PMCID string | Title string | Sentences string |
|---|---|---|
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Yellow and red asterisks: non-risk and risk allele, respectively. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | b Scatter dot plot showing (black dots) 65 SNPs with significant allele-specific effects on reporter activity in the MPRA and (gray dots) 822 SNPs without them. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Arrows indicate the functional SNPs identified in this study (two near IRF6, rs11119348 and rs661849, and one near FOXE1, rs10984103). |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Dashed lines indicate the 95th percentile of the reporter activity of scrambled elements; on both axes, zero, i.e., log2(1) represents the reporter activity of the empty vector. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | The number of SNPs tested, and the number with allele-specific effects, in the MPRA at each locus In the MPRA results we assessed the enhancer activity of each element and whether the SNP allele affected it. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Of the 887 elements evaluated, one or both variants of 480 (54.1%) had activity significantly different from that of the basal promoter (false discovery rate (FDR) <0.01); 180 (20.3%) of them exhibited higher activity than that of the basal promoter (Supplementary Data 6 and Supplementary Fig. 1b). |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | As expected, the average activity of 84 scrambled genomic test elements was very close to that to the basal promoter (Supplementary Fig. 1c and Supplementary Data 7). |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Unexpectedly, the enhancer activities of the test elements were only weakly correlated with levels of histone H3 lysine 27 acetylation (H3K27Ac), a mark of active enhancers, in primary normal human epidermal keratinocytes (NHEK) (Pearson correlation coefficient r = 0.1126) (Supplementary Fig. 1d). |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Further, the enhancer activities of four elements from within a known oral epithelium enhancer (IRF6 MCS9.7) were not higher than that of the basal promoter (Supplementary Fig. 1c). |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | These unexpected findings may reflect the semi-epithelial phenotype of this cell line, discussed below. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Nonetheless, 65 (7%) of the SNPs tested had allele-specific enhancer activity in the MPRA (Fig. 1b and Supplementary Data 6) (FDR <0.05), nominating these SNPs as candidates to be functional. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | These SNPs were enriched among elements whose activity was different from that of the basal promoter (P < 0.0001, one-sided Fisher’s exact test). |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Each of the eight loci had at least one of the 65 MPRA-nominated SNPs; notably, at the 1q32/IRF6 locus, there were 46 such SNPs (Figs. 1b, 2a, Table 1, and Supplementary Data 6). |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | We filtered MPRA-nominated SNPs for those lying within enhancers active in embryonic oral epithelium. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | In one approach to identify such enhancers, we used NHEKs as a model for embryonic oral epithelium and the activity-by-contact (ABC) method to identify enhancers for the presumed effector genes. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | This method incorporates cell type-specific RNA-seq, DNAse hypersensitivity, and H3K27Ac ChIP-seq datasets, all of which are available for NHEKs, and averaged HiC chromatin-contact data from ten ENCODE cell types. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Two apparent enhancers for IRF6 and one for FOXE1 contained MPRA-nominated SNPs (i.e., rs11119348 and rs661849 near IRF6, rs10984103 near FOXE1) (Fig. 2a–c). |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | The first candidate SNP near IRF6, rs11119348, lies within IRF6 MCS9.7. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | This element is the site of a rare single-base-pair duplication that appears to cause Van der Woude syndrome. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | It also contains three other common SNPs (i.e., in addition to rs11119348) that are associated with non-syndromic OFC, including rs642961, the focus of an earlier study. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | We tested all four of these common SNPs (separately) in the MPRA but only rs11119348 had allele-specific effects in it (Supplementary Fig. 1c). |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | We will refer to this as the “IRF6 −10 kb SNP” (Fig. 2b).Fig. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | 2Two SNPs (rs11119348 and rs661849) at IRF6 and one SNP (rs10984103) at FOXE1 as regulatory variants.a–c UCSC browser views of the human genome, GRCh37/hg19, focused on the OFC-associated SNPs at a, b IRF6 and c FOXE1. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | OFC SNPs, SNPs identified by GWAS and evaluated by MPRA at each locus. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | One SNP at the FOXE1 locus, which was tested in the MPRA but lacked significant effects in it, rs1877431, is outside of the range presented here. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | MPRA-sig SNPs, SNPs with allele-specific effects in the MPRA. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | IRF6 enhancers, FOXE1 enhancers, and enhancers for the indicated gene identified using the activity-by-contact (ABC) method and datasets from NHEK (see Methods). |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Gray arrows, GWAS-meta-analysis P values of the indicated SNPs. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Two-sided P values are calculated with an inverse-variance weighted fixed-effects meta-analysis of a transmission disequilibrium test without adjustment for multiple testing and a logistic regression (that had 18 principal components of ancestry as covariates). |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | GM12878 B-cell derived cell line, H1-ESC embryonic stem cells, K562 myelogenous leukemia cell line, HepG2 liver cancer cell line, HUVEC human umbilical vein endothelial cells, HMEC human mammary epithelial cells, HSMM human skeletal muscle myoblasts, NHEK normal human epidermal keratinocytes, NHLF normal human lung fibroblasts. