Diversity and impact of rare variants in genes encoding the platelet G protein-coupled receptors

Summary

Platelet responses to activating agonists are influenced by common population variants within or near G protein-coupled receptor (GPCR) genes that affect receptor activity. However, the impact of rare GPCR gene variants is unknown. We describe the rare single nucleotide variants (SNVs) in the coding and splice regions of 18 GPCR genes in 7,595 exomes from the 1,000-genomes and Exome Sequencing Project databases and in 31 cases with inherited platelet function disorders (IPFDs). In the population databases, the GPCR gene target regions contained 740 SNVs (318 synonymous, 410 missense, 7 stop gain and 6 splice region) of which 70 % had global minor allele frequency (MAF) < 0.05 %. Functional annotation using six computational algorithms, experimental evidence and structural data identified 156/740 (21 %) SNVs as potentially damaging to GPCR function, most commonly in regions encoding the transmembrane and C-terminal intracellular receptor domains. In 31 index cases with IPFDs (Gi-pathway defect n=15; secretion defect n=11; thromboxane pathway defect n=3 and complex defect n=2) there were 256 SNVs in the target regions of 15 stimulatory platelet GPCRs (34 unique; 12 with MAF< 1 % and 22 with MAF≥ 1 %). These included rare variants predicting R122H, P258T and V207A substitutions in the P2Y₁₂ receptor that were annotated as potentially damaging, but only partially explained the platelet function defects in each case. Our data highlight that potentially damaging variants in platelet GPCR genes have low individual frequencies, but are collectively abundant in the population. Potentially damaging variants are also present in pedigrees with IPFDs and may contribute to complex laboratory phenotypes.

Keywords

Receptors - G-protein-coupled - genetic variation - blood platelets - blood platelet disorders

