Variant to gene mapping for carpal tunnel syndrome risk loci implicates skeletal muscle regulatory elements.

BACKGROUND: Carpal tunnel syndrome (CTS) is a common disorder caused by compression of the median nerve in the wrist, resulting in pain and numbness throughout the hand and forearm. While multiple behavioural and physiological factors influence CTS risk, a growing body of evidence supports a strong genetic contribution. Recent genome-wide association study (GWAS) efforts have reported 53 independent signals associated with CTS. While GWAS can identify genetic loci conferring risk, it does not determine which cell types drive the genetic aetiology of the trait, which variants are "causal" at a given signal, and which effector genes correspond to these non-coding variants. These obstacles limit interpretation of potential disease mechanisms. METHODS: We analysed CTS GWAS findings in the context of chromatin conformation between gene promoters and accessible chromatin regions across cellular models of bone, skeletal muscle, adipocytes and neurons. We identified proxy variants in high LD with the lead CTS sentinel SNPs residing in promoter connected open chromatin in the skeletal muscle and bone contexts. FINDINGS: We detected significant enrichment for heritability in skeletal muscle myotubes, as well as a weaker correlation in human mesenchymal stem cell-derived osteoblasts. In myotubes, our approach implicated 117 genes contacting 60 proxy variants corresponding to 20 of the 53 GWAS signals. In the osteoblast context we implicated 30 genes contacting 24 proxy variants coinciding with 12 signals, of which 19 genes shared. We subsequently prioritized BZW2 as a candidate effector gene in CTS and implicated it as novel gene that perturbs myocyte differentiation in vitro. INTERPRETATION: Taken together our results suggest that the CTS genetic component influences the size, integrity, and organization of multiple tissues surrounding the carpal tunnel, in particular muscle and bone, to predispose the nerve to being compressed in this disease setting. FUNDING: This work was supported by NIH Grant UM1 DK126194 (SFAG and WY), R01AG072705 (SFAG & KDH) and the Center for Spatial and Functional Genomics at CHOP (SFAG & ADW). SFAG is supported by the Daniel B. Burke Endowed Chair for Diabetes Research. WY is supported by the Perelman School of Medicine of the University of Pennsylvania.

Foxp3 depends on Ikaros for control of regulatory T cell gene expression and function.

Ikaros is a transcriptional factor required for conventional T cell development, differentiation, and anergy. While the related factors Helios and Eos have defined roles in regulatory T cells (Treg), a role for Ikaros has not been established. To determine the function of Ikaros in the Treg lineage, we generated mice with Treg-specific deletion of the Ikaros gene (Ikzf1). We find that Ikaros cooperates with Foxp3 to establish a major portion of the Treg epigenome and transcriptome. Ikaros-deficient Treg exhibit Th1-like gene expression with abnormal production of IL-2, IFNg, TNFa, and factors involved in Wnt and Notch signaling. While Ikzf1-Treg-cko mice do not develop spontaneous autoimmunity, Ikaros-deficient Treg are unable to control conventional T cell-mediated immune pathology in response to TCR and inflammatory stimuli in models of IBD and organ transplantation. These studies establish Ikaros as a core factor required in Treg for tolerance and the control of inflammatory immune responses.

GWAS-informed data integration and non-coding CRISPRi screen illuminate genetic etiology of bone mineral density.

Over 1,100 independent signals have been identified with genome-wide association studies (GWAS) for bone mineral density (BMD), a key risk factor for mortality-increasing fragility fractures; however, the effector gene(s) for most remain unknown. Informed by a variant-to-gene mapping strategy implicating 89 non-coding elements predicted to regulate osteoblast gene expression at BMD GWAS loci, we executed a single-cell CRISPRi screen in human fetal osteoblast 1.19 cells (hFOBs). The BMD relevance of hFOBs was supported by heritability enrichment from cross-cell type stratified LD-score regression involving 98 cell types grouped into 15 tissues. 24 genes showed perturbation in the screen, with four (ARID5B, CC2D1B, EIF4G2, and NCOA3) exhibiting consistent effects upon siRNA knockdown on three measures of osteoblast maturation and mineralization. Lastly, additional heritability enrichments, genetic correlations, and multi-trait fine-mapping revealed that many BMD GWAS signals are pleiotropic and likely mediate their effects via non-bone tissues that warrant attention in future screens.

