A novel [288delC] mutation in exon 2 of GPIIb associated with type I Glanzmann thrombasthenia

Universidad de Las Palmas de Gran Canaria, Las Palmas, Canary Islands, Spain
British Journal of Haematology (Impact Factor: 4.71). 11/2000; 111(1):96-103. DOI: 10.1046/j.1365-2141.2000.02336.x
Source: PubMed


This work reports the molecular genetic analysis of two patients who suffer mucocutaneous haemorrhages, prolonged bleeding time and failure of platelets to aggregate, either spontaneously or in response to agonists. The absence of platelet surface glycoprotein (GP)IIb-IIIa complexes confirmed the clinical diagnosis of Glanzmann's thrombasthenia (GT). Polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) analysis of exon 2 of GPIIb showed polymorphic bands caused by the homozygous deletion of a cytosine at position 288 relative to the translation start site. causing a shifting of the reading frame and appearance of a premature termination codon. The heterozygous relatives showed a reduced platelet content of GPIIb-IIIa, and a correlation was found between the levels of GPIIb mRNA and surface expression of GPIIb-IIIa complexes. Unlike other mRNAs carrying a nonsense mutation, (288Cdel)GPIIb does not force alternative splicing of GPIIb mRNA. As expected, co-transfection of Chinese hamster ovary (CHO) cells with cDNAs encoding GPIIIa and (288delC)GPIIb failed to enhance the surface exposure of GPIIIa. It is concluded that the (288delC)GPIIb mutation is responsible for the thrombasthenic phenotype of the patients. In addition, it has also been determined that heterodimerization of GPIIb-IIIa requires the integrity of exons 2 and 3 of GPIIb.

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    ABSTRACT: We report the molecular, genetic and functional analysis of a case of thrombasthenic phenotype. The proband showed absence of platelet glycoprotein (GP)IIb and very low content of GPIIIa, and both his parents showed a marked reduction in the levels of platelet GPIIb-IIIa. Single-stranded conformational polymorphism-polymerase chain reaction (SSCP-PCR) analysis and direct sequencing of PCR-amplified GPIIb exon-12 revealed the presence of a G-->A transition at position 1063 with the expected substitution of glutamate 324 with lysine (K). This mutation did not alter the level of GPIIb mRNA. Co-expression of normal or mutant [324K] GPIIb with normal human GPIIIa in Chinese hamster ovary (CHO) cells failed to show surface exposure of [324K]GPIIb-IIIa complexes. Pulse-chase and immunoprecipitation analysis demonstrated that [324K]GPIIb cDNA was translated into proGPIIb, but neither mutant GPIIb heavy chain (GPIIbH) nor [324K]GPIIb-GPIIIa complexes were detected, suggesting that this mutation is the underlying molecular basis for the thrombasthenic phenotype. Mutation analysis demonstrated that 324E of GPIIb could be replaced by other negatively charged or polar amino acids (AAs) without impairing the surface expression of GPIIb-IIIa. However, substitution of 324E of GPIIb for a positively charged AA other than K prevented the expression of GPIIb-IIIa complexes. These observations suggest that a domain encompassing 324E of GPIIb is essential for heterodimerization with GPIIIa and its substitution for a positively charged residue precludes normal subunit association.
    Full-text · Article · Dec 2000 · British Journal of Haematology
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