Get access

Capillary Electrophoresis Analysis of Conventional Splicing Assays: IARC Analytical and Clinical Classification of 31 BRCA2 Genetic Variants


  • Contract grant sponsors: EAV was supported by research grants PI10/2910 (Instituto de Salud Carlos III), CSI004A10-2 (Consejería de Educación, Regional Government of Castilla y León) and BIO39/VA27/10 (Consejería de Sanidad, Regional Government of Castilla y León). Alberto Acedo is supported by the European Social Fund and Regional Government of Castilla y León (P.O. 2007-2013). ZGC is supported by the Genetic Counseling Program (Regional Government of Valencia). SGE is funded by a Miguel Servet contract (CP10/00617, Instituto de Salud Carlos III). HUVH-VHIO research was supported by a Miguel Servet research grant CP10/00617, Instituto de Salud Carlos III). MH was supported by research grants PI 09/00859 and PI 12/00539 (Instituto de Salud Carlos III). Instituto de Salud Carlos III research grants are partially supported by European Regional Development FEDER Funds.

  • Communicated by David E. Goldgar


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.

Get access to the full text of this article