Studies of ectopic and in vitro–expressed FVII mRNA. (A) RT-PCR amplification of white blood cell mRNA obtained from patient 2 carrying the 9729del4 mutation. PCR products (primers 1-5) were separated by 8% PAGE (left) or by 11% PAGE after Bcl I restriction (right). NC, negative control. (B) RT-PCR amplification of FVII mRNA expressed in cells transfected with constructs containing the three mutations (9729del4, 9726+5A, 9726+7G) or the normal sequence (N). Upper part, ethidium bromide staining of fragments amplified with primers 3 and 4; M, size marker. Lower part, autoradiographs of the same fragments labeled by ³²P and restricted by TaqI. Gels were overexposed. (C) Schematic diagrams of FVII cDNAs (exons 6-8) and of fragments amplified from the ectopic mRNA (primers 1-5) or from the mRNA expressed in transfected cells (primers 3-4). The restriction sites used in (A) (Bcl I) and (B) (Taq I) are also indicated together with the sizes of fragments, which are given in bp. Length of abnormally spliced transcripts and of their restricted fragments are reported in parentheses. The 182-bp and 186-bp fragments were obtained from the 9729del4 and 9726+5A constructs, respectively. The 144-bp fragment was obtained by Taq I restriction of the 186-bp amplified fragment. The gray box indicates the repeat inserted in the abnormally spliced transcripts.