4.6 Article

A rapid solubility assay of protein domain misfolding for pathogenicity assessment of rare DNA sequence variants

Journal

GENETICS IN MEDICINE
Volume 22, Issue 10, Pages 1642-1652

Publisher

ELSEVIER SCIENCE INC
DOI: 10.1038/s41436-020-0842-1

Keywords

sequence variant; protein misfolding; Kv11; 1; LMNA; P53

Funding

  1. National Institutes of Health (NIH) [R01 HL07887]
  2. American Heart Association (AHA) Competitive Catalyst Renewal Grant [17CCRG33700289]
  3. AHA Midwest Affiliate Predoctoral Fellowship
  4. National Research Service Award (NRSA) postdoctoral fellowship [F32 HL128091]
  5. NIH [R01 HL139738-01, R01 HL060723]

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Purpose DNA sequencing technology has unmasked a vast number of uncharacterized single-nucleotide variants in disease-associated genes, and efficient methods are needed to determine pathogenicity and enable clinical care. Methods We report an E. coli-based solubility assay for assessing the effects of variants on protein domain stability for three disease-associated proteins. Results First, we examined variants in the Kv11.1 channel PAS domain (PASD) associated with inherited long QT syndrome type 2 and found that protein solubility correlated well with reported in vitro protein stabilities. A comprehensive solubility analysis of 56 Kv11.1 PASD variants revealed that disruption of membrane trafficking, the dominant loss-of-function disease mechanism, is largely determined by domain stability. We further validated this assay by using it to identify second-site suppressor PASD variants that improve domain stability and Kv11.1 protein trafficking. Finally, we applied this assay to several cancer-linked P53 tumor suppressor DNA-binding domain and myopathy-linked Lamin A/C Ig-like domain variants, which also correlated well with reported protein stabilities and functional analyses. Conclusion This simple solubility assay can aid in determining the likelihood of pathogenicity for sequence variants due to protein misfolding in structured domains of disease-associated genes as well as provide insights into the structural basis of disease.

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