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Capillary Electrophoresis Analysis of Conventional Splicing Assays: IARC Analytical and Clinical Classification of 31 BRCA 2 Genetic Variants
Author(s) -
Garibay Gorka Ruiz,
Acedo Alberto,
GarcíaCasado Zaida,
GutiérrezEnríquez Sara,
Tosar Alicia,
Romero Atocha,
Garre Pilar,
Llort Gemma,
Thomassen Mads,
Díez Orland,
PérezSegura Pedro,
DíazRubio Eduardo,
Velasco Eladio A.,
Caldés Trinidad,
Hoya Miguel
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.22456
Subject(s) - minigene , biology , rna splicing , alternative splicing , genetics , computational biology , locus (genetics) , gene , exon , rna
Rare sequence variants in “high‐risk” disease genes, often referred as unclassified variants ( UV s), pose a serious challenge to genetic testing. However, UV s resulting in splicing alterations can be readily assessed by in vitro assays. Unfortunately, analytical and clinical interpretation of these assays is often challenging. Here, we explore this issue by conducting splicing assays in 31 BRCA 2 genetic variants. All variants were assessed by RT ‐ PCR followed by capillary electrophoresis and direct sequencing. If assays did not produce clear‐cut outputs ( C lass‐2 or C lass‐5 according to analytical I nternational A gency for R esearch on C ancer guidelines), we performed q PCR and/or minigene assays. The latter were performed with a new splicing vector (p SAD ) developed by authors of the present manuscript (patent # P 201231427 CSIC ). We have identified three clinically relevant C lass‐5 variants (c.682‐2 A > G , c.7617+1 G > A , and c.8954‐5 A > G ), and 27 analytical C lass‐2 variants (not inducing splicing alterations). In addition, we demonstrate that rs9534262 (c.7806‐14 T > C ) is a BRCA 2 splicing quantitative trait locus.