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.

Case report and functional verification of a novel mutation in the interferon regulatory transcription factor 6 gene in a family with orofacial clefts.

BACKGROUND: This study aimed to identify the causative genetic variant in a Chinese family with orofacial clefts. METHODS: We retrospectively analyzed the clinical information of a family with orofacial clefts. Then, we performed an etiological genetic analysis of the family using whole exome sequencing analysis and Sanger sequencing. We created a hybrid code-shifting mutation cell line (293T-462het) and evaluated its impact on cell proliferation, migration, and apoptosis, as well as E-cadherin and vimentin expression. RESULTS: Whole exome sequencing revealed a novel heterozygous variant c.1386del (p.A462Pfs*28) in the interferon regulatory transcription factor 6 (IRF6) gene in a family with orofacial clefts. Sanger sequencing further confirmed that this heterozygous variant was the genetic cause of orofacial clefts in this family. The c.1386del variant of IRF6 was classified as likely pathogenic. The heterozygous mutation IRF6 (c.1386del) enhanced cell proliferation and migration while inhibiting cell apoptosis and regulating the expression of E-cadherin and vimentin. CONCLUSION: This study identified a novel c.1386del mutation in the IRF6 gene and explored how this mutation leads to lip and palate defects. Our results provide a solid theoretical foundation for future genetic detection of these orofacial defects.

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

BACKGROUND: 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. METHODS: 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). RESULTS: 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). CONCLUSION: 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.

A novel IRF6 gene mutation impacting the regulation of TGFß2-AS1 in the TGFß 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 TGFß2-AS1 expression. The findings highlight the mutation's influence on TGFß2-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.

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.

Potentially functional genetic variants in interferon regulatory factor family genes are associated with colorectal cancer survival.

Interferon regulatory factor (IRF) family genes play a critical role in colorectal cancer (CRC) development and impact patient survival. This study evaluated the influence of functional single nucleotide polymorphisms (SNPs) in IRF genes on CRC survival, including functional predictions and experimental validations. Multivariate Cox regression analysis identified three linked SNPs as significant survival predictors, with the rs141112353 T/T genotype in the 3'UTR region of IRF6 significantly associated with decreased survival (HR = 1.60, P = 6E-04). Expression quantitative trait loci (eQTL) analysis indicated that the rs141112353 TA > T alteration reduced IRF6 expression. Dual luciferase assays showed lower activity for the T allele in the presence of hsa-miR-548ap-3p. Data from The Cancer Genome Atlas (TCGA) and other databases confirmed lower IRF6 levels in CRC tissues, correlating with worse survival and inversely with M2 macrophage infiltration. In vitro, IRF6 overexpression inhibited CRC cell proliferation and M2 macrophage polarization by downregulating MIF expression. These findings suggest that the IRF6 rs141112353 TA > T variant significantly affects CRC survival, potentially by enhancing miR-548-ap-3p binding affinity.

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

BACKGROUND: Non-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. METHODS: We 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. RESULTS: A 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. CONCLUSIONS: Our 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.

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

BACKGROUND: Ovarian 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. METHODS: The 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. RESULTS: The 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. CONCLUSION: IRF6 is closely correlated with OC development and progression and could be considered a novel biomarker and therapeutic target for OC patients.

Review on the Role of IRF6 in the Pathogenesis of Non-syndromic Orofacial Clefts.

Non-syndromic orofacial clefts (NSOCs) are the most common craniofacial malformation. In the complex aetiology and pathogenesis of NSOCs, genetic factors play a crucial role and IRF6, located at chromosome 1q32.2, is the best documented NSOC susceptibility gene. IRF6 is a key factor in oral maxillofacial development and known to contribute the most in NSOCs. It is essential to conduct a complete review of the existing results on IRF6 to further understand its role in the pathogenesis of NSOCs. Thus, the present authors summarised the research progress on the mechanism of IRF6 in NSOCs from both genetic and functional perspectives in this review.

