Mutations In Interferon Regulatory Factor Research Articles

Lymphoproliferative disorder – Castleman’s disease with early childhood-onset in a child (a clinical case)

Castleman’s disease (angiofollicular lymphoma, giant lymph node hyperplasia, Castleman’s pseudotumor) is an understudied orphan lymphoproliferative disease with a long period of asymptomatic course and a high risk of malignancy, and variability of its clinical features can cause difficulties in diagnosis. The aim of the study. To acquaint clinicians and increase the effectiveness of early diagnosis and treatment of Castleman’s disease (CD) in children to prevent malignancy and improve prognosis. Results. The article presents a clinical case of CD in a 3.5-year-old boy with a long history of fever, stunted growth, sweating, arthralgia, anemia, high laboratory inflammatory factors, elevated IL-6. There was a complex diagnostic search except for infectious, immunodeficiency, autoimmune conditions. The diagnosis of hyaline-vascular type of CD was confirmed by immunohistochemical examination after lymph node excisional biopsy. Rapid positive dynamics was noted after radical removal of the lymph node conglomerate. At follow-up after 8 months, the patient met criteria for clinical and laboratory remission. Conclusions. Castleman’s disease should be included in the search algorithm for lymphoproliferative diseases. Determining the role of genetic mutations in interferon regulatory factor (IRF8) is of scientific interest to clarify the etiology of this disease.

IRF6 is Required for Cell‐Extracellular Matrix Adhesions

Patients with mutations in Interferon Regulatory Factor 6 (IRF6) display increased post-surgical wound healing complications. IRF6 is a transcription factor that functions as a master regulator of keratinocyte differentiation.. We demonstrated that IRF6 is also necessary for keratinocyte migration. Cellular adhesions are essential structures for cell migration. They mediate attachments between cells to the extracellular matrix (ECM), or between cells themselves, and these two different types of adhesions are known to regulate each other. Keratinocytes deficient for IRF6 show defective subcellular localization of components of cell-cell adhesions. Hence, we hypothesized that IRF6 also regulates cell-ECM adhesions, specifically focal adhesions. To test our hypothesis, we took advantage of human keratinocyte cell lines knocked down for IRF6 (shIRF6) and evaluated their ability to attach to plastic or Collagen IV. We found that shIRF6 keratinocytes show a delay attaching to Collagen IV compared to wild-type cells. However, no differences were observed on plastic. We further evaluated the ability of these cell lines to attach to the ECM by exposing keratinocytes in culture to centripetal forces and quantify their detachment from the plates. We found higher detachment of shIRF6 keratinocytes on Collagen IV but no differences on plastic. Plastic is stiffer than Collagen IV, hence, we hypothesized that the stiffness of the ECM would affect how IRF6 mediates cell-ECM adhesions. Using polyacrylamide gels of different stiffness, we found that shIRF6 keratinocytes have a significant reduction in their migrational speed on soft gels (1.5 kPa) compared to wild-types. Reduction of IRF6 also caused more deformation of the ECM. To test if increased cellular contractility mediated by the actomyosin cytoskeleton was driving the observed deformation, we performed immunostaining for Myosin IIb and filamentous actin. We found no visible differences in filamentous actin. However, Myosin IIb was significantly increased in shIRF6 compared with wild types, both on soft and stiff ECMs. Cell-ECM attachments are mediated by focal adhesion protein complexes including vinculin and beta1-integrin amongst others. We found that both were significantly reduced in shIRF6 keratinocytes regardless of the stiffness of the ECM. Overall, our data demonstrate that IRF6 is required for cell-ECM adhesion, potentially by regulating the localization of focal adhesion components at the cell membrane. Furthermore, IRF6 may promote cell-ECM adhesions differently depending on the stiffness of the ECM. As wounded tissue is stiffer than healthy tissue, these results are clinically significant and could contribute to the understanding of wound healing defects in patients with IRF6 mutations.

Clinical Findings and Outcomes of Palatal Repair in 9 Patients With Van der Woude Syndrome.

