Hereditary breast/ovarian cancer syndrome is caused by germline deleterious mutations
in BRCA1 and BRCA2. A major problem of genetic testing and counseling is the finding
of variants of uncertain significance (VUS). We sought to ascertain the pathogenicity
of 25 BRCA1 and BRCA2 VUS identified in Portuguese families during genetic testing.
We performed cosegregation analysis of VUS with cancer in families, evaluated their
frequency in unaffected controls, and looked for loss of heterozygosity in tumors.
In addition, three different bioinformatic algorithms were used (Interactive Biosoftware,
ESEfinder, and PolyPhen). Finally, six VUS located in exon-intron boundaries were
analyzed by RT-PCR. We found that seven variants segregated with the disease, six
variants co-occurred with a pathogenic mutation in the same gene, and four variants
co-occurred with a deleterious mutation in the other BRCA gene. By RT-PCR, we observed
that four variants (BRCA1 c.4484G>T, BRCA2 c.682-2A>C, BRCA2 c.8488-1G>A, and BRCA2
c.8954-5A>G) disrupted splicing. After the combined analysis, we were able to classify
4 splicing variants as pathogenic mutations, 16 variants as neutral, and 3 variants
as polymorphisms; only 2 variants remained classified as VUS. This work highlights
the contribution of DNA, RNA, and in silico data to assess the pathogenicity of BRCA1/2
VUS, which, in turn, allows more accurate genetic counseling and clinical management
of the families carrying them.
Copyright © 2014 American Society for Investigative Pathology and the Association
for Molecular Pathology. Published by Elsevier Inc. All rights reserved.