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

Objective: Craniofacial 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. Data: This review adhered to the guidelines outlined in the PRISMA statement. Nine studies were included in the analysis. Sources: This 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 selection: After 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. Results: The 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. Conclusion: In 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 relevance: this review provides a productive avenue for understanding the powerful and conserved zebrafish model for investigating the pathogenesis of human cleft lip and palate.

LncRNA AL645608.3 mediates malignant progression of acute myeloid leukemia.

OBJECTIVE: To investigate the role of lncRNA AL645608.3 in the malignant progression of acute myeloid leukemia (AML) cells and explore relevant molecular mechanisms. METHODS: The expression level of AL645608.3 was measured in AML cell lines (THP-1, HL-60, KG-1, and AML-193) via real-time quantitative polymerase chain reaction (RT-qPCR). Small hairpin RNA (shRNA) and open reading frame of AL645608.3 were cloned into lentiviral vectors and were infected into THP-1 and AML-193 cells. The expression of casitas B-lineage lymphoma (CBL), interferon regulatory factor 6 (IRF6), and interferon beta 1 (IFNB1) was detected through RT-qPCR, and western blot. Co-immunoprecipitation (Co-IP) on IRF6 was conducted. Matrix metalloprotease-9 (MMP-9) activity was evaluated via gelatin zymography assay. RESULTS: LncRNA AL645608.3 was expressed in the four AML cell lines (THP-1, HL-60, KG-1, and AML-193). Silencing AL645608.3 mitigated the expression of IRF6 and IFNB1 but elevated the expression of CBL in THP-1 cells. Oppositely, AL645608.3 overexpression up-regulated the expression of IRF6 and IFNB1 but decreased the expression of CBL in AML-193 cells. Co-IP results proved that AL645608.3 could directly mediate IRF6 activity in THP-1 and AML-193 cells. MMP-9 activity was decreased by AL645608.3 knockdown and was improved by AL645608.3 overexpression in AML-193 cells. CONCLUSION: AL645608.3 is expressed in different AML cell lines, and mediates the expression of CBL, IRF6, IFNB1, and MMP-9. These findings might deepen our comprehension of the molecular mechanisms underlying AML.

Association between IRF6, TP63, GREM1 Gene Polymorphisms and Non-Syndromic Orofacial Cleft Phenotypes in Vietnamese Population: A Case-Control and Family-Based Study.

This study aims to identify potential variants in the TP63-IRF6 pathway and GREM1 for the etiology of non-syndromic orofacial cleft (NSOFC) among the Vietnamese population. By collecting 527 case-parent trios and 527 control samples, we conducted a stratified analysis based on different NSOFC phenotypes, using allelic, dominant, recessive and over-dominant models for case-control analyses, and family-based association tests for case-parent trios. Haplotype and linkage disequilibrium analyses were also conducted. IRF6 rs2235375 showed a significant association with an increased risk for non-syndromic cleft lip and palate (NSCLP) and cleft lip with or without cleft palate (NSCL/P) in the G allele, with pallele values of 0.0018 and 0.0003, respectively. Due to the recessive model (p = 0.0011) for the NSCL/P group, the reduced frequency of the GG genotype of rs2235375 was associated with a protective effect against NSCL/P. Additionally, offspring who inherited the G allele at rs2235375 had a 1.34-fold increased risk of NSCL/P compared to the C allele holders. IRF6 rs846810 and a G-G haplotype at rs2235375-rs846810 of IRF6 impacted NSCL/P, with p-values of 0.0015 and 0.0003, respectively. In conclusion, our study provided additional evidence for the association of IRF6 rs2235375 with NSCLP and NSCL/P. We also identified IRF6 rs846810 as a novel marker associated with NSCL/P, and haplotypes G-G and C-A at rs2235375-rs846810 of IRF6 associated with NSOFC.

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.

Analysis of candidate genes for cleft lip ± cleft palate using murine single-cell expression data.

