An efficient pipeline for the generation and functional analysis of human BRCA2 variants of uncertain significance.
Hum Mutat
; 35(11): 1382-91, 2014 Nov.
Article
em En
| MEDLINE
| ID: mdl-25146914
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.
Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Temas:
Geral
/
Prevencao_e_fatores_de_risco
/
Hereditariedade
Base de dados:
MEDLINE
Assunto principal:
Variação Genética
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Testes Genéticos
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Proteína BRCA2
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Estudos de Associação Genética
Tipo de estudo:
Guideline
/
Prognostic_studies
Limite:
Animals
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Female
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Humans
Idioma:
En
Revista:
Hum Mutat
Assunto da revista:
GENETICA MEDICA
Ano de publicação:
2014
Tipo de documento:
Article
País de afiliação:
Holanda