IRF6 Research Articles

Programmed RNA editing with an evolved bacterial adenosine deaminase.

Programmed RNA editing presents an attractive therapeutic strategy for genetic disease. In this study, we developed bacterial deaminase-enabled recoding of RNA (DECOR), which employs an evolved Escherichia coli transfer RNA adenosine deaminase, TadA8e, to deposit adenosine-to-inosine editing to CRISPR-specified sites in the human transcriptome. DECOR functions in a variety of cell types, including human lung fibroblasts, and delivers on-target activity similar to ADAR-overexpressing RNA-editing platforms with 88% lower off-target effects. High-fidelity DECOR further reduces off-target effects to basal level. We demonstrate the clinical potential of DECOR by targeting Van der Woude syndrome-causing interferon regulatory factor 6 (IRF6) insufficiency. DECOR-mediated RNA editing removes a pathogenic upstream open reading frame (uORF) from the 5' untranslated region of IRF6 and rescues primary ORF expression from 12.3% to 36.5%, relative to healthy transcripts. DECOR expands the current portfolio of effector proteins and opens new territory in programmed RNA editing.

Breast Cancer Stem Cells Upregulate IRF6 in Stromal Fibroblasts to Induce Stromagenesis.

The microenvironment of a cancer stem cell (CSC) niche is often found in coexistence with cancer-associated fibroblasts (CAFs). Here, we show the first in-depth analysis of the interaction between primary triple-negative breast cancer stem cells (BCSCs) with fibroblasts. Using 2D co-culture models with specific seeding ratios, we identified stromal fibroblast aggregation at the BCSC cluster periphery, and, on closer observation, the aggregated fibroblasts was found to encircle BCSC clusters in nematic organization. In addition, collagen type I and fibronectin accumulation were also found at the BCSC-stromal periphery. MACE-Seq analysis of BCSC-encapsulating fibroblasts displayed the transformation of stromal fibroblasts to CAFs and the upregulation of fibrosis regulating genes of which the Interferon Regulatory Factor 6 (IRF6) gene was identified. Loss of function experiments with the IRF6 gene decreased fibroblast encapsulation around BCSC clusters in 2D co-cultures. In BCSC xenografts, fibroblast IRF6 expression led to an increase in the stromal area and fibroblast density in tumors, in addition to a reduction in necrotic growth. Based on our findings, we propose that fibroblast IRF6 function is an important factor in the development of the stromal microenvironment and in sustaining the BCSC tumor niche.

Role of MTHFR, IRF6, PAX7 and TP63 SNPs in susceptibility to non-syndromic orofacial cleft, a candidate gene study in a Portuguese population.

Non-syndromic orofacial cleft (NSOC) is a complex phenotype, involving multiple genetic and environmental factors. Association studies exploring the genetic susceptibility to this prevalent oral malformation show variability of results in different populations. Using a candidate gene approach, we aimed to verify the role of four single-nucleotide polymorphisms (SNPs) in the susceptibility to NSOC in Portuguese patients. A total of 254 non-consanguineous individuals of Portuguese were recruited, including 120 patients with NSOC and 134 controls. About 92% of these patients had non-syndromic cleft lip with or without cleft palate (NSCL/P) and 8% had only non-syndromic cleft palate (NSCP). SNPs in the MTHFR (rs1801133), IRF6 (rs642961), PAX7 (rs742071) and TP63 (rs9332461) genes were studied, using a real-time approach with TaqMan probes. Allelic, genotypic, dominant, recessive and over-dominant models were explored using a chi-squared test. Adjusted p-value was calculated for multiple comparisons using the Benjamini-Hochberg false discovery rate (FDR). All SNPs were in Hardy-Weinberg equilibrium. For MTHFR, IRF6, and PAX7 SNPs, no statistically significant difference was highlighted for any of the evaluated models. For TP63 SNP, data fitted an over-dominant model, with a protective effect for heterozygotes (OR 1.897; CI 95% [1.144-3.147]; p < .016, when comparing controls vs. cases), but significance was lost when applying adjusted p-value for multiple comparisons (4 × 5 tests). In this Portuguese population, there was no evidence of an association between the evaluated SNPs and NSOC. For TP63 SNP, the possibility of a protective effect of heterozygotes should be further investigated.

High incidence and geographic distribution of cleft palate cases in Finland are associated with a regulatory variant in IRF6.