Introduction: Cleft lip ± cleft palate (CL/P) is one of the most common birth defects. Although research has identified multiple genetic risk loci for different types of CL/P (i.e., syndromic or non-syndromic forms), determining the respective causal genes and understanding the relevant functional networks remain challenging. The recent introduction of single-cell RNA sequencing (scRNA-seq) has provided novel opportunities to study gene expression patterns at cellular resolution. The aims of our study were to: (i) aggregate available scRNA-seq data from embryonic mice and provide this as a resource for the craniofacial community; and (ii) demonstrate the value of these data in terms of the investigation of the gene expression patterns of CL/P candidate genes. Methods and Results: First, two published scRNA-seq data sets from embryonic mice were re-processed, i.e., data representing the murine time period of craniofacial development: (i) facial data from embryonic day (E) E11.5; and (ii) whole embryo data from E9.5-E13.5 from the Mouse Organogenesis Cell Atlas (MOCA). Marker gene expression analyses demonstrated that at E11.5, the facial data were a high-resolution representation of the MOCA data. Using CL/P candidate gene lists, distinct groups of genes with specific expression patterns were identified. Among others we identified that a co-expression network including Irf6, Grhl3 and Tfap2a in the periderm, while it was limited to Irf6 and Tfap2a in palatal epithelia, cells of the ectodermal surface, and basal cells at the fusion zone. The analyses also demonstrated that additional CL/P candidate genes (e.g., Tpm1, Arid3b, Ctnnd1, and Wnt3) were exclusively expressed in Irf6+ facial epithelial cells (i.e., as opposed to Irf6- epithelial cells). The MOCA data set was finally used to investigate differences in expression profiles for candidate genes underlying different types of CL/P. These analyses showed that syndromic CL/P genes (syCL/P) were expressed in significantly more cell types than non-syndromic CL/P candidate genes (nsCL/P). Discussion: The present study illustrates how scRNA-seq data can empower research on craniofacial development and disease.

A Complex Intrachromosomal Rearrangement Disrupting IRF6 in a Family with Popliteal Pterygium and Van der Woude Syndromes.

Clefts of the lip and/or palate (CL/P) are considered the most common form of congenital anomalies occurring either in isolation or in association with other clinical features. Van der woude syndrome (VWS) is associated with about 2% of all CL/P cases and is further characterized by having lower lip pits. Popliteal pterygium syndrome (PPS) is a more severe form of VWS, normally characterized by orofacial clefts, lower lip pits, skin webbing, skeletal anomalies and syndactyly of toes and fingers. Both syndromes are inherited in an autosomal dominant manner, usually caused by heterozygous mutations in the Interferon Regulatory Factor 6 (IRF6) gene. Here we report the case of a two-generation family where the index presented with popliteal pterygium syndrome while both the father and sister had clinical features of van der woude syndrome, but without any point mutations detected by re-sequencing of known gene panels or microarray testing. Using whole genome sequencing (WGS) followed by local de novo assembly, we discover and validate a copy-neutral, 429 kb complex intra-chromosomal rearrangement in the long arm of chromosome 1, disrupting the IRF6 gene. This variant is copy-neutral, novel against publicly available databases, and segregates in the family in an autosomal dominant pattern. This finding suggests that missing heritability in rare diseases may be due to complex genomic rearrangements that can be resolved by WGS and de novo assembly, helping deliver answers to patients where no genetic etiology was identified by other means.

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.

A Comprehensive Genetic Analysis of Slovenian Families with Multiple Cases of Orofacial Clefts Reveals Novel Variants in the Genes IRF6, GRHL3, and TBX22.

