Type II Glanzmann thrombasthenia in a compound heterozygote for the alpha IIb gene. A novel missense mutation in exon 27

Abstract

BACKGROUND AND OBJECTIVES: Glanzmann thrombasthenia is an autosomal recessive bleeding disorder characterized by a life-long hemorrhagic tendency and absent or severely reduced platelet aggregation in response to agonists, caused by quantitative or qualitative abnormalities in the platelet fibrinogen receptor, integrin alphaIIb beta3. The aim of this study was to identify the molecular genetic defect and determine its functional consequences in a patient with type II Glanzmann thrombasthenia. DESIGN AND METHODS: The expression of platelet alphaIIb beta3 was determined by flow cytometry and western blotting. Mutations were identified by sequencing both cDNA and genomic DNA. Functional characterization was assessed by exontrap and transient transfection analysis. RESULTS: Flow cytometry and western blot analysis revealed markedly reduced levels of platelet alphaIIb beta3, which may account for the residual fibrinogen binding detected upon platelet activation. Sequencing of genomic DNA revealed the presence of two mutations in the alphaIIb gene: a C1750T transition in the last codon of exon 17 changing Arg553 to STOP, and a C2829T transition in exon 27 that changes Pro912 to Leu. Sequence analysis of reversely transcribed alphaIIb mRNA did not detect cDNA from the C1750T mutant allele, and revealed a significant increase of the physiological splicing out of exon 28 in the cDNA carrying the C2829T mutation. Transient expression of [912Leu]alphaIIb in CHO-b3 cells showed a marked reduction in the rate of surface expression of alphaIIb beta3. INTERPRETATION AND CONCLUSIONS: The results suggest that the thrombasthenic phenotype is the result of reduced availability of alphaIIb-mRNA, enhanced expression of exon 28-deleted transcripts, and defective processing of [912Leu]alphaIIb.