Identification of the genetic defect responsible for Osaka-12.

(A) Nucleotide sequence analysis of α_IIb cDNA from Osaka-12. Platelet α_IIb mRNA was amplified by RT-PCR. Nucleotide sequence of the amplified fragments was determined by using Taq DyeDeoxy Terminator Cycle Sequencing kit and an ABI 373A DNA sequencer. (B) Allele-specific restriction enzyme analysis of α_IIb cDNA. The region around exon 4 of the α_IIb mRNA was amplified by PCR, followed by digestion with ScaI. ScaI digestion of the PCR products yielded 517-bp, 264-bp, and 316-bp fragments in the healthy allele. The T>C substitution abolished one of the restriction sites forScaI. Resultant fragments were electrophoresed in a 1.5% agarose gel. Marker indicates 100-bp DNA ladder. (C) Allele-specific restriction enzyme analysis of α_IIb genomic DNA. The region around exon 4 of the α_IIb gene was amplified by PCR using primers IIb3945S and IIbE4, followed by digestion withScaI. ScaI digestion of the PCR products yielded 241-bp and 75-bp fragments in the healthy allele. The T>C substitution abolished the restriction site for ScaI. Resultant fragments were electrophoresed in a 6% polyacrylamide gel. Marker indicates φX174 digested with HaeIII.