Functional evaluation of five BRCA2 unclassified variants identified in a Sri Lankan cohort with inherited cancer syndromes using a mouse embryonic stem cell-based assay

Breast cancer is the most common cancer in women and a leading cause of cancer morbidity and mortality in Sri Lanka [1]. Latest epidemiological reports indicate that breast cancer accounts for 13.1% of all cancers and 24% of all female cancers in the country [2]. These figures highlight the importance of identifying individuals at risk of breast cancer early so that appropriate management and preventive measures could be undertaken to reduce the morbidity and mortality associated with this disease. The advent of next-generation sequencing (NGS)-based genomic testing has facilitated rapid, precise genetic diagnosis and management of patients with inherited cancer syndromes.

In 2015, using the Illumina MiSeq NGS platform and an in-house developed validated bioinformatics pipeline, multi-gene cancer panel testing and clinical exome sequencing were successfully implemented at our center for the genetic evaluation of patients with inherited cancer syndromes [3, 4]. However, the implementation of NGS-based genomic testing into our routine clinical cancer practice has simultaneously yielded a multitude of rare germline variants in cancer predisposing genes that are known as variants of unknown clinical significance (VUS). This poses significant challenges for both patients and clinicians, especially with regard to risk assessment, genetic counseling, and clinical decision making. Such variants might not contribute to risk assessment and may at times prompt anxiety and overtreatment. In this regard, the non-representation of genetic variants found in the Sri Lankan population in public databases is an additional drawback and challenge. To overcome this limitation, often times we resort to careful assessment of the three-generation pedigrees and testing the particular variant in other affected and unaffected family members, for further confirmation and to identify a clear pattern of co-segregation in the family members. However, the only means to precisely delineate the exact biological significance of these variants is through functional studies. This study aims to describe the functional assays which were conducted to determine the functional significance of five VUS identified in Sri Lankan families with inherited cancer syndromes.

We retrospectively analyzed the clinical and genetic test data of consecutive patients from families with two or more patients with inherited cancer syndromes who underwent NGS-based testing between January 2015 and December 2018 which were maintained prospectively in a database. Ethical clearance for the study was obtained from the Ethics Review Committee of the Faculty of Medicine, University of Colombo [EC-13-182]. Written informed consent was obtained from all the study participants. The genetic variants were classified using the five-class system as pathogenic, likely pathogenic, VUS, likely benign, or benign according to the lab classification criteria. This criteria relies on the guidelines of the American College of Medical Genetics and Genomics (ACMG) and the Association of Molecular Pathology [5]. All retained variants underwent thorough assessment and review of available evidence (e.g., population frequency databases, published literature, case/control and functional studies, internal co-occurrence and co-segregation data, evolutionary conservation, and in silico functional predictions) to arrive at a final variant classification. The variants in the BRCA1 and BRCA2 genes identified in this cohort are summarized in Table 1. We focused our studies on five VUS identified in the BRCA2 gene in this cohort for further investigations to determine their functional significance using a mouse embryonic stem (mES) cell-based assay.

Mouse embryonic stem (mES) cell-based assays provide a simple and reliable assay to test the functional significance of BRCA2 VUS [6]. The assay is based on the observation that BRCA2 is essential for mES cell viability. The ability of human BRCA2 to rescue the lethality of Brca2-deficient mES cells and the sensitivity of viable cells to various DNA damaging agents are used to evaluate the functional significance of the variants [6]. We used this approach to determine the functional significance of five unclassified BRCA2 germline variants [NM_000059.3:c.784G>A|NP_000050.2:p.Ala262Thr|rs397507393; NM_000059.3:c.2353A>G|NP_000050.2:p.Ile785Val|rs747748537; NM_000059.3:c.2488A>G|NP_000050.2:p.Asn830Asp|rs574039421; NM_000059.3:c.6231G>C|NP_000050.2:p.Lys2077Asn|rs541826447;

NM_000059.3:c.9117G>A|NP_000050.2:p.Pro3039Pro|rs28897756] identified in Sri Lankan families with hereditary breast and ovarian cancer syndrome (Fig. 1a).

The desired BRCA2 variants were generated in bacterial artificial chromosomes (BAC) and expressed in mES cells (Fig. 1b). We failed to detect the full-length protein expression of BRCA2 p.Pro3039Pro variant by Western analysis. This silent mutation at the coding level resulted in skipping of exon 23 and production of a smaller non-functional protein (Fig. 1c, d). This variant is known to disrupt a splice donor site and has previously been shown in in vitro studies to cause aberrant mRNA processing with skipping of exon 23 [7]. Our finding further corroborates the earlier observations and clearly establishes the pathogenicity of this variant. p.Pro3039Pro has previously been reported in families with breast and/or ovarian cancer [8‐12].

The p.Ile785Val, p.Asn830Asp, and p.Lys2077Asn variants are likely to be neutral as they rescued mES cell lethality and were indistinguishable from wild-type (WT) BRCA2 in their sensitivity to DNA damaging agents such as cisplatin, camptothecin, mitomycin C, methyl methanesulfonate (MMS), olaparib (poly ADP-ribose polymerase inhibitor), and γ-irradiation (IR) as measured by XTT cell-proliferation assay (Fig. 1e, Table 2). Interestingly p.Ala262Thr was also indistinguishable from WT in all assays except it exhibited specific but mild sensitivity to olaparib among the drugs tested. This was further confirmed by clonogenic survival assay (Fig. 1f). We did not observe any defect in RAD51 recruitment at 3, 6, and 10 h after 10 Gy IR (Fig. 1g, h). However, using a green fluorescent protein (GFP)-based reporter assay [13], we observed a reduction in homologous recombination (HR) efficiency of p.Ala262Thr compared to WT BRCA2 (Fig. 1i) suggesting that RAD51 foci formation may not be sensitive enough to detect minor reduction in HR efficiency.

These findings highlight the value of mES cell-based assays for determining the functional significance of unclassified variants and further extends the spectrum of germline pathogenic variants in the BRCA2 gene with functional evidence. Our findings revealed a mild and specific effect of p.Ala262Thr variant on sensitivity to olaparib as well as impact on HR. To date, most pathogenic mutations in BRCA2 have been identified in the C-terminal DNA binding domain of BRCA2 [14, 15]. A second DNA binding domain has been identified near the N-terminus of BRCA2 between residues 250 and 500, which contains a putative zinc-finger (zf) PARP-like domain between residues 265 and 349 [16]. DNA binding studies have shown its ability to bind to various DNA structures including ssDNA. Interestingly, unlike the C-terminal domain, it also exhibits dsDNA binding activity, which is predicted to facilitate the interaction of BRCA2 to dsDNA/ssDNA junctions during HR. Our preliminary studies with p.Ala262Thr variant did not reveal a defect in binding of BRCA2 to the chromatin (data not shown). Future studies will be aimed at understanding if p.Ala262Thr has any effect on RPA-dependent strand exchange ability of RAD51. A defect in RPA-dependent strand exchange will explain the specific effect of this variant on HR and olaparib sensitivity [16].