title: A massively parallel pipeline to clone DNA variants and examine molecular phenotypes of human disease mutations.
abstract: 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.
authors.affiliation: 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.
authors.forename: Xiaomu
authors.initials: X
authors.lastname: Wei
authors.affiliation: 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.
authors.forename: Jishnu
authors.initials: J
authors.lastname: Das
authors.affiliation: Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, New York, United States of America; Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York, United States of America.
authors.forename: Robert
authors.initials: R
authors.lastname: Fragoza
authors.affiliation: Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, New York, United States of America.
authors.forename: Jin
authors.initials: J
authors.lastname: Liang
authors.affiliation: Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, New York, United States of America; Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York, United States of America.
authors.forename: Francisco M
authors.initials: FM
authors.lastname: Bastos de Oliveira
authors.affiliation: 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.
authors.forename: Hao Ran
authors.initials: HR
authors.lastname: Lee
authors.affiliation: 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.
authors.forename: Xiujuan
authors.initials: X
authors.lastname: Wang
authors.affiliation: Institute of Medical Genetics, Cardiff University, Heath Park, Cardiff, United Kingdom.
authors.forename: Matthew
authors.initials: M
authors.lastname: Mort
authors.affiliation: Institute of Medical Genetics, Cardiff University, Heath Park, Cardiff, United Kingdom.
authors.forename: Peter D
authors.initials: PD
authors.lastname: Stenson
authors.affiliation: Institute of Medical Genetics, Cardiff University, Heath Park, Cardiff, United Kingdom.
authors.forename: David N
authors.initials: DN
authors.lastname: Cooper
authors.affiliation: Department of Medicine, Weill Cornell College of Medicine, New York, New York, United States of America.
authors.forename: Steven M
authors.initials: SM
authors.lastname: Lipkin
authors.affiliation: Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, New York, United States of America; Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York, United States of America.
authors.forename: Marcus B
authors.initials: MB
authors.lastname: Smolka
authors.affiliation: 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.
authors.forename: Haiyuan
authors.initials: H
authors.lastname: Yu
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism; D000483:Alleles; D002853:Chromatography, Liquid; D003001:Cloning, Molecular / Q000379:methods*; D056915:DNA Copy Number Variations; D004252:DNA Mutational Analysis / Q000379:methods*; D059472:Exome; D005786:Gene Expression Regulation; D015723:Gene Library; D057809:HEK293 Cells; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D000070957:MutL Protein Homolog 1; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D010641:Phenotype; D010957:Plasmids / Q000235:genetics; D054730:Protein Interaction Domains and Motifs; D055550:Protein Stability; D012441:Saccharomyces cerevisiae / Q000235:genetics; D053719:Tandem Mass Spectrometry
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C501486:MLH1 protein, human; D009687:Nuclear Proteins; D000070957:MutL Protein Homolog 1