Characterisation of ITGB3 and ITGA2B gene defects associated with Glanzmann thrombasthenia

Glanzmann thrombasthenia (GT) is a recessively inherited bleeding disorder caused by quantitative and, or, qualitative deficiencies of the platelet integrin αIIbβ3, which binds fibrinogen to mediate platelet aggregation at sites of vascular injury. The α- and β-subunits of αIIbβ3 are encoded by ITGA2B and ITGB3 respectively, and many genetic defects resulting in reduced expression or dysfunction of αIIbβ3 have been described. A previous survey of UK GT patients that was carried out by our group identified 14 uncharacterised nonsynonymous alterations in ITGA2B and ITGB3 that predicted amino acid substitutions in different domains of αIIb and β3. This study used combined in silico and in vitro approaches to confirm the pathogenicity of 13 of these alterations; eight ITGB3 variants predicting p.Trp11Arg, p.Pro189Ser, p.Glu200Lys, p.Trp264Leu, p.Ser317Phe, p.Cys547Trp, p.Cys554Arg and p.Ile665Thr substitutions in β3 and five ITGA2B variants predicting p.Asp396Asn, p.Leu492Pro, p.Ile596Thr, p.Asn670Lys and p.Glu698Asp substitutions in αIIb. With the exception of the β3_p.Glu200Lys and αIIb_p.Glu698Asp variants, in silico analysis predicted all variants to be pathogenic. Compared to cells expressing wild-type (WT) αIIbβ3, there was an almost complete absence of surface αIIbβ3 in cells expressing the p.Trp11Arg, p.Pro189Ser, Trp264Leu and Ser317Phe β3 variants (p<0.0001). In contrast, the β3_p.IIe665Thr variant was expressed at similar levels to WT, but showed reduced ability to bind an antibody that is specific for the active conformation of the receptor, PAC1 (p<0.01) and also fibrinogen (p<0.05), while the β3_p.Glu200Lys variant does not appear to cause dysfunction to αIIbβ3. Interestingly, cells expressing the p.Cys547Trp and p.Cys554Arg β3 variants showed moderate reductions in surface expression of αIIbβ3 (p<0.0001) that exhibited spontaneous activation (p<0.0001;p<0.001). The majority of the substitutions in β3 resulted in greater than 90% reductions (p<0.0001) in membrane expression of αvβ3 with the exception of the p.Glu200Lys and p.Pro189Ser substitutions which resulted in 78% (p<0.0001) and 21% (p<0.001) reductions in membrane expression of αvβ3, respectively. There was a severe reduction in membrane αIIbβ3 on cells expressing the p.Asp396Asn, the p.Leu492Pro and the p.Ile596Thr αIIb variants (p<0.0001) while the p.Asn670Lys αIIb was expressed at similar levels to wild-type αIIb. Interestingly, the p.Leu492Pro and p.Asn670Lys variants showed reduced ability to bind PAC1 (p<0.0001;p<0.01) and fibrinogen (p<0.0001;p<0.01). The c.2094G>T transversion in ITGA2B, predicted to cause a p.Glu698Asp substitution in αIIb, was associated with a ii splicing defect, resulting in the deletion of exon 20 from the ITGA2B RNA , and the loss of αIIbβ3 expression. These findings confirm the pathogenicity and demonstrate the underlying mechanisms associated with GT for the majority of the variants studied. Interestingly, while the β3_p.Glu200Lys variant does not appear to cause αIIbβ3 dysfunction, it does result in a reduction in αvβ3 (vitronectin) receptor expression, though it remains unknown how this defect results in GT.