Interferon Regulatory Factor 6 Gene Research Articles

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.

Identification of Genetic Effects of ACADVL and IRF6 Genes with Milk Production Traits of Holstein Cattle in China.

With the development of high-throughput sequencing, RNA sequencing has been widely used in the identification of candidate genes for complex traits in livestock, and the functional genes and mutations with large genetic effects on milk production traits can provide molecular information for marker-assisted selection to increase the selection accuracy and accelerate genetic gain in dairy cattle. Our previous study on the liver transcriptome of Holstein cows found that acyl-CoA dehydrogenase (ACADVL) and interferon regulatory factor 6 (IRF6) are differentially expressed between dry and peak lactation periods, as well as that they are involved in lipid metabolism and the proliferation and differentiation of mammary epithelial cells. Thus, the two genes were considered candidates for milk traits. Hence, this study further collected 1186 Holstein cows from 110 sire families to investigate their genetic associations with milk yield and composition traits. By resequencing the entire exons and 2000 bp of the 5' and 3' flanking regions of the two genes, we identified eight SNPs in ACADVL and eight SNPs in IRF6. Subsequent single-locus association analyses showed that the eight SNPs in ACADVL were all significantly associated with milk fat yield, fat percentage, and protein yield (p values ≤ 0.0001-0.0414), and the eight SNPs in IRF6 were associated with milk, fat, and protein yields in the first or second lactation (p values ≤ 0.0001-0.0467). Using Haploview 4.2, one haplotype block with eight of the SNPs in ACADVL (D' = 0.99-1.00) and two haplotype blocks in IRF6 with three of the SNPs in each were observed (D' = 0.98-1.00). Similarly, the haplotype combinations of ACADVL were significantly associated with milk yield, fat percentage, fat yield, and protein yield in the two lactations (p values ≤ 0.0001-0.0125), and those of IRF6 were associated with five milk traits (p values ≤ 0.0001-0.0263). Furthermore, with the JASPAR software, it was predicted that the SNPs 19:g.26933503T>C in ACADVL and 16:g.73501985G>A in IRF6 changed the transcription factor binding sites of ZEB1, PLAGL2, and RHOXF1, implying their impacts on the expressions of the corresponding genes. Our findings demonstrated that the ACADVL and IRF6 genes have significant genetic effects on milk yield and composition traits, and the valuable SNPs might be used as genetic markers for genomic selection programs in dairy cattle.

Negative regulation of interferon regulatory factor 6 (IRF6) in interferon and NF-κB signalling pathways of common carp (Cyprinus carpio L.)

BackgroundInterferon (IFN) regulatory factors (IRFs) is a kind of transcription factors, which play an important role in regulating the expression of type I IFN and related genes. In mammals, IRF6 is not relevant with IFN expression, while zebrafish IRF6 was reported to be a positive regulator of IFN expression and could be phosphorylated by both MyD88 and TBK1. However, the role of IRF6 in the immune response and IFN transcription of common carp is unknown.ResultsIn the present study, the cDNA of IRF6 gene (CcIRF6) was cloned from common carp using RACE technique, with a total length of 1905 bp, encoding 471 amino acid residues, which possesses two functional domains of DBD and IAD. Similarity analysis showed that CcIRF6 had more than 50% similarity with IRFs of other vertebrates, and had the highest similarity with grass carp and zebrafish, among which the DBD domain was much more conserved. The phylogenetic analysis showed that CcIRF6 is in the branch of Osteichthyes and has the closest relationship with grass carp. In healthy common carp, the CcIRF6 was expressed in all the examined tissues, with the highest level in the oral epithelium, and the lowest level in the head kidney. After intraperitoneal injection of poly(I:C) or Aeromonas hydrophila, the expression of CcIRF6 increased in spleen, head kidney, foregut and hindgut of common carp. Moreover, poly(I:C), LPS, PGN and flagellin induced the expression of CcIRF6 in peripheral leukocytes and head kidney leukocytes of common carp in vitro. In EPC cells, CcIRF6 inhibited the expression of some IFN-related genes and pro-inflammatory cytokines, and dual luciferase reporter assay showed that CcIRF6 reduced the activity of IFN and NF-κB reporter genes.ConclusionsThe present study suggests that CcIRF6 is involved in the antiviral and antibacterial immune response of common carp, and negatively regulate the expression of IFN and NF-κB signalling pathways, which provides a theoretical basis for the study and prevention of fish disease pathogenesis.

