Functional Analysis of Human Breast Cancer Susceptibility Gene Variants

Division Of Basic Sciences - Nci

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Abstract

BACKGROUND AND SIGNIFICANCE Among the various risk factors responsible for the development of breast cancer, the best-established indicator is inheritance of a mutant BRCA1 or BRCA2 gene. BRCA1 and BRCA2 code for proteins that consist of 1863 and 3418 amino acids, respectively. Both BRCA1 and BRCA2 play a critical role in maintaining the genomic integrity because they are required for the repair of double stranded DNA breaks. Individuals with a personal or family history of early onset and/or bilateral breast and/or ovarian cancer, or a history of male breast cancer are offered sequencing-based genetic tests to screen for mutations in BRCA1 and BRCA2. Sequencing-based genetic tests have resulted in the identification of large number of unique variants in these genes. Most variants are rare and very limited epidemiological data is available for most variants. Consequently, they remain unclassified and are referred to as variants of uncertain significance or VUS. Currently, BRCA Exchange lists more than 40000 unique BRCA variants. Of these, less than 8000 variants have been classified by the Evidence Based Network for the Interpretation of Germline Mutant alleles (ENIGMA) expert panel. There is a clear need to develop new approaches to determine the pathogenicity of variants and clinically annotate and classify them. To address this, several in silico prediction models as well as functional assays have been developed. These assays examine the ability of BRCA1 and BRCA2 variants to support cell viability, sensitivity to DNA damaging agents including PARP inhibitors, repair double stand breaks by homologous recombination (HR) or exhibit transcriptional activity. ES cell based functional assay We have developed a functional assay based on the critical role of BRCA2 in the survival and proliferation of Brca2-null mouse embryonic stem cells (mESC). Variants are generated in human BRCA2 cloned in a bacterial artificial chromosome (BAC) by recombineering-based gene editing method. These variants are expressed under the control of their own promoter in mESC, which ensures expression at physiological levels. We have generated a mESC line, PL2F7, for functional evaluation of BRCA2 VUSs. In PL2F7 cells, one allele of Brca2 is functionally null (ko) and the second is a conditional (cko) allele flanked by two loxP sites. The two loxP sites recombine in the presence of CRE, a functional HPRT mini gene is generated, enabling selection of recombinant ES-cells in HAT media (HATr). Loss of Brca2 results in mESC lethality. We have shown that this lethality can be rescued by a BAC expressing wild-type BRCA2 (WT). These mESCs can be used for functional evaluation of BRCA2 VUS by examining their impact on cell viability and sensitivity to DNA damaging agents. Pathogenic variants that have a significant defect in BRCA2 function and fail to result in viable HATr Brca2-null cells. The neutral variants are functionally indistinguishable from WT in cell viability as well as sensitivity to DNA damaging agents. The hypomorphic variants may exhibit severe to mild reduction in cell viability as well as sensitivity to DNA damaging agents due to the partial loss of BRCA2 function. Those variants that have severe effects are likely to be pathogenic and those that exhibit mild defects are likely to be intermediate or low risk variants. A major challenge for all functional assays has been the ability to integrate the functional data in the risk assessment models that are based on posterior probability calculations. This requires converting the functional assay data into probabilities of impact on function (PIF). In collaboration with Dr. Edwin Iversen of Duke University, we have developed a VarCall model that not only combines the impact of six different DNA damaging agents but also combines the results of cell viability efficiency with sensitivity to these DNA damaging agents. We have used our model to successfully determine the pathogenicity of 113 BRCA2 variants including 54 previously uncharacterized variants. We have found the results of our VarCall model to be consistent with the classification reported by other laboratories as well those listed in the ClinVar database. Medium Throughput NGS-based Approach While the BAC-recombineering based methods has been streamlined, there are multiple steps-including generation of the desired SNVs in the BAC and then electroporating it into mESCs to confirm its successful integration and near-physiological expression, which makes the approach time consuming. This has impeded the number of variants that can be examined at a time. To overcome this, we have developed a next generation sequencing (NGS)-based medium-throughput approach for functional evaluation of BRCA2 variants. Instead of clonogenic or XTT-based cell survival assays, pools of mESC expressing 25-30 BRCA2 variants from the same exon are analyzed by NGS. We have generated 223 BRCA2 variants spanning 5 different exons in BACs. These BACs were individually electroporated into PL2F7 mESC. We have developed computational tools to analyze the NGS data and statistical models to determine the probability of impact on function (PIF) of the variants to functionally classify these variants. A manuscript describing the classification of these 223 variant will be submitted soon for publication. CRISPR-based High Throughput Saturation Genome Editing to classify BRCA2 VUS Since 2008, we have functionally classified over 400 BRCA2 variants. Although the functional classification using this approach has proven to be extremely reliable, the generation of SNVs in mESCs is time-consuming and labor intensive with limited potential to multiplex these variants. To overcome this shortcoming, we have developed a CRISPR-Cas9-based saturation genome editing (SGE) approach to perform high throughput assays that will allow us to rapidly examine the functional impact of variants on cell viability and drug sensitivity. Generation of mESC line for functional evaluation of variants by Saturation genome editing To perform functional evaluation of BRCA2 VUS by CRISPR mediated SGE, we have generated a mESC clone with a single copy of full-length human BRCA2 into PL2F7 cells. We deleted the endogenous Brca2 conditional allele allowing the cells to be dependent on the human BRCA2 transgene. We have initiated a saturation genome editing based approach to generate functional data for all possible SNVs in the coding sequence of BRCA2. We are generating these variants in the humanized mESC and examining their impact on cell viability and sensitivity to olaparib and cisplatin using next generation sequencing. As a proof-of-principle, we have saturated Exon 13 and multiplexed these variants for their functional categorization. We have recovered 252 SNVs out of 264 possible missense variants. Twelve variants were excluded due to low read counts and discordance between the replicates We generated a function score (FS) representing the frequency of variants in the final pool relative to the initial pool. The FSs of all variants were then used in a statistical model to calculate the probability of impact on function (PIF) of the variants. We are now focusing on exons 15-26 that code for the C-terminal DNA binding domain spanning residues 2479-3167 of BRCA2. Since October 2022, we have generated more than 6000 variants and completed the analysis of 5080 variants of which more than 840 are VUS. We plan to complete the analysis of the remaining variants from the CTD domain and proceed to other key domains of BRCA2 in the coming months.