IRF6 Research Articles

The IRF6 AP-2α binding site polymorphism relate to the severity of non-syndromic orofacial cleft of Indonesian patients.

IRF6 AP-2α binding site polymorphism is known as IRF6 rs642961. It has been associated with a nonsyndromic orofacial cleft (NS OFC). This study aimed to determine the IRF6 rs642961 as a risk factor associated with NS OFC and its phenotypes. The case-control design used for 264 subjects consists of 158 NS CLP subjects (42 CU CLP, 34 CB CLP, 33 CLO, 49 CPOs) and 106 healthy controls. The DNA is extracted from venous blood. The segment of IRF6 rs642961 amplified by polymerase chain reaction (PCR) followed by restriction fragment length of polymorphisms (RFLPs) used the MspI digestion enzyme. The qPCR method to identify the mRNA expression levels of the IRF6 gene rs642961 was analyzed by the Livak method. The study results show that in NS CB CLP phenotype as the most severe phenotype of NS OFC, the Odds Ratio (OR) of A mutant allele was 5.094 (CI=1.456-17.820; P=0.011) and the OR of AA homozygous mutant genotype was 13.481 (CI=2.648-68.635; P=0.001). There are different levels of mRNA expression changes from NS OFC and its phenotypes. It is substantial among the 2-ΔΔCt and the group of AA, GA, and GG genotypes (P<0.05); in the NS CPO phenotype, it shows IRF6 mRNA under-expression in GA, AA genotypes while in other phenotypes it shows IRF6 mRNA overexpression. The IRF6 AP-2α binding site polymorphism is strongly associated with the severity of NS OFC, and this polymorphism has a functional role in affecting IRF6 mRNA expression that is variable in each phenotype.

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.

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.

Consistent downregulation of the cleft lip/palate-associated genes IRF6 and GRHL3 in carcinomas.

Interferon Regulatory Factor 6 (IRF6) and Grainyhead Like Transcription Factor 3 (GRHL3) are transcription factors that orchestrate gene regulatory networks required for the balance between keratinocyte differentiation and proliferation. Absence of either protein results in the lack of a normal stratified epidermis with keratinocytes failing to stop proliferating and to terminally differentiate. Numerous pathological variants within IRF6 and GRHL3 have been identified in orofacial cleft-affected individuals and expression of the two transcription factors has been found to be often dysregulated in cancers. However, whether orofacial cleft-associated IRF6 and GRHL3 variants in patients might also affect their cancer risk later in life, is not clear yet. The fact that the role of IRF6 and GRHL3 in cancer remains controversial makes this question even more challenging. Some studies identified IRF6 and GRHL3 as oncogenes, while others could attribute tumor suppressive functions to them. Trying to solve this apparent conundrum, we herein aimed to characterize IRF6 and GRHL3 function in various types of carcinomas. We screened multiple cancer and normal cell lines for their expression, and subsequently proceeded with functional assays in cancer cell lines. Our data uncovered consistent downregulation of IRF6 and GRHL3 in all types of carcinomas analyzed. Reduced levels of IRF6 and GRHL3 were found to be associated with several tumorigenic properties, such as enhanced cell proliferation, epithelial mesenchymal transition, migration and reduced differentiation capacity. Based on our findings, IRF6 and GRHL3 can be considered as tumor suppressor genes in various carcinomas, which makes them potential common etiological factors for cancer and CLP in a fraction of CLP-affected patients.

TWIST1 interacts with β/δ-catenins during neural tube development and regulates fate transition in cranial neural crest cells.

