PMCID string | Title string | Sentences string |
|---|---|---|
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | f Scattered dot plot of relative levels of IRF6 mRNA in control versus ETS2-depleted HEKn assessed by qRT-PCR. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Expression levels of IRF6 are normalized against ACTB. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Data are represented as mean ± SD from three replicates. |
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 | g Model showing binding of ETS2 to the IRF6 −22 kb enhancer, which is favored by the risk allele and which reduces IRF6 expression. |
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 Consensus ETS2 binding motif from the JASPAR database of transcription factor DNA-binding preferences (Matrix ID: MA1484.1) and alignment of the variant site in several mammals. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | The risk allele (C) is the reference allele but has a lower frequency than the non-risk allele (T) in most populations. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | The risk allele improves the match to the ETS2 binding site which remains partial. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | b, c Percent input identified by ChIP-qPCR for anti-ETS2 and anti-H3K27Ac respectively in iOE cells heterozygous for rs661849 using primers specific to the IRF6 −22 kb enhancer site or, as a negative control, to a region 103.7 kb upstream IRF6 transcription start site lacking ATAC-Seq and H3K27Ac ChIP-Seq signals in HIOEC or NHEK. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Error bars refer to three ChIP replicates and expressed as mean ± SD. |
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 | NS non-significant. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | d Sequencing of anti-ETS2 and anti-H3K27Ac ChIP-PCR product of cells heterozygous for rs661849 using the indicated antibody. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | e Scattered dot plot of relative luciferase activity of the IRF6 −22 kb reporter construct (longest version) harboring the risk allele of rs661849 in control versus ETS2-depleted primary neonatal keratinocytes (HEKn). |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Data are represented as mean ± SD from three 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 | f Scattered dot plot of relative levels of IRF6 mRNA in control versus ETS2-depleted HEKn assessed by qRT-PCR. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Expression levels of IRF6 are normalized against ACTB. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Data are represented as mean ± SD from three replicates. |
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 | g Model showing binding of ETS2 to the IRF6 −22 kb enhancer, which is favored by the risk allele and which reduces IRF6 expression. |
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 | Similarly, we observed that the risk allele of the FOXE1 24 kb SNP diminished the predicted affinity for four transcription factors (GATA2, FOXH1, GCM2, and NRL) and elevated it for three transcription factors (TFAP2A, SP2, and KLF12) (Supplementary Data 18, 19). |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | While we did not attempt chromatin immunoprecipitation (ChIP) with antibodies to transcription factors at this locus, ChIP with an anti-H3K27Ac antibody precipitated more of the risk allele of the FOXE1 24 kb SNP from heterozygous iOE cells (Supplementary Fig. 20a–c), consistent with higher activity of the enhancer and higher expression of FOXE1 in cells with the risk genotype. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Finally, to assess whether the IRF6 −10 kb (rs11119348) and IRF6 -22 kb (rs661849) SNPs can explain the heritable risk for CL/P associated with 1q32/IRF6 we conducted conditional analyses. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | We previously found evidence for multiple independent signals at IRF6 by LD-clumping, and different patterns of association with OFC subtypes have been reported by several studies. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | We therefore performed conditional analyses separately for cleft lip only (CL) and cleft lip and palate (CLP) cases to explore the contributions of each SNP to each phenotype (Fig. 6a–h and Supplementary Data 20, 21). |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | As expected, the IRF6 locus was associated with CL (lead SNP rs12403599, P value = 3.29 × 10) (Fig. 6a) and CLP (lead SNP rs2076149, P value = 5.24 × 10) (Fig. 6e and Supplementary Data 22). |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Conditioning on either the IRF6 -22 kb or IRF6 −10 kb SNPs explained some of the signal at the IRF6 locus for both CL and CLP but neither SNP alone reduced the association peak fully, as evidenced by P values and odds ratios indicating residual evidence of association (Supplementary Data 22). |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Importantly, the impact of conditioning on either SNP alone was different for CL than for CLP due to differences in linkage disequilibrium between the functional SNPs and other SNPs at the locus associated with each phenotype. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Neither the IRF6 −10 kb SNP nor the IRF6 -22 kb SNP, separately or in combination, completely accounted for the signal tagged by lead SNP rs2076149 in CL, which saw some increases in P value but only minor changes in effect size in each analysis. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | For CLP, the IRF6 −10 kb SNP accounted for more of the signal than the IRF6 -22 kb SNP and conditioning on both SNPs showed no remaining evidence of association at the 1q32/IRF6 locus by both P value and effect size. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Together, we conclude that both SNPs account for the majority of the association signal at 1q32/IRF6 observed in this dataset for CL and CLP.Fig. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | 6The haplotypes containing rs11119348 and rs661849 together explain much of the CL association, and most of the CLP association, in Europeans at the 1q32/IRF6 locus.a–h Locus Zoom plots of a–d cleft lip only and e–h cleft lip and palate; a, e unconditioned; b, f conditioned on rs661849 (IRF6 −22 kb); c, g conditioned on rs1119348 (IRF6 −10 kb); d, h conditioned on both SNPs simultaneously. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Two-sided P values are calculated with a logistic regression with sex without adjustment for multiple testing and ten principal components of ancestry as covariates (and the genotype of the SNP being conditioned on). |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Points are color-coded based on linkage disequilibrium (r) in Europeans for each SNP with rs661849. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | a–h Locus Zoom plots of a–d cleft lip only and e–h cleft lip and palate; a, e unconditioned; b, f conditioned on rs661849 (IRF6 −22 kb); c, g conditioned on rs1119348 (IRF6 −10 kb); d, h conditioned on both SNPs simultaneously. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Two-sided P values are calculated with a logistic regression with sex without adjustment for multiple testing and ten principal components of ancestry as covariates (and the genotype of the SNP being conditioned on). |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Points are color-coded based on linkage disequilibrium (r) in Europeans for each SNP with rs661849. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | To understand how common variants predispose individuals to orofacial cleft (OFC) here we tested 887 OFC-associated SNPs in a massively parallel reporter assay (MPRA) as has been done in other studies seeking to identify SNPs that affect enhancer activity. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Because enhancers are often cell type specific, we limited the study to SNPs from loci where the presumed effector gene is expressed in oral epithelium (as opposed to oral mesenchyme or brain) and used a cell line model of this tissue for the MPRA. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Filtering SNPs with allele-specific effects in the MPRA for those lying in chromatin with evidence of enhancer activity in relevant cell types, or in explants of human embryonic faces, promoted the candidacy of MPRA-nominated SNPs near FOXE1, IRF6, MAFB, TFAP2A, and TP63. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | We then used luciferase reporter assays to confirm the findings of the MPRA at six SNPs. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | At five of these six SNPs, either or both variants had activity different from that of the basal promoter (FDR <0.01), the exception being a SNP at TP63. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Finally, we engineered the genome of iPSCs to be homozygous for the risk or non-risk allele of the three top-candidate SNPs (separately) and differentiated the cells into embryonic oral epithelium. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | All three SNPs had allele-specific effects on expression levels of the respective effector gene. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | The risk alleles of both functional SNPs at IRF6 decreased the expression level of IRF6, which is consistent with loss-of-function mutations in IRF6 causing Van der Woude syndrome. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | The risk allele of the functional SNP at FOXE1 increased the expression level of FOXE1. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Both gain- and loss-of-function mutations in FOXE1 are detected in individuals with Bamforth–Lazarus Syndrome (which includes OFC) and, similarly, both a loss-of-function mutation in Foxe1 and over-expression of Foxe1 can cause cleft palate in mice. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | We identified transcription factors whose binding is affected by the SNP allele for both SNPs at IRF6, and showed that they regulate the expression of the enhancers harboring the SNPs, and of IRF6 expression levels, in the predicted direction. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Finally, we returned to the GWAS data and found that the haplotypes containing the two SNPs at IRF6 explain most of the CLP association signal at this locus in the cohorts evaluated. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | In summary, we found strong evidence for three functional SNPs and suggestive evidence for three more. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | An objective in identifying functional SNPs is to determine the transcription factors whose binding is impacted by the SNPs allele, as the genes encoding such TFs may be associated with OFC risk. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Supporting this possibility, earlier we found that a functional SNP in the enhancer IRF6 MCS9.7 affects binding of IRF6 itself , and here we found that the IRF6 −10 kb SNP affects the binding affinity of FOXE1, encoded by a gene implicated in risk for syndromic and nonsyndromic OFC. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | We infer that FOX family members expressed in oral epithelium are candidates to be OFC risk genes. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | In this light, it is intriguing that chromatin immunoprecipitation with sequencing (ChIP-seq) revealed binding of FOXA1 and FOXA2 in bronchial epithelial cells at the IRF6 −10 kb enhancer, and knocking down expression of either transcription factor led to upregulation of IRF6. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Another implication is that there may be a greater-than-additive effect on OFC risk in individuals harboring risk-associated haplotypes at both IRF6 and FOXE1. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Unfortunately, current GWAS data are underpowered to reveal such gene-by-gene interactions. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | It would be interesting to test for genetic interactions between IRF6 and FOXE1 in model organisms or iPSCs. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | The risk allele of the IRF6 −22 kb SNP creates a low-affinity ETS family binding site, and ChIP-qPCR indicated that the risk allele promotes binding of ETS2. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Low-affinity ETS binding sites are important for the regulation of SHH in the limb. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Mice embryos homozygous for a strong loss-of-function mutation in ETS2 die by E8.5; heterozygous mutants exhibit craniosynostosis, probably reflecting a role for ETS2 in osteogenesis within the fibroblast growth factor (FGF) signaling pathway. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Mutations in ETS2 have been detected in patients with craniosynostosis, and with a developmental disorder that includes cleft palate in some individuals. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | It is expressed in differentiated keratinocytes and glandular epithelium and regulates the differentiation of trophoblast stem cells. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | We infer that ETS2 and is paralogs, in particular ETS1, which inhibits differentiation of keratinocytes, are potential contributors to isolated OFC, and that genetic interaction between IRF6 and ETS2 in OFC patients and animal models are worth exploring. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | The IRF6 -10 kb SNP is the third example of a SNP with relatively strong evidence of directly influencing risk for cleft lip, cleft palate, or both within the same evolutionarily conserved enhancer of IRF6, MCS9.7. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Of note, this enhancer was identified through investigation of a non-syndromic CL/P-associated SNP, rs642961, also with some evidence of being functional. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | In stable transgenic mice, MCS9.7 (from the human genome) drove reporter expression in oral epithelium; in stable transgenic zebrafish, it drove reporter expression in periderm in the entire embryo, including in the oral cavity. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | While the risk-associated allele of rs642961 disrupts binding of the transcription factor AP2-α (TFAP2A) in an electrophoretic mobility shift assay, current and previous MPRA and luciferase results all did not detect allele-specific effects of rs642961 on enhancer activity. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | It is possible that the IRF6 −22 kb SNP, which is in linkage disequilibrium with rs642961, is the functional SNP underlying the association of rs642961 with OFC; supporting this notion both IRF6 −22kb SNP and rs642961 are more strongly associated with CL than with CLP. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | By contrast, a rare, single-nucleotide insertion in MCS9.7 segregating with cleft palate and other characteristic symptoms in a Brazilian family with Van der Woude syndrome strongly affected the enhancer activity of MCS9.7 . |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | In addition, we recently found that a SNP in MCS9.7, rs570516915, is associated with CP only in populations from Finland and Estonia and that the risk allele reduces IRF6 expression level in engineered induced oral epithelial cells. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | The current study shows that IRF6 -10 kb (rs11119348), associated with CL and CLP and in the same enhancer, affects IRF6 expression potentially by altering the binding of FOXE1. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Taken together, these studies emphasize the extraordinary importance of a single enhancer in genetic predisposition for orofacial cleft, and raise the question of how different variants in this enhancer elevate risk for cleft lip with or without cleft palate or for cleft palate only. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | There remains a need for high-throughput methods to screen functional SNPs in OFC and other common disorders. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | The MPRA we performed required the synthesis of a library of oligos. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | This step was avoided in an MPRA that used reporter constructs from sheared chromatin from individuals of distinct genotypes; this study managed to score more than 50 million SNPs in two cell types. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | However, the success of MPRAs at identifying functional SNPs at many loci is likely to rely on the cell line deployed being a reasonable model of the embryonic tissue where the SNP affects disease risk in vivo. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | While results from an MPRA helped us identify six OFC-associated SNPs that alter enhancer activity in luciferase reporter assays, we only pursued three of them with the in-depth experiments, confirming that they can alter IRF6 or FOXE1 expression in induced oral epithelium cells. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | It will be interesting to assess whether additional MPRA-nominated SNPs within active enhancers near IRF6 are indeed functional, and if they affect IRF6 expression in combination. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | However, the step of engineering the genome in iPSCs and creating induced oral epithelium or oral mesenchyme remains laborious and expensive. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | One promising approach to prioritize non-coding SNPs is chromatin-accessibility QTL analyses but these, too, require appropriate cell line models. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | It will also be interesting to assess the effect of functional SNPs on the expression of genes downstream of the directly affected transcription factor, and to establish OFC-prone animal models where the phenotypic effects of single SNPs might be evident. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | In many GWAS loci, the effector gene is unclear, which increases the challenge of identifying functional SNPs; in addition, validated functional SNPs may contribute to disease etiology by directly affecting the expression of genes other than the apparent effector gene. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Despite these challenges, identifying functional SNPs remains an essential step in translating the findings of GWAS into an understanding of how common genetic variants contribute to the pathogenesis of OFC and other congenital anomalies. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Our research complies with all relevant ethical regulations. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | All GWAS data used in this study have been previously published and were anonymized/deidentified to all authors on this manuscript. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | The WTC-11 cell line (UCSFi001-A (RRID:CVCL_Y803)) was ethically derived (https://hpscreg.eu/cell-line/UCSFi001-A#ethics). |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | The details of the donor are provided at the human pluripotent stem cell registry, and the derivation of the cells underwent ethical review by a committee at the University of California, San Francisco. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | The IRB protocol and approval numbers are both 10-02521. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | We performed a genome-wide meta-analysis of CL/P, CP, and all OFCs, from European and Asian ancestries from two consortia. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | The details of the sample collections, genotyping, and genotype quality control (QC) procedures have been described previously. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Briefly, the original studies recruited individuals with OFCs, their unaffected relatives, and unrelated controls (individuals with no known family history of OFCs or other craniofacial anomalies; N = 1626) from 18 sites across 13 countries from North America, Central or South America, Asia, Europe and Africa. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Samples from the GENEVA consortium were genotyped for 589,945 SNPs on the Illumina Human610-Quadv.1_B BeadChip and imputed to the 1000 Genomes Phase 1 release (June 2011) reference panel. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Samples from the POFC consortium were genotyped for approximately 580,000 SNPs from the Illumina HumanCore+Exome array, of which ~539,000 SNPs passed quality control filters recommended by the Center for Inherited Disease Research (CIDR) and the Genetics Coordinating Center (GCC) at the University of Washington. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | These data were then phased with SHAPEIT2 and imputed with IMPUTE2 to the 1000 Genomes Project Phase 3 release (September 2014) reference panel. |
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