Variant-to-function analysis of the childhood obesity chr12q13 locus implicates rs7132908 as a causal variant within the 3' UTR of FAIM2.

The ch12q13 locus is among the most significant childhood obesity loci identified in genome-wide association studies. This locus resides in a non-coding region within FAIM2; thus, the underlying causal variant(s) presumably influence disease susceptibility via cis-regulation. We implicated rs7132908 as a putative causal variant by leveraging our in-house 3D genomic data and public domain datasets. Using a luciferase reporter assay, we observed allele-specific cis-regulatory activity of the immediate region harboring rs7132908. We generated isogenic human embryonic stem cell lines homozygous for either rs7132908 allele to assess changes in gene expression and chromatin accessibility throughout a differentiation to hypothalamic neurons, a key cell type known to regulate feeding behavior. The rs7132908 obesity risk allele influenced expression of FAIM2 and other genes and decreased the proportion of neurons produced by differentiation. We have functionally validated rs7132908 as a causal obesity variant that temporally regulates nearby effector genes and influences neurodevelopment and survival.

Perturbation of the insomnia WDR90 genome-wide association studies locus pinpoints rs3752495 as a causal variant influencing distal expression of neighboring gene, PIG-Q.

Although genome-wide association studies (GWAS) have identified loci for sleep-related traits, they do not directly uncover the underlying causal variants and corresponding effector genes. The majority of such variants reside in non-coding regions and are therefore presumed to impact cis-regulatory elements. 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 phosphatidyl inositol glycan (PIG)-Q as a functionally relevant gene at the insomnia "WDR90" GWAS locus. However, importantly that effort did not characterize the corresponding underlying causal variant. Specifically, our previous 3D genomic datasets nominated a shortlist of three neighboring single nucleotide polymorphisms (SNPs) in strong linkage disequilibrium within an intronic enhancer region of WDR90 that contacted the open PIG-Q promoter. We sought to investigate the influence of these SNPs collectively and then individually on PIG-Q modulation to pinpoint the causal "regulatory" variant. Starting with gross level perturbation, deletion of the entire region in NPCs via CRISPR-Cas9 editing and subsequent RNA sequencing revealed expression changes in specific PIG-Q transcripts. Results from individual luciferase reporter assays for each SNP in iPSCs revealed that the region with the rs3752495 risk allele (RA) induced a ~2.5-fold increase in luciferase expression. Importantly, rs3752495 also exhibited an allele-specific effect, with the RA increasing the luciferase expression by ~2-fold versus the non-RA. In conclusion, our variant-to-function approach and in vitro validation implicate rs3752495 as a causal insomnia variant embedded within WDR90 while modulating the expression of the distally located PIG-Q.

Shared and unique 3D genomic features of substance use disorders across multiple cell types.

Recent genome-wide association studies (GWAS) have revealed shared genetic components among alcohol, opioid, tobacco and cannabis use disorders. However, the extent of the underlying shared causal variants and effector genes, along with their cellular context, remain unclear. We leveraged our existing 3D genomic datasets comprising high-resolution promoter-focused Capture-C/Hi-C, ATAC-seq and RNA-seq across >50 diverse human cell types to focus on genomic regions that coincide with GWAS loci. Using stratified LD regression, we determined the proportion of genomewide SNP heritability attributable to the features assayed across our cell types by integrating recent GWAS summary statistics for the relevant traits: alcohol use disorder (AUD), tobacco use disorder (TUD), opioid use disorder (OUD) and cannabis use disorder (CanUD). Statistically significant enrichments (P<0.05) were observed in 14 specific cell types, with heritability reaching 9.2-fold for iPSC-derived cortical neurons and neural progenitors, confirming that they are crucial cell types for further functional exploration. Additionally, several pancreatic cell types, notably pancreatic beta cells, showed enrichment for TUD, with heritability enrichments up to 4.8-fold, suggesting genomic overlap with metabolic processes. Further investigation revealed significant positive genetic correlations between T2D with both TUD and CanUD (FDR<0.05) and a significant negative genetic correlation with AUD. Interestingly, after partitioning the heritability for each cell type's cis-regulatory elements, the correlation between T2D and TUD for pancreatic beta cells was greater (r=0.2) than the global genetic correlation value. Our study provides new genomic insights into substance use disorders and implicates cell types where functional follow-up studies could reveal causal variant-gene mechanisms underpinning these disorders.