Fig. 6: WGCNA network hub-gene enrichment of variant-restricted genes uncover disease risk of EBF3 for craniofacial development.

a Histogram of the number of gene-scrambled modules that have protein–protein interaction (ppi) enrichment at a Bonferroni-adjusted one-sided p value <0.05. The vertical orange line marks the number of modules that have significant ppi in the observed WGCNA network (permutation p value = 0.01). b Histogram of (LOEUF) deciles of hub genes and randomly selected non-hub genes from all modules in the WGCNA network. Deciles range from decile 1 (d1), which represents the most constrained genes, to d10, genes that are the most tolerant to putative loss-of-function (pLoF) variation. The bars for the expected overlap represent the median and lines represent the standard deviation calculated from 1000 iterations of randomly selected genes available from gnomAD (n = 17,277). c Network showing some of the top-scoring genes (Supplementary Data 6) in the black module, along with known disease genes. The edges represent the Pearson correlation (>0.9) between genes. Blue edges connect genes that are highly correlated to EBF3. Genes are represented by nodes which are characterized by shape, fill color, border color, and size. A black fill indicates the gene has craniofacial-specific expression (Gini ≥0.5). Known disease genes are marked with a Fuschia border. If the gene has been assigned three or more CFSEs it has a diamond shape, one CFSE is square, and 0 are circles. A node with a large size is a hub gene. Genes which have several of these criteria are displayed with larger and bold text. d CNCC Hi-C interaction plot of the EBF3 locus, data from¹²¹. Below is a zoomed-in genome browser shot of EBF3 locus showing 25-state ChromHMM segmentation from a culture model of CNCCs, primary embryonic CF tissue, embryonic heart and fetal cortex, and CS13 through CS20 of primary human craniofacial tissue. Two orofacial clefting GWAS SNPs reside in and near the TAD boundary. e Significance and fold enrichments of overlaps of genes from our prioritization strategy (Prioritized), CF Gini genes (Gini), WGCNA hubs (Hubs), and genes targeted by CFSEs (CFSE) with genes identified with at least one DNM (All notation) or multiple DNMs (Multi notation) from orofacial clefting trios.