Association of single nucleotide polymorphisms in the IRF6 gene with nonsyndromic cleft lip with or without cleft palate in Kinh Vietnamese patients

The interferon regulatory factor 6 (IRF6) gene, which causes Van der Woude syndrome, is associated with nonsyndromic cleft lip with or without cleft palate (NSCL/P). Single nucleotide polymorphisms (SNPs) in the IRF6 gene were identified as susceptibility indicators of this defect in several populations. To further evaluate its role in this birth defect, we conducted this study with the aim of identifying allele frequencies, genotype frequencies, and associations of 5 SNPs (rs2235373, rs2235371, rs2235375, rs2013162, and rs2236907) in the IRF6 gene with NSCL/P in Kinh Vietnamese patients. A total of 132 patients with NSCL/P and 132 healthy individuals were included in our study group. Direct sequencing was performed to genotype the tag SNPs. Genetic models were used to compare genotype and allele frequencies between the case and control groups. In the recessive model, the genotypes C/C of rs2236907, C/C of rs2013162, G/G of rs2235375, and A/A of rs2235373 were associated with an increased risk of NSCL/P, whereas there was no clear association between rs2235371 and the malformation in any genetic model. When subgroup analysis was performed, we observed a similar risk trend in the cleft lip and palate, cleft palate only and cleft lip only phenotypes. In haplotype analysis, haplotype models of 5 tag SNPs were associated with increased risks of this defect in all phenotypic models (ORGCGCC/CCAA = 23.64, 95% CI 12.28-45.49, p < 0.0001). These findings point to a considerable contribution of rs2236907, rs2013162, rs2235373, and rs2235375 to the NSCL/P defect in Kinh Vietnamese individuals.

Novel de novo missense mutation in the interferon regulatory factor 6 gene in an Italian infant with IRF6-related disorder

BackgroundCongenital maxillomandibular syngnathia is a rare craniofacial anomaly leading to difficulties in feeding, breathing and ability to thrive. The fusion may consist of soft tissue union (synechiae) to hard tissue union. Isolated cases of maxillomandibular fusion are extremely rare, it is most often syndromic in etiology.Case presentationClinical management of a female newborn with oromaxillofacial abnormities (synechiae, cleft palate, craniofacial dysmorphisms, dental anomaly) and extraoral malformations (skinfold overlying the nails of both halluces, syndactyly, abnormal external genitalia) is presented. The associated malformations addressed to molecular genetic investigations revealing an interferon regulatory factor 6 (IRF6)-related disorder (van der Woude syndrome/popliteal pterygium syndrome). A novel de novo heterozygous mutation in exon 4 of IRF6 gene on chromosome 1q32.2, precisely c.262A > G (p.Asn88Asp), was found. Similarities are discussed with known asparagine missense mutations in the same codon, which may alter IRF6 gene function by reduced DNA-binding ability. A concomitant maternal Xp11.22 duplication involving two microRNA genes could contribute to possible epigenetic effects.ConclusionsOur reported case carrying a novel mutation can contribute to expand understandings of molecular mechanisms underlying synechiae and orofacial clefting and to correct diagnosing of incomplete or overlapping features in IRF6-related disorders. Additional multidisciplinary evaluations to establish the phenotypical extent of the IRF6-related disorder and to address family counseling should not only be focused on the surgical corrections of syngnathia and cleft palate, but also involve comprehensive otolaryngologic, audiologic, logopedic, dental, orthopedic, urological and psychological evaluations.

