Novel point mutations in the αIIb subunit (Phe289 → Ser, Glu324 → Lys and Gln747 → Pro) causing thrombasthenic phenotypes in four Japanese patients

Hironobu Ambo, Tetsuji Kamata, Makoto Handa, Yohko Kawai, Atsushi Oda, Mitsuru Murata, Yoshikazu Takada, Yasuo Ikeda

Research output: Contribution to journalArticle

18 Citations (Scopus)

Abstract

We analysed the molecular basis of Glanzmann thrombasthenia (GT) in four Japanese patients with type I or type II disease. Polymerase chain reaction (PCR) and subsequent direct sequencing of platelet RNA and genomic DNA revealed three single nucleotide substitutions of the αIIb gene, which were confirmed by allele-specific PCR or restriction analysis. One patient with type I GT had a T to C base substitution in exon 11 resulting in a Phe (TTT)- 289 to Ser (TCT) mutation (F289s) of the subunit. Another type I patient had a G to A base substitution in exon 12 resulting in a Glu (GAA)-324 to Lys (AAA) mutation (E324K). Interestingly, two unrelated patients with type II GT shared an A to C base substitution in exon 23, a region previously not associated with GT, resulting in a Gin (CAA)-747 to Pro (CCA) mutation (Q747P). To analyse the effects of these mutations on αlIbβ3 surface expression, the wild-type αIIb cDNA or mutant αIIb cDNAs were transfected into Chinese hamster ovary (CHO) cells together with a wild-type β3 cDNA. Flow cytometric analysis using an anti-αIIbβ3 complex antibody revealed that 50.6% of CHO cells with wild-type αIIbβ3 expressed complexes, whereas only 1.6%, 7.7% and 31.3% of cells, with αIIb(F289S)β3 αIIb(E324K)β3 and αIIb(Q747P)β3 expressed complexes, respectively. Our data indicate that these three novel point mutations in the αIIb subunit may hamper surface expression of the αIIbβ3 complex, thus resulting in the quantitative GT phenotypes of platelets from these patients.

Original languageEnglish
Pages (from-to)829-840
Number of pages12
JournalBritish Journal of Haematology
Volume102
Issue number3
DOIs
Publication statusPublished - 1998
Externally publishedYes

Fingerprint

Thrombasthenia
Point Mutation
Phenotype
Exons
Mutation
Complementary DNA
Cricetulus
Ovary
Blood Platelets
RNA Sequence Analysis
Polymerase Chain Reaction
Nucleotides
Alleles
Antibodies
DNA
Genes

Keywords

  • αIIbβ3 integrin
  • Gene analysis
  • Glanzmann thrombasthenia
  • Platelets
  • Point mutation

ASJC Scopus subject areas

  • Hematology

Cite this

Novel point mutations in the αIIb subunit (Phe289 → Ser, Glu324 → Lys and Gln747 → Pro) causing thrombasthenic phenotypes in four Japanese patients. / Ambo, Hironobu; Kamata, Tetsuji; Handa, Makoto; Kawai, Yohko; Oda, Atsushi; Murata, Mitsuru; Takada, Yoshikazu; Ikeda, Yasuo.

In: British Journal of Haematology, Vol. 102, No. 3, 1998, p. 829-840.

Research output: Contribution to journalArticle

Ambo, Hironobu ; Kamata, Tetsuji ; Handa, Makoto ; Kawai, Yohko ; Oda, Atsushi ; Murata, Mitsuru ; Takada, Yoshikazu ; Ikeda, Yasuo. / Novel point mutations in the αIIb subunit (Phe289 → Ser, Glu324 → Lys and Gln747 → Pro) causing thrombasthenic phenotypes in four Japanese patients. In: British Journal of Haematology. 1998 ; Vol. 102, No. 3. pp. 829-840.
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abstract = "We analysed the molecular basis of Glanzmann thrombasthenia (GT) in four Japanese patients with type I or type II disease. Polymerase chain reaction (PCR) and subsequent direct sequencing of platelet RNA and genomic DNA revealed three single nucleotide substitutions of the αIIb gene, which were confirmed by allele-specific PCR or restriction analysis. One patient with type I GT had a T to C base substitution in exon 11 resulting in a Phe (TTT)- 289 to Ser (TCT) mutation (F289s) of the subunit. Another type I patient had a G to A base substitution in exon 12 resulting in a Glu (GAA)-324 to Lys (AAA) mutation (E324K). Interestingly, two unrelated patients with type II GT shared an A to C base substitution in exon 23, a region previously not associated with GT, resulting in a Gin (CAA)-747 to Pro (CCA) mutation (Q747P). To analyse the effects of these mutations on αlIbβ3 surface expression, the wild-type αIIb cDNA or mutant αIIb cDNAs were transfected into Chinese hamster ovary (CHO) cells together with a wild-type β3 cDNA. Flow cytometric analysis using an anti-αIIbβ3 complex antibody revealed that 50.6{\%} of CHO cells with wild-type αIIbβ3 expressed complexes, whereas only 1.6{\%}, 7.7{\%} and 31.3{\%} of cells, with αIIb(F289S)β3 αIIb(E324K)β3 and αIIb(Q747P)β3 expressed complexes, respectively. Our data indicate that these three novel point mutations in the αIIb subunit may hamper surface expression of the αIIbβ3 complex, thus resulting in the quantitative GT phenotypes of platelets from these patients.",
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T1 - Novel point mutations in the αIIb subunit (Phe289 → Ser, Glu324 → Lys and Gln747 → Pro) causing thrombasthenic phenotypes in four Japanese patients