Cell fate determination is a necessary and tightly regulated process for producing different cell types and structures during development. Cranial neural crest cells (CNCCs) are unique to vertebrate embryos and emerge from the neural plate borders into multiple cell lineages that differentiate into bone, cartilage, neurons and glial cells. We have previously reported that Irf6 genetically interacts with Twist1 during CNCC-derived tissue formation. Here, we have investigated the mechanistic role of Twist1 and Irf6 at early stages of craniofacial development. Our data indicate that TWIST1 is expressed in endocytic vesicles at the apical surface and interacts with β/δ-catenins during neural tube closure, and Irf6 is involved in defining neural fold borders by restricting AP2α expression. Twist1 suppresses Irf6 and other epithelial genes in CNCCs during the epithelial-to-mesenchymal transition (EMT) process and cell migration. Conversely, a loss of Twist1 leads to a sustained expression of epithelial and cell adhesion markers in migratory CNCCs. Disruption of TWIST1 phosphorylation in vivo leads to epidermal blebbing, edema, neural tube defects and CNCC-derived structural abnormalities. Altogether, this study describes a previously uncharacterized function of mammalian Twist1 and Irf6 in the neural tube and CNCCs, and provides new target genes for Twist1 that are involved in cytoskeletal remodeling.

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.

141 A noncanonical role for IRF6 in promoting cellular adhesions

We recently demonstrated that Interferon Regulatory Factor 6 (IRF6) is a transcription factor essential for keratinocyte adhesion. Specifically, it is required for the recycling and delivery of E-cadherin to the plasma membrane. Interestingly, the total E-cadherin levels were not altered in the absence of IRF6, and under culture conditions used to test keratinocyte adhesion behavior, IRF6 was not detected in the nucleus. IRF6 protein includes a DNA binding domain (DBD), a protein binding domain (PBD), and a serine rich autoinhibitory region (SRR).

LncRNA ENSMUST_147219 mediates the progression of ischemic acute kidney injury by targeting the miR-221-5p/IRF6 axis

Although previous studies have revealed that long noncoding RNAs (lncRNAs) regulate the progression of ischemic acute kidney injury (AKI), the exact role and mechanism of lncRNA ENSMUST_147219 in ischemic AKI are not clear. In the present study, lncRNA ENSMUST_147219 was induced by ischemic injury in vitro and in vivo. Functionally, lncRNA ENSMUST_147219 mediated apoptosis in mouse proximal tubule‐derived cell line (BUMPT). Mechanistically, lncRNA ENSMUST_147219 sponged the microRNA (miR)-221-5p to upregulate the expression of interferon regulatory factor 6 (IRF6) to drive apoptosis. Finally, knockdown of lncRNA ENSMUST_147219 markedly attenuated the ischemic AKI by targeting the miR-221-5p/IRF6 axis. Collectively, our data demonstrated that lncRNA ENSMUST_147219 promoted the development of ischemic AKI by regulating the miR-221-5p/IRF6 pathway, which could be considered a new therapeutic target for ischemic AKI.

Rab11A forms a complex with Interferon Regulatory Factor 6 in recycling endosomes

Interferon Regulatory Factor 6 (IRF6) is a transcription factor essential for regulating keratinocyte migration, proliferation and differentiation. Structurally, the IRF6 protein includes a DNA binding domain (DBD), a protein binding domain (PBD), and a serine rich autoinhibitory region (SRR). Historically, IRF6 has been assumed to play its role by regulating gene expression, however growing evidence suggest that some of its functions may be dependent on protein‐protein interactions. Although the effect of such interactions on the regulated proteins remains to be elucidated, some groups have proposed a protective role for IRF6 preventing protein degradation. Alternatively, we hypothesize that IRF6 may also play a scaffolding function regulating the subcellular localization of effector proteins. We recently demonstrated that IRF6 is required for the delivery of E‐cadherin to the plasma membrane. More specifically, we found that recycling of E‐cadherin was defective in absence of IRF6. Interestingly, the total E‐cadherin levels were not altered in the absence of IRF6, and under culture conditions used to test keratinocyte adhesion behavior, IRF6 was not detected in the nucleus. To explore the molecular mechanism used by IRF6 to regulate E‐cadherin at the plasma membrane, we investigated whether IRF6 colocalized with NME1 and NME2, two proteins known to internalize E‐cadherin towards recycling endosomes and regulate the secretory pathway, respectively. We found that in our experimental conditions NME2 does not colocalize with IRF6. However, by immunofluorescence, IRF6 and NME1 colocalize and their mutual coimmunoprecipitation suggest they are part of the same complex. Because NME1 is necessary for E‐cadherin recycling, we hypothesized that IRF6‐NME1 regulates recycling endosome function. To test our hypothesis, we evaluated the interaction between E‐cadherin, IRF6 and Rab11A, a required promoter of recycling endosome. Our results show that Rab11A colocalizes with all these proteins in recycling endosomes, and that IRF6, Rab11A and NME1 coimmunoprecipitated with each other, suggesting that they belong to the same complex. Overall, our results suggest that IRF6 participate in a complex with Rab11A and NME1, potentially modulating recycling endosome function and promoting the recyling of Ecadherin.

