Abstract
Establishing a universally applicable protocol to assess the impact of BRCA1 variants of uncertain significance (VUS) expression is a problem which has yet to be resolved despite major progresses have been made. The numerous difficulties which must be overcome include the choices of cellular models and functional assays. We hypothesised that the use of induced pluripotent stem (iPS) cells might facilitate the standardisation of protocols for classification, and could better model the disease process. We generated eight iPS cell lines from patient samples expressing either BRCA1 pathogenic variants, non-pathogenic variants, or BRCA1 VUSs. The impact of these variants on DNA damage repair was examined using a ɣH2AX foci formation assay, a Homologous Repair (HR) reporter assay, and a chromosome abnormality assay. Finally, all lines were tested for their ability to differentiate into mammary lineages in vitro. While the results obtained from the two BRCA1 pathogenic variants were consistent with published data, some other variants exhibited differences. The most striking of these was the BRCA1 variant Y856H (classified as benign), which was unexpectedly found to present a faulty HR repair pathway, a finding linked to the presence of an additional variant in the ATM gene. Finally, all lines were able to differentiate first into mammospheres, and then into more advanced mammary lineages expressing luminal- or basal-specific markers. This study stresses that BRCA1 genetic analysis alone is insufficient to establish a reliable and functional classification for assessment of clinical risk, and that it cannot be performed without considering the other genetic aberrations which may be present in patients. The study also provides promising opportunities for elucidating the physiopathology and clinical evolution of breast cancer, by using iPS cells.
Highlights
Worldwide, among females, breast cancer is the most commonly diagnosed cancer (*24%), and is still the leading cause of cancer-related deaths in women, closely followed by lung cancer
Skin samples were obtained from pathogenic BRCA1 variant carriers and variants of uncertain significance (VUS) BRCA1 carriers undergoing either risk-reducing mastectomy (K381X, A1708E) or tumour-removal surgery (C61G, G462R, Y856H, D1733G, Q1811K, V1687G) (Fig 1A and 1B)
NonBRCA1 variant control induced pluripotent stem (iPS) cells were generated by reprogramming fibroblasts obtained from St John’s Institute of Dermatology and the HipSci Biobank
Summary
Among females, breast cancer is the most commonly diagnosed cancer (*24%), and is still the leading cause of cancer-related deaths in women, closely followed by lung cancer (respectively 15.5% and 13.7%; [1, 2]). The breast cancers form a highly heterogeneous group, and are clinically challenging to diagnose and manage. Variants in the genes BRCA1 or BRCA2 are known to confer a high lifetime risk of developing breast cancer, because the presence of certain heterozygous BRCA variants increases the risk of breast (~80% for BRCA1) and ovarian cancer (~40% for BRCA1), identifying BRCA1 and BRCA2 as highpenetrance breast cancer predisposition genes [3]. BRCA1 and BRCA2 are the most common genes associated with increased risk in hereditary breast and ovarian cancer. BRCA1 is a tumour suppressor gene coding for a large protein containing multiple functional domains, which interacts with multiple other proteins [4, 5]. The BRCA1 gene is an essential component of DNA damage repair via the Homologous Recombination (HR) pathway, which is an error-free repair mechanism, and crucial for cellular survival. BRCA1 is involved in chromosome segregation and mismatch repair, so that it plays a critical role in the maintenance of genome integrity [6]
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