Alternative splicing because of c.7235G > A (p.R2336H) mutation. (A) RT-PCR analysis of the Brca2^KO/KO ES cells expressing WT transgene, and Brca2^CKO/KO and Brca2^KO/KO ES cells expressing p.R2336H variant human BRCA2 transgene. FL indicates fragment of a full-length product; Δexon12, transcript with deletion of exon 12 (this is a natural, alternatively spliced form of BRCA2³² ); Δexon 13, transcript with deletion of exon 13; and Δexons 12 and 13, transcript with deletion of exons 12 and 13. (B) Schematic diagram of the effect of p.R2336H (c.7235G > A) mutation on splicing. Exon numbers are indicated above the boxes representing the different exons. Arrows indicate the positions of the primers. (♦) represents the stop codon generated because of exon skipping. (C) Sequence analysis of different RT-PCR products from Brca2^KO/KO ES cells expressing p.R2336H mutation. Left panel: Full-length transcript with the presence of mutation marked in red. Middle panel: Skipping of exon 13. Right panel: Skipping of exons 12 and 13. (D) Real-time RT-PCR analysis of the full-length transcript in mouse ES cells expressing either WT or p.R2336H BRCA2 variant. Left panel: Scheme of mature RNA quantified by real-time RT-PCR. Arrows indicate the primers. (E) WB showing the expression of WT and p.R2336H BRCA2 transgenes. c-myc antibody was used to detect BRCA2. For loading control, β-actin blot is shown. (F) Binding of U1 snRNA to the 5′ splice site. Uppercase letters correspond to the exon, and lowercase letters represent intron. Arrow indicates the change because of p.R2336H mutation. (G) RHCglo minigene reporter construct containing BRCA2 exon 13 variants (WT or p.R2336H) along with the flanking intron segments shown on top. Genomic fragments were PCR amplified using the primers containing flanking SalI and XbaI sites from the corresponding BACs and cloned into RHCglo reporter construct. After transient transfection into COS7 cells, RT-PCR was done using the primers pF and pR as indicated by arrows. RT-PCR analysis of BRCA2 exon 13 exclusion/inclusion in RHCglo minigene is shown below.