Identification of new and known polymorphisms in glycoprotein IIb and IIIa genes by denaturing gradient gel electrophoresis.
Glycoprotein IIb and IIIa contain antigenic determinants involved in the potential production of allo- or autoantibodies directed against platelets, that may result in severe thrombocytopenia. Most of these epitopes appear to be supported by single nucleotide substitutions. We have used denaturing gradient gel electrophoresis (DGGE) to identify sequence variations within the promoter and the coding regions of the glycoprotein IIb and glycoprotein IIIa genes. Using genomic DNA from 60 unrelated normal individuals, we have amplified short domains that encompass the coding sequences and the exon-intron boundaries of both genes that were further separated according to their melting behaviour during the denaturant electrophoretic migration. Only the fragments with an abnormal migration pattern were sequenced. We confirmed the sensitivity of this method by recognizing both previously described Human Platelet Antigen polymorphisms and mutations affecting either the glycoprotein IIb or the glycoprotein IIIa genes in thrombasthenic patients. We also identified four other polymorphisms. Two were located in the glycoprotein IIb gene, involving intron 21 (C<-->G at nucleotide 10480) and first codon of exon 30 (codon GTC<-->GTT coding for residue Val 990), and two in the glycoprotein IIIa gene (exon 6 CCC<-->CCT coding for residue Pro 268; intron 14 C<--> T at position 37126). The screening of the GPIIIa promoter also revealed three different polymorphisms located at position-468 (A/T polymorphism), -425 (A/C polymorphism) and-400 (A/C polymorphism), which could influence the expression of the complex at the cell surface. Denaturing gradient gel electrophoresis appears to be a sensitive and specific technique for identifying polymorphisms and mutations in the GPIIb and GPIIIa genes.
Negrier
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Centre de Traitement de l'Hémophilie, Laboratoire d'Hémostatse, Hôpital Edouard Herriot, Lyon, France. negrier@laennec.univ-lyon1.fr
Grenier
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Attali
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Dechavanne
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Vinciguerra
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