Investigating the Effects of IRF6 on Focal Adhesions in Keratinocytes

Focal adhesions are one of the two main types of adhesions between cells and the extra‐cellular matrix (ECM). Focal adhesions are dynamic complexes anchored to the actin cytoskeleton through intracellular adaptor proteins, and to the ECM through heterodimers of α and β integrins, forming a transmembrane link between the intra‐ and extra‐cellular space. β‐1 integrin is the primary β subunit found in keratinocytes, and its corresponding heterodimers show affinity for a wide range of ECM proteins including collagen, fibronectin, and laminin. Collagen is the main structural protein in cutaneous ECM (the dermis), while fibronectin is a glycoprotein found in the ECM that binds integrins and plays a crucial role in wound healing. Interferon regulatory factor 6 (IRF6) is a transcription factor expressed by epithelial cells that regulates keratinocyte differentiation, cell‐cell adhesions, and migration. It is well known that focal adhesions are required for cell migration, however the role of IRF6 in regulating focal adhesions is unknown. Therefore, we hypothesize that IRF6 regulates focal adhesion formation. To determine if IRF6 levels affect attachment to the ECM, we seeded wildtype and IRF6 knockdown keratinocytes on collagen IV and plastic and quantified the number of cells attached to the plate after thirty and sixty minutes. No differences were found between wild‐type and IRF6 knockdown keratinocytes on plastic. However, less IRF6 knockdown keratinocytes were attached on collagen IV compared to wildtypes. To explore how IRF6 affects focal adhesions, we plated IRF6 knockdown and wild‐type keratinocytes on different types of ECM that were representative of both normal and wounded conditions of the dermis. Immunofluorescent staining for β‐1 integrin and phalloidin was performed, followed by quantitative analysis of focal adhesion length and intensity. Initial observations indicate that IRF6 knockdown keratinocytes consistently have longer focal adhesions compared to the wildtype. Variations of β‐1 fluorescent intensity was also observed in wild‐type cells compared to IRF6 knockdown keratinocytes. These preliminary results suggest a role for IRF6 in regulating focal adhesions, providing a potential mechanism for the underlying defect observed in the migration of IRF6 deficient keratinocytes.

The genetic factors contributing to the risk of cleft lip-cleft palate and their clinical utility.

Cleft lip and cleft palate (CL/P) are among the most common congenital malformations in neonates and have syndromic or nonsyndromic forms. Nonsyndromic forms of malformation are being reported to be associated with chromosomal DNA modification by teratogenic exposure and to complex genetic contributions of multiple genes. Syndromic forms are shown to be related to chromosomal aberrations or monogenic diseases. There is a growing body of data illustrating the association of several genes with risk of developing this malformation, including genetic defects in T-box transcription factor-22 (TBX22), interferon regulatory factor-6 (IRF6), and poliovirus receptor-like-1 (PVRL1), responsible for X-linked cleft palate, cleft lip/palate-ectodermal dysplasia syndrome, and Van der Woude and popliteal pterygium syndromes, respectively. Genetic variants in MTR, PCYT1A, ASS1, SLC 25A13, GSTM1, GSTT1, SUMO1 BHMT1, and BHMT2 are being reported to be linked with CL/P risk. The etiology of nonsyndromic CLP is still remained to be unknown, although mutations in candidate genes have been found. Here, we provide an overview about the potential variants to be associated with CL/P for identification of the relative risk of CLP with respect to the basis of genetic background and environmental factors (e.g., dietary factors, alcohol use).

