Fig 1.
Structure and functional analysis of the minigene MGBR2_ex14-20.
A) Structure of the minigene MGBR2_ex14-20 (slashes indicate shortened introns): [IVS14 (328 pb)–EX14 (428 pb)–IVS14 (1139 pb)—EX15 (182 pb)–IVS15 (358 pb) // IVS15 (333 pb)–EX16 (188 pb)–IVS16 (234 pb) // IVS16 (181 pb)–EX17 (171 pb)–IVS17 (485 pb)–EX18 (355 pb)–IVS18 (314 pb) // IVS18 (235 pb)–EX19 (156 pb)–IVS19 (398 pb)–EX20 (145 pb)–IVS20 (207 pb)]. Boxes highlight the 4 different cloning steps of this minigene. The expected splicing reactions in eukaryotic cells are indicated by arrows. B) Splicing functional analysis of the empty vector pSAD and the final (14–20) and intermediate (16–18, 16–20) constructs in MCF-7 cells. The four electropherograms were overlaid. cDNAs were amplified with vector exon specific primers SD6-PSPL3_RTFW and RTpSAD-RV (arrows within vector exons V1 and V2 above). Full-length transcripts are shown as blue peaks and the Genescan Liz-1200 size standard is shown as orange/faint peaks. Fragment sizes (bp) and relative fluorescent units are indicated on the x- and y-axes, respectively.
Fig 2.
Map of splicing enhancers for recognition of exons 17 and 18.
A) Functional mapping of ESEs of exons 17 (left) and 18 (right) by exonic microdeletions (30-nt above) of the wild type minigene MGBR2_ex14-20. cDNA was amplified with primers of BRCA2 exon 16 (RTBR2_ex16-FW) and vector exon V2 (RTpSAD-RV; amplicon size = 1012 nt) and run in 1.5% agarose gel. Arrows indicate abnormal transcripts. B) Functional analysis of 10-nt microdeletions of exon 18 (left) and single nucleotide substitutions (right) c.7984A>T that disrupts SF2-I, and c.8001C>T and 8003G>A that target SF-II motif (see ESE map below). The triple mutant is the combination c.[7984A>T;8001C>T;8003G>A], and the double one is c.[8001C>T;8003G>A]. C) HSF predictions of putative ESE motifs of exon 17 (above) between cDNA positions 7944 and 7973 and exon 18 (below) between cDNA positions 7979–8008 and 8004–8033. Intronic sequences are in lower case. SF2* motif of exon 17 is detected by the specific SF2/ASF (IgM-BRCA1) algorithm of ESEfinder. Red and pink microdeletions alter splicing. Artificial SF2- single-nucleotide substitutions are indicated above exon 18 sequence.
Table 1.
Splicing outcomes of BRCA2 exons 17 and 18 variants.
Fig 3.
Analysis of transcripts induced by DNA variants from exon 17.
cDNA was amplified with primers RTBR2_ex16-FW (BRCA2 exon 16) (blue peaks) and FAM-labelled RTpSAD-RV (vector exon V2) and electrophoresed on a DNA sequencer with Genescan LIZ 1200 as size standard (orange/faint peaks). Arrows indicate minor aberrant transcripts. Screenshots of electropherograms visualized with the Peak Scanner software v1.0 are shown. Fragment sizes (bp) and relative fluorescent units are indicated on the x- and y-axes, respectively.
Fig 4.
Analysis of transcripts induced by DNA variants from exon 18.
cDNA was amplified with primers RTBR2_ex16-FW (BRCA2 exon 16) (blue peaks) and FAM-labelled RTpSAD-RV (exon V2 of minigene) and electrophoresed on a DNA sequencer with Genescan LIZ 1200 as size standard (orange/faint peaks). Arrows indicate minor aberrant transcripts. Screenshots of electropherograms visualized with the Peak Scanner software v1.0 are shown. Fragment sizes (bp) and relative fluorescent units are indicated on the x- and y-axes, respectively. Electropherograms of c.8007A>G, c.8072C>T and c.8249_8250del are not represented since they show similar patterns (partial exon 18 skipping) to other DNA variants (e.g. c.8009C>G).
Fig 5.
Schematic representation of splicing events and transcripts generated by wild type and mutant minigenes.
Exons and splicing reactions exons are represented by boxes and broken lines, respectively. Anomalous/skipped exons and aberrant events are shown in red.