In Finland the frequency of isolated cleft palate (CP) is higher than that of isolated cleft lip with or without cleft palate (CL/P). This trend contrasts to that in other European countries but its genetic underpinnings are unknown. We performed a genome-wide association study for orofacial clefts, which include CL/P and CP, in the Finnish population. We identified rs570516915, a single nucleotide polymorphism that is highly enriched in Finns and Estonians, as being strongly associated with CP ( P = 5.25 × 10 -34 , OR = 8.65, 95% CI 6.11-12.25), but not with CL/P ( P = 7.2 × 10 -5 ), with genome-wide significance. The risk allele frequency of rs570516915 parallels the regional variation of CP prevalence in Finland, and the association was replicated in independent cohorts of CP cases from Finland ( P = 8.82 × 10 -28 ) and Estonia ( P = 1.25 × 10 -5 ). The risk allele of rs570516915 disrupts a conserved binding site for the transcription factor IRF6 within a previously characterized enhancer upstream of the IRF6 gene. Through reporter assay experiments we found that the risk allele of rs570516915 diminishes the enhancer activity. Oral epithelial cells derived from CRISPR-Cas9 edited induced pluripotent stem cells demonstrate that the CP-associated allele of rs570516915 concomitantly decreases the binding of IRF6 and the expression level of IRF6 , suggesting impaired IRF6 autoregulation as a molecular mechanism underlying the risk for CP.

Neural crest and periderm-specific requirements of Irf6 during neural tube and craniofacial development.

IRF6 is a key genetic determinant of syndromic and non-syndromic cleft lip and palate. The ability to interrogate post-embryonic requirements of Irf6 has been hindered, as global Irf6 ablation in the mouse causes neonatal lethality. Prior work analyzing Irf6 in mouse models defined its role in the embryonic surface epithelium and periderm where it is required to regulate cell proliferation and differentiation. Several reports have also described Irf6 gene expression in other cell types, such as muscle, and neuroectoderm. However, analysis of a functional role in non-epithelial cell lineages has been incomplete due to the severity and lethality of the Irf6 knockout model and the paucity of work with a conditional Irf6 allele. Here we describe the generation and characterization of a new Irf6 floxed mouse model and analysis of Irf6 ablation in periderm and neural crest lineages. This work found that loss of Irf6 in periderm recapitulates a mild Irf6 null phenotype, suggesting that Irf6-mediated signaling in periderm plays a crucial role in regulating embryonic development. Further, conditional ablation of Irf6 in neural crest cells resulted in an anterior neural tube defect of variable penetrance. The generation of this conditional Irf6 allele allows for new insights into craniofacial development and new exploration into the post-natal role of Irf6.

A novel IRF6 gene mutation impacting the regulation of TGFβ2-AS1 in the TGFβ pathway: A mechanism in the development of Van der Woude syndrome.

Several mutations in the IRF6 gene have been identified as a causative link to VWS. In this investigation, whole-exome sequencing (WES) and Sanger sequencing of a three-generation pedigree with an autosomal-dominant inheritance pattern affected by VWS identified a unique stop-gain mutation-c.748C>T:p.R250X-in the IRF6 gene that co-segregated exclusively with the disease phenotype. Immunofluorescence analysis revealed that the IRF6-p.R250X mutation predominantly shifted its localization from the nucleus to the cytoplasm. WES and protein interaction analyses were conducted to understand this mutation's role in the pathogenesis of VWS. Using LC-MS/MS, we found that this mutation led to a reduction in the binding of IRF6 to histone modification-associated proteins (NAA10, SNRPN, NAP1L1). Furthermore, RNA-seq results show that the mutation resulted in a downregulation of TGFβ2-AS1 expression. The findings highlight the mutation's influence on TGFβ2-AS1 and its subsequent effects on the phosphorylation of SMAD2/3, which are critical in maxillofacial development, particularly the palate. These insights contribute to a deeper understanding of VWS's molecular underpinnings and might inform future therapeutic strategies.

Case Report of Recurrent Popliteal Pterygium Syndrome

AbstractA 23 week pregnant woman with a history of a child with facial and limb malformations underwent a fetal ultrasound revealing similar abnormalities in the current fetus. Genetic testing confirmed a new IRF6 gene mutation consistent with popliteal pterygium syndrome type 1. This case highlights the potential for recurrence and the role of genetic testing in prenatal diagnosis.

A novel noncanonical function for IRF6 in the recycling of E-cadherin.