The interferon regulatory factor 6 promotes cisplatin sensitivity in colorectal cancer

ABSTRACT Colorectal cancer (CRC) is one of the most common malignancies and causes of cancer-related mortality worldwide. Cell proliferation and tumor metastasis as well as chemoresistance are correlated with poor survival of CRC. The interferon regulatory factor 6 (IRF6) is functioned as a tumor suppressor gene in several cancers and is associated with risk of CRC. We explored the role of IRF6 in CRC in the present study. The protein expressions of IRF6 in human CRC tissues, normal para-carcinoma tissue and liver metastases from CRC were measured. Cell proliferation, chemotherapeutic sensitivity, cell apoptosis, migration and invasion including the related markers along with IRF6 expression were explored. Our results indicated that IRF6 expression in CRC and liver metastasis were lower than normal tissues, which were correlated positively with E-cadherin and negatively with Ki67 expression in CRC tissue. IRF6 promoted CRC cell sensitivity to cisplatin to suppress cell proliferation, migration and invasion as well as aggravate cell apoptosis. Our study suggested that IRF6 may enhance chemotherapeutic sensitivity of cisplatin mediated by affecting cell proliferation, migration and invasion along with apoptosis through regulating E-cadherin and Ki67, while the identified molecular mechanisms remain to be further explored.

Genetic comorbidity of hypertension and bronchial asthma

Objective. To assess the association of single nucleotide polymorphisms of genes potentially involved in the comorbidity of bronchial asthma (BA) and essential hypertension (HTN) in patients with different time onset of the diseases.Design and methods. Genotyping of 92 SNPs was performed using MALDI-TOF mass spectrometry in patients with BA and HTN (n = 97) and healthy individuals (n = 153). The group of patients with comorbid pathology was divided into two subgroups depending on the time of onset of symptoms of BA relative to HTN, and the prevalence of all studied SNPs was compared in each subgroup relative to the control.Results. The variant rs11590807 regulating expression for UTP25, TRAF3IP3, C1orf74, HSD11B1-AS 1, IRF6 genes in the heart, blood vessels, and lung is associated with BA and HTN, regardless of the time onset of each of these diseases. Associations of other variants are specific with respect for each subgroup of comorbid diseases. The rs1010461 variant, which regulates the expression of RNASE4 and ANG genes, is linked with HTN as the first phenotype of the comorbidity. The rs769214, rs11032700, rs11032699, rs484214, and rs480575 variants, which regulate the expression of CAT gene, are associated with BA as the first phenotype of disease comorbidity.Conclusions. We found specific associations of the studied polymorphic variants in the development of comorbid phenotypes of BA and HTN, which differ in the time of manifestation of each of the diseases relative to each other.

Investigation of Molecular Iridium Fluorides IrFn (n=1-6): A Combined Matrix-Isolation and Quantum-Chemical Study.

The photo‐initiated defluorination of iridium hexafluoride (IrF6) was investigated in neon and argon matrices at 6 K, and their photoproducts are characterized by IR and UV‐vis spectroscopies as well as quantum‐chemical calculations. The primary photoproducts obtained after irradiation with λ=365 nm are iridium pentafluoride (IrF5) and iridium trifluoride (IrF3), while longer irradiation of the same matrix with λ=278 nm produced iridium tetrafluoride (IrF4) and iridium difluoride (IrF2) by Ir−F bond cleavage or F2 elimination. In addition, IrF5 can be reversed to IrF6 by adding a F atom when exposed to blue‐light (λ=470 nm) irradiation. Laser irradiation (λ=266 nm) of IrF4 also generated IrF6, IrF5, IrF3 and IrF2. Alternatively, molecular binary iridium fluorides IrF n (n=1–6) were produced by co‐deposition of laser‐ablated iridium atoms with elemental fluorine in excess neon and argon matrices under cryogenic conditions. Computational studies up to scalar relativistic CCSD(T)/triple‐ζ level and two‐component quasirelativistic DFT computations including spin‐orbit coupling effects supported the formation of these products and provided detailed insights into their molecular structures by their characteristic Ir−F stretching bands. Compared to the Jahn‐Teller effect, the influence of spin‐orbit coupling dominates in IrF5, leading to a triplet ground state with C 4v symmetry, which was spectroscopically detected in solid argon and neon matrices.

Induction of Salivary Gland-Like Tissue by Induced Pluripotent Stem Cells In Vitro.

To investigate the in vitro induction of salivary gland-like tissue by ips cells in an interferon regulatory factor 6 (IRF6) overexpression and parotid conditioned medium environment. Urine-derived ips cells were isolated, identified, transfected with IRF6 and cultured in parotid conditioned medium to induce ips cells into salivary gland differentiation, morphological changes of ips cells were observed, CCK-8 was used to determine the cell proliferation efficiency and transcriptome sequencing was used to detect the expression of genes related to parotid gland formation. Immunofluorescence staining showed that the isolated ips cells were positive for NANOG, SSEA4 and OCT4 and had embryonic-like stem cell characteristics; CCK-8 showed that there was no statistical difference in the proliferation efficiency between the IRF6+ induced group and the simple induced group after induction of ips cells into salivary glands. The results of transcriptome sequencing showed that there were a total of 643 differentially expressed genes, including 365 up-regulated genes and 278 down-regulated genes in the IRF6+ induced group compared to the blank control group, and the salivary gland related genes HAPLN1, CCL2, MSX2, ANXA1, CYP11A1, HES1 and LUM were all highly expressed in the IRF6+ induced group. IRF6 promotes salivary gland differentiation in urine-derived iPSCs, and its mechanism of promoting differentiation may be that IRF6 upregulates the expression of HAPLN1, CCL2, MSX2, ANXA1, CYP11A1, HES1 and LUM to promote epithelial differentiation.

Van der Woude syndrome

Orofacial clefts are common birth defects and Van der Woude syndrome (VWS) is the most common form of orofacial cleft syndrome, accounting for approximately 2% of patients with a cleft.[1,2] The cardinal features of VWS are lower lip pits associated with cleft lip and/or palate.[2-4] Lip pits are reported to occur in over 80% of individuals with VWS.[2] Most reported cases of VWS have been linked to chromosome 1q32-q41.[5-7] The interferon regulatory factor 6 (IRF6) gene, which is located at 1q32-p41 region, has been implicated in several studies.[2,8] There are over 300 IRF6 variants that have been identified in patients with VWS, with approximately 50% of these being missense variants.[9] This paper describes a 7.5-year-old male patient that is heterozygous for a missense variant in C.101A &gt;C p.(Lys34Thr) which is likely to be pathogenic.[9] This patient has the cardinal features for VWS but also has duodenal atresia. Neither the truncating variant identified in this patient nor other variants associated with VWS have been previously linked to duodenal atresia.

Blood biomarkers representing maternal-fetal interface tissues used to predict early-and late-onset preeclampsia but not COVID-19 infection