• References

• 1 Jones CI, Bray S, Garner SF. et al. A functional genomics approach reveals novel quantitative trait loci associated with platelet signaling pathways. Blood 2009; 114: 1405-1416.
• 2 Johnson AD, Yanek LR, Chen MH. et al. Genome-wide meta-analyses identifies seven loci associated with platelet aggregation in response to agonists. Nature Gen 2010; 42: 608-613.
• 3 Dupont A, Fontana P, Bachelot-Loza C. et al. An intronic polymorphism in the PAR-1 gene is associated with platelet receptor density and the response to SFLLRN. Blood 2003; 101: 1833-1840.
• 4 Muehlschlegel JD, Perry TE, Liu KY. et al. Polymorphism in the protease-activated receptor-4 gene region associates with platelet activation and perioperative myocardial injury. Am J Hematol 2012; 87: 161-166.
• 5 Fontana P, Gandrille S, Remones V. et al. Identification of functional polymorphisms of the thromboxane A2 receptor gene in healthy volunteers. Thromb Haemost 2006; 96: 356-360.
• 6 Zee RY, Michaud SE, Diehl KA. et al. Purinergic receptor P2Y, G-protein coupled, 12 gene variants and risk of incident ischemic stroke, myocardial infarction, and venous thromboembolism. Atherosclerosis 2008; 197: 694-699.
• 7 Small KM, Brown KM, Seman CA. et al. Complex haplotypes derived from noncoding polymorphisms of the intronless alpha2A-adrenergic gene diversify receptor expression. Proc Natl Acad Sci USA 2006; 103: 5472-5477.
• 8 Genomes Project C, Abecasis GR, Altshuler D. et al. A map of human genome variation from population-scale sequencing. Nature 2010; 467: 1061-1073.
• 9 Li Y, Vinckenbosch N, Tian G. et al. Resequencing of 200 human exomes identifies an excess of low-frequency non-synonymous coding variants. Nature Gen 2010; 42: 969-972.
• 10 Tennessen JA, Bigham AW, O’Connor TD. et al. Evolution and functional impact of rare coding variation from deep sequencing of human exomes. Science 2012; 337: 64-69.
• 11 Daly ME, Dawood BB, Lester WA. et al. Identification and characterization of a novel P2Y 12 variant in a patient diagnosed with type 1 von Willebrand disease in the European MCMDM-1VWD study. Blood 2009; 113: 4110-4113.
• 12 Fontana G, Ware J, Cattaneo M. Haploinsufficiency of the platelet P2Y12 gene in a family with congenital bleeding diathesis. Haematologica 2009; 94: 581-584.
• 13 Mumford AD, Dawood BB, Daly ME. et al. A novel thromboxane A2 receptor D304N variant that abrogates ligand binding in a patient with a bleeding diathesis. Blood 2010; 115: 363-369.
• 14 Mumford AD, Nisar S, Darnige L. et al. Platelet dysfunction associated with the novel Trp29Cys thromboxane A(2) receptor variant. J Thromb Haemost 2013; 11: 547-554.
• 15 Nisar S, Daly ME, Federici AB. et al. An intact PDZ motif is essential for correct P2Y12 purinoceptor traffic in human platelets. Blood 2011; 118: 5641-5651.
• 16 Nisar SP, Lordkipanidze M, Jones ML. et al. A novel thromboxane A2 receptor N42S variant results in reduced surface expression and platelet dysfunction. Thromb Haemost 2014; 111: 923-932.
• 17 Patel YM, Lordkipanidze M, Lowe GC. et al. A novel mutation in the p2y receptor and a function-reducing polymorphism in par-1 in a patient with chronic bleeding. J Thromb Haemost. 2014. Epub ahead of print
• 18 Rowley JW, Oler AJ, Tolley ND. et al. Genome-wide RNA-seq analysis of human and mouse platelet transcriptomes. Blood 2011; 118: e101-e111.
• 19 Dawood BB, Lowe GC, Lordkipanidze M. et al. Evaluation of participants with suspected heritable platelet function disorders including recommendation and validation of a streamlined agonist panel. Blood 2012; 120: 5041-5049.
• 20 Dawood BB, Wilde J, Watson SP. Reference curves for aggregation and ATP secretion to aid diagnose of platelet-based bleeding disorders: effect of inhibition of ADP and thromboxane A(2) pathways. Platelets 2007; 18: 329-345.
• 21 Jones ML, Murden SL, Bem D. et al. Rapid genetic diagnosis of heritable platelet function disorders with next-generation sequencing: proof-of-principle with Hermansky-Pudlak syndrome. J Thromb Haemost 2012; 10: 306-309.
• 22 Bendl J, Stourac J, Salanda O. et al. PredictSNP: Robust and Accurate Consensus Classifier for Prediction of Disease-Related Mutations. PLoS Comp Biol 2014; 10: e1003440.
• 23 Colombo M, De Vecchi G, Caleca L. et al. Comparative in vitro and in silico analyses of variants in splicing regions of BRCA1 and BRCA2 genes and characterization of novel pathogenic mutations. PloS One 2013; 08: e57173.
• 24 Ballesteros JA, Weinstein H. Analysis and refinement of criteria for predicting the structure and relative orientations of transmembranal helical domains. Biophys J 1992; 62: 107-109.
• 25 Venkatakrishnan AJ, Deupi X, Lebon G. et al. Molecular signatures of G-protein-coupled receptors. Nature 2013; 494: 185-194.
• 26 Zhang C, Srinivasan Y, Arlow DH. et al. High-resolution crystal structure of human protease-activated receptor 1. Nature. 2012; 492: 387-392.
• 27 Xu F, Wu H, Katritch V. et al. Structure of an agonist-bound human A2A adenosine receptor. Science 2011; 332: 322-327.
• 28 Zhang K, Zhang J, Gao ZG. et al. Structure of the human P2Y receptor in complex with an antithrombotic drug. Nature. 2014. Epub ahead of print
• 29 Sauna ZE, Kimchi-Sarfaty C. Understanding the contribution of synonymous mutations to human disease. Nature Rev Gen 2011; 12: 683-691.
• 30 Lander ES, Linton LM, Birren B. et al. Initial sequencing and analysis of the human genome. Nature 2001; 409: 860-921.
• 31 Fryxell KJ, Moon WJ. CpG mutation rates in the human genome are highly dependent on local GC content. Mol Biol Evol 2005; 22: 650-658.
• 32 Kozlowski P, de Mezer M, Krzyzosiak WJ. Trinucleotide repeats in human genome and exome. Nucl Acid Res 2010; 38: 4027-4039.
• 33 Marchese A, Paing MM, Temple BR. et al. G protein-coupled receptor sorting to endosomes and lysosomes. Annu Rev Pharmacol Toxicol 2008; 48: 601-629.
• 34 Remijn JA, MJ IJ, Strunk AL. et al. Novel molecular defect in the platelet ADP receptor P2Y12 of a patient with haemorrhagic diathesis. Clin Chem Lab Med 2007; 45: 187-189.
• 35 Salon JA, Lodowski DT, Palczewski K. The significance of G protein-coupled receptor crystallography for drug discovery. Pharmacol Rev 2011; 63: 901-937.
• 36 Cattaneo M, Zighetti ML, Lombardi R. et al. Molecular bases of defective signal transduction in the platelet P2Y12 receptor of a patient with congenital bleeding. Proc Natl Acad Sci USA 2003; 100: 1978-1983.
• 37 Cattaneo M, Lecchi A, Lombardi R. et al. Platelets from a patient heterozygous for the defect of P2CYC receptors for ADP have a secretion defect despite normal thromboxane A2 production and normal granule stores: further evidence that some cases of platelet ‘primary secretion defect’ are heterozygous for a defect of P2CYC receptors. Arterioscl Thromb Vasc Biol 2000; 20: E101-E106.