期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2015
卷号：112
期号：15
页码：E1898-E1907
DOI：10.1073/pnas.1422238112
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：SignificanceNext-generation sequencing is identifying millions of novel gene variants, presenting challenges to researchers and clinicians. Variations in the genes ITGA2B and ITGB3 affect integrin IIb{beta}3, leading to the bleeding disorder Glanzmann thrombasthenia. We analyzed novel missense variants on [~]32,000 alleles of ITGA2B and ITGB3 and found missense variants affecting [~]10% of the amino acids in each protein in [~]1.3% of the population. Almost all variants are rare, indicating recent entry into the population. Two novel variants we predicted would be deleterious profoundly affected recombinant protein expression. At cut-off values that correctly predicted at least 69% of the known Glanzmann thrombasthenia mutations as deleterious, three variant prediction algorithms predicted that at least 27% of the novel variants are deleterious. Next-generation sequencing is transforming our understanding of human genetic variation but assessing the functional impact of novel variants presents challenges. We analyzed missense variants in the integrin IIb{beta}3 receptor subunit genes ITGA2B and ITGB3 identified by whole-exome or -genome sequencing in the ThromboGenomics project, comprising [~]32,000 alleles from 16,108 individuals. We analyzed the results in comparison with 111 missense variants in these genes previously reported as being associated with Glanzmann thrombasthenia (GT), 20 associated with alloimmune thrombocytopenia, and 5 associated with aniso/macrothrombocytopenia. We identified 114 novel missense variants in ITGA2B (affecting [~]11% of the amino acids) and 68 novel missense variants in ITGB3 (affecting [~]9% of the amino acids). Of the variants, 96% had minor allele frequencies (MAF) < 0.1%, indicating their rarity. Based on sequence conservation, MAF, and location on a complete model of IIb{beta}3, we selected three novel variants that affect amino acids previously associated with GT for expression in HEK293 cells. IIb P176H and {beta}3 C547G severely reduced IIb{beta}3 expression, whereas IIb P943A partially reduced IIb{beta}3 expression and had no effect on fibrinogen binding. We used receiver operating characteristic curves of combined annotation-dependent depletion, Polyphen 2-HDIV, and sorting intolerant from tolerant to estimate the percentage of novel variants likely to be deleterious. At optimal cut-off values, which had 69-98% sensitivity in detecting GT mutations, between 27% and 71% of the novel IIb or {beta}3 missense variants were predicted to be deleterious. Our data have implications for understanding the evolutionary pressure on IIb{beta}3 and highlight the challenges in predicting the clinical significance of novel missense variants.
