title: A three amino acid deletion in glycoprotein IIIa is responsible for type I Glanzmann's thrombasthenia: importance of residues Ile325Pro326Gly327 for beta3 integrin subunit association.
abstract: Glanzmann's thrombasthenia (GT) is a recessive autosomal bleeding disorder characterized by abnormal platelet aggregation due to a qualitative or quantitative defect of the glycoprotein (GP) IIb-IIIa complex (integrin alphaIIb beta3). We describe a new mutation in the GPIIIa gene responsible for type I GT in a consanguineous Algerian family. A discordance between phenotyping and genotyping of the GPIIIa-related HPA-1 platelet alloantigen system in three family members heterozygous for the disease suggested a genetic defect in the GPIIIa gene and a normal GPIIb gene. Sequence analysis of amplified genomic DNA fragments showed a 6-bp deletion in exon 7 of the GPIIIa gene resulting in the amino acid deletion/substitution (Ile325pro326Gly327 --> Met) and creating a new BspHI restriction site. Expression of the mutated integrin beta3 subunit cDNA in Chinese hamster ovary cells showed that the cDNA gene was transcribed into a full-length beta3 protein with an apparent molecular weight identical to wild-type beta3 and accumulated as a single-chain molecule in the cell cytoplasm. The absence of heterodimeric complex formation of the mutant beta3 protein with endogenous alpha v was shown by immunoprecipitation experiments, intracellular immunofluorescent labeling, and a semiquantitative enzyme-linked immunosorbent assay using the alpha vbeta3 complex-specific monoclonal antibodies LM609 and 23C6. Substitution of the methionine residue by a proline, present at position 326 of wild-type beta3, did not restore the ability of the recombinant mutant beta3 protein to associate with alpha v , suggesting that the Ile-Pro-Gly motif is located in a beta3 domain important for integrin subunit interaction. The association of a BspHI restriction site with this newly identified mutation has allowed allele-specific restriction analysis of Algerian GT individuals and the identification of two new unrelated type I patients exhibiting the same mutation, suggesting that the described mutation might be significant in this population and that BspHI restriction analysis will provide a useful screening assay for antenatal diagnosis and genetic counselling.
authors.affiliation: Service d'Immunologie Leucoplaquettaire, GIP-INTS, Paris, France.
authors.forename: M C
authors.initials: MC
authors.lastname: Morel-Kopp
authors.forename: C
authors.initials: C
authors.lastname: Kaplan
authors.forename: V
authors.initials: V
authors.lastname: Proulle
authors.forename: V
authors.initials: V
authors.lastname: Jallu
authors.forename: C
authors.initials: C
authors.lastname: Melchior
authors.forename: O
authors.initials: O
authors.lastname: Peyruchaud
authors.forename: M H
authors.initials: MH
authors.lastname: Aurousseau
authors.forename: N
authors.initials: N
authors.lastname: Kieffer
mesh_terms: D000462:Algeria / Q000208:ethnology; D000595:Amino Acid Sequence; D000818:Animals; D015703:Antigens, CD / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics*; D001483:Base Sequence; D016466:CHO Cells; D003241:Consanguinity; D006224:Cricetinae; D017931:DNA Primers; D005260:Female; D005602:France; D005998:Glycine; D006801:Humans; D007223:Infant; D039661:Integrin beta3; D007532:Isoleucine; D008297:Male; D010375:Pedigree; D010980:Platelet Membrane Glycoproteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics*; D016133:Polymerase Chain Reaction; D018807:Polymorphism, Single-Stranded Conformational; D011392:Proline; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D017384:Sequence Deletion; D013915:Thrombasthenia / Q000097:blood / Q000235:genetics*; D014162:Transfection
chemical_list: D015703:Antigens, CD; D017931:DNA Primers; D039661:Integrin beta3; D010980:Platelet Membrane Glycoproteins; D011994:Recombinant Proteins; D007532:Isoleucine; D011392:Proline; D005998:Glycine