Fig. 3: Two SNPs at IRF6 and one SNP at FOXE1 have allele-specific effects on enhancer activities and expression levels of the adjacent OFC risk-associated genes.
From: Identification of functional non-coding variants associated with orofacial cleft
a Bar chart of MPRA and luciferase reporter activities for the indicated SNPs at various loci in GMSM-K using elements of the indicated length centered on the SNP. MPRA data are represented as mean ± standard error of the mean (SEM) from the indicated number of replicates. Statistical significance (P value, two-tailed) of the difference between major and minor allele’s reporter activity is determined by Welch’s t-test, followed by Benjamini–Hochberg false discovery rate correction. P (MPRA) = 0.0452 (rs11110348), 0.0057 (rs661849), 0.9097 (rs642961), 0.6942 (rs4844939), 0.4487 (rs12104876), 0.0238 (rs75436877), 0.7100 (rs79482068), 0.0734 (rs79792381), 0.0125 (rs201265), 0.1206 (rs10124184), 0.0067 (rs10984103), 0.0458 (rs4812449), 0.2945 (rs57369620). For luciferase reporter activities, data are represented as mean ± standard deviation (SD) from four independent experiments. Statistical significance (P value, two-tailed) is determined by Student’s t-test. P (luciferase) = 0.0006 (rs11110348), <0.0001 (rs661849), 0.3822 (rs642961), <0.0001 (rs4844939), 0.14 (rs12104876), <0.0001 (rs75436877), 0.0693 (rs79482068), 0.5115 (rs79792381), 0.002 (rs201265), <0.0001 (rs10124184), <0.0001 (rs10984103), <0.0001 (rs4812449), 0.1421 (rs57369620). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, NS non-significant. b–d Scattered dot plots of relative luciferase activity (using the longer elements described in Results) for non-risk and risk alleles of rs11119348, rs661849 and rs10984103 in primary neonatal keratinocytes (HEKn). Value of 1 is that of the empty pGL3 promoter vector. Data are represented as mean ± standard deviation (SD) from four independent experiments. Statistical significance (P value, two-tailed) is determined by Student’s t-test and P value is indicated on the plot. e–g Scattered dot plot of relative levels of e, f IRF6 or g FOXE1 mRNA in edited induced oral epithelium (iOE) cells homozygous for the non-risk or risk alleles of each SNP, as indicated, assessed by qRT-PCR. Expression levels are normalized against those of ACTB, GAPDH, HPRT1, UBC and CDH1. Data were represented as mean ± SD from e, f six replicates or g nine replicates of cells harboring each genotype, as indicated in the plot. Each dot represents three technical qPCR replicates. Statistical significance (P value, two-tailed) is determined by Student’s t-test and P value is indicated on the plot. Source data are provided as a Source Data file.
