A massively parallel pipeline to clone DNA variants and examine molecular phenotypes of human disease mutations. Academic Article uri icon

Overview

MeSH

  • Adaptor Proteins, Signal Transducing
  • Alleles
  • Chromatography, Liquid
  • Exome
  • Gene Expression Regulation
  • Gene Library
  • HEK293 Cells
  • High-Throughput Nucleotide Sequencing
  • Humans
  • MutL Protein Homolog 1
  • Nuclear Proteins
  • Plasmids
  • Protein Interaction Domains and Motifs
  • Protein Stability
  • Saccharomyces cerevisiae
  • Tandem Mass Spectrometry

MeSH Major

  • Cloning, Molecular
  • DNA Copy Number Variations
  • DNA Mutational Analysis
  • Mutagenesis, Site-Directed
  • Mutation
  • Phenotype

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.

publication date

  • December 2014

has subject area

  • Adaptor Proteins, Signal Transducing
  • Alleles
  • Chromatography, Liquid
  • Cloning, Molecular
  • DNA Copy Number Variations
  • DNA Mutational Analysis
  • Exome
  • Gene Expression Regulation
  • Gene Library
  • HEK293 Cells
  • High-Throughput Nucleotide Sequencing
  • Humans
  • MutL Protein Homolog 1
  • Mutagenesis, Site-Directed
  • Mutation
  • Nuclear Proteins
  • Phenotype
  • Plasmids
  • Protein Interaction Domains and Motifs
  • Protein Stability
  • Saccharomyces cerevisiae
  • Tandem Mass Spectrometry

Research

keywords

  • Journal Article

Identity

Language

  • eng

PubMed Central ID

  • PMC4263371

Digital Object Identifier (DOI)

  • 10.1371/journal.pgen.1004819

PubMed ID

  • 25502805

Additional Document Info

start page

  • e1004819

volume

  • 10

number

  • 12