publication . Other literature type . Preprint . Article . 2018

Multiplex Assessment of Protein Variant Abundance by Massively Parallel Sequencing

Vanessa Gray; Ronald Hause; Martin Kircher; Kenneth Matreyek; Melissa Chiasson; Beth Martin;
Open Access
  • Published: 16 Jan 2018
  • Publisher: bioRxiv
Abstract
<jats:title>ABSTRACT</jats:title><jats:p>Determining the pathogenicity of human genetic variants is a critical challenge, and functional assessment is often the only option. Experimentally characterizing millions of possible missense variants in thousands of clinically important genes will likely require generalizable, scalable assays. Here we describe Variant Abundance by Massively Parallel Sequencing (VAMP-seq), which measures the effects of thousands of missense variants of a protein on intracellular abundance in a single experiment. We apply VAMP-seq to quantify the abundance of 7,595 single amino acid variants of two proteins, PTEN and TPMT, in which functi...
Subjects
free text keywords: Genetics, Article
Funded by
NIH| F-CAP: Functionalization of Variants in Clinically Actionable Pharmacogenes
Project
  • Funder: National Institutes of Health (NIH)
  • Project Code: 5R24GM115277-03
  • Funding stream: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES
,
NIH| Large-Scale Methods for Assessing the Consequences of Mutations in Proteins
Project
  • Funder: National Institutes of Health (NIH)
  • Project Code: 1R01GM109110-01
  • Funding stream: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES
,
NIH| CANCER CENTER SUPPORT GRANT
Project
  • Funder: National Institutes of Health (NIH)
  • Project Code: 5P30CA021765-12
  • Funding stream: NATIONAL CANCER INSTITUTE
,
NIH| MEDICAL GENETICS
Project
  • Funder: National Institutes of Health (NIH)
  • Project Code: 5T32GM007454-15
  • Funding stream: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES
,
NIH| Interdisciplinary Training in Cancer Research Training Grant
Project
  • Funder: National Institutes of Health (NIH)
  • Project Code: 2T32CA080416-11A1
  • Funding stream: NATIONAL CANCER INSTITUTE
60 references, page 1 of 4

Shirts, BH, Pritchard, CC, Walsh, T. Family-Specific Variants and the Limits of Human Genetics. Trends Mol Med. 2016; 22: 925-934 [OpenAIRE] [PubMed]

Lek, M. Analysis of protein-coding genetic variation in 60,706 humans. Nature. 2016; 536: 285-291 [OpenAIRE] [PubMed]

Landrum, MJ. ClinVar: Public archive of relationships among sequence variation and human phenotype. Nucleic Acids Res. 2014; 42: 980-985

Fowler, DM, Stephany, JJ, Fields, S. Measuring the activity of protein variants on a large scale using deep mutational scanning. Nat Protoc. 2014; 9: 2267-2284 [OpenAIRE] [PubMed]

Gasperini, M, Starita, L, Shendure, J. The power of multiplexed functional analysis of genetic variants. Nat Protoc. 2016; 11: 1782-1787 [OpenAIRE] [PubMed]

Manolio, TA. Commentary Bedside Back to Bench: Building Bridges between Basic and Clinical Genomic Research. Cell. 2017; 169: 6-12 [OpenAIRE] [PubMed]

Starita, LM. Massively Parallel Functional Analysis of BRCA1 RING Domain Variants. Genetics. 2015; 200: 413-422 [OpenAIRE] [PubMed]

Majithia, AR. Prospective functional classification of all possible missense variants in PPARG. Nat Genet. 2016; 48: 1570-1575 [OpenAIRE] [PubMed]

Yue, P, Li, Z, Moult, J. Loss of protein structure stability as a major causative factor in monogenic disease. J Mol Biol. 2005; 353: 459-473 [PubMed]

Redler, RL, Das, J, Diaz, JR, Dokholyan, NV. Protein Destabilization as a Common Factor in Diverse Inherited Disorders. J Mol Evol. 2016; 82: 11-16 [OpenAIRE] [PubMed]

Berger, AH, Knudson, AG, Pandolfi, PP. A continuum model for tumour suppression. Nature. 2011; 476: 163-169 [OpenAIRE] [PubMed]

Lee, MS. Comprehensive analysis of missense variations in the BRCT domain of BRCA1 by structural and functional assays. Cancer Res. 2010; 70: 4880-4890 [OpenAIRE] [PubMed]

Tai, HL, Krynetski, EY, Schuetz, EG, Yanishevski, Y, Evans, WE. Enhanced proteolysis of thiopurine S-methyltransferase (TPMT) encoded by mutant alleles in humans (TPMT*3A, TPMT*2): mechanisms for the genetic polymorphism of TPMT activity. Proc Natl Acad Sci U S A. 1997; 94: 6444-9 [OpenAIRE] [PubMed]

Kim, I, Miller, CR, Young, DL, Fields, S. High-throughput analysis of in vivo protein stability. Mol Cell Proteomics. 2013; 12: 3370-8 [OpenAIRE] [PubMed]

Klesmith, JR, Bacik, JP, Wrenbeck, EE, Michalczyk, R, Whitehead, TA. Trade-offs between enzyme fitness and solubility illuminated by deep mutational scanning. Proc Natl Acad Sci U S A. 2017; 114: 2265-2270 [OpenAIRE] [PubMed]

