Genome Annotation by Shotgun Inactivation of a Native Gene in Hemizygous Cells: Application toBRCA2with Implication of Hypomorphic Variants

Abstract

The greatest interpretive challenge of modern medicine may be to functionally annotate the vast variation of human genomes. Demonstrating a proposed approach, we created a library of BRCA2 exon 27 shotgun-mutant plasmids including solitary and multiplex mutations to generate human knockin clones using homologous recombination. This 55-mutation, 13-clone syngeneic variance library (SyVaL) comprised severely affected clones having early-stop nonsense mutations, functionally hypomorphic clones having multiple missense mutations emphasizing the potential to identify and assess hypomorphic mutations in novel proteomic and epidemiologic studies, and neutral clones having multiple missense mutations. Efficient coverage of nonessential amino acids was provided by mutation multiplexing. Severe mutations were distinguished from hypomorphic or neutral changes by chemosensitivity assays (hypersensitivity to mitomycin C and acetaldehyde), by analysis of RAD51 focus formation, and by mitotic multipolarity. A multiplex unbiased approach of generating all-human SyVaLs in medically important genes, with random mutations in native genes, would provide databases of variants that could be functionally annotated without concerns arising from exogenous cDNA constructs or interspecies interactions, as a basis for subsequent proteomic domain mapping or clinical calibration if desired. Such gene-irrelevant approaches could be scaled up for multiple genes of clinical interest, providing distributable cellular libraries linked to public-shared functional databases.

WEE1 inhibition in pancreatic cancer cells is dependent on DNA repair status in a context dependent manner

“…As previously published, both FANCC - and BRCA2 -deficient cells were more sensitive to MMC treatment ( Supplementary Fig. S2B and Supplementary Table S6 ) 32 44 45 . Surprisingly, we observed that BRCA2 (+/−), BRCA2 (−/−) and FANCC (−/−) clones were more sensitive to MK-1775 than parental clones ( Supplementary Fig.…”