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

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 alpha IIb 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 (T (T) under bar T)-289 to Ser (T (C) under bar T) mutation (F289S) of the subunit, Another type I patient had a G to A base substitution in exon 12 resulting in a Glu ((G) under bar AA)-324 to Lys ((A) under bar AA) 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 Gln (C (A) under bar A)-747 to Pro (C (C) under bar A) mutation (Q747P), To analyse the effects of these mutations on alpha IIb beta 3 surface expression, the wild-type alpha IIb cDNA or mutant alpha IIb cDNAs were transfected into Chinese hamster ovary (CHO) cells together with a wild-type beta 3 cDNA. Flow cytometric analysis using an anti-alpha IIb beta 3 complex antibody revealed that 50.6% of CHO cells with wild-type alpha IIb beta 3 expressed complexes, whereas only 1.6%, 7.7% and 31.3% of cells, with alpha IIb(F289S)beta 3, alpha IIb(E324K)beta 3 and alpha IIb(Q747P)beta 3 expressed complexes, respectively. Our data indicate that these three novel point mutations in the alpha IIb subunit may hamper surface expression of the alpha IIb beta 3 complex, thus resulting in the quantitative GT phenotypes of platelets from these patients.