An Efficient Pipeline for the Generation and Functional Analysis of Human BRCA2 Variants of Uncertain Significance

作者：Hendriks Giel; Morolli Bruno; Calleja Fabienne M G R; Plomp Anouk; Mesman Romy L S; Meijers Matty; Sharan Shyam K; Vreeswijk Maaike P G; Vrieling Harry^*

来源：Human Mutation, 2014, 35(11): 1382-1391.

DOI：10.1002/humu.22678

• BRCA2
• VUS
• functional testing
• BAC recombineering
• positive-negative selection

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 %26quot;variants of uncertain significance%26quot; (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.

• 出版日期2014-11