A Novel IRF6 Frameshift Mutation in a Large Chinese Pedigree With Van der Woude syndrome.

Van der Woude syndrome (VWS) is one of the most common craniofacial anomalies, causing significant functional and psychological burden to the patients. This study aimed to identify the genetic cause of VWS in a Chinese family. Whole genome sequencing(WGS) was performed to screen for pathogenic mutations. Various Bioinformatics tools were used to assess the pathogenicity of the variants. Cosegregation analysis of the candidate variant was carried out. Interpretation of variants was performed according to the American College of Medical Genetics and Genomics guidelines. A novel frameshift duplication c.373_374dupAA (p.Asn125Lys fs*43) was identified in exon 4 of the interferon regulatory factor 6 (IRF6) gene in all 3 affected members, which were not found in unaffected family members. The novel mutation leads to a frameshift and a premature stop codon which caused putative truncated protein. Protein alignment indicated high evolutionary conservation of the p.N125 residue, and this mutation was predicted by online tools to be damaging and deleterious. This study demonstrates that the novel mutation c.373_374dupAA (p.Asn125Lysfs*43) in the IRF6 gene corresponds to the VWS in this family. The discovery of this pathogenic variant enriches the genotypic spectrum of IRF6 gene and contributes to genetic diagnosis and counseling of families with VWS.

A Novel Van der Woude Syndrome-Causing IRF6 Variant Is Subject to Incomplete Non-sense-Mediated mRNA Decay Affecting the Phenotype of Keratinocytes.

Van der Woude syndrome (VWS) is a genetic syndrome that leads to typical phenotypic traits, including lower lip pits and cleft lip/palate (CLP). The majority of VWS-affected patients harbor a pathogenic variant in the gene encoding for the transcription factor interferon regulatory factor 6 (IRF6), a crucial regulator of orofacial development, epidermal differentiation and tissue repair. However, most of the underlying mechanisms leading from pathogenic IRF6 gene variants to phenotypes observed in VWS remain poorly understood and elusive. The availability of one VWS individual within our cohort of CLP patients allowed us to identify a novel VWS-causing IRF6 variant and to functionally characterize it. Using VWS patient-derived keratinocytes, we reveal that most of the mutated IRF6_VWS transcripts are subject to a non-sense-mediated mRNA decay mechanism, resulting in IRF6 haploinsufficiency. While moderate levels of IRF6_VWS remain detectable in the VWS keratinocytes, our data illustrate that the IRF6_VWS protein, which lacks part of its protein-binding domain and its whole C-terminus, is noticeably less stable than its wild-type counterpart. Still, it maintains transcription factor function. As we report and characterize a so far undescribed VWS-causing IRF6 variant, our results shed light on the physiological as well as pathological role of IRF6 in keratinocytes. This acquired knowledge is essential for a better understanding of the molecular mechanisms leading to VWS and CLP.

Mutations in P63 and IRF-6 Present with Overlapping Craniofacial Defects: A study from Lebanon

Interferon Regulatory Factor 6 (IRF-6) and p63 are two vital transcription factors implicated in normal craniofacial development. In this report, we present a family with Van Der Woude Syndrome (VWS) with a mutation in exon 9 of IRF-6 gene and a phenotypically overlapping case of Rapp-Hodgkin Syndrome (RHS) resulting from a mutation in the p63 gene. Members from both families presented with congenital lip pits and cleft lip/palate. The RHS case had additional ectodermal features that underscore the upstream nature of p63 in the complex p63-IRF-6 interactive pathway.

Association Between an Interferon Regulatory Factor 6 Gene Polymorphism and Nonsyndromic Cleft Palate Risk.