Van der Woude syndrome (VWS), an autosomal dominant condition associated with clefts of the lip and/or palate and lower lip pits, is caused by mutations in interferon regulatory factor 6 gene. It is reported to be the most common syndromic cleft worldwide. This case series presents the phenotypic characteristics and treatment outcomes in a group of 9 patients diagnosed with VWS. A retrospective review was performed on records of patients given a diagnosis of VWS presenting to the Department of Plastic Surgery of Sohag University between July 2009 and November 2019. Data analyses included age and sex of affected patients, type of the cleft, associated anomalies, presence of lower lip pits, and history of lower lip pits/cleft in the family and treatment outcomes. The study identified 9 patients (male = 2, female = 7). Age at first presentation ranged between 1 week and 7 years (mean = 1.3 years). Four patients had bilateral cleft lip and palate and 1 patient had unilateral cleft lip and palate (UCLP), whereas 4 patients had isolated cleft palate. Bilateral lower lip pits were presented in 8 patients, whereas the ninth patient has unilateral pit. Family history was positive in 3 patients. Most patients in this study developed after palatal repair speech problems with an increased rate of secondary surgical procedures needed for correction of velopharyngeal insufficiency. All reviewed cases of VWS presented with lower lip pits and cleft palate with or without cleft lip. A positive family history of similar conditions was demonstrated in 33.3% of our patients. This study suggests an increased incidence of secondary palatal surgeries for velopharyngeal insufficiency in patients with VWS; this warrant further studies including control group of nonsyndromic cleft patients.

Interferon regulatory factor 6 is required for proper wound healing in vivo.

Van der Woude syndrome (VWS) is the most common form of syndromic orofacial cleft caused predominantly by mutations in Interferon Regulatory Factor 6 (IRF6). We previously reported that individuals with VWS have increased risk of wound healing complications following cleft repair compared with individuals with nonsyndromic orofacial clefts (nonsyndromic cleft lip and palate-NSCLP). In vitro, absence of IRF6 leads to impaired keratinocyte migration and embryonic wound healing. However, there is currently no data on tissue repair in adult animals and cells with reduced levels of IRF6 like in VWS. Excisional wounds of Irf6+/- and wild-type animals were analyzed 4 and 7 days post-wounding. Although all wounds were reepithelialized after 7 days, the epidermal and wound volume of repaired wounds was larger in Irf6+/- . These data were supported by increased keratinocyte proliferation in the neoformed epidermis and a less mature granulation tissue with increased cytokine levels. This effect was not cell autonomous, as Irf6+/- neonatal keratinocytes in vitro did not exhibit defects in scratch wound closure or proliferation. Keratinocytes from individuals with VWS also migrated similarly to keratinocytes from NSCLP individuals. These data support a role for IRF6 in wound healing by regulating keratinocyte proliferation, granulation tissue maturation, and cytokine levels.

A novel IRF6 mutation causing non-syndromic cleft lip with or without cleft palate in a pedigree.

Non-syndromic cleft lip with or without cleft palate (NSCLP) is the most common congenital craniofacial malformation, and its harmful influence on affected individuals is apparent. Despite many studies, the causative genes and their mechanisms are not completely clear. We recruited a Han Chinese NSCLP family and explored the causative variant in this pedigree. We performed whole-exome sequencing on two patients. Bioinformatics screening and analysis were used to identify the mutation. We also performed species conservation analysis, mutation function predictions, and homology protein modelling to evaluate the influence of the mutation. We identified a rare mutation in interferon regulatory factor 6 (IRF6) (c.26G>A; p.Arg9Gln) as a candidate of causative mutation. This mutation was predicted to be deleterious. The codon is conserved in many species. The residue change caused by this mutation would affect the structure of IRF6 to a degree. Our study suggested that the rare IRF6 variant is probably the pathogenic mutation in this family. Our result adds evidence that IRF6 variants play a role in the aetiology of orofacial clefts.

IRF6 is the mediator of TGFβ3 during regulation of the epithelial mesenchymal transition and palatal fusion.

Mutation in interferon regulatory factor 6 (IRF6) is known to cause syndromic and non-syndromic cleft lip/palate in human. In this study, we investigated the molecular mechanisms related to IRF6 during palatal fusion using palatal shelves organ culture. The results showed that ablation of Irf6 resulted in a delay in TGFβ3-regulated palatal fusion. Ectopic expression of IRF6 was able to promote palatal fusion and rescue shTgfβ3-induced fusion defect. These findings indicate that IRF6 is involved in TGFβ3-mediated palatal fusion. Molecular analysis revealed that ectopic expression of IRF6 increased the expression of SNAI2, an epithelial mesenchymal transition (EMT) regulator, and diminished the expression of various epithelial markers, such as E-cadherin, Plakophilin and ZO-1. In addition, knockdown of Irf6 expression decreased SNAI2 expression, and restored the expression of ZO-1 and Plakophilin that were diminished by TGFβ3. Blocking of Snai2 expression delayed palatal fusion and abolished the IRF6 rescuing effect associated with shTgfβ3-induced fusion defect. These findings indicate that TGFβ3 increases IRF6 expression and subsequently regulates SNAI2 expression, and IRF6 appears to regulate EMT during palatal fusion via SNAI2. Taken together, this study demonstrates that IRF6 is a mediator of TGFβ3, which regulates EMT and fusion process during the embryonic palate development.

