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. 2015 Apr;150(2):289-98.
doi: 10.1007/s10549-015-3313-7. Epub 2015 Feb 28.

Splicing analysis of 14 BRCA1 missense variants classifies nine variants as pathogenic

Lise B Ahlborn  1 , Mette Dandanell, Ane Y Steffensen, Lars Jønson, Finn C Nielsen, Thomas V O Hansen
Affiliations

Splicing analysis of 14 BRCA1 missense variants classifies nine variants as pathogenic

Lise B Ahlborn et al. Breast Cancer Res Treat. 2015 Apr.

Abstract

Pathogenic germline mutations in the BRCA1 gene predispose carriers to early onset breast and ovarian cancer. Clinical genetic screening of BRCA1 often reveals variants with uncertain clinical significance, complicating patient and family management. Therefore, functional examinations are urgently needed to classify whether these uncertain variants are pathogenic or benign. In this study, we investigated 14 BRCA1 variants by in silico splicing analysis and mini-gene splicing assay. All 14 alterations were missense variants located within the BRCT domain of BRCA1 and had previously been examined by functional analysis at the protein level. Results from a validated mini-gene splicing assay indicated that nine BRCA1 variants resulted in splicing aberrations leading to truncated transcripts and thus can be considered pathogenic (c.4987A>T/p.Met1663Leu, c.4988T>A/p.Met1663Lys, c.5072C>T/p.Thr1691Ile, c.5074G>C/p.Asp1692His, c.5074G>A/p.Asp1692Asn, c.5074G>T/p.Asp1692Tyr, c.5332G>A/p.Asp1778Asn, c.5332G>T/p.Asp1778Tyr, and c.5408G>C/p.Gly1803Ala), whereas five BRCA1 variants had no effect on splicing (c.4985T>C/p.Phe1662Ser, c.5072C>A/p.Thr1691Lys, c.5153G>C/p.Trp1718Ser, c.5154G>T/p.Trp1718Cys, and c.5333A>G/p.Asp1778Gly). Eight of the variants having an effect on splicing (c.4987A>T/p.Met1663Leu, c.4988T>A/p.Met1663Lys, c.5074G>C/p.Asp1692His, c.5074G>A/p.Asp1692Asn, c.5074G>T/p.Asp1692Tyr, c.5332G>A/p.Asp1778Asn, c.5332G>T/p.Asp1778Tyr, and c.5408G>C/p.Gly1803Ala) were previously determined to have no or an uncertain effect on the protein level, whereas one variant (c.5072C>T/p.Thr1691Ile) were shown to have a strong effect on the protein level as well. In conclusion, our study emphasizes that in silico splicing prediction and mini-gene splicing analysis are important for the classification of BRCA1 missense variants located close to exon/intron boundaries.

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Figures

Fig. 1
Fig. 1
Overview of BRCA1 constructs. The different exons were cloned into the pSPL3 vector including a minimum of 250 bp intronic sequence. The exon–intron boundary sequence is shown and the mutated nucleotide is marked in bold for each BRCA1 construct. BRCA1 constructs covering: a exon 16, b exon 17, c exon 19, d exon 21, e exon 22, and f exon 23. IVS: intervening sequence, SA: splice acceptor site, SD: splice donor site, bp: basepair
Fig. 2
Fig. 2
Mini-gene splicing analysis of BRCA1 variants. COS-7 cells were transfected with wild-type or mutant vectors in duplicate. Total RNA was isolated, RT-PCR analysis was performed, and PCR products were separated by agarose gel electrophoresis and visualized by ethidium bromide staining. Gel band intensities were quantified (Quant.) using the Image Lab 2.0 software. The sizes of the DNA marker (M) are indicated to the left. All PCR products were verified by Sanger sequencing. a The BRCA1 c.4985T>C/p.Phe1662Ser variant generated a strong 488-bp band corresponding to wild-type exon 16 (unaltered splicing) as well as a weak 177-bp band lacking exon 16 also present in the wild-type. b The BRCA1 c.4987A>T/p.Met1663Leu, c.4988T>A/p.Met1663Lys, c.5072C>T/p.Thr1691Ile, c.5074G>C/p.Asp1692His, c.5074G>A/p.Asp1692Asn, and c.5074G>T/p.Asp1692Tyr variants all resulted in one strong band of 177 bp lacking exon 17. Moreover, c.4988T>A/p.Met1663Lys and c.5072C>T/p.Thr1691Ile also revealed a very weak wild-type band including exon 17, while c.5074G>A/p.Asp1692Asn, c.5074G>C/p.Asp1692His, and c.5074G>T/p.Asp1692Tyr also generated a weak band comprising of 418 bp containing 153 bp of intron 17. The c.5072C>A/p.Thr1691Lys variant had no major effect on splicing compared to the wild-type exon 17 (unaltered splicing). c The BRCA1 c.5153G>C/p.Trp1718Ser and c.5154G>T/p.Trp1718Cys alterations both generated a 218-bp PCR product corresponding to wild-type exon 19 (unaltered splicing). d The BRCA1 c.5332G>A/p.Asp1778Asn and c.5332G>T/p.Asp1778Tyr variants both resulted in one strong band of 177 bp lacking exon 21. e The BRCA1 c.5333A>G/p.Asp1778Gly variant produced a 251-bp product corresponding to wild-type exon 22 (unaltered splicing). f The BRCA1 c.5408G>C/p.Gly1803Ala variant resulted in one strong band of 177 bp lacking exon 23
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