro, Manuela Pinheiro, Carla Pinto, Rui Henrique, Manuel R. Teixeira The Journal of Molecular Diagnostics Volume 16, Issue 3, Pages 324-334 (May 2014) DOI: 10.1016/j.jmoldx.2014.01.005 Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 A: RT-PCR analysis of BRCA1 c.4484G>T shows the changes in the levels of alternative transcripts produced in carriers. B: Sequencing electropherography of carriers shows that the full-length transcript is produced only by the normal allele (b1). Sequencing of the alternative transcripts (b2 and b3) reveal exon 14 skipping and the deletion of exons 14 and 15, respectively. Note that there is a band in the agarose gel, between b2 and b3, that could not be sequenced. The Journal of Molecular Diagnostics 2014 16, 324-334DOI: (10.1016/j.jmoldx.2014.01.005) Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 A: Aberrant splicing induced by BRCA2 c.682-2A>C. B: Schematics of all the transcripts obtained and sequenced. The transcript b1, present in carriers and controls, corresponds to the full-length sequence. Two additional aberrant transcripts were obtained in carriers: b2 shows the deletion of the first 73 bp of exon 9, and b3 shows the skipping of the entire exon 9. The Journal of Molecular Diagnostics 2014 16, 324-334DOI: (10.1016/j.jmoldx.2014.01.005) Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 A: RT-PCR analysis of the BRCA2 c.8488-1G>A mutation. B: Schematics of all the transcripts obtained by sequencing of the agarose gel bands. In the patient, b1 presents the deletion of the first 12 bp of exon 20 and the alternative retention of 64 bp from intron 20, b2 consists of the deletion of the first 12 bp of exon 20, and b3 represents exon 20 skipping. In controls, the three transcripts retain 64 bp from intron 20 (b4), full-length transcript (b5), and have exon 19 deletion (b6). The Journal of Molecular Diagnostics 2014 16, 324-334DOI: (10.1016/j.jmoldx.2014.01.005) Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 4 A: RT-PCR analysis of the BRCA2 c.8954-5A>G mutation. B: Schematics of all the transcripts obtained by sequencing of the agarose gel bands. Mutation carriers present an aberrant splicing with the out-of-frame retention of the last four bases of intron 22 (b1 and b3, which also presented the deletion of exon 22), the full-length transcript (b2), the skipping of exon 22 and the same retention of 4 bp (b3), and the deletion of exon 22 and the first 51 bp of exon 23 (b4). On the other hand, the controls present three transcripts: the higher-molecular-weight b5 is the full-length sequence, b6 is the same as b2 from carriers, and the lightest one, b7, represents the skipping of exon 22. The Journal of Molecular Diagnostics 2014 16, 324-334DOI: (10.1016/j.jmoldx.2014.01.005) Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 5 Pedigree of the family carrying BRCA2 c.2T>G shows the nonsegregation of the variant in a niece of the index case affected with breast cancer at 44 years of age. Under the symbols the current age of each individual is indicated, as well as the tumor type and age at diagnosis in those affected. Unaffected individuals are indicated with white symbols and the oblique line indicates that the patient is deceased. Plus and minus signals represent family members with and without the c.2T>G variant. The Journal of Molecular Diagnostics 2014 16, 324-334DOI: (10.1016/j.jmoldx.2014.01.005) Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions