[Show abstract][Hide abstract] ABSTRACT: Interferon Regulatory Factor 6 regulates keratinocyte proliferation and differentiation. In this study, we tested the hypothesis that Irf6 regulates cellular migration and adhesion. Irf6-deficient embryos at 10.5 days post conception failed to close their wound compared to wild type. In vitro, Irf6-deficient murine embryonic keratinocytes were delayed in closing a scratch wound. Live imaging of the scratch showed a deficient directional migration and reduced speed in cells lacking Irf6. To understand the underlying molecular mechanisms, cell-cell and cell-matrix adhesions were investigated. We show that wild type and Irf6-deficient keratinocytes adhere similarly to all matrices after 60 min. However, Irf6-deficient keratinocytes were consistently larger and more spread, a phenotype that persisted during the scratch. Interestingly, Irf6-deficient keratinocytes exhibited an increased network of stress fibers and active RhoA compared to wild type. Blocking ROCK, a downstream effector of RhoA, rescued the scratch wound delay. Arhgap29, a Rho GTPase Activating Protein was reduced in Irf6-deficient keratinocytes. Together these data suggest that Irf6 functions through the RhoA pathway to regulate cellular migration.
[Show abstract][Hide abstract] ABSTRACT: The purpose of this needs assessment was to determine community leader perceptions of health-related needs and resources available to rural-dwelling older adults as part of a community-academic partnership in the rural Midwest. A community advisory board, in accordance with community-based participatory research principles, was influential in study design and implementation. Key informant interviews (N = 30) were conducted with community leaders including professionals from schools, businesses, churches, and health care as well as government officials. Thematic analysis revealed "Family Is Central," "Heritage," "Strength," and "Longevity" as important themes related to older adults and their health care needs within the community. "Close-knit" and "Church Is Central" were also identified as important aspects of elder care. Community leaders perceived the "Rural Economy," "Distance to Resources," and "Seasonal Resources" as significant barriers for older adults. This work contributes important insights into community leaders' perceptions of health needs and challenges faced by older adults in rural settings.
Western Journal of Nursing Research 04/2014; 37(5). DOI:10.1177/0193945914530046 · 1.38 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Previous studies identified a cluster of individuals with an autosomal recessive form of deafness that reside in a small region of mid-Michigan. We hypothesized that afffected members from this community descend from a defined founder population. Using public records and personal interviews, we constructed a genealogical database that includes the afffected individuals and their extended families as descendants of 461 settlers who emigrated from the Eifel region of Germany between 1836 and 1875. The genealogical database represents a 13-generation pedigree that includes 27,747 descendants of these settlers. Among these descendants, 13,784 are presumed living. Many of the extant descendants reside in a 90-square-mile area, and 52% were born to parents who share at least one common ancestor. Among those born to related parents, the median kinship coeffficient is 3.7 × 10-3. While the pedigree contains 2,510 founders, 344 of the 461 settlers accounted for 67% of the genome in the extant population. These data suggest that we identified a new population isolate in North America and that, as demonstrated for congenital hearing loss, this rural mid-Michigan community is a new resource to discover heritable factors that contribute to common health-related conditions.
Human Biology 02/2014; 86(1):59-68. DOI:10.3378/027.086.0103 · 1.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: DNA variation in Interferon Regulatory Factor 6 (IRF6) causes Van der Woude syndrome (VWS), the most common syndromic form of cleft lip and palate. However, an etiologic variant in IRF6 has been found in only 70% of VWS families. To test whether DNA variants in regulatory elements cause VWS, we sequenced three conserved elements near IRF6 in 70 VWