Skip to main content
. 2014 Mar 26;22(12):1362–1368. doi: 10.1038/ejhg.2014.40

Figure 1.

Figure 1

Mini-gene splicing analysis of BRCA1 VUS/putative splice 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. The sizes of the DNA marker (M) are indicated to the left. All PCR products were verified by sequencing. (a) The different exons were cloned into the pSPL3 vector. IVS, intervening sequence; SA, splice acceptor sit; SD, splice donor site. (b) The BRCA1 c.80+1G>A variant resulted in a 177-bp band corresponding to a transcript lacking exon 2. (c) The BRCA1 c.132C>T variant produced a 227-bp PCR product by usage of a cryptic splice donor site 4 bp within exon 3. (d) The BRCA1 c.213-1G>A variant resulted in a 325-bp transcript by usage of a cryptic splice acceptor site 59 bp within intron 5. (e) The BRCA1 c.670+1delG variant produced a 253-bp transcript by usage of a cryptic splice donor site 1 bp within exon 10, whereas the BRCA1 c.670+16G>A variant produced a 254-bp PCR product that corresponds to wild-type exon 10, although the intensity of the bands was changed. (f) The BRCA1 c.4185+1G>A variant resulted in a 177-bp band corresponding to a transcript lacking exon 12. (g) The BRCA1 c.5075-1G>C variant resulted in a 177-bp band corresponding to a transcript lacking exon 18. (h) The BRCA1 c.-19-22_-19-21dupAT variant produced a 276-bp band corresponding to wild-type exon 2 (unaltered splicing). (i) The BRCA1 c.302-15C>G variant resulted in a 317-bp product corresponding to wild-type exon 7 (unaltered splicing). (j) The BRCA1 c.547+14delG variant produced a 283-bp PCR product that corresponds to wild-type exon 8 (unaltered splicing). (k) The BRCA1 c.4676-20A>G variant produced a 488-bp transcript corresponding to wild-type exon 16 and a weak 177-bp PCR product that corresponds to a transcript lacking exon 16. (l) The BRCA1 c.4987-21G>T variant produced a 265-bp PCR product that corresponds to wild-type exon 17 (unaltered splicing). (m) The BRCA1 c.5278-14C>G variant produced a 232-bp PCR product that corresponds to wild-type exon 21 (unaltered splicing).