Interferon Regulatory Factor 6 (IRF6) is a transcription factor essential for keratinocyte cell-cell adhesions. Previously, we found that recycling of E-cadherin was defective in the absence of IRF6, yet total E-cadherin levels were not altered, suggesting a previously unknown, nontranscriptional function for IRF6. IRF6 protein contains a DNA binding domain (DBD) and a protein binding domain (PBD). The transcriptional function of IRF6 depends on its DBD and PBD, however, whether the PBD is necessary for the interaction with cytoplasmic proteins has yet to be demonstrated. Here, we show that an intact PBD is required for recruitment of cell-cell adhesion proteins at the plasma membrane, including the recycling of E-cadherin. Colocalizations and coimmunoprecipitations reveal that IRF6 forms a complex in recycling endosomes with Rab11, Myosin Vb, and E-cadherin, and that the PBD is required for this interaction. These data indicate that IRF6 is a novel effector of the endosomal recycling of E-cadherin and demonstrate a non-transcriptional function for IRF6 in regulating cell-cell adhesions.

Interferon regulatory factor 6 (IRF6) determines intestinal epithelial cell development and immunity

Intestinal epithelial cell (IEC) responses to interferon (IFN) favor antiviral defense with minimal cytotoxicity, but IEC-specific factors that regulate these responses remain poorly understood. Interferon regulatory factors (IRFs) are a family of nine related transcription factors, and IRF6 is preferentially expressed by epithelial cells, but its roles in IEC immunity are unknown. In this study, CRISPR screens found that Irf6 deficiency enhanced IFN-stimulated antiviral responses in transformed mouse IECs but not macrophages. Furthermore, KO ofIrf6in IEC organoids resulted in profound changes to homeostasis and immunity gene expression. Irf6 KO organoids grew more slowly, and single-cell RNA sequencing indicated reduced expression of genes in epithelial differentiation and immunity pathways. IFN-stimulated gene expression was also significantly different inIrf6KO organoids, with increased expression of stress and apoptosis-associated genes. Functionally, the transcriptional changes in Irf6 KO organoids were associated with increased cytotoxicity upon IFN treatment or inflammasome activation. These data indicate a previously unappreciated role for IRF6 in IEC biology, including regulation of epithelial development and moderation of innate immune responses to minimize cytotoxicity and maintain barrier function.

Identification and validation of IRF6 related to ovarian cancer and biological function and prognostic value

BackgroundOvarian cancer (OC) is a severe gynecological malignancy with significant diagnostic and therapeutic challenges. The discovery of reliable cancer biomarkers can be used to adjust diagnosis and improve patient care. However, serous OC lacks effective biomarkers. We aimed to identify novel biomarkers for OC and their pathogenic causes.MethodsThe present study used the differentially expressed genes (DEGs) obtained from the “Limma” package and WGCNA modules for intersection analysis to obtain DEGs in OC. Three hub genes were identified—claudin 3 (CLDN3), interferon regulatory factor 6 (IRF6), and prostasin (PRSS8)—by searching for hub genes through the PPI network and verifying them in GSE14407, GSE18520, GSE66957, and TCGA + GTEx databases. The correlation between IRF6 and the prognosis of OC patients was further confirmed in Kaplan-Miller Plotter. RT-qPCR and IHC confirmed the RNA and protein levels of IRF6 in the OC samples. The effect of IRF6 on OC was explored using transwell invasion and scratch wound assays. Finally, we constructed a ceRNA network of hub genes and used bioinformatics tools to predict drug sensitivity.ResultsThe joint analysis results of TCGA, GTEx, and GEO databases indicated that IRF6 RNA and protein levels were significantly upregulated in serous OC and were associated with OS and PFS. Cell function experiments revealed that IRF6 knockdown inhibited SKOV3 cell proliferation, migration and invasion.ConclusionIRF6 is closely correlated with OC development and progression and could be considered a novel biomarker and therapeutic target for OC patients.

Detection of Single Nucleotide Polymorphism (SNP) rs2235371 of IRF6 Gene in Patients with Non-Syndromic Cleft Lip and Palate

The orofacial clefts are the most common structural birth defects among newborn babies. They consist of two groups, namely Cleft Palate (CP) and Cleft Lip (CL). Infants born with clefts have considerable problems during communication, swallowing, and face certain challenges due to facial deformity. These problems remain throughout the life if not treated and entail long-lasting effects on the health and social life of an individual. Untreated infants need special care as they have higher morbidity and mortality rates. The primary objective of this study is to detect the Single Nucleotide Polymorphism (SNP) rs2235371 within the IRF6 gene and assess its potential association in patients diagnosed with Non-Syndromic Cleft Lip/Palate (NSCL/P). The participants were selected through a non-probability sampling technique, specifically the purposive sampling method. In accordance with this approach, a total of 50 patients afflicted with NSCL/P, representing both the genders, were sampled. Furthermore, to establish a comparative baseline, an additional 50 healthy individuals were also recruited to serve as the control group. After taking their informed consent, blood was collected in EDTA vials. PCR (Polymerase Chain Reaction) and RFLP were performed on each sample to find SNP. After sample collection, processing, and analysis, SPSS (20.00 version) was used to analyze all previously generated data. After sample analysis, 72% of CL/P (cleft lip and palate) patients were found to have SNP with or without a family history. The association of SNP rs2235371 with disease was measured by applying chi-square. The results provided no evidence of the association between given SNP and disease. As compared to previous research, this study showed a low frequency of SNP rs2235371 in patients with NSCL/P. To get a better understanding of the association of SNP rs2235371 with NSCL/P, large-scale research with an increased sample size should be conducted.

Biophysical and functional characterization of N-terminal domain of Human Interferon Regulatory Factor 6.

Interferon regulatory factor 6 (IRF6) has a key function in palate fusion during palatogenesis during embryonic development, and mutations in IRF6 cause orofacial clefting disorders. The in silico analysis of IRF6 is done to obtain leads for the domain boundaries and subsequently the sub-cloning of the N-terminal domain of IRF6 into the pGEX-2TK expression vector and successfully optimized the overexpression and purification of recombinant glutathione S-transferase-fused NTD-IRF6 protein under native conditions. After cleavage of the GST tag, NTD-IRF6 was subjected to protein folding studies employing Circular Dichroism and Intrinsic fluorescence spectroscopy at variable pH, temperature, and denaturant. CD studies showed predominantly alpha-helical content and the highest stability of NTD-IRF6 at pH 9.0. A comparison of native and renatured protein depicts loss in the secondary structural content. Intrinsic fluorescence and quenching studies have identified that tryptophan residues are majorly present in the buried areas of the protein and a small fraction was on or near the protein surface. Upon the protein unfolding with a higher concentration of denaturant urea, the peak of fluorescence intensity decreased and red shifted, confirming that tryptophan residues are majorly present in a more polar environment. While regulating IFNβ gene expression during viral infection, the N-terminal domain binds to the promoter region of Virus Response Element-Interferon beta (VRE-IFNβ). Along with the protein folding analysis, this study also aimed to identify the DNA-binding activity and determine the binding affinities of NTD-IRF6 with the VRE-IFNβ promoter region. The protein-DNA interaction is specific as demonstrated by gel retardation assay and the kinetics of molecular interactions as quantified by Biolayer Interferometry showed a strong affinity with an affinity constant (KD) value of 7.96 × 10-10 M. NTD-IRF6 consists of a mix of α-helix and β-sheets that show temperature-dependent cooperative unfolding between 40 °C and 55 °C. Urea-induced unfolding shows moderate tolerance to urea as the mid-transition concentration of urea (Cm) is 3.2 M. The tryptophan residues are majorly buried as depicted by fluorescence quenching studies. NTD-IRF6 has a specific and high affinity toward the promoter region of VRE-IFNβ.

The application of zebrafish model in the study of cleft lip and palate development: A systematic review

ObjectiveCraniofacial growth and development are more than a scientific curiosity; it is of tremendous interest to clinicians. Insights into the genetic etiology of cleft lip and palate development are essential for improving diagnosis and treatment planning. The purpose of this systematic review was to utilize a zebrafish model to highlight the role of the IRF6 gene in cleft lip and palate development in humans. DataThis review adhered to the guidelines outlined in the PRISMA statement. Nine studies were included in the analysis. SourcesThis study used major scientific databases such as MEDLINE, EMBASE, Web of Science, and the Zebrafish Information Network and yielded 1275 articles. Two reviewers performed the screening using COVIDENCE™ independently, and a third reviewer resolved any conflicts. Study selectionAfter applying the inclusion and exclusion criteria and screening, nine studies were included in the analysis. The Systematic Review Center for Laboratory Animal Experimentation's (SYRCLE's) risk-of-bias tool was used to assess the quality of the included studies. ResultsThe main outcome supports the role of the IRF6 gene in zebrafish periderm development and embryogenesis, and IRF6 variations result in cleft lip and palate development. The overall SYRCLE risk of bias was low-medium. ConclusionIn conclusion, this review indicated the critical role of the IRF6 gene and its downstream genes (GRHL3, KLF17, and ESRP1/2) in the development of cleft lip and palate in zebrafish models. Genetic mutation zebrafish models provide a high level of insights into zebrafish craniofacial development. Clinical relevancethis review provides a productive avenue for understanding the powerful and conserved zebrafish model for investigating the pathogenesis of human cleft lip and palate.

