(A) Evaluation of exon 13 splice variants in unaffected and affected family members. Reverse transcription of 1 μg of RNA was completed using Superscript II RNase H^- Reverse Transcriptase and oligo dT primer. cDNA was PCR amplified using Ex Taq Polymerase and primers F5e13F and F5e13R (indicated by arrows in B). The 2 left lanes show normal, unaffected RNA RT-PCR; the 2 right lanes show RNA from affected individuals. (B) Diagram of splicing event. Pre-mRNA of FV-short created by a splicing donor site at position 2441 and an acceptor site at position 4547 of exon 13 of the coagulation F5 gene. This event results in the in-frame removal of 2106 coding base pairs from exon 13 that encode amino acids 756–1458. In this process, a new asparagine or aspartic acid residue would be created at this junction in normal or affected individuals, respectively. The sequence surrounding the splice site is separated into codons that encode wild-type FV with the corresponding normal protein sequence, and the splice sites are indicated by a backslash. This mutation changes the 5′ splice site from TA/GT to TG/GT. Diagram of gene is not to scale. (C) Sequencing results of the RT-PCR from affected patient RNA depicting the expression of A2440G in the splice donor site. These results are derived from the transcript-specific RT-PCR product (seen in Figure 3A) of an affected patient using FVShortF and FVShortR primers.