Wei Xiaomu, Das Jishnu, Fragoza Robert, Liang Jin, Bastos de Oliveira Francisco M, Lee Hao Ran, Wang Xiujuan, Mort Matthew, Stenson Peter D, Cooper David N, Lipkin Steven M, Smolka Marcus B, Yu Haiyuan
Department of Medicine, Weill Cornell College of Medicine, New York, New York, United States of America; Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, New York, United States of America.
Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, New York, United States of America; Department of Biological Statistics and Computational Biology, Cornell University, Ithaca, New York, United States of America.
PLoS Genet. 2014 Dec 11;10(12):e1004819. doi: 10.1371/journal.pgen.1004819. eCollection 2014 Dec.
Understanding the functional relevance of DNA variants is essential for all exome and genome sequencing projects. However, current mutagenesis cloning protocols require Sanger sequencing, and thus are prohibitively costly and labor-intensive. We describe a massively-parallel site-directed mutagenesis approach, "Clone-seq", leveraging next-generation sequencing to rapidly and cost-effectively generate a large number of mutant alleles. Using Clone-seq, we further develop a comparative interactome-scanning pipeline integrating high-throughput GFP, yeast two-hybrid (Y2H), and mass spectrometry assays to systematically evaluate the functional impact of mutations on protein stability and interactions. We use this pipeline to show that disease mutations on protein-protein interaction interfaces are significantly more likely than those away from interfaces to disrupt corresponding interactions. We also find that mutation pairs with similar molecular phenotypes in terms of both protein stability and interactions are significantly more likely to cause the same disease than those with different molecular phenotypes, validating the in vivo biological relevance of our high-throughput GFP and Y2H assays, and indicating that both assays can be used to determine candidate disease mutations in the future. The general scheme of our experimental pipeline can be readily expanded to other types of interactome-mapping methods to comprehensively evaluate the functional relevance of all DNA variants, including those in non-coding regions.
理解DNA变异的功能相关性对于所有外显子组和基因组测序项目都至关重要。然而，目前的诱变克隆方案需要桑格测序，因此成本高昂且 labor-intensive。我们描述了一种大规模平行的定点诱变方法，即“克隆测序”，利用下一代测序快速且经济高效地产生大量突变等位基因。使用克隆测序，我们进一步开发了一种比较相互作用组扫描流程，整合了高通量绿色荧光蛋白、酵母双杂交（Y2H）和质谱分析，以系统地评估突变对蛋白质稳定性和相互作用的功能影响。我们使用该流程表明，蛋白质-蛋白质相互作用界面上的疾病突变比远离界面的突变更有可能破坏相应的相互作用。我们还发现，在蛋白质稳定性和相互作用方面具有相似分子表型的突变对比具有不同分子表型的突变对更有可能导致相同的疾病，这验证了我们的高通量绿色荧光蛋白和Y2H分析在体内的生物学相关性，并表明这两种分析未来都可用于确定候选疾病突变。我们实验流程的总体方案可以很容易地扩展到其他类型的相互作用组映射方法，以全面评估所有DNA变异的功能相关性，包括非编码区域的变异。