A comprehensive consolidation of data on the relationship between IRF6 polymorphisms and non-syndromic cleft lip/palate susceptibility: From 79 case-control studies

BackgroundNon-syndromic cleft lip with or without cleft palate (NSCL/P) is a prevalent craniofacial birth defect on a global scale. A number of candidate genes have been identified as having an impact on NSCL/P. However, the association between interferon regulatory factor 6 (IRF6) polymorphisms and NSCL/P has yielded inconsistent results, prompting the need for a meta-analysis to obtain more accurate estimates. MethodsWe conducted a thorough screening of all relevant articles published up until November 15, 2023, in online bibliographic databases. The statistical analysis of the collected data was performed using the Comprehensive Meta-Analysis (Version 4.0) software. ResultsA total of 79 case-control studies, comprising 14,003 cases and 19,905 controls, were included in our analysis. The combined data indicated that the IRF6 rs642961 and rs2235371 polymorphisms were associated with an increased risk of NSCL/P in the overall population. However, no significant association was found between the rs2013162 and rs2235375 polymorphisms and the risk of NSCL/P in the overall population. Furthermore, subgroup analyses revealed significant correlations between the IRF6 rs642961, rs2235371, and rs2235375 polymorphisms and the risk of NSCL/P based on ethnic background and country of origin. Nevertheless, the rs2013162 polymorphism plays a protective role in Caucasians and mixed populations. ConclusionsOur collective data indicates a significant association between the rs642961 and rs2235371 polymorphisms and the risk of NSCL/P in the overall population. The rs2235375 polymorphism could influence the susceptibility to NSCL/P based on ethnic background. Meanwhile, the rs2013162 polymorphism provides protective effects in Caucasian, mixed populations, and the Brazilian population.

Correlation between methylation of interferon regulatory factor 6 gene promoter in renal tissues and overall survival of patients with Kidney renal clear cell carcinoma

To assess the prognostic value of methylation of interferon regulatory factor 6 (IRF6) gene promoter in patients diagnosed with Kidney renal clear cell carcinoma (KIRC). The primary lesions of fifty KIRC patients who were diagnosed at the First Affiliated Hospital of Nanjing Medical University from January 2016 to January 2020 were collected. The expression of IRF6 protein was determined with an immunohistochemical method. The correlation between the level of IRF6 expression and survival and/or metastasis status was analyzed. The mRNA and protein levels of the IRF6 in KIRC and normal renal tissues were compared by using bioinformatic tools. The difference in the methylation rate of the IRF6 gene promoter between tumor and adjacent tissues was analyzed by searching the online databases. Statistical analysis was carried out for the methylation status of the IRF6 gene promoter region to select those negatively correlated with the overall survival (OS) among the patients. In vitro experiments were conducted with cell lines to verify the correlation between the status of promoter methylation and transcription level of the IRF6 gene. The mRNA and protein levels of the IRF6 gene in KIRC tissues were significantly lower than those of the normal controls, and this was more prominent in patients who had died or developed metastasis. The extent of IRF6 gene promoter methylation in the KIRC tissues was much higher compared with that of the adjacent normal renal tissues. There was a significant negative correlation between the methylation of the IRF6 gene promoter and mRNA level of the IRF6 (R = -0.52). The higher methylation degree in the IRF6 gene promoter regions cg12034118 and cg16030177, the shorter the OS and worse prognosis in the patients. Only twenty CpG sites in cg12034118 were confirmed to be highly methylated in KIRC cell lines. The transcription level of the IRF6 gene was upregulated in a time- and dose-dependent manner after the treatment with demethylation reagent 5-azadeoxycytidine. The methylation of IRF6 gene promoter in the renal tissues of KIRC patients is closely correlated with the OS. Cg12034118 may provide a promising biomarker for laboratory detection, and its high methylation rate has certain reference value for the prognosis.

