RNA analysis for 5312—19A>C sequence variation. (A) DNA sequence from intron 30 and exon 31 showing the putative branch site, an alternative branch site, and the acceptor splice (AS) site. The 5312–19A>C sequence variation is underlined in the putative branch site. (B) RNA isolated from the platelets of the index case of family 2 and from a healthy person was reverse transcribed and PCR amplified from exons 30-32. Only one VWF cDNA product of 311 bp was observed in both persons, indicating correct splicing (lane 1, patient; lane 2, healthy person; lane 3, PCR blank; lane 4, 100-bp ladder). (C) Sequence analysis revealed a correctly spliced product, with exon 30 spliced directly to exon 31. (D) RNA from the index case of family 2 and a healthy person was reverse transcribed and PCR amplified from exons 28-29 (amplified product contains aa 1584). The PCR product was subsequently digested with KpnI, and 3 bands of 1181 bp (uncut), 780 bp, and 401 bp were observed for the patient, demonstrating that both alleles are stably expressed as RNA (lane 1). Only one band is observed in the healthy person because he does not have the mutation (lane 2). Lane 3, 100-bp ladder. (E) RNA isolated from COS-7 cells transfected with pBKVWF29-32A, pBKVWF29-32C, or both (which contain either the A or C at 5312) was reverse transcribed. Only one VWF product of 567 bp was amplified from the wild-type (lane 1), cotransfected (lane 2), or homozygous sequence variant (lane 3) cDNAs. Lane 4 is a 100-bp ladder.