Although the aetiology of non-syndromic orofacial clefts (nsOFCs) is usually multifactorial, syndromic OFCs (syOFCs) are often caused by single mutations in known genes. Some syndromes, e.g., Van der Woude syndrome (VWS1; VWS2) and X-linked cleft palate with or without ankyloglossia (CPX), show only minor clinical signs in addition to OFC and are sometimes difficult to differentiate from nsOFCs. We recruited 34 Slovenian multi-case families with apparent nsOFCs (isolated OFCs or OFCs with minor additional facial signs). First, we examined IRF6, GRHL3, and TBX22 by Sanger or whole exome sequencing to identify VWS and CPX families. Next, we examined 72 additional nsOFC genes in the remaining families. Variant validation and co-segregation analysis were performed for each identified variant using Sanger sequencing, real-time quantitative PCR and microarray-based comparative genomic hybridization. We identified six disease-causing variants (three novel) in IRF6, GRHL3, and TBX22 in 21% of families with apparent nsOFCs, suggesting that our sequencing approach is useful for distinguishing syOFCs from nsOFCs. The novel variants, a frameshift variant in exon 7 of IRF6, a splice-altering variant in GRHL3, and a deletion of the coding exons of TBX22, indicate VWS1, VWS2, and CPX, respectively. We also identified five rare variants in nsOFC genes in families without VWS or CPX, but they could not be conclusively linked to nsOFC.

Causal Variations at IRF6 Gene Identified in Van der Woude Syndrome Pedigrees.

The purpose of this study is to analyze the clinical characteristics of patients with Van der Woude syndrome (VWS) and to detect variations in each patient. Finally, the combination of genotype and phenotype can make a clear diagnosis of VWS patients with different phenotype penetrance.Five Chinese VWS pedigree were enrolled. Whole exome sequencing of the proband was performed, and the potential pathogenic variation was further verified by Sanger sequencing in the patient and their parents. The human mutant IRF6 coding sequence was generated from the human full-length IRF6 plasmid by site-directed mutagenesis and cloned into the GV658 vector, RT-qPCR and Western blot were used to detect the expression of IRF6.We found one de novo nonsense variation (p. Gln118Ter) and three novel missense variations (p. Gly301Glu, p. Gly267Ala, and p. Glu404Gly) co-segregated with VWS. RT-qPCR analysis revealed that p. Glu404Gly significantly reduced the expression level of IRF6 mRNA. Western blot of cell lysates confirmed that IRF6 p. Glu404Gly abundance levels were lower than those for IRF6 wild type.This discovery of the novel variation (IRF6 p. Glu404Gly) expands the spectrum of known variations in VWS in Chinese humans. Genetic results combined with clinical phenotypes and differential diagnosis points from other diseases can make a definitive diagnosis and provide genetic counseling for families.

The NOTCH-RIPK4-IRF6-ELOVL4 Axis Suppresses Squamous Cell Carcinoma.

Receptor-interacting serine/threonine protein kinase 4 (RIPK4) and its kinase substrate the transcription factor interferon regulatory factor 6 (IRF6) play critical roles in the development and maintenance of the epidermis. In addition, ourselves and others have previously shown that RIPK4 is a NOTCH target gene that suppresses the development of cutaneous and head and neck squamous cell carcinomas (HNSCCs). In this study, we used autochthonous mouse models, where the expression of Pik3caH1047R oncogene predisposes the skin and oral cavity to tumor development, and show that not only loss of Ripk4, but also loss of its kinase substrate Irf6, triggers rapid SCC development. In vivo rescue experiments using Ripk4 or a kinase-dead Ripk4 mutant showed that the tumor suppressive function of Ripk4 is dependent on its kinase activity. To elucidate critical mediators of this tumor suppressive pathway, we performed transcriptional profiling of Ripk4-deficient epidermal cells followed by multiplexed in vivo CRISPR screening to identify genes with tumor suppressive capabilities. We show that Elovl4 is a critical Notch-Ripk4-Irf6 downstream target gene, and that Elovl4 loss itself triggers SCC development. Importantly, overexpression of Elovl4 suppressed tumor growth of Ripk4-deficient keratinocytes. Altogether, our work identifies a potent Notch1-Ripk4-Irf6-Elovl4 tumor suppressor axis.

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

BACKGROUND: 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. METHOD: 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. RESULTS: 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. CONCLUSIONS: 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.