Genetic Factors of Teeth Impaction: Polymorphic and Haplotype Variants of PAX9, MSX1, AXIN2, and IRF6 Genes.

In recent research, there has been a growing awareness of the role of genetic factors in the positioning and eruption of teeth in the maxilla and mandible. This study aimed to evaluate the potential of specific polymorphic markers of single nucleotide polymorphisms (SNPs) located within the PAX9, MSX1, AXIN2, and IRF6 genes to determine the predisposition to tooth impaction. The study participants were divided into two groups: the first group consisted of individuals with at least one impacted secondary tooth. In contrast, the second group (control group) had no impacted teeth in their jaws. To analyze the genes, real-time PCR (polymerase chain reaction) and TaqMan probes were utilized to detect the selected polymorphisms. The findings suggest that disruptions in the structure and function of the mentioned genetic factors such as polymorphic and haplotype variants of PAX9, MSX1, AXIN2, and IRF6 genes, which play a direct role in tooth and periodontal tissue development, might be significant factors in tooth impaction in individuals with genetic variations. Therefore, it is reasonable to hypothesize that tooth impaction may be influenced, at least in part, by the presence of specific genetic markers, including different allelic variants of the PAX9, AXIN2, and IRF6 genes, and especially MSX1.

P152 Allele-associated chromatin interactions in primary immune cells uncover mechanisms of gene regulation

Abstract Background/Aims Genome-wide association studies studies have identified many genetic variants associated with the risk of developing common rheumatological conditions. These variants primarily affect regulatory elements of the genome which can affect the expression of genes located far away from their genomic location through chromatin interaction mechanisms. Furthermore, common genetic variants have been associated with specific changes in chromatin interactions in cell lines. Studying chromatin interactions can improve our understanding of these diseases. Here, we aim to study chromatin interactions and how they are affected by genetic variation in primary immune cells that are relevant for rheumatological conditions. Methods We generated promoter Capture Hi-C (pCHi-C) data for primary CD4+ T cells and CD14+ monocytes isolated from peripheral blood of 10 systemic sclerosis patients and 5 healthy controls. We developed a new computational pipeline to explore how genetic variation affects chromatin interactions. Briefly, genotypes were called from the pCHi-C data using a modified GLIMPSE pipeline. Genotypes were phased using the integrated phasing pipeline which combined population haplotype information with the pCHi-C data. Reads were then assigned to the correct allele using SNPsplit and assigned to chromatin loops (CHiCAGO score &amp;gt; 5). A test for allelic imbalance was carried out using a binomial test, and results were consolidated using Fisher’s method. Results We identified 171 and 139 allele-associated loops (FDR &amp;lt; 0.05) in CD4+ T cells and CD14+ monocytes respectively. These interactions are cell-type specific and highly associated with expression quantitative trait loci (eQTLs). We found that 80% of the variants involved in allele-associated loops were eQTLs for at least one gene. These interactions link to the promoters of 62 and 57 distal genes in CD4+ T cells and CD14+ monocytes respectively. Of these, 38 and 31 respectively were also eQTL for those genes. Many of these allele-associated interactions affect genomic regions that are highly significant for the function of these cell populations. For example, a region with variants affecting T cell-specific chromatin interactions is the TRAF3IP3/IRF6/UTP25 locus. In this region there are separate groups of variants which affect gene expression with a complex mechanism. A group of variants overlapping the UTP25 promoter increases interaction strength with the TRAF3IP3 and IRF6 gene bodies. Interestingly, these are also associated with an increase in UTP25 expression but a decrease in TRAF3IP3 and IRF6 expression. Another group of variants located near the IRF6 promoter is associated with increased interactions with the UTP25 promoter. In contrast, these variants are associated with a reduction of UTP25 expression but an increase in TRAF3IP3 and IRF6 expression. Conclusion We created a new pipeline that allows the identification of allele-associated chromatin interactions in primary cells. These new data will be helpful in uncovering mechanisms of gene regulation in loci highly significant to autoimmune conditions. Disclosure D. González-Serna: None. C. Shi: None. M. Kerick: None. J. Hankinson: None. J. Ding: None. A. McGovern: None. M. Tutino: None. G. Villanueva Martin: None. N. Ortego-Centeno: None. J. Luis Callejas: None. J. Martin: None. G. Orozco: None.