Background: Involvement of interferon regulatory factor 6 (IRF6) gene polymorphisms in nonsyndromic cleft palate (NSCP) risk remains controversial. This investigation was performed to evaluate the relationship between IRF6 gene polymorphisms and NSCP risk. Materials and Methods: Two hundred forty-one patients with NSCP (including 103 complete trio families) were recruited, and 242 unaffected individuals were included as controls. Polymorphisms for the IRF6 rs2235371, rs801619, rs642961, rs44844880, and rs8049367 loci were characterized in both groups. Furthermore, eligible studies were identified from the databases through June 1, 2017, and were included in a meta-analysis to enhance the robustness of our conclusions. Results: The IRF6 rs2235371 A allele and AA genotype in the case group were found at higher frequencies than in the control group (A allele: p < 0.0016; AA genotype: p < 0.0049). The IRF6 rs801619 AA genotype and G allele were associated with NSCP risk (G allele: p < 0.0061; AA genotype: p < 0.0195). At the IRF6 rs642961, rs44844880, and rs8049367 loci genotype and allele frequencies were not statistically different between the NSCP group and normal controls. In the meta-analysis, the IRF6 A/G gene polymorphism (rs2235371) and IRF6 A/G gene polymorphism (rs642961) were associated with NSCP risk in the general population, whereas the IRF6 A/C gene polymorphism (rs2013162) was not. Conclusion: The IRF6 A/G gene polymorphisms at rs2235371 and rs642961, but not the IRF6 A/C gene polymorphism rs2013162, were associated with NSCP risk.

Interaction between interferon regulatory factor 6 and glycine receptor beta shows a protective effect on developing nonsyndromic cleft lip with or without cleft palate in the Han Chinese population.

Single-nucleotide polymorphisms (SNPs) in protein-coding regions of genes which were previously reported to be associated with nonsyndromic cleft lip, with or without palate involvement (NSCL/P), were investigated. Twelve candidate loci [platelet-derived growth factor C (PDGFC), platelet-derived growth factor subunit A (PDGFA), platelet-derived growth factor receptor alpha (PDGFRA), glycine receptor alpha 2 (GLRA2), glycine receptor beta (GLRB), ATP binding cassette subfamily A member 4 (ABCA4), MAF bZIP transcription factor B (MAFB), interferon regulatory factor 6 (IRF6), CCDC26 long non-coding RNA (CCDC26), paired box 7 (PAX7), ventral anterior homeobox 1 (VAX1), and netrin 1 (NTN1)] covering 1.5Mbp were sequenced in 136 NSCL/P patients and 54 healthy controls. Twenty-five genomic variants identified were further validated in another 400 NSCL/P and 200 controls. Two SNPs in IRF6 showed a protective effect against the development of NSCL/P (rs12405750, OR=0.54, 95% CI: 0.41-0.69; and rs2235371, OR=0.55, 95% CI: 0.43-0.71). The missense variant, rs2235371, alters the conserved amino acid valine to isoleucine at codon 274 (V274I). We observed that SNPs at IRF6 (rs2235371 and rs12405750) and GLRB (rs73856838 and rs72685584) show consistent interaction effects. The association between the missense SNP rs2235371 in gene IRF6 and NSCL/P suggests that this SNP may play an important role as a risk factor for NSCL/P in the Han Chinese populations. The marginal signal near 4q31 detected in previous genome-wide association studies might be caused by an interaction between the IRF6 and GLRB genes. This interaction needs to be further validated by experimentation in follow-up studies.

Association Studies Between Regulatory Regions of IRF6/TP63 Genes and Nonsyndromic Oral Clefts.

To evaluate genetic variants within the regulatory regions of interferon regulatory factor 6 (IRF6) and TP63 for the etiology of nonsyndromic oral clefts risk factors. We performed allelic transmission disequilibrium test analysis on 5 eligible single-nucleotide polymorphisms (SNPs) and SNP haplotypes using the Family-Based Association Test. The study sample consisted of 334 case-parent trios of nonsyndromic oral clefts from Taiwanese population, separated into nonsyndromic cleft lip/palate (NSCL/P) and nonsyndromic cleft palate only (NSCPO) groups. We found all 3 selected SNPs of the IRF6 gene show significant association with nonsyndromic oral clefts (rs2235371, P = 5.10E-07; rs642961, P = .00194; and rs77542756, P = 9.08E-07). Haplotype analyses identified 3 possible SNP combination haplotypes in the IRF6 gene and found that C-G-G showed significant undertransmission (P = .058), whereas 2 other haplotypes, T-G-A and C-A-G (P = 2.71E-06 and P = 5.00E-04, respectively), were significantly overtransmitted to the NSCL/P children but not to the NSCPO children. For the TP63 gene, we failed to detect evidence of nonsyndromic oral cleft association in the 2 SNPs within the TP63 large intron 1 region. We used a family-based analysis in 334 Taiwanese case-parent trios to evaluate selected SNPs of IRF6 genes and TP63 genes for a risk of orofacial clefting. This study provides additional evidence for an association between IRF6 and NSCL/P, including the genetic variants within the 5'-noncoding region of the gene. We also confirmed that NSCL/P and NSCPO individuals belong to different groups. For the TP63, our data did not favor the direct involvement of TAp63 isoforms during orofacial development.

Interferon Regulatory Factor 6 (IRF6) and Gene – Environment Interactions in Non-Syndromic Orofacial Cleft Cases in Saudi Arabia-A Case Control Study

The association between interferon regulatory factor 6 (IRF6) and nonsyndromic orofacial cleft (NSOFC) is affected by ethnicity. Also, gene-environment interactions (GEI) may play an important role in its etiology.Objectives: This case-control study investigated whether IRF6 gene variants were associated with NSOFC in Saudi Arabian population and whether the gene was affected by maternal environmental exposures.Methods: We extracted DNA from saliva samples obtained from 171 infant–parent triad cases and 189 matched controls (age, gender, and location) from January 2010–December 2011; this study included a total of 11 referral hospitals in Saudi Arabia. IRF6 (rs2013162, rs2235375, and rs2235371) polymorphisms were genotyped using restriction-digestion polymerase chain reaction. Data on environmental exposures, for GEI analyses, were collected through questionnaire-led interviews with parents.Results: We found statistically significant over transmission of the common IRF6 rs2013162 allele among cleft lip with or without palate CL(P) cases. No associations were found for either of the other two IRF6 SNPs. Maternal exposure to antipyretics, folic acid, fever, antibiotics, illnesses, common cold/flu, paternal water pipe smoking, stress, x-rays, and/or chemicals could significantly interact with the maternal IRF6 (rs2013162 and rs2235375) gene variants, affecting the likelihood of having an offspring with NSOFC.Conclusion: The common allele at IRF6 rs2013162 was significantly over transmitted among CL(P) cases. This study provides hypotheses for future investigations into genetic and environmental factors and their interaction in the etiology of NSOFC.

Association between genotype and phenotype of virulence gene in Van der Woude syndrome families.

Members from two Van der Woude syndrome (VWS) families were screened to determine the prevalence of interferon regulatory factor 6 (IRF6) as a disease‑causing gene and to analyze the interrelationships between patient genotype and phenotype. The peripheral blood of 24 members from two VWS families and 200 control samples were collected. The family members were interviewed for medical histories and other clinical abnormalities using questionnaires. Polymerase chain reaction was directly performed on the peripheral blood to screen for the coding region of the IRF6 gene. Of the 24 family members, a total of 6patients had mutations of IRF6 gene. c.1234C>T (p.R412X) heterozygous mutation was detected in 3 members of family 1. In families 2 and 3, members carried the c.1210G>A (p.E404K) heterozygous mutations. The other members of the families, were wild type (wt/wt) for IRF6. Genetic testing demonstrated that the disease mutations c.1234C>T and c.1210G>A co‑segregated with the two families' pathogenic mutations. The existence of genetic heterogeneity and the complexity of the clinical phenotype was demonstrated in Chinese VWS patients.

