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.
Subject(s)
Platelet Glycoprotein GPIIb-IIIa Complex/genetics , Point Mutation , Thrombasthenia/genetics , Animals , Base Sequence , Blood Platelets/metabolism , CHO Cells , Chickens , Child, Preschool , Cricetinae , Flow Cytometry , Homozygote , Humans , Male , Mice , Molecular Sequence Data , Polymorphism, Single-Stranded Conformational , Sequence Analysis, DNA , Sequence Homology, Nucleic Acid , Thrombasthenia/blood , XenopusSubject(s)
Thrombophilia/genetics , Venous Thrombosis/genetics , Child , DNA Mutational Analysis , Factor V/genetics , Family Health , Homozygote , Humans , Male , Methylenetetrahydrofolate Reductase (NADPH2) , Oxidoreductases Acting on CH-NH Group Donors/genetics , Pedigree , Point Mutation , Prothrombin/genetics , Restriction MappingABSTRACT
This work reports the molecular genetic study of a patient who suffered from Glanzmann thrombasthenia (GT). Structural analysis of the glycoprotein (GP) IIb and GPIIIa genes showed the presence of a homozygous G1846-->T transversion in exon 11 of GPIIIa that changes Glu616-->Stop. Cytometric and immunochemical analysis indicated that platelet GPIIb-IIIa was absent in the proband but present at normal levels in the heterozygous relatives. The following observations indicate that this mutation is responsible for the thrombasthenic phenotype of the proband. (1) We failed to detect mutations other than [T1846]GPIIIa in the coding region of both GPIIb and GPIIIa genes. (2) The G1846-->T mutation was observed in either parent and a brother of the proband, but none of 100 unrelated individuals carried this defect. (3) Pulse-chase and immunoprecipitation analysis of GPIIb-IIIa complexes in cells transiently cotransfected with cDNAs encoding normal GPIIb and [T1846]GPIIIa showed neither maturation of GPIIb nor complex formation and surface exposure of GPIIb-triangle upGPIIIa. These observations indicate that the sequence from Glu616 to Thr762 in GPIIIa is essential for heterodimerization with GPIIb. Polymerase chain reaction-based analysis demonstrated the presence of normal levels of full-length GPIIIa-mRNA in the proband and in heterozygous relatives. In addition, a shortened transcript, with a 324-nucleotide deletion, resulting from in-frame skipping of exons 10 and 11, was detectable upon reamplification of the DNA. Thus, unlike other nonsense mutations, [T1846]GPIIIa does not lead to abnormal processing or reduction in the number of transcripts with the termination codon.