IRF6 and FGF1 polymorphisms in non-syndromic cleft lip with or without cleft palate in the Polish population.

Non-syndromic cleft lip with or without cleft palate (NSCL/P) is the most common developmental defect that significantly affects the morphology and function of the stomatognathic system in children. The etiology of these birth defects is multifactorial, and single nucleotide polymorphisms (SNPs) in IRF6 and FGF1 have been associated with NSCL/P. This study aimed to evaluate whether SNPs in IRF6, namely rs2013162, rs642961, rs2235373, and rs34010 in FGF1, are associated with NSCL/P occurrence in the Polish population. The study included 627 participants: 209 children with NSCL/P and 418 healthy controls. DNA was isolated from saliva in the study group and from umbilical cord blood in controls. Genotyping of polymorphisms was performed using quantitative PCR. There was no statistically significant association of IRF6 gene variants with NSCL/P occurrence, although for rs2013162, AA genotype, odds ratio (OR) = 1.16 and for AC genotype, OR = 0.83; for rs642961, AA genotype, OR = 0.84 and for AG genotype, OR = 1.41; and for rs2235373, AA genotype, OR = 0.79 and for AG, OR = 0.85. In the instance of rs34010 polymorphism in FGF1, the presence of the AA genotype was statistically significant in reducing the risk of NSCL/P (OR = 0.31, p = 0.001). Genetic variation in FGF1 is an important risk marker of NSCL/P in the Polish population, which cannot be stated for the polymorphisms in the IRF6 gene.

Genetic Risk Assessment of Nonsyndromic Cleft Lip with or without Cleft Palate by Linking Genetic Networks and Deep Learning Models.

Recent deep learning algorithms have further improved risk classification capabilities. However, an appropriate feature selection method is required to overcome dimensionality issues in population-based genetic studies. In this Korean case-control study of nonsyndromic cleft lip with or without cleft palate (NSCL/P), we compared the predictive performance of models that were developed by using the genetic-algorithm-optimized neural networks ensemble (GANNE) technique with those models that were generated by eight conventional risk classification methods, including polygenic risk score (PRS), random forest (RF), support vector machine (SVM), extreme gradient boosting (XGBoost), and deep-learning-based artificial neural network (ANN). GANNE, which is capable of automatic input SNP selection, exhibited the highest predictive power, especially in the 10-SNP model (AUC of 88.2%), thus improving the AUC by 23% and 17% compared to PRS and ANN, respectively. Genes mapped with input SNPs that were selected by using a genetic algorithm (GA) were functionally validated for risks of developing NSCL/P in gene ontology and protein-protein interaction (PPI) network analyses. The IRF6 gene, which is most frequently selected via GA, was also a major hub gene in the PPI network. Genes such as RUNX2, MTHFR, PVRL1, TGFB3, and TBX22 significantly contributed to predicting NSCL/P risk. GANNE is an efficient disease risk classification method using a minimum optimal set of SNPs; however, further validation studies are needed to ensure the clinical utility of the model for predicting NSCL/P risk.

Novel IRF6 variant in orofacial cleft patients from Durban, South Africa.

To date, there are over 320 variants identified in the IRF6 gene that cause Van der Woude syndrome or popliteal pterygium syndrome. We sequenced this gene in a South African orofacial cleft cohort to identify the causal IRF6 variants in our population. Saliva samples from 100 patients with syndromic and non-syndromic CL ± P were collected. Patients were recruited from the cleft clinics at two public, tertiary hospitals in Durban, South Africa (SA), namely Inkosi Albert Luthuli Central Hospital (IALCH) and KwaZulu-Natal Children's Hospital (KZNCH). We prospectively sequenced the exons of IRF6 in 100 orofacial cleft cases, and where possible, we also sequenced the parents of the individuals to determine the segregation pattern. Two variants were identified; one novel (p.Cys114Tyr) and one known (p.Arg84His) missense variant in IRF6 gene were identified. The patient with the p.Cys114Tyr variant was non-syndromic with no clinical VWS phenotype expected of individuals with IRF6 coding variants, and the patient with the p.Arg84His had phenotypic features of popliteal pterygium syndrome. The p.Arg84His variant segregated in the family, with the father also being affected. This study provides evidence that IRF6 variants are found in the South African population. Genetic counselling is essential for affected families, particularly in the absence of a known clinical phenotype since it helps with the plans for future pregnancies.