Van der Woude syndrome: A review of 11 cases seen at the Lagos University Teaching Hospital

Van der Woude syndrome (VWS), an autosomal dominant condition associated with clefts of the lip and/or palate and lower lip pits and is caused by mutations in interferon regulatory factor six gene. It is reported to be the most common syndromic cleft world-wide. Non-penetrance for the lip pit phenotype is found in at least 10% of affected individuals and those without the pits are phenocopies for non-syndromic clefting. The aim of this study is to present the phenotypic characteristic of VWS seen at the Lagos University Teaching Hospital (LUTH) cleft clinic. A review of cases of patients with VWS that attended the cleft lip and palate clinic at the LUTH Idi-Araba, Lagos, from January 2007 to December 2012 was conducted. Data analyses included sex of affected patients, types of cleft, presence of lower lip pits and history of lower lip pits/cleft in the family. A total of 11 cases were seen during the period (male = 4; female = 7). Age at presentation ranged between 1 week and 12 years, with majority (n = 10) less than 2 years of age. Bilateral cleft lip and palate (BCLP) was seen in six patients, isolated soft palatal cleft (n = 3) and unilateral cleft lip and alveolus (n = 1) and cleft of hard and soft palate (n = 1). Bilateral lower lips were presented in 10 out of the 11 cases. The mother of the only patient without lip pits presented with bilateral lower lip pits. No family history of cleft/lip pits was elicited in 10 other cases. Most of the cases of VWS presented with BCLP and lower lip pits. Non-penetrance for the lip pits was seen in one out of 11 cases. Our study emphasizes the need to screen family members in all cleft cases, especially clinically diagnosed non-syndromic cases who may be VWS with no lip pits. Future studies are required to investigate the genetic causes of this syndrome in our population.

Dominant Mutations in GRHL3 Cause Van der Woude Syndrome and Disrupt Oral Periderm Development

Mutations in interferon regulatory factor 6 (IRF6) account for ∼70% of cases of Van der Woude syndrome (VWS), the most common syndromic form of cleft lip and palate. In 8 of 45 VWS-affected families lacking a mutation in IRF6, we found coding mutations in grainyhead-like 3 (GRHL3). According to a zebrafish-based assay, the disease-associated GRHL3 mutations abrogated periderm development and were consistent with a dominant-negative effect, in contrast to haploinsufficiency seen in most VWS cases caused by IRF6 mutations. In mouse, all embryos lacking Grhl3 exhibited abnormal oral periderm and 17% developed a cleft palate. Analysis of the oral phenotype of double heterozygote (Irf6(+/-);Grhl3(+/-)) murine embryos failed to detect epistasis between the two genes, suggesting that they function in separate but convergent pathways during palatogenesis. Taken together, our data demonstrated that mutations in two genes, IRF6 and GRHL3, can lead to nearly identical phenotypes of orofacial cleft. They supported the hypotheses that both genes are essential for the presence of a functional oral periderm and that failure of this process contributes to VWS.

Treatment Outcomes in Patients With Van der Woude and Popliteal Pterygium Syndromes Compared to Patients With Nonsyndromic Cleft Lip/Palate

Van der Woude syndrome (VWS) and popliteal pterygium syndrome (PPS) are allelic autosomal dominant disorders caused by mutations in interferon regulatory factor 6 (IRF6). They comprise the single most common syndromic cause of cleft lip and/or palate (CL/P), accounting for 2% of all cases. VWS is characterized by CL/P and lower lip sinuses; PPS has the same features as VWS plus popliteal webbing, genital malformations, and syngnathia. The aim of this study is to summarize the clinical characteristics and treatment outcomes for patients with VWS/PPS and compare them to those for patients with nonsyndromic CL/P.

