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An Efficient Pipeline for the Generation and Functional Analysis of Human BRCA2 Variants of Uncertain Significance
Author(s) -
Hendriks Giel,
Morolli Bruno,
Calléja Fabienne M.G.R.,
Plomp Anouk,
Mesman Romy L.S.,
Meijers Matty,
Sharan Shyam K.,
Vreeswijk Maaike P.G.,
Vrieling Harry
Publication year - 2014
Publication title -
human mutation
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 162
eISSN - 1098-1004
pISSN - 1059-7794
DOI - 10.1002/humu.22678
Subject(s) - biology , gene , computational biology , genetics , exon , dna sequencing
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 (m ES ) 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.