Rapid functional analysis of computationally complex rare human IRF6 gene variants using a novel zebrafish model.

Large-scale sequencing efforts have captured a rapidly growing catalogue of genetic variations. However, the accurate establishment of gene variant pathogenicity remains a central challenge in translating personal genomics information to clinical decisions. Interferon Regulatory Factor 6 (IRF6) gene variants are significant genetic contributors to orofacial clefts. Although approximately three hundred IRF6 gene variants have been documented, their effects on protein functions remain difficult to interpret. Here, we demonstrate the protein functions of human IRF6 missense gene variants could be rapidly assessed in detail by their abilities to rescue the irf6 -/- phenotype in zebrafish through variant mRNA microinjections at the one-cell stage. The results revealed many missense variants previously predicted by traditional statistical and computational tools to be loss-of-function and pathogenic retained partial or full protein function and rescued the zebrafish irf6 -/- periderm rupture phenotype. Through mRNA dosage titration and analysis of the Exome Aggregation Consortium (ExAC) database, IRF6 missense variants were grouped by their abilities to rescue at various dosages into three functional categories: wild type function, reduced function, and complete loss-of-function. This sensitive and specific biological assay was able to address the nuanced functional significances of IRF6 missense gene variants and overcome many limitations faced by current statistical and computational tools in assigning variant protein function and pathogenicity. Furthermore, it unlocked the possibility for characterizing yet undiscovered human IRF6 missense gene variants from orofacial cleft patients, and illustrated a generalizable functional genomics paradigm in personalized medicine.

Silencing of interferon regulatory factor gene 6 in melanoma.

BackgroundMethylation of a CpG island (CGI; a dense cluster of CpGs) located in the 5' region of a gene suppresses transcription of that gene. Interferon regulatory factor 6 (IRF6) is associated with the expression of interferon, which is used as an effective adjuvant therapy for melanoma, and is regarded as a tumor suppressor. However, little is known about the methylation status of the IRF6 gene in melanoma.ObjectiveThe purpose was to determine the methylation status of the CGI located in the 5' region of IRF6 (5' IRF6 CGI) in melanoma.MethodsQuantitative real-time methylation-specific PCR (RT-MSP) and bisulfite sequencing were performed to examine IRF6 gene methylation status. Quantitative real-time reverse transcription-PCR (RT-PCR) was performed to examine IRF6 expression.ResultsThe methylation level of the 5' IRF6 CGI was completely inversely correlated with cell sensitivity to interferon-β in eight examined melanoma cell lines. These methylation levels were high in the melanoma cell lines with suppression of IRF6 expression and were low in the cell lines with IRF6 expression. The methylation levels of the 5' IRF6 CGI ranged widely from 0.0% to 65.4% in 21 clinical melanoma samples but showed a narrow range of low levels between 0.0% to 7.2% in 24 clinical melanocytic nevus samples. These methylation levels were not associated with clinical parameters except for melanoma subtypes.ConclusionIRF6 is aberrantly silenced by DNA methylation of the 5' IRF6 CGI in melanoma. The methylation status of IRF6 is potentially associated with the sensitivity of melanoma to interferon.

Presence of sequence and SNP variation in the IRF6 gene in healthy residents of Guangdong Province

AbstractObjectiveThis study was to investigate the single nucleotide polymorphism (SNP) in the interferon regulatory factor 6 (IRF6) gene in healthy residents of Guangdong Province, China, for further analysis of their associations with the development of cleft lip with or without palate (CL/P).MethodologyDNA was extracted from blood samples of 13 healthy residents. IRF6 genes were sequenced and analyzed by alignment to the reference sequences in GenBank.ResultsThe IRF6 genes containing 45.21% GC were 25016–25046 bp in length, and had 2215-bp exons and a 1404-bp coding region. There were 65 SNPs, including 58 SNPs in the 5′-untranslated region or introns and 7 SNPs in the exons. These sites had two alleles, including 39 transition sites and 26 transversion sites. Five novel SNPs were identified. c. 459G&gt;T and c. 820G&gt;A in the coding region represented silent and Val274Ile mutations, respectively. The SNPs made two sequences (GenBank HQ875393 and JF346417).ConclusionsThe sequences and SNP profiles of the IRF6 gene in healthy residents of Guangdong Province are consistent with the one in GenBank but with slight variations. Our findings form a basis for further investigation of the association between IRF6 gene polymorphisms and CL/P in residents of Guangdong Province.

