RT-NPCR to detect F8 intron-1 inversion mutations. (A) Schematic representation of wild-type F8 (WT-F8) mRNA from the signal peptide (SP) sequence through exon 3. Primers (gray arrows and font) were designed to hybridize within F8 signal peptide (SP) sequence (violet) and F8 exons 1 (yellow), 2 (brown), and 3 (light blue). (B) WT-F8 mRNA transcript. RT-NPCR produces 509-bp (black line, often not visible on the gel) and 413-bp (gray bar) bands that are both diagnostic of an intact F8 exon 1 to 2 junction sequence. (C) RT-NPCR Inv1 test 1, representative result. MW, molecular weight ladder; N, HA, C, normal control, HA-Inv1, and carrier-Inv1 subjects, respectively. The 413-bp band is amplified from the N and C samples. (The 509-bp outer RT-NPCR product is also seen on this gel.) (D) Schematic representation of the chimeric F8-VBP1 mRNA from an individual with an Inv1 mutation, showing the transcript from the F8 SP sequence to the 5′ region of the VBP1 gene. Primers were designed to hybridize within F8 exon 1 (yellow), and VBP1 exon 2 (turquoise). Note that 2 facultative exons between F8 exon 1 and VBP1 exon 1 are also contained in this chimeric transcript formed as a consequence of the inversion mutation. Outer primers exon 1 FWD and VBP1 REV1 were designed to amplify a 590-bp product. Inner primers exon 1 FWD2 and VBP1 REV2 were designed to amplify a 500-bp product. (E) RT-NPCR produces 590-bp (black line, often not visible on the gel) and 500-bp (gray bar) bands that are both diagnostic of the chimeric RNA resulting from an Inv1 mutation. (F) RT-NPCR Inv1 test 2, representative result. MW, molecular weight ladder; N, HA, C, normal control, HA-Inv1, and carrier-Inv1 subjects, respectively. The 500-bp band is amplified from the HA and C samples.