Comprehensive In Vivo Interrogation Reveals Phenotypic Impact of Human Enhancer Variants

Abstract

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.

Figure 1:. EnSERT allows large-scale and robust enhancer variant assessment.

(A) Overview of the method. The conventional enhancer-reporter assay relies on random integration of the transgene into the mouse genome upon zygote microinjection (top), which results in a low transgenic rate and limited reproducibility due to randomectopic effects. EnSERT (bottom) uses CRISPR/Cas9 to direct transgene integration to a specific genomic location, which results in higher reproducibility, no ectopic effects, and higher efficiency. Shown on the right are independently injected LacZ-stained embryos that resulted from random integration (top) or enSERT (bottom) of a transgene containing a human forebrain enhancer (hs200) or human limb enhancer (ZRS). Arrows denote reproducible enhancer activity at E11.5. (B) The human ZRS limb enhancer of SHH is located approximately 1 Mb from its target promoter. When tested in a transgenic mouse reporter assay, ZRS activates reporter expression in the posterior margins of both fore- and hindlimb buds (ZPA) of E11.5 mouse embryos (left). Sequence variants in ZRS cause Shh misexpression in the anterior limb bud (middle), which results in polydactyly in multiple vertebrate species (right; shown here: polydactylous cat; photo by Jonna Austin). (C) EnSERT reproducibly detects the anterior lacZ misexpression (red arrowheads) caused by the ‘Cuban’ allele of the human ZRS enhancer (see Figure S3A for details).Numbers of embryos with LacZ staining in the anterior limb bud (red) over the total number of transgenic embryos screened (black) are indicated. Only transgenic embryos that carried at least two copies of the reporter transgene at the H11 locus were considered in the analysis (see STAR methods for details). TFBS, transcription factor binding site.

Figure 2:. Systematic assessment of all variants in the human ZRS enhancer.

Enhancer activities for each of the 36 ZRS variant alleles implicated in preaxial polydactyly, including 21 human variants reported in the literature, nine ZRS mutations from other vertebrate species, and eight additional human variants identified in this study (cyan boxes). Shown are representative forelimb buds of transgenic E11.5 mouse embryos. Human ZRS enhancer (789 bp; chr7:156,791,087-156,791,875; hg38) variants are shown as blue bars, whereas variants discovered at orthologous positions in the ZRS enhancer of other species are shown as yellow bars. Red arrows indicate ectopic anterior LacZ staining. For positions with multiple reported variants, results for only one variant are shown (401A>C and 404G>A), but the respective other variants at the same position also show anterior expression gain (Table S2). Numbers of embryos with LacZ staining in the anterior limb bud (red) over the total number of transgenic embryos screened are indicated. See Table S2 for details.

Figure 3:. Variant knock-in mice accurately reproduce human phenotypes.

(A) CRISPR/Cas9-mediated human variant knock-in into the mouse ZRS enhancer. Schematic of the mouse Shh locus is shown (left, not to scale). The ZRS is located in intron five of the Lmbr1 gene (intron-exon structure not shown), 850 kb away from the promoter of Shh. A CRISPR/Cas9-modified mouse Shh locus with a human ZRS variant is shown below. A representative image of a wild-type E18.5 mouse hindlimb skeletal preparation, stained for bone (red) and cartilage (blue), is shown on the right; f, fibula; t, tibia; a, autopod; 1-5, digit numbers. (B-C) LacZ staining in the hindlimbs of transgenic E11.5 mouse embryos containing human ZRS enhancer alleles (first column). Red arrows indicate ectopic LacZ staining in the anterior limb bud. Hindlimb skeletal preparations from E18.5 mice (second column), with genotyping sequence traces confirming the variant knock-ins at the endogenous mouse ZRS enhancer locus (third column) shown. Numbers indicate how many embryos exhibited the representative limb phenotype (B: polydactyly, C: wild-type) over the total number of embryos with the targeted genotype. Red asterisk: extra digit. See Figure S4 and STAR Methods for all knock-in mice and details.

Figure 4:. Systematic mutagenesis of the ZRS enhancer.

Enhancer mutagenesis strategy and results. Shown is the human ZRS enhancer in which we introduced either 40 base pair substitutions (left, 5% mutagenesis) or 16 base pair substitutions (right, 2% mutagenesis). The known variants from Figure 2 and nucleotides immediately neighboring them were excluded (Table S1). Pie charts below summarize results for all mutagenesis alleles tested in each of the groups. Schematic illustration of limb buds with corresponding LacZ staining is shown.

Figure 5:. Identification of novel pathogenic variants.

(A) Dissection of gain-of-function compound mutant. Representative LacZ-stained forelimbs of transgenic E11.5 mouse embryos (right) containing mutagenized human enhancer alleles (left) are shown. Red arrows indicate ectopic LacZ staining in the anterior portion of the limb bud. Numbers of embryos with LacZ staining in the anterior limb bud over the total number of transgenic embryos screened are indicated. (B) Dissection of loss-of-function compound mutant. All individual variants resulted in normal enhancer activity. Two of 16 constructs with normal activity are shown as examples. Numbers of embryos with LacZ staining in the posterior margins of limb buds over the total number of transgenic embryos screened are indicated. See STAR Methods for details.