Association of single-nucleotide polymorphisms, rs2235371 and rs2013162, in the IRF6 gene with non-syndromic cleft palate in northeast China

The aim of this study was to determine the association between two SNPs (rs2235371 and rs2013162) in the interferon regulatory factor 6 (IRF6) gene and non-syndromic cleft palate (NSCP) in northeast China. We genotyped these two SNPs in 104 NSCP cases, as well as in 178 parents and 300 controls. Case-control and case-parent analyses were performed using χ2 tests and family-based association tests (FBAT). Results indicated that there were significant differences in both genotypic and allelic distributions between patients and controls at rs2235371 and rs2013162 in the IRF6 gene. Case-parent analysis revealed over-transmission of the C allele in rs2235371 and the A allele in rs2013162. Lastly, FBAT showed over-transmission of the CA haplotype. This study demonstrated that the two SNPs, rs2235371 and rs2013162, are strongly associated with NSCP in the northeast Chinese population.

Abstract 3840: A new prognostic marker: interferon regulatory factor 6 in renal cell carcinoma

Abstract According to our previous methylated-CpG island recovery assay (MIRA) and RNA expression array findings, the results indicated methylated status of Interferon regulatory factor 6 (IRF6) could be observed in most of renal cell carcinoma (RCC) cases (40.6%), and presented a negative correlation with gene expression, clear cell type of RCCs especially. As known that IRF6 is one of members of IRF family. It has been described might play an important role in epidermal cells, and correlated with cell proliferation and differentiation. The regulation of feedback loop between IRF6 and ΔNp63, which is known could control the mechanism of keratinocytes differentiation and palate closure, has also been noted. In addition, the tumor suppressor activity of IRF6 had been also demonstrated in squamous cell carcinoma associated with inhibition of tumor cell invasion and migration. The aim of this study is to clarify the clinical significance and role of IRF6 in RCC. We hypothesized low expression of IRF6 via DNA methylation might lead to irregular differentiation of RCC cells, and then further trigger cells proliferated out of control. In total, 105 pairs of clinical RCCs patients and eight RCC cell lines (included chromophobe type RCC98, sacomatoid type RCC52, clear cell type Caki-1, HH332, HOKN-9, RCC100, papillary type HH050 and adenocarcinoma 786-O), as well as Human Embryonic Kidney 293 cell used as control have involved in current study. Firstly, we performed real-time PCR on all cases. Secondly, 5-aza-2′deoxycytidine was treated on all type of RCC cell lines; it is a way of de-methylated DNA. Western blot detections were then to confirm whether the expression level of IRF6 restored in RCC cells. The IRF6 gene expressed levels differ from normal and RCC tissues were applied by the paired- T test, and shown by -ΔCT. Generally, the variant and lower level gene expression of the IRF6 could be observed from all different type of RCC cell lines, except RCC98 and RCC100 cells. After treated with 5-aza-2′deoxycytidine on RCC cell lines, the obvious expression of IRF6 was restored in all clear cell RCC cell lines. The mean - ΔCT of normal tissue was -8.0, and RCC tissue was -11.5, significantly IRF6 expressed levels differ from normal and tumor tissues could be noted (p = 0.013). The IRF6 gene was usually expressed lower level in RCC due to methylation. Our findings demonstrated the expression of IRF6 could be easily resorted by de-methylated in RCC cells, and it might further inhibit cell proliferation in the tumoral progression, and might consider as a prognostic marker to predict patients’ outcomes in RCC. However, the further cell viability and correlation with the clinical information should be further analyzed in the future. Citation Format: Chih-Yang Wei, Ying-Tzu Chen, See-Tong Pang, Wen-Hui Weng. A new prognostic marker: interferon regulatory factor 6 in renal cell carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3840. doi:10.1158/1538-7445.AM2015-3840

A novel RIPK4-IRF6 connection is required to prevent epithelial fusions characteristic for popliteal pterygium syndromes.

