5 years ago

Receptor homodimerization plays a critical role in a novel dominant negative P2RY12 variant identified in a family with severe bleeding

Joel Mackay, David Rabbolini, William Stevenson, James L Hutchinson, Sara Gabrielli, Christopher M Ward, Riyaad Aungraheeta, Tatjana Kilo, Stuart J Mundell, Marie-Christine Morel-Kopp, Qiang Chen
Background Although inherited platelet disorders are still underdiagnosed worldwide, advances in molecular techniques are improving disease diagnosis and patient management. Objective To identify and characterize the mechanism underlying the bleeding phenotype in a Caucasian family with an autosomal dominant P2RY12 variant. Methods Full blood count, platelet aggregometry, flow cytometry and western-blotting were performed before NGS. Detailed molecular analysis of the identified P2Y12R receptor's variant was subsequently performed in mammalian cells over-expressing receptor constructs. Results All three referred individuals had markedly impaired ADP-induced platelet aggregation with primary wave only despite normal total and surface P2Y12R expression. By NGS, a single P2RY12:c.G794C substitution (p.R265P) was identified in all affected individuals and confirmed by Sanger sequencing. Mammalian cell experiments with the R265P-P2Y12R variant showed normal receptor surface expression versus wild-type (WT). Agonist-stimulated R265P-P2Y12R function (both signaling and surface receptor loss) was reduced versus WT. Critically, the R265P-P2Y12R variant acted in dominant negative manner with agonist-stimulated WT-P2Y12R activity reduced by variant co-expression suggesting dramatic loss of WT homodimers. Importantly, platelet P2RY12 cDNA cloning and sequencing in 2 affected individuals also revealed a 3-fold mutant mRNA overexpression, decreasing even further the likelihood of WT homodimer formation. R265 located within extracellular loop 3 (EL3) is one of four amino acids important for the receptor functional integrity, maintaining the binding pocket conformation and allowing rotation following ligand binding. Conclusion This novel dominant negative variant confirms the important role of R265 in EL3 in the functional integrity of the P2Y12R and suggests that pathological heterodimer formation may underlie this family bleeding phenotype. This article is protected by copyright. All rights reserved.

Publisher URL: http://onlinelibrary.wiley.com/resolve/doi

DOI: 10.1111/jth.13900

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