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Functional characterization of DNA variants from exons 17 and 18 of the BRCA2 gene

AuthorsFraile-Bethencourt, Eugenia; Díez-Gómez, Beatriz; Velásquez-Zapata, Valeria; Acedo, Alberto ; Sanz, David J.; Hernández-Moro, Cristina; Marcos García, G.; Infante, Mar ; Durán, Mercedes ; Velasco, Eladio
Issue Date2016
CitationEuropean Human Genetics Conference (2016)
AbstractA large fraction of pathogenic BRCA2 variants impairs mRNA splicing in hereditary breast/ovarian cancer. Missense, synonymous or in frame-deletion/insertion variants are usually classified as variants of uncertain significance. The best method to identify splicing aberrations is based on the study of patient RNA, but that is often difficult to obtain. Minigene-based technology is an alternative approach to test candidate splicing variants without the need of patient samples. To study this process we constructed a large minigene in the pSAD plasmid with exons 14 to 20 (MGBR2_ex14-20) to keep the genomic context. Here, we focus on exons 17 y 18 because of the atypical GC donor in exon 17. The intronic GT dinucleotide is the most conserved element of the donor splice signal. However, in a small fraction of the donor sites (<1%), GT is replaced by GC that are rather located in alternatively spliced introns. A total of 226 variants were analysed with NNSplice and HSF so that 48 of them were selected to assay. They were introduced into MGBR2_ex14-20 by site-directed mutagenesis. Minigenes were transfected into HeLa and MCF7 cells to examine their transcripts. We found that 22 variants (45.8%) produced abnormal transcripts. Moreover, micro-deletion assays showed that the 3' region of exon 17 and the 5' of exon 18 were essential for exon recognition and might contain splicing enhancer sequences. Actually, five reported variants mapping in these intervals disrupted splicing.
DescriptionResumen del póster presentado a la European Human Genetics Conference, celebrada en Barcelona (España) del 21 al 24 de mayo de 2016.
Appears in Collections:(IBGM) Comunicaciones congresos
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