AU - Ambo, Hironobu

AU - Kamata, Tetsuji

AU - Handa, Makoto

AU - Kawai, Yohko

AU - Oda, Atsushi

AU - Murata, Mitsuru

AU - Takada, Yoshikazu

AU - Ikeda, Yasuo

PY - 1998

Y1 - 1998

N2 - We analysed the molecular basis of Glanzmann thrombasthenia (GT) in four Japanese patients with type I or type II disease. Polymerase chain reaction (PCR) and subsequent direct sequencing of platelet RNA and genomic DNA revealed three single nucleotide substitutions of the αIIb gene, which were confirmed by allele-specific PCR or restriction analysis. One patient with type I GT had a T to C base substitution in exon 11 resulting in a Phe (TTT)- 289 to Ser (TCT) mutation (F289s) of the subunit. Another type I patient had a G to A base substitution in exon 12 resulting in a Glu (GAA)-324 to Lys (AAA) mutation (E324K). Interestingly, two unrelated patients with type II GT shared an A to C base substitution in exon 23, a region previously not associated with GT, resulting in a Gin (CAA)-747 to Pro (CCA) mutation (Q747P). To analyse the effects of these mutations on αlIbβ3 surface expression, the wild-type αIIb cDNA or mutant αIIb cDNAs were transfected into Chinese hamster ovary (CHO) cells together with a wild-type β3 cDNA. Flow cytometric analysis using an anti-αIIbβ3 complex antibody revealed that 50.6% of CHO cells with wild-type αIIbβ3 expressed complexes, whereas only 1.6%, 7.7% and 31.3% of cells, with αIIb(F289S)β3 αIIb(E324K)β3 and αIIb(Q747P)β3 expressed complexes, respectively. Our data indicate that these three novel point mutations in the αIIb subunit may hamper surface expression of the αIIbβ3 complex, thus resulting in the quantitative GT phenotypes of platelets from these patients.

AB - We analysed the molecular basis of Glanzmann thrombasthenia (GT) in four Japanese patients with type I or type II disease. Polymerase chain reaction (PCR) and subsequent direct sequencing of platelet RNA and genomic DNA revealed three single nucleotide substitutions of the αIIb gene, which were confirmed by allele-specific PCR or restriction analysis. One patient with type I GT had a T to C base substitution in exon 11 resulting in a Phe (TTT)- 289 to Ser (TCT) mutation (F289s) of the subunit. Another type I patient had a G to A base substitution in exon 12 resulting in a Glu (GAA)-324 to Lys (AAA) mutation (E324K). Interestingly, two unrelated patients with type II GT shared an A to C base substitution in exon 23, a region previously not associated with GT, resulting in a Gin (CAA)-747 to Pro (CCA) mutation (Q747P). To analyse the effects of these mutations on αlIbβ3 surface expression, the wild-type αIIb cDNA or mutant αIIb cDNAs were transfected into Chinese hamster ovary (CHO) cells together with a wild-type β3 cDNA. Flow cytometric analysis using an anti-αIIbβ3 complex antibody revealed that 50.6% of CHO cells with wild-type αIIbβ3 expressed complexes, whereas only 1.6%, 7.7% and 31.3% of cells, with αIIb(F289S)β3 αIIb(E324K)β3 and αIIb(Q747P)β3 expressed complexes, respectively. Our data indicate that these three novel point mutations in the αIIb subunit may hamper surface expression of the αIIbβ3 complex, thus resulting in the quantitative GT phenotypes of platelets from these patients.

KW - αIIbβ3 integrin

KW - Gene analysis

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KW - Platelets

KW - Point mutation

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