The Arg633His substitution responsible for the private platelet antigen Gro(a) unravelled by SSCP analysis and direct sequencing.
We have previously described the private or family platelet antigen, Gro(a), which was identified in a case of neonatal alloimmune thrombocytopenia. The Gro(a) antigen was found to be located on the GP IIIa (beta3) subunit of the GP IIb/IIIa complex, the most prominent fibrinogen receptor of platelets. Initial experiments to characterize the Gro(a) antigen at the molecular genetic level were unsuccessful. We therefore decided to use a different strategy to unravel the molecular basis of this antigen. Platelet GP IIIa mRNA of a Gro(a+) and a Gro(a-) donor was amplified with suitable primers in a reverse transcriptase-polymerase chain reaction (RT-PCR) and subjected to single-strand conformational polymorphism (SSCP) analysis. Three regions of the amplified GP IIIa cDNA derived from the Gro(a+) donor showed a different SSCP pattern when compared to that of the Gro(a-) donor. Direct nucleotide sequence analysis of these three segments revealed that two of them contained silent substitutions, A1163C, A1553G and G1565A. The first and the latter changes were described previously. In the third segment a G1996A mutation was found, predicting an arginine --> histidine substitution at position 633 of the mature glycoprotein. PCR-ASRA (allele-specific restriction enzyme analysis) performed on cDNA as well as on genomic DNA with the restriction enzyme MaeIII showed that the His633 form of GPIIIa is restricted to the Gro(a+) phenotype. The observed mutation is three amino acids upstream of the mutation underlying the HPA-8/Sr system (Arg636Cys), suggesting this region of GP IIIa to be susceptible for mutations. Moreover, the presence of a silent mutation and two low-frequency forms of the silent polymorphisms strongly suggests that the G1996A mutation did not occur in a direct ancestral allele.
Simsek
S
S
Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, University of Amsterdam.
Folman
C
C
van der Schoot
C E
CE
von dem Borne
A E
AE
D000911:Antibodies, Monoclonal / Q000276:immunology; D016824:Antigens, Human Platelet / Q000235:genetics* / Q000276:immunology; D005784:Gene Amplification; D006801:Humans; D007519:Isoantigens / Q000235:genetics; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000235:genetics*; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D018807:Polymorphism, Single-Stranded Conformational; D017421:Sequence Analysis
D000911:Antibodies, Monoclonal; D016824:Antigens, Human Platelet; D007519:Isoantigens; D019039:Platelet Glycoprotein GPIIb-IIIa Complex