Wound Complications After Cleft Repair in Children With Van der Woude Syndrome

Van der Woude syndrome (VWS; OMIM 119300) is an autosomal-dominant condition associated with clefts of the lip and/or palate and lower lip pits and is caused by mutations in interferon regulatory factor 6 (IRF6). The standard of practice for children born with cleft lip/palate is surgical repair, which requires proper wound healing. We tested the hypothesis that children with VWS are more likely to have wound complications after cleft repair than children with nonsyndromic cleft lip/palate (NSCLP). Furthermore, we hypothesized that children with VWS have more surgical procedures. A retrospective, case-controlled study was performed. Seventeen children with VWS and 68 matched controls with NSCLP were scored for the presence of wound complications after cleft repair, for the severity of complications, and for number of surgeries from age 0 to 10. Of the 17 children with VWS, 8 had wound complications. Of 68 controls, 13 had wound complications (P = 0.02). Of 8 wound complications in the VWS group, 6 were major, whereas of 13 complications in the control group, 9 were major (P = 0.04). Most wound complications were fistulae and occurred in isolated cleft palate and bilateral cleft lip. The mean number of surgeries in the VWS group was 3.0 compared with 2.8 in the control group (P = 0.67). Our studies suggest that children with VWS have an increased risk for wound complications after cleft repair compared with children with NSCLP. Furthermore, these data support a role for IRF6 in wound healing.

Lower Lip Pits in a Patient With van der Woude Syndrome

van der Woude syndrome (VWS) is a congenital malformation characterized by lower lip pits with or without cleft lip or cleft palate. It is an autosomal-dominant inherited disorder with variable expression in clinical manifestation. Microdeletion in chromosome bands 1q32-q41 and recently identified mutation in interferon regulatory factor 6 gene (IRF6) have been reported to cause VWS. We report a case of VWS with lower lip pits as its main clinical manifestation without associated cleft in lip or palate. No mutation or deletion was found in the IRF6 gene or promoter site, indicating the heterogeneity of this defect.

Identification of a Van der Woude syndrome mutation in the cleft palate 1 mutant mouse

Mutations in Interferon Regulatory Factor 6 (IRF6) have been identified in two human allelic syndromes with cleft lip and/or palate: Van der Woude (VWS) and Popliteal Pterygium syndromes (PPS). Furthermore, common IRF6 haplotypes and single nucleotide polymorphisms (SNP) alleles are strongly associated with nonsyndromic clefting defects in multiple ethnic populations. Mutations in the mouse often provide good models for the study of human diseases and developmental processes. We identified the cleft palate 1 (clft1) mouse mutant in a forward genetic screen for phenotypes modeling human congenital disease. In the clft1 mutant, we have identified a novel missense point mutation in the mouse Irf6 gene, which confers an amino acid alteration that has been found in a VWS family. Phenotypic comparison of clft1 mutants to previously reported Irf6 mutant alleles demonstrates the Irf6(clft1) allele is a hypomorphic allele. The cleft palate seen in these mutants appears to be due to abnormal adhesion between the palate and tongue. The Irf6(clft1) allele provides the first mouse model for the study of an etiologic IRF6 missense mutation observed in a human VWS family.

The potential role for Interferon regulatory factor 6 in keratinocyte adhesion and/or migration

Mutations in Interferon Regulatory Factor (IRF6) cause two orofacial clefting syndromes, Van der Woude (VWS) and Popliteal Pterygium (PPS). In addition to an oral defect, recent data show that VWS patients are more likely to have wound complications after corrective cleft surgery than patients with isolated cleft. Thus we hypothesize that Irf6 is critical to wound closure. To test this, we used keratinocytes from Irf6 null mice. We created in vitro scratches and observed a significant delay in scratch closure. To determine the cause of delay, we examined the cellular structure of Irf6−/− keratinocytes by staining for filamentous actin and found that these cells have more stress fibers. In addition, we examined cellular adhesion by staining Irf6−/− keratinocytes for E‐cadherin and vinculin. We found that these cells form junctions, but cell‐cell contacts appear in an abnormal pattern, and there appear to be more focal adhesions. Finally, we analyzed proliferation of Irf6−/− keratinocytes and found no deficiency. Together, our data suggests Irf6−/− keratinocytes exhibit characteristics of less motile cells. Thus, we speculate that the defect in Irf6−/− keratinocyte scratch closure is due to aberrant migration and/or adhesion, and that cellular proliferation is not inhibitory to closure. Broadly, these data suggest that Irf6 is necessary for events critical to proper cutaneous wound healing.Grant Funding Source: NIH‐NIAMS AR 055313

