IDENTIFICATION OF A NONSENSE MUTATION AT AMINO-ACID 584-ARGININE OF PLATELET GLYCOPROTEIN IIB IN PATIENTS WITH TYPE-I GLANZMANN THROMBASTHENIA

Using Southern blot, the restriction digests of genomic DNAs in 11 patients with Glanzmann thrombasthenia from 10 unrelated kindreds were probed with a full-length GPIIb cDNA. An addiitonal 2.3 kb Taq I fragment and two 1.65 kb and 0.65 kb fragments with reduced band intensity were found in the genes of two affected siblings from a family originating from the city of Huang Yan in the Zhejiang province. The Taq I digest of the abnormal gene was further probed with three portions of GPIIb cDNA, revealing that the heterozygous mutation was present in the region around exons 15-17 of the GPIIb gene. Two primers for polymerase chain reaction (PCR) were then designed, and a 394 bp PCR product was generated and sequenced, indicating that a stop codon (TGA) was substituted for an Arg codon (CGA) at amino acid position 584 of GPIIb, and resulted in a premature termination of translation and production of a shortened protein. The Western blot analysis showed that GPIIIa at the platelet surface was apparently deficient, it may be ascribed to the rapid turn-over of GPIIIa uncomplexed with the truncated GPIIb. The abnormal 2.3 kb Taq I fragment was used as a specific genetic marker to detect the carrier status of the patient family. The abnormal allele was proved to be derived from the mother, the two affected siblings are double heterozygotes, and one clinically unaffected daughter has also inherited this defective allele, while the father carries another recessive abnormal allele unidentified.