title: Double heterozygosity for a novel missense mutation of Ile304 to Asn in addition to the missense mutation His280 to Pro in the integrin beta3 gene as a cause of the absence of platelet alphaIIbbeta3 in Glanzmann's thrombasthenia.
abstract: BACKGROUND
  Glanzmann's thrombasthenia (GT) is a hereditary bleeding disorder characterized by a defect in the expression or the function of alphaIIbbeta3.
  
  
  OBJECTIVE
  The purpose of the present study was to identify genetic defects in a GT patient.
  
  
  METHODS
  The expression of alphaIIbbeta3 was determined by flow cytometric analysis and Western blotting. We analyzed the cDNA sequences of both alphaIIb and beta3, and performed transfection experiments using COS7 cells to confirm that a specific mutation was responsible for the GT case.
  
  
  RESULTS
  Flow cytometric analysis and Western blotting showed remarkably reduced expression of alphaIIbbeta3. Sequence analysis of the patient's cDNA indicated a new missense mutation that led to the amino acid substitution of Ile304 (ATC) with Asn (AAC) in exon 6 of the beta3 gene. This was in addition to the missense mutation of His280 (CAT) to Pro (CCT) in exon 5, which had been previously reported. The missense mutation of Ile304 (ATC) to Asn (AAC) in beta3 was found to be responsible for this GT case. This was because transfection experiments using COS7 cells indicated that alphaIIbbeta3 possessing Asn304 in beta3 was not expressed on the surface of the transfected cells. In addition, immunoprecipitation analysis demonstrated that alphaIIbbeta3 was absent inside the transfected COS7 cells possessing Asn304 in beta(3).
  
  
  CONCLUSIONS
  In this study, we describe a new missense mutation (ATC to AAC) at position 1009 in exon 6 that leads to an amino acid substitution (Ile304 to Asn) in beta3, which is responsible for this GT case.
authors.affiliation: Osaka Red Cross Center, Morinomiya, Joto-ku, Osaka, Japan. seiken@al.mbn.or.jp
authors.forename: S
authors.initials: S
authors.lastname: Tanaka
authors.forename: T
authors.initials: T
authors.lastname: Hayashi
authors.forename: K
authors.initials: K
authors.lastname: Yoshimura
authors.forename: M
authors.initials: M
authors.lastname: Nakayama
authors.forename: T
authors.initials: T
authors.lastname: Fujita
authors.forename: T
authors.initials: T
authors.lastname: Amano
authors.forename: Y
authors.initials: Y
authors.lastname: Tani
mesh_terms: D000328:Adult; D000818:Animals; D001216:Asparagine / Q000235:genetics*; D015153:Blotting, Western; D019556:COS Cells; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000378:metabolism; D005091:Exons; D005260:Female; D005434:Flow Cytometry; D005822:Genetic Vectors; D006579:Heterozygote; D006639:Histidine / Q000235:genetics*; D006801:Humans; D047468:Immunoprecipitation; D039661:Integrin beta3 / Q000235:genetics*; D007532:Isoleucine / Q000235:genetics*; D008297:Male; D020125:Mutation, Missense; D010375:Pedigree; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000235:genetics*; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length; D011392:Proline / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D013915:Thrombasthenia / Q000235:genetics*; D014162:Transfection
chemical_list: D017931:DNA Primers; D018076:DNA, Complementary; D039661:Integrin beta3; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D007532:Isoleucine; D006639:Histidine; D001216:Asparagine; D011392:Proline