Prevalence and nonrandom distribution of exonic mutations in interferon regulatory factor 6 in 307 families with Van der Woude syndrome and 37 families with popliteal pterygium syndrome

PurposeInterferon regulatory factor 6 encodes a member of the IRF family of transcription factors. Mutations in interferon regulatory factor 6 cause Van der Woude and popliteal pterygium syndrome, two related orofacial clefting disorders. Here, we compared and contrasted the frequency and distribution of exonic mutations in interferon regulatory factor 6 between two large geographically distinct collections of families with Van der Woude and between one collection of families with popliteal pterygium syndrome. MethodsWe performed direct sequence analysis of interferon regulatory factor 6 exons on samples from three collections, two with Van der Woude and one with popliteal pterygium syndrome. ResultsWe identified mutations in interferon regulatory factor 6 exons in 68% of families in both Van der Woude collections and in 97% of families with popliteal pterygium syndrome. In sum, 106 novel disease-causing variants were found. The distribution of mutations in the interferon regulatory factor 6 exons in each collection was not random; exons 3, 4, 7, and 9 accounted for 80%. In the Van der Woude collections, the mutations were evenly divided between protein truncation and missense, whereas most mutations identified in the popliteal pterygium syndrome collection were missense. Further, the missense mutations associated with popliteal pterygium syndrome were localized significantly to exon 4, at residues that are predicted to bind directly to DNA. ConclusionThe nonrandom distribution of mutations in the interferon regulatory factor 6 exons suggests a two-tier approach for efficient mutation screens for interferon regulatory factor 6. The type and distribution of mutations are consistent with the hypothesis that Van der Woude is caused by haploinsufficiency of interferon regulatory factor 6. On the other hand, the distribution of popliteal pterygium syndrome-associated mutations suggests a different, though not mutually exclusive, effect on interferon regulatory factor 6 function.

Missense mutations that cause Van der Woude syndrome and popliteal pterygium syndrome affect the DNA-binding and transcriptional activation functions of IRF6.

Cleft lip and cleft palate (CLP) are common disorders that occur either as part of a syndrome, where structures other than the lip and palate are affected, or in the absence of other anomalies. Van der Woude syndrome (VWS) and popliteal pterygium syndrome (PPS) are autosomal dominant disorders characterized by combinations of cleft lip, CLP, lip pits, skin-folds, syndactyly and oral adhesions which arise as the result of mutations in interferon regulatory factor 6 (IRF6). IRF6 belongs to a family of transcription factors that share a highly conserved N-terminal, DNA-binding domain and a less well-conserved protein-binding domain. To date, mutation analyses have suggested a broad genotype–phenotype correlation in which missense and nonsense mutations occurring throughout IRF6 may cause VWS; in contrast, PPS-causing mutations are highly associated with the DNA-binding domain, and appear to preferentially affect residues that are predicted to interact directly with the DNA. Nevertheless, this genotype–phenotype correlation is based on the analysis of structural models rather than on the investigation of the DNA-binding properties of IRF6. Moreover, the effects of mutations in the protein interaction domain have not been analysed. In the current investigation, we have determined the sequence to which IRF6 binds and used this sequence to analyse the effect of VWS- and PPS-associated mutations in the DNA-binding domain of IRF6. In addition, we have demonstrated that IRF6 functions as a co-operative transcriptional activator and that mutations in the protein interaction domain of IRF6 disrupt this activity.

Irf6 is a key determinant of the keratinocyte proliferation-differentiation switch

The epidermis is a highly organized structure, the integrity of which is central to the protection of an organism. Development and subsequent maintenance of this tissue depends critically on the intricate balance between proliferation and differentiation of a resident stem cell population; however, the signals controlling the proliferation-differentiation switch in vivo remain elusive. Here, we show that mice carrying a homozygous missense mutation in interferon regulatory factor 6 (Irf6), the homolog of the gene mutated in the human congenital disorders Van der Woude syndrome and popliteal pterygium syndrome, have a hyperproliferative epidermis that fails to undergo terminal differentiation, resulting in soft tissue fusions. We further demonstrate that mice that are compound heterozygotes for mutations in Irf6 and the gene encoding the cell cycle regulator protein stratifin (Sfn; also known as 14-3-3sigma) show similar defects of keratinizing epithelia. Our results indicate that Irf6 is a key determinant of the keratinocyte proliferation-differentiation switch and that Irf6 and Sfn interact genetically in this process.