60 references, page 1 of 4
Abstract
<jats:title>ABSTRACT</jats:title><jats:p>Determining the pathogenicity of human genetic variants is a critical challenge, and functional assessment is often the only option. Experimentally characterizing millions of possible missense variants in thousands of clinically important genes will likely require generalizable, scalable assays. Here we describe Variant Abundance by Massively Parallel Sequencing (VAMP-seq), which measures the effects of thousands of missense variants of a protein on intracellular abundance in a single experiment. We apply VAMP-seq to quantify the abundance of 7,595 single amino acid variants of two proteins, PTEN and TPMT, in which functi...
Subjects
free text keywords: Genetics, Article
Funded by
NIH| F-CAP: Functionalization of Variants in Clinically Actionable Pharmacogenes
Project
  • Funder: National Institutes of Health (NIH)
  • Project Code: 5R24GM115277-03
  • Funding stream: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES
,
NIH| Large-Scale Methods for Assessing the Consequences of Mutations in Proteins
Project
  • Funder: National Institutes of Health (NIH)
  • Project Code: 1R01GM109110-01
  • Funding stream: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES
,
NIH| CANCER CENTER SUPPORT GRANT
Project
  • Funder: National Institutes of Health (NIH)
  • Project Code: 5P30CA021765-12
  • Funding stream: NATIONAL CANCER INSTITUTE
,
NIH| MEDICAL GENETICS
Project
  • Funder: National Institutes of Health (NIH)
  • Project Code: 5T32GM007454-15
  • Funding stream: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES
,
NIH| Interdisciplinary Training in Cancer Research Training Grant
Project
  • Funder: National Institutes of Health (NIH)
  • Project Code: 2T32CA080416-11A1
  • Funding stream: NATIONAL CANCER INSTITUTE
60 references, page 1 of 4

Shirts, BH, Pritchard, CC, Walsh, T. Family-Specific Variants and the Limits of Human Genetics. Trends Mol Med. 2016; 22: 925-934 [OpenAIRE] [PubMed]

Lek, M. Analysis of protein-coding genetic variation in 60,706 humans. Nature. 2016; 536: 285-291 [OpenAIRE] [PubMed]

Landrum, MJ. ClinVar: Public archive of relationships among sequence variation and human phenotype. Nucleic Acids Res. 2014; 42: 980-985

Fowler, DM, Stephany, JJ, Fields, S. Measuring the activity of protein variants on a large scale using deep mutational scanning. Nat Protoc. 2014; 9: 2267-2284 [OpenAIRE] [PubMed]

Gasperini, M, Starita, L, Shendure, J. The power of multiplexed functional analysis of genetic variants. Nat Protoc. 2016; 11: 1782-1787 [OpenAIRE] [PubMed]

Manolio, TA. Commentary Bedside Back to Bench: Building Bridges between Basic and Clinical Genomic Research. Cell. 2017; 169: 6-12 [OpenAIRE] [PubMed]

Starita, LM. Massively Parallel Functional Analysis of BRCA1 RING Domain Variants. Genetics. 2015; 200: 413-422 [OpenAIRE] [PubMed]

Majithia, AR. Prospective functional classification of all possible missense variants in PPARG. Nat Genet. 2016; 48: 1570-1575 [OpenAIRE] [PubMed]

Yue, P, Li, Z, Moult, J. Loss of protein structure stability as a major causative factor in monogenic disease. J Mol Biol. 2005; 353: 459-473 [PubMed]

Redler, RL, Das, J, Diaz, JR, Dokholyan, NV. Protein Destabilization as a Common Factor in Diverse Inherited Disorders. J Mol Evol. 2016; 82: 11-16 [OpenAIRE] [PubMed]

Berger, AH, Knudson, AG, Pandolfi, PP. A continuum model for tumour suppression. Nature. 2011; 476: 163-169 [OpenAIRE] [PubMed]

Lee, MS. Comprehensive analysis of missense variations in the BRCT domain of BRCA1 by structural and functional assays. Cancer Res. 2010; 70: 4880-4890 [OpenAIRE] [PubMed]

Tai, HL, Krynetski, EY, Schuetz, EG, Yanishevski, Y, Evans, WE. Enhanced proteolysis of thiopurine S-methyltransferase (TPMT) encoded by mutant alleles in humans (TPMT*3A, TPMT*2): mechanisms for the genetic polymorphism of TPMT activity. Proc Natl Acad Sci U S A. 1997; 94: 6444-9 [OpenAIRE] [PubMed]

Kim, I, Miller, CR, Young, DL, Fields, S. High-throughput analysis of in vivo protein stability. Mol Cell Proteomics. 2013; 12: 3370-8 [OpenAIRE] [PubMed]

Klesmith, JR, Bacik, JP, Wrenbeck, EE, Michalczyk, R, Whitehead, TA. Trade-offs between enzyme fitness and solubility illuminated by deep mutational scanning. Proc Natl Acad Sci U S A. 2017; 114: 2265-2270 [OpenAIRE] [PubMed]

60 references, page 1 of 4
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