title: An efficient pipeline for the generation and functional analysis of human BRCA2 variants of uncertain significance.
abstract: The implementation of next-generation sequence analysis of disease-related genes has resulted in an increasing number of genetic variants with an unknown clinical significance. The functional analysis of these so-called "variants of uncertain significance" (VUS) is hampered by the tedious and time-consuming procedures required to generate and test specific sequence variants in genomic DNA. Here, we describe an efficient pipeline for the generation of gene variants in a full-length human gene, BRCA2, using a bacterial artificial chromosome. This method permits the rapid generation of intronic and exonic variants in a complete gene through the use of an exon-replacement strategy based on simple site-directed mutagenesis and an effective positive-negative selection system in E. coli. The functionality of variants can then be assessed through the use of functional assays, such as complementation of gene-deficient mouse-embryonic stem (mES) cells in the case of human BRCA2. Our methodology builds upon an earlier protocol and, through the introduction of a series of major innovations, now represents a practical proposition for the rapid analysis of BRCA2 variants and a blueprint for the analysis of other genes using similar approaches. This method enables rapid generation and reliable classification of VUS in disease-related genes, allowing informed clinical decision-making.
authors.affiliation: Department of Toxicogenetics, Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: Giel
authors.initials: G
authors.lastname: Hendriks
authors.forename: Bruno
authors.initials: B
authors.lastname: Morolli
authors.forename: Fabienne M G R
authors.initials: FM
authors.lastname: Calléja
authors.forename: Anouk
authors.initials: A
authors.lastname: Plomp
authors.forename: Romy L S
authors.initials: RL
authors.lastname: Mesman
authors.forename: Matty
authors.initials: M
authors.lastname: Meijers
authors.forename: Shyam K
authors.initials: SK
authors.lastname: Sharan
authors.forename: Maaike P G
authors.initials: MP
authors.lastname: Vreeswijk
authors.forename: Harry
authors.initials: H
authors.lastname: Vrieling
mesh_terms: D000818:Animals; D024682:BRCA2 Protein / Q000235:genetics* / Q000378:metabolism*; D002460:Cell Line; D053595:Embryonic Stem Cells / Q000378:metabolism; D005260:Female; D015870:Gene Expression; D056726:Genetic Association Studies / Q000379:methods*; D005820:Genetic Testing / Q000379:methods*; D014644:Genetic Variation; D006801:Humans; D051379:Mice; D016297:Mutagenesis, Site-Directed; D012326:RNA Splicing; D012641:Selection, Genetic
chemical_list: D024682:BRCA2 Protein
keywords: BAC recombineering; BRCA2; VUS; functional testing; positive-negative selection