Molecular genetic analysis of a compound heterozygote for the glycoprotein(GP) IIb gene associated with Glanzmann's thrombasthenia: Disruption of the 674-687 disulfide bridge in GPIIb prevents surface exposure of GPIIb-IIIacomplexes

This work was aimed at elucidating the molecular genetic lesion(s) responsi ble for the thrombasthenic phenotype of a patient whose low platelet conten t of glycoprotein (GP) Ilb-IIIa indicated that it was a case of type II Gla nzmann's thrombasthenia (GT), The parents did not admit consanguinity and s howed a reduced platelet content of GPIIb-IIIa, Polymerase chain reaction ( PCR)-single-stranded conformational polymorphism analysis of genomic DNA sh owed no mutations in the patient's GPIIIa and two novel mutations in the GP IIb gene: one of them was a heterozygous splice junction mutation, a C-->A transversion, at position +2 of the exon 5-intron 5 boundary [IVS5(+2)C-->A ] inherited from the father. The predicted effect of this mutation, inserti on of intron 5 (76 bp) into the GPIIb-mRNA, was confirmed by reverse transc ription-PCR analysis of platelet mRNA. The almost complete absence of this mutated form of GPIIb-mRNA suggests that it is very unstable. Virtually all of the proband's GPIIb-mRNA was accounted for by the allele inherited from the mother showing a T-213-->C transition that changes Cys(674)-->Arg(674) disrupting the 674-687 intramolecular disulfide bridge. The proband showed a platelet accumulation of proGPIIb and minute amounts of GPIIb and GPIIIa . Moreover, transfection and immunoprecipitation analysis demonstrated that [Arg(674)]GPIIb is capable of forming a heterodimer complex with GPIIIa, b ut the rate of subunit maturation and the surface exposure of GPIIb-IIIa ar e strongly reduced. Thus, the intramolecular 674-687 disulfide bridge in GP IIb is essential for the normal processing of GPIIb-IIIa complexes. The add itive effect of these two GPIIb mutations provides the molecular basis for the thrombasthenic phenotype of the proband. (C) 1999 by The American Socie ty of Hematology.