Comprehensive In Vivo Interrogation Reveals Phenotypic Impact of Human Enhancer Variants

Cell. 2020 Mar 19;180(6):1262-1271.e15. doi: 10.1016/j.cell.2020.02.031. Epub 2020 Mar 12.


Establishing causal links between non-coding variants and human phenotypes is an increasing challenge. Here, we introduce a high-throughput mouse reporter assay for assessing the pathogenic potential of human enhancer variants in vivo and examine nearly a thousand variants in an enhancer repeatedly linked to polydactyly. We show that 71% of all rare non-coding variants previously proposed as causal lead to reporter gene expression in a pattern consistent with their pathogenic role. Variants observed to alter enhancer activity were further confirmed to cause polydactyly in knockin mice. We also used combinatorial and single-nucleotide mutagenesis to evaluate the in vivo impact of mutations affecting all positions of the enhancer and identified additional functional substitutions, including potentially pathogenic variants hitherto not observed in humans. Our results uncover the functional consequences of hundreds of mutations in a phenotype-associated enhancer and establish a widely applicable strategy for systematic in vivo evaluation of human enhancer variants.

Keywords: CRISPR/Cas9; Sonic hedgehog; ZRS; cis-regulatory element; enhancer; genome editing; limb development; mutation; polydactyly; rare non-coding variant.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, N.I.H., Intramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Enhancer Elements, Genetic / genetics*
  • Enhancer Elements, Genetic / physiology
  • Gene Expression Regulation, Developmental / genetics
  • Gene Knock-In Techniques / methods
  • Hedgehog Proteins / genetics
  • Hedgehog Proteins / metabolism
  • High-Throughput Screening Assays / methods*
  • Humans
  • Mice
  • Mutation
  • Phenotype
  • Polydactyly / genetics*
  • Polydactyly / metabolism
  • RNA, Untranslated / genetics


  • Hedgehog Proteins
  • RNA, Untranslated