Perforin mutations in the patient with CAEBV and in his parents. (A) The perforin gene contains 3 exons and the open reading frame is encoded by the second and third exons. Exons 2 and 3 were amplified by PCR from genomic DNA using the indicated primers (arrowheads),20 cloned in plasmid pCR2.1, and sequenced. Mutations were found in both alleles of exon 3 in the patient. Four of 6 clones had a G to C mutation at nucleotide 1229 and 2 clones had a T to C mutation at nucleotide 577, indicating that each allele had a separate mutation. (B) Chromatograms of RT-PCR products. RT-PCR was performed using mRNA isolated from stimulated PBMCs of the patient, his parents, and a healthy donor. RT-PCR products were directly sequenced (not having been cloned) and asterisks indicate mutations.
