Aberrant FANCB splice products and their quantitation. (A) Reverse transcription PCR (RT-PCR) analysis of fibroblast cell line RNA from individual 7 with c.949C>T (p.Q317*) variant in exon 3. The gel shows, along with the predicted size product, an additional smaller product (arrow) that lacked the last 74 bp from the end of exon 3 (c.878_951del74; p.G294Cfs3*). Both products encode truncated proteins. RNA from a fibroblast cell line from an unaffected individual was used as WT control. (B) RT-PCR analysis of fibroblast cell line RNA from individuals 4 and 19 carrying splice junction c.1496+1G>A and missense c.1435T>G (p.W479G) variants, respectively. Individual 4 RNA shows exclusively exon 7 skipping (c.1327_1496del170; p.K443Vfs*4) (arrow). Individual 19 also shows exon 7 skipping along with a normal size product. (C) Schematics of quantitative fluorescence RT-PCR (qf-RT-PCR) for relative quantification of transcripts with and without exon 7. Two FANCB-specific forward primers (FPs) that bind to either exon 7-8 junction or exon 6-8 junction and a reverse primer (RP) that binds to exon 8 were designed to amplify products from transcripts with (top) or without exon 7 (bottom), respectively. The product with exon 7 is 3 bp longer than that from the product without exon 7. FAM-labeled M13 FP (M13F-FAM) was included to generate fluorescently labeled products. (D) Representative qf-RT-PCR profiles from individuals 4 and 19. Product size and intensity are on the x- and y-axes, respectively; both numbers are shown under each peak. The left panel shows products from ACTB, an internal transcript control. The right panel shows FANCB products. The fragment size for products with exon 7 (black arrow) and without exon 7 (red arrow) appeared around 399 and 396 bp, respectively. Data from WT, individual 4, and individual 19 are presented on the top, middle, and bottom rows, respectively. (E) Percentage of transcripts with or without exon 7; average from 8 independent assays.