Comprehensive In Vivo Interrogation Reveals Phenotypic Impact of Human Enhancer Variants

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. [Display omitted] •enSERT—a highly efficient CRISPR/Cas9-mediated site-specific transgenic mouse assay•In vivo assessment of all rare variants linked to polydactyly in a human enhancer•In vivo testing showed normal enhancer activity for 30% of presumed pathogenic variants•Systematic mutagenesis of this human enhancer identifies novel pathogenic variants Development of a scalable in vivo mouse enhancer-reporter assay and its use to systematically interrogate the canonical human ZRS enhancer, which drives Sonic hedgehog expression in mammalian limb development, broadly illuminates enhancer variant pathogenicity.