Receptor-interacting protein kinase 4 (RIPK4)-deficient mice have epidermal defects and fusion of all external orifices. These are similar to Bartsocas-Papas syndrome and popliteal pterygium syndrome (PPS) in humans, for which causative mutations have been documented in the RIPK4 and IRF6 (interferon regulatory factor 6) gene, respectively. Although genetically distinct, these syndromes share the anomalies of marked pterygia, syndactyly, clefting and hypoplastic genitalia. Despite the strong resemblance of these two syndromes, no molecular connection between the transcription factor IRF6 and the kinase RIPK4 was known and the mechanism underlying the phenotype was unclear. Here we describe that RIPK4 deficiency in mice causes epithelial fusions associated with abnormal periderm development and aberrant ectopic localization of E-cadherin on the apical membrane of the outer peridermal cell layers. In Xenopus, RIPK4 depletion causes the absence of ectodermal epiboly and concomitant gastrulation defects that phenocopy ectopic expression of dominant-negative IRF6. We found that IRF6 controls RIPK4 expression and that wild-type, but not kinase-dead, RIPK4 can complement the gastrulation defect in Xenopus caused by IRF6 malfunctioning. In contrast to the mouse, we observed only minor effects on cadherin membrane expression in Xenopus RIPK4 morphants. However, gastrulation defects were associated with a virtual absence of cortical actin in the ectodermal cells that face the blastocoel cavity and this was phenocopied in embryos expressing dominant-negative IRF6. A role for RIPK4 in actin cytoskeleton organization was also revealed in mouse epidermis and in human epithelial HaCaT cells. In conclusion, we showed that in mice RIPK4 is implicated in cortical actin organization and in E-cadherin localization or function, which can explain the characteristic epithelial fusions observed in PPSs. In addition, we provide a novel molecular link between IRF6 and RIPK4 that unifies the different PPSs to a common molecular pathway.

Sequencing of the interferon regulatory factor 6 (IRF6) gene and correlation to its phenotypes in familial non-syndromic cleft lip and palate in North Indian population

The incidence of orofacial clefting varies depending upon geographical location, ethnicity, sex and even socioeconomic status. The study aimed to test the contribution of interferon regulatory factor 6 (IRF6) gene allelic variation to the incidence of non-syndromic cleft lip and/or cleft palate (CL/P) from the population of North India. Patients from 12 North Indian families with non-syndromic cleft lip with or without cleft palate (NSCL/P) with at least two members affected were clinically evaluated and tested. DNA was extracted from the blood, and three exonic part of the IRF6 gene (exons 2, 5 and 7) were amplified by PCR. Direct sequencing was performed by PCR product, and sequencing results were compared with the reference DNA sequence of the IRF6 gene. Three SNPs (rs861019, rs2013162 and rs2235372) in 4 out of 12 families were observed. SNP rs861019 was detected in family no. 4 (F4) around exon 2, SNP 2013162 was detected in family no. 10 (F10) and SNP 2235372 was detected in family nos. 5 and 9 (F5 and F9). This study demonstrates that the three SNPs within the IRF6 gene are associated with NSCL/P, suggesting IRF6 gene sequence variability and variation in this gene, providing first genetic clue to the NSCL/P phenotype in North Indian families. This is our initial step to demonstrate the involvement of the IRF6 gene in NSCL/P patients in North India. Further extensive study and statistical analysis are needed to validate our result. Level of Evidence: Level IV, diagnostic study.