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

Wei X, Das J, Fragoza R, Liang J, Bastos de Oliveira FM, Lee HR, Wang X, Mort M, Stenson PD, Cooper DN, Lipkin SM, Smolka MB, Yu H - PLoS Genet. (2014)

Bottom Line: 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.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.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.

View Article: PubMed Central - PubMed

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.

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.

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Effect of disease mutations on protein stability and protein-protein interactions.(a) Western blotting with anti-GFP antibody confirming the protein expression levels of wild-type Rrm2b, Actn2, Hprt1, Pnp, Tpk1, Gnmt, Gale, Fbp1, Klhl3, Tp53, Pnp, Smad4, and corresponding mutant alleles. β-tubulin and γ-tubulin were used as loading controls. Red denotes “interface residue” mutations, orange denotes “interface domain” mutations and blue denotes “away from the interface” mutations. (b) Likelihood of disruption of interactions by “interface residue”, “interface domain” and “away from the interface” mutations – overall and for stable mutants only; likelihood of a disease mutation disrupting a given interaction in the absence of structural information. Error bars indicate +SE. (N = 204 mutations).
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pgen-1004819-g004: Effect of disease mutations on protein stability and protein-protein interactions.(a) Western blotting with anti-GFP antibody confirming the protein expression levels of wild-type Rrm2b, Actn2, Hprt1, Pnp, Tpk1, Gnmt, Gale, Fbp1, Klhl3, Tp53, Pnp, Smad4, and corresponding mutant alleles. β-tubulin and γ-tubulin were used as loading controls. Red denotes “interface residue” mutations, orange denotes “interface domain” mutations and blue denotes “away from the interface” mutations. (b) Likelihood of disruption of interactions by “interface residue”, “interface domain” and “away from the interface” mutations – overall and for stable mutants only; likelihood of a disease mutation disrupting a given interaction in the absence of structural information. Error bars indicate +SE. (N = 204 mutations).

Mentions: For the 204 mutations on proteins with co-crystal structures, we first examined whether the mutant proteins can be stably expressed in human cells. To do this, we tagged every wild-type and mutant protein with GFP at the C-terminus using high-throughput Gateway cloning (Fig. 1b). The GFP constructs were transfected into HEK293T cells and fluorescence intensities were measured by a plate reader (Fig. 3c; Materials and Methods). All fluorescence intensity readings were also confirmed manually under a microscope. Compared with the corresponding wild-type proteins, the expression levels of 3 of the 27 “interface residue” mutants, 8 of the 99 “interface domain” mutants and 6 of the 77 “away from the interface” mutants are significantly diminished (Fig. 3c; Materials and Methods; S2 Table). To validate these findings, we also performed Western blotting for 8 random mutants that are stably expressed and 8 random mutants with significantly diminished expression levels (Fig. 4a). Western blotting results confirm our GFP intensity readings.


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

Wei X, Das J, Fragoza R, Liang J, Bastos de Oliveira FM, Lee HR, Wang X, Mort M, Stenson PD, Cooper DN, Lipkin SM, Smolka MB, Yu H - PLoS Genet. (2014)

Effect of disease mutations on protein stability and protein-protein interactions.(a) Western blotting with anti-GFP antibody confirming the protein expression levels of wild-type Rrm2b, Actn2, Hprt1, Pnp, Tpk1, Gnmt, Gale, Fbp1, Klhl3, Tp53, Pnp, Smad4, and corresponding mutant alleles. β-tubulin and γ-tubulin were used as loading controls. Red denotes “interface residue” mutations, orange denotes “interface domain” mutations and blue denotes “away from the interface” mutations. (b) Likelihood of disruption of interactions by “interface residue”, “interface domain” and “away from the interface” mutations – overall and for stable mutants only; likelihood of a disease mutation disrupting a given interaction in the absence of structural information. Error bars indicate +SE. (N = 204 mutations).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4263371&req=5

pgen-1004819-g004: Effect of disease mutations on protein stability and protein-protein interactions.(a) Western blotting with anti-GFP antibody confirming the protein expression levels of wild-type Rrm2b, Actn2, Hprt1, Pnp, Tpk1, Gnmt, Gale, Fbp1, Klhl3, Tp53, Pnp, Smad4, and corresponding mutant alleles. β-tubulin and γ-tubulin were used as loading controls. Red denotes “interface residue” mutations, orange denotes “interface domain” mutations and blue denotes “away from the interface” mutations. (b) Likelihood of disruption of interactions by “interface residue”, “interface domain” and “away from the interface” mutations – overall and for stable mutants only; likelihood of a disease mutation disrupting a given interaction in the absence of structural information. Error bars indicate +SE. (N = 204 mutations).
Mentions: For the 204 mutations on proteins with co-crystal structures, we first examined whether the mutant proteins can be stably expressed in human cells. To do this, we tagged every wild-type and mutant protein with GFP at the C-terminus using high-throughput Gateway cloning (Fig. 1b). The GFP constructs were transfected into HEK293T cells and fluorescence intensities were measured by a plate reader (Fig. 3c; Materials and Methods). All fluorescence intensity readings were also confirmed manually under a microscope. Compared with the corresponding wild-type proteins, the expression levels of 3 of the 27 “interface residue” mutants, 8 of the 99 “interface domain” mutants and 6 of the 77 “away from the interface” mutants are significantly diminished (Fig. 3c; Materials and Methods; S2 Table). To validate these findings, we also performed Western blotting for 8 random mutants that are stably expressed and 8 random mutants with significantly diminished expression levels (Fig. 4a). Western blotting results confirm our GFP intensity readings.

Bottom Line: 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.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.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.

View Article: PubMed Central - PubMed

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.

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.

Show MeSH
Related in: MedlinePlus