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | CS 13-20, Carnegie stages for human embryonic face explants. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Colored bars, inferred chromatin state from combinatorial analysis of multiple chromatin mark datasets. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Orange and yellow, active and weak enhancer element, respectively; bright red, active promoter; light red, weak promoter; purple, inactive/poised promoter; blue, insulator; light green, weakly transcribed; gray, Polycomb repressed; light gray, heterochromatin/repetitive. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Additional color bars in the facial explants dataset, green, transcription; green-yellow, transcription weak enhancer; purple, bivalent promoter; light purple, poised promoter. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | a–c UCSC browser views of the human genome, GRCh37/hg19, focused on the OFC-associated SNPs at a, b IRF6 and c FOXE1. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | OFC SNPs, SNPs identified by GWAS and evaluated by MPRA at each locus. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | One SNP at the FOXE1 locus, which was tested in the MPRA but lacked significant effects in it, rs1877431, is outside of the range presented here. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | MPRA-sig SNPs, SNPs with allele-specific effects in the MPRA. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | IRF6 enhancers, FOXE1 enhancers, and enhancers for the indicated gene identified using the activity-by-contact (ABC) method and datasets from NHEK (see Methods). |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Gray arrows, GWAS-meta-analysis P values of the indicated SNPs. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Two-sided P values are calculated with an inverse-variance weighted fixed-effects meta-analysis of a transmission disequilibrium test without adjustment for multiple testing and a logistic regression (that had 18 principal components of ancestry as covariates). |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | GM12878 B-cell derived cell line, H1-ESC embryonic stem cells, K562 myelogenous leukemia cell line, HepG2 liver cancer cell line, HUVEC human umbilical vein endothelial cells, HMEC human mammary epithelial cells, HSMM human skeletal muscle myoblasts, NHEK normal human epidermal keratinocytes, NHLF normal human lung fibroblasts. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | CS 13-20, Carnegie stages for human embryonic face explants. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Colored bars, inferred chromatin state from combinatorial analysis of multiple chromatin mark datasets. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Orange and yellow, active and weak enhancer element, respectively; bright red, active promoter; light red, weak promoter; purple, inactive/poised promoter; blue, insulator; light green, weakly transcribed; gray, Polycomb repressed; light gray, heterochromatin/repetitive. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Additional color bars in the facial explants dataset, green, transcription; green-yellow, transcription weak enhancer; purple, bivalent promoter; light purple, poised promoter. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | The second candidate SNP in this locus, rs661849, resides within an evolutionarily conserved sequence 21.7 kb upstream of the IRF6 promoter and within the UTP25 promoter (Fig. 2b). |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | There are no other OFC-associated SNPs within this element. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | We will refer to rs661849 as the “IRF6 −22 kb SNP”. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | This SNP is an expression QTL (eQTL) in GTEx for several genes and tissues in the region, including IRF6 in cerebellum (P value = 1.5e-8), and is a splicing QTL (sQTL) for IRF6 in sun-exposed skin (P value = 8.3e-9) and for neighboring gene TRAF3IP3 in whole blood. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Of note, rs642961, mentioned above, is in linkage disequilibrium with the IRF6 −22 kb SNP (R = 0.769) but not with the IRF6 −10 kb SNP (R = 0.033) (based on data from the 1000 Genomes project) (Supplementary Fig. 2 and Supplementary Data 8). |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | The candidate SNP within a FOXE1 enhancer, rs10984103, lies within a predicted enhancer 23.8 kb downstream of the FOXE1 transcription start site (Fig. 2c). |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | This SNP is an eQTL for FOXE1 in multiple tissues, including the testis, esophageal mucosa, nerve, and cerebellum. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | We will refer to this SNP as the “FOXE1 24 kb SNP”. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | In a second, broader approach to identify potential embryonic oral epithelium enhancers that is agnostic to the identity of the effector gene we used chromatin-mark evidence of enhancers active (1) in NHEK (ENCODE), (2) in human immortalized oral epithelial cells (HIOEC), which are potentially a better model of embryonic oral epithelium than NHEK, or (3) in human embryonic faces. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Within elements marked as enhancers in one or more of these contexts there were fourteen MPRA-nominated SNPs, including the three discussed above (Fig. 2b, c); the additional SNPs were detected in the 1q32/IRF6, 6p24.3/TFAP2A, 3q28/TP63, and 20q12/MAFB loci (Supplementary Data 9 and Supplementary Figs. 3–9). |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | In summary, intersecting MPRA-nominated SNPs with predicted enhancers strengthened the candidacy of 14 SNPs as being functional (Table 1 and Supplementary Data 9). |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | We next tested a subset of the MPRA results in traditional luciferase reporter assays in GMSM-K cells, using reporter elements arbitrarily chosen to be 701 bp long and centered on the SNP (Fig. 3a and Supplementary Data 10, 11). |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | For these tests, we picked six SNPs with allele-specific effects in the MPRA and seven SNPs without them. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | The former were all among the fourteen candidates discussed above and included IRF6 −10 kb, IRF6 −22 kb, FOXE1 24 kb, rs4812449 (near MAFB), rs201265 (near TFAP2A), and rs75436877 (near TP63); the latter included rs642961 within IRF6 MCS9.7. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | All six SNPs with allele-specific effects in the MPRA also had them in the luciferase reporter assays, although in two cases, FOXE1 24 kb and rs201265 (near TFAP2A), the direction of effect was reversed (Fig. 3a and Supplementary Data 11, 12). |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Discordance in the direction of effects of SNP alleles between traditional reporter assays and MPRA has been reported in other studies and presumably reflects that the short elements used in MPRAs are not full-sized enhancers (addressed below). |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Among the seven SNPs that did not have allele-specific effects in the MPRA, five also did not have them in the luciferase reporter assays. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | These five included rs642961 within IRF6 MCS9.7, in agreement with a previous report. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | In summary, for eleven of thirteen SNPs tested, luciferase reporter assays agreed with MPRA regarding whether enhancer activity was affected by a SNP allele; this rate of concordance between luciferase and MPRA results is significant (P = 0.0163, one-sided Fisher’s exact test) and matches or exceeds that of other studies using MPRAs to detect functional non-coding SNPs. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Fig. 3Two 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.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. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | MPRA data are represented as mean ± standard error of the mean (SEM) from the indicated number of replicates. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | 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. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | 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). |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | For luciferase reporter activities, data are represented as mean ± standard deviation (SD) from four independent experiments. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Statistical significance (P value, two-tailed) is determined by Student’s t-test. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | 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). * |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, NS non-significant. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | 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). |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Value of 1 is that of the empty pGL3 promoter vector. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Data are represented as mean ± standard deviation (SD) from four independent experiments. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Statistical significance (P value, two-tailed) is determined by Student’s t-test and P value is indicated on the plot. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | 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. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Expression levels are normalized against those of ACTB, GAPDH, HPRT1, UBC and CDH1. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | 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. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Each dot represents three technical qPCR replicates. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Statistical significance (P value, two-tailed) is determined by Student’s t-test and P value is indicated on the plot. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Source data are provided as a Source Data file. |
PMC12267437 | 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. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | MPRA data are represented as mean ± standard error of the mean (SEM) from the indicated number of replicates. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | 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. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | 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). |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | For luciferase reporter activities, data are represented as mean ± standard deviation (SD) from four independent experiments. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Statistical significance (P value, two-tailed) is determined by Student’s t-test. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | 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). * |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, NS non-significant. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | 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). |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Value of 1 is that of the empty pGL3 promoter vector. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Data are represented as mean ± standard deviation (SD) from four independent experiments. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Statistical significance (P value, two-tailed) is determined by Student’s t-test and P value is indicated on the plot. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | 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. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Expression levels are normalized against those of ACTB, GAPDH, HPRT1, UBC and CDH1. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | 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. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Each dot represents three technical qPCR replicates. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Statistical significance (P value, two-tailed) is determined by Student’s t-test and P value is indicated on the plot. |
Subsets and Splits
No community queries yet
The top public SQL queries from the community will appear here once available.