Shyam K. Sharan

Degradation of 5hmC-marked stalled replication forks by APE1 causes genomic instability

Loss of DNA demethylase TET2 protects stalled replication forks from degradation and confers chemoresistance.

Bypass of premature stop codons and generation of functional BRCA2 by exon skipping

A pathogenic mutation in BRCA2 significantly increases the risk of breast and ovarian cancers making it imperative to examine the functional consequences of variants of uncertain clinical significance. Variants that are predicted to result in a truncated protein are unambiguously classified as pathogenic. We have previously shown how a pathogenic splice site variant known to generate a premature termination codon (PTC) in exon 9 and a nonsense mutation at exon 7, can generate functional BRCA2 by skipping exons 4-7 and restoring the reading frame. Using a well-established mouse embryonic stem cell-based assay, we functionally characterize here one splice site mutation and 11 pathogenic BRCA2 variants that are either nonsense mutation or generate PTC in different exons ranging from exons 4 to 7. Our study shows that five variants can restore the open reading frame by exon skipping and generate a functional protein. This suggests further need to exercise prudence when classifying clearly pathogenic variants.

Variants of uncertain clinical significance in hereditary breast and ovarian cancer genes: best practices in functional analysis for clinical annotation

Germline DNA tests to identify pathogenic variants in genes linked to hereditary breast and ovarian cancer susceptibility have become widely available. However, the clinical utility of genetic testing depends on reliable evidence-based classification of sequence variants. Determination of pathogenicity traditionally relies on painstaking pedigree-based segregation analyses. However, the rapid increase in usage of germline DNA tests has led to the discovery of a large number of variants of uncertain clinical significance (VUS). For most VUS there is insufficient information for segregation analysis and therefore assessment of functional consequences is increasingly being used to support clinical annotation. Functional assays need to be accurate, robust, and reproducible to be used for clinical purposes. Here we use the lessons learned from

Characterization of BRCA2 R3052Q variant in mice supports its functional impact as a low-risk variant

AbstractPathogenic variants in BRCA2 are known to significantly increase the lifetime risk of developing breast and ovarian cancers. Sequencing-based genetic testing has resulted in the identification of thousands of BRCA2 variants that are considered to be variants of uncertain significance (VUS) because the disease risk associated with them is unknown. One such variant is p.Arg3052Gln, which has conflicting interpretations of pathogenicity in the ClinVar variant database. Arginine at position 3052 in BRCA2 plays an important role in stabilizing its C-terminal DNA binding domain. We have generated a knock-in mouse model expressing this variant to examine its role on growth and survival in vivo. Homozygous as well as hemizygous mutant mice are viable, fertile and exhibit no overt phenotype. While we did not observe any hematopoietic defects in adults, we did observe a marked reduction in the in vitro proliferative ability of fetal liver cells that were also hypersensitive to PARP inhibitor, olaparib. In vitro studies performed on embryonic and adult fibroblasts derived from the mutant mice showed significant reduction in radiation induced RAD51 foci formation as well as increased genomic instability after mitomycin C treatment. We observed mis-localization of a fraction of R3052Q BRCA2 protein to the cytoplasm which may explain the observed in vitro phenotypes. Our findings suggest that BRCA2 R3052Q should be considered as a hypomorphic variant.

Sequencing-based functional assays for classification of BRCA2 variants in mouse ESCs

Sequencing of genes, such as BRCA1 and BRCA2, is recommended for 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. Such sequencing efforts have resulted in the identification of more than 17,000 BRCA2 variants. The functional significance of most variants remains unknown; consequently, they are called variants of uncertain clinical significance (VUSs). We have previously developed mouse embryonic stem cell (mESC)-based assays for functional classification of BRCA2 variants. We now developed a next-generation sequencing (NGS)-based approach for functional evaluation of BRCA2 variants using pools of mESCs expressing 10-25 BRCA2 variants from a given exon. We use this approach for functional evaluation of 223 variants listed in ClinVar. Our functional classification of BRCA2 variants is concordant with the classification reported in ClinVar or those reported by other orthogonal assays.

5hmC enhances PARP trapping and restores PARP inhibitor sensitivity in chemoresistant BRCA1/2-deficient cells

Mutations in BRCA1 and BRCA2 genes are the leading cause of hereditary breast and ovarian cancer. BRCA1/2-mutant cells are defective in repairing damaged DNA by homologous recombination and are characterized by hypersensitivity to PARP inhibitors. PARP inhibitors can trap PARP proteins on the chromatin, a mechanism that can contribute to the death of BRCA1/2-deficient cells. The FDA has approved multiple PARP inhibitors for the treatment of metastatic breast and ovarian cancers, yet despite the success of PARP inhibitors in treating BRCA1/2-mutant cancers, drug resistance is a major challenge. Here, we report that 5hmC enhances PARP1 trapping on the chromatin in olaparib-treated cells. Elevated PARP trapping generates replication gaps, leading to the restoration of PARP inhibitor sensitivity in chemoresistant BRCA1/2-deficient cells. Our findings suggest that combining 5hmC with olaparib can restore the sensitivity of chemoresistant BRCA1/2-deficient cells.