title: Capillary electrophoresis analysis of conventional splicing assays: IARC analytical and clinical classification of 31 BRCA2 genetic variants.
abstract: Rare sequence variants in "high-risk" disease genes, often referred as unclassified variants (UVs), pose a serious challenge to genetic testing. However, UVs 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 BRCA2 genetic variants. All variants were assessed by RT-PCR followed by capillary electrophoresis and direct sequencing. If assays did not produce clear-cut outputs (Class-2 or Class-5 according to analytical International Agency for Research on Cancer guidelines), we performed qPCR and/or minigene assays. The latter were performed with a new splicing vector (pSAD) developed by authors of the present manuscript (patent #P201231427 CSIC). We have identified three clinically relevant Class-5 variants (c.682-2A>G, c.7617+1G>A, and c.8954-5A>G), and 27 analytical Class-2 variants (not inducing splicing alterations). In addition, we demonstrate that rs9534262 (c.7806-14T>C) is a BRCA2 splicing quantitative trait locus.
authors.affiliation: Laboratorio de Oncología Molecular, Instituto de Investigación Sanitaria San Carlos (IdISSC), Madrid, Spain.
authors.forename: Gorka Ruiz
authors.initials: GR
authors.lastname: de Garibay
authors.forename: Alberto
authors.initials: A
authors.lastname: Acedo
authors.forename: Zaida
authors.initials: Z
authors.lastname: García-Casado
authors.forename: Sara
authors.initials: S
authors.lastname: Gutiérrez-Enríquez
authors.forename: Alicia
authors.initials: A
authors.lastname: Tosar
authors.forename: Atocha
authors.initials: A
authors.lastname: Romero
authors.forename: Pilar
authors.initials: P
authors.lastname: Garre
authors.forename: Gemma
authors.initials: G
authors.lastname: Llort
authors.forename: Mads
authors.initials: M
authors.lastname: Thomassen
authors.forename: Orland
authors.initials: O
authors.lastname: Díez
authors.forename: Pedro
authors.initials: P
authors.lastname: Pérez-Segura
authors.forename: Eduardo
authors.initials: E
authors.lastname: Díaz-Rubio
authors.forename: Eladio A
authors.initials: EA
authors.lastname: Velasco
authors.forename: Trinidad
authors.initials: T
authors.lastname: Caldés
authors.forename: Miguel
authors.initials: M
authors.lastname: de la Hoya
mesh_terms: D017398:Alternative Splicing; D024682:BRCA2 Protein / Q000235:genetics*; D019075:Electrophoresis, Capillary; D005091:Exons; D024522:Genes, BRCA2; D005820:Genetic Testing / Q000379:methods*; D014644:Genetic Variation; D006801:Humans; D020641:Polymorphism, Single Nucleotide; D040641:Quantitative Trait Loci; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA
chemical_list: D024682:BRCA2 Protein; C551750:BRCA2 protein, human
keywords: BRCA2; UVs; capillary electrophoresis; minigene; qPCR; splicing