BRCA1 VUS: A functional analysis to differentiate pathogenic from benign variants identified in clinical diagnostic panels for breast cancer

Rita Adubeiro Lourenço, Miguel Lança, Octávia Monteiro Gil, Joana Cardoso, Teresa Lourenço, José B. Pereira-Leal, António Sebastião Rodrigues, José Rueff, Susana Nunes Silva

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Genetic testing for susceptibility genes through next‑generation sequencing (NGS) has become a widely used technique. Using this, a number of genetic variants have been identified, several of which are variants of unknown significance (VUS). These VUS can either be pathogenic or benign. However, since their biological effect remains unclear, functional assays are required to classify their functional nature. As the use of NGS becomes more mainstream as a diagnostic tool in clinical practice, the number of VUS is expected to increase. This necessitates their biological and functional classification. In the present study, a VUS was identified in the BRCA1 gene (NM_007294.3:c.1067A>G) in two women at risk for breast cancer, for which no functional data has been reported. Therefore, peripheral lymphocytes were isolated from the two women and also from two women without the VUS. DNA from all samples were sequenced by NGS of a breast cancer clinical panel. Since the BRCA1 gene is involved in DNA repair and apoptosis, the functional assays chromosomal aberrations, cytokinesis‑blocked micronucleus, comet, γH2AX, caspase and TUNEL assays were then conducted on these lymphocytes after a genotoxic challenge by ionizing radiation or doxorubicin to assess the functional role of this VUS. The micronucleus and TUNEL assays revealed a lower degree of DNA induced‑damage in the VUS group compared with those without the VUS. The other assays showed no significant differences between the groups. These results suggested that this BRCA1 VUS is likely benign, since the VUS carriers were apparently protected from deleterious chromosomal rearrangements, subsequent genomic instability and activation of apoptosis.

Original languageEnglish
Article number136
JournalMolecular Medicine Reports
Issue number1
Publication statusPublished - 1 Jul 2023


  • BRCA1
  • breast cancer
  • functional analysis of VUS
  • genotoxic challenge
  • next‑generation sequencing
  • variants of unknown significance (VUS)


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