Mutation analysis in FPD/AML pedigrees.

Electropherograms from affected and control individuals showing the A>G substitution leading to the K83E missense mutation in pedigree 1 (A), the C>A substitution resulting in a nonsense mutation Y260X in pedigree 3 (B), and the deletion of an A at the +3 position of the splice donor site of intron 4 in pedigree 2 (IVS4 + 3delA, C). Arrows indicate the point of the mutations. (D) Schematic representation of the cryptic splice site within exon 4 used in the mutated transcript in pedigree 2 and the RT-PCRs used to analyze the splicing. A, B, and C represent forward primers used in the RT-PCR experiment. Primer A is specific to the mutated form, and primer B is specific to the wild-type form. Primer C was used as control. The same reverse primer was used in each reaction. The 23 nucleotides of exon 4 excluded from the mutant transcript are indicated by the black box. (E) RT-PCR on patient and control RNA as described in panel D. The primer specific to the mutated form does not show any amplification on control RNA. (F) Sequence analysis of the mutant exon 4–exon 5 junction.