Perturbation of the insomnia WDR90 GWAS locus pinpoints rs3752495 as a causal variant influencing distal expression of neighboring gene, PIG-Q

Although genome wide association studies (GWAS) have been crucial for the identification of loci associated with sleep traits and disorders, the method itself does not directly uncover the underlying causal variants and corresponding effector genes. The overwhelming majority of such variants reside in non-coding regions and are therefore presumed to impact the activity of cis-regulatory elements, such as enhancers. Our previously reported variant-to-gene mapping effort in human induced pluripotent stem cell (iPSC)-derived neural progenitor cells (NPCs), combined with validation in both Drosophila and zebrafish, implicated PIG-Q as a functionally relevant gene at the insomnia WDR90 locus. However, importantly that effort did not characterize the corresponding underlying causal variant at this GWAS signal. Specifically, our genome-wide ATAC-seq and high-resolution promoter-focused Capture C datasets generated in this cell setting brought our attention to a shortlist of three tightly neighboring single nucleotide polymorphisms (SNPs) in strong linkage disequilibrium in a candidate intronic enhancer region of WDR90 that contacted the open PIG-Q promoter. The objective of this study was to investigate the influence of the proxy SNPs collectively and then individually on PIG-Q modulation and to pinpoint the causal "regulatory" variant among the three SNPs. Starting at a gross level perturbation, deletion of the entire region harboring all three SNPs in human iPSC-derived neural progenitor cells via CRISPR-Cas9 editing and subsequent RNA sequencing revealed expression changes in specific PIG-Q transcripts. Results from more refined individual luciferase reporter assays for each of the three SNPs in iPSCs revealed that the intronic region with the rs3752495 risk allele induced a [~]2.5-fold increase in luciferase expression (n=10). Importantly, rs3752495 also exhibited an allele specific effect, with the risk allele increasing the luciferase expression by [~]2-fold compared to the non-risk allele. In conclusion, our variant-to-function approach and subsequent in vitro validation implicates rs3752495 as a causal insomnia risk variant embedded at the WDR90-PIG-Q locus.