AlphaIIbbeta3 integrin: new allelic variants in Glanzmann thrombasthenia, effects on ITGA2B and ITGB3 mRNA splicing, expression, and structure-function.

Glanzmann thrombasthenia (GT) is an autosomal recessive inherited bleeding disorder characterized by an impaired platelet aggregation due to defects in integrin alphaIIbbeta3 (ITGA2B, ITGB3), a fibrinogen receptor. Mutations from 24 GT patients and two carriers of various origins, Caucasian, North-African and Asian were characterized. Promoter and exon sequences of alphaIIb and beta3 genes were amplified and directly sequenced. Among 29 identified mutations, 17 new allelic variants resulting from nonsense, missense and deletion/insertion mutations were described. RNA alterations were evaluated by using Web servers. The alphaIIb p.S926L, p.V903F, and beta3 p.C38Y, p.M118R, p.G221D substitutions prevented complex expression at the surface of COS-7 cells by altering the alphaIIb or the beta3 subunit structure. As shown by free energy analyses applied on the resolved structure of alphaIIbbeta3 and structural modeling of the mutant, the p.K253M substitution of beta3 helped to define a key role of the K253 in the interaction of the alphaIIb beta-propeller and the beta3 beta-I domains. finally, the alphaIIb p.Q595H substitution allowed cell surface expression of the complex but its corresponding c.2800G>T mutation is predicted to alter normal RNA splicing. In conclusion, our study yielded the discovery of 17 new GT allelic variants, revealed the key role of K253 of alphaIIb for the alphaIIbbeta3 complex formation and provides an additional example of an apparently missense mutation causing a splicing defect.

Recurrence of a Phe31Ser mutation in the Gla domain of blood coagulation factor X, in unrelated Algerian families: a founder effect?

The presence of gene lesions in blood coagulation factor X (FX) was investigated in eight FX-deficient patients with severe bleeding symptoms, originating from five unrelated Algerian families (FX coagulant activity <1%, FX antigen ranging from 2% to 16%). A missense mutation (p.Phe31Ser) in the Gla domain was found in homozygous form for all patients but one, who is a compound heterozygote for the Phe31Ser mutation and for a non-sense mutation, Tyr130Term in EGF-2 domain. The haplotypes of FX alleles were determined by the following allelic variants located in the promoter: g.1323_1330delTTGTGA (A1/A2), g.1449T>C, g.1451C>A, upstream to exon 3: g.17257C>T and downstream to exon 3: g.17396A>C. The A1-C-A-T-C haplotype was found on each allele bearing the Phe31Ser mutation in the eight FX deficient patients contrasting with its low frequency (8%) in a control Algerian population (in which the Phe31Ser substitution was absent). The patients came from the same geographical area of Algeria (5/8 are certainly from Kabyle origin) and the haplotype analysis suggests a founder effect. Transient expression study reveals that, for the mutant FX-Phe31Ser, FX antigen level was 60% in conditioned media and 140% in cell lysates compared with the wild type FX. The partial retention and intracellular accumulation of the mutant FX might be due to impaired folding and/or conformational changes, and the discrepancies observed between the FX antigen level in COS-7 cell supernatant (60%) and in the patients plasma (2-16%) to an in vivo increased clearance of the secreted unstable FX mutant.