BackgroundA well-known blood biomarker (soluble fms-like tyrosinase-1 [sFLT-1]) for preeclampsia, i.e., a pregnancy disorder, was found to predict severe COVID-19, including in males. True biomarker may be masked by more-abrupt changes related to endothelial instead of placental dysfunction. This study aimed to identify blood biomarkers that represent maternal-fetal interface tissues for predicting preeclampsia but not COVID-19 infection. MethodsThe surrogate transcriptome of tissues was determined by that in maternal blood, utilizing four datasets (n = 1354) which were collected before the COVID-19 pandemic. Applying machine learning, a preeclampsia prediction model was chosen between those using blood transcriptome (differentially expressed genes [DEGs]) and the blood-derived surrogate for tissues. We selected the best predictive model by the area under the receiver operating characteristic (AUROC) using a dataset for developing the model, and well-replicated in datasets both with and without an intervention. To identify eligible blood biomarkers that predicted any-onset preeclampsia from the datasets but that were not positive in the COVID-19 dataset (n = 47), we compared several methods of predictor discovery: (1) the best prediction model; (2) gene sets of standard pipelines; and (3) a validated gene set for predicting any-onset preeclampsia during the pandemic (n = 404). We chose the most predictive biomarkers from the best method with the significantly largest number of discoveries by a permutation test. The biological relevance was justified by exploring and reanalyzing low- and high-level, multiomics information. ResultsA prediction model using the surrogates developed for predicting any-onset preeclampsia (AUROC of 0.85, 95 % confidence interval [CI] 0.77 to 0.93) was the only that was well-replicated in an independent dataset with no intervention. No model was well-replicated in datasets with a vitamin D intervention. None of the blood biomarkers with high weights in the best model overlapped with blood DEGs. Blood biomarkers were transcripts of integrin-α5 (ITGA5), interferon regulatory factor-6 (IRF6), and P2X purinoreceptor-7 (P2RX7) from the prediction model, which was the only method that significantly discovered eligible blood biomarkers (n = 3/100 combinations, 3.0 %; P =.036). Most of the predicted events (73.70 %) among any-onset preeclampsia were cluster A as defined by ITGA5 (Z-score ≥ 1.1), but were only a minority (6.34 %) among positives in the COVID-19 dataset. The remaining were predicted events (26.30 %) among any-onset preeclampsia or those among COVID-19 infection (93.66 %) if IRF6 Z-score was ≥-0.73 (clusters B and C), in which none was the predicted events among either late-onset preeclampsia (LOPE) or COVID-19 infection if P2RX7 Z-score was <0.13 (cluster C). Greater proportions of predicted events among LOPE were cluster A (82.85 % vs 70.53 %) compared to early-onset preeclampsia (EOPE). The biological relevance by multiomics information explained the biomarker mechanism, polymicrobial infection in any-onset preeclampsia by ITGA5, viral co-infection in EOPE by ITGA5-IRF6, a shared prediction with COVID-19 infection by ITGA5-IRF6-P2RX7, and non-replicability in datasets with a vitamin D intervention by ITGA5. ConclusionsIn a model that predicts preeclampsia but not COVID-19 infection, the important predictors were genes in maternal blood that were not extremely expressed, including the proposed blood biomarkers. The predictive performance and biological relevance should be validated in future experiments.

Association of rs2013162 and rs2235375 Polymorphisms in IRF6 Gene with Susceptibility to Non-Syndromic Cleft Lip and Palate.

Background:Non-syndromic cleft lip occurs by the interaction of environmental and genetic factors. The purpose of the current study was to analyze the association of Single Nucleotide Polymorphisms (SNPs) in IRF6 and NSCL/P in an Iranian population.Methods:A group of 105 children with NSCL/P and 185 normal controls were included in the current study. Genotyping of IRF6 rs2013162 and rs2235375 was performed by Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) method.Results:A substantial association of AA and CA genotypes in rs2013162 with the risk of NSCL/P (AA vs. CC; OR=2.36; 95%CI [1.05–5.29], p=0.004; and CA vs. CC; OR=0.47; 95%CI [0.28–0.79], p=0.018) was found. However, there were no important associations between A allele and risk of NSCL/P (p=0.980). According to logistic regression analysis results, subjects with GG genotype and G allele in rs2235375 polymorphism had increased risk of NSCL/P.Conclusion:The IRF6 polymorphisms are associated with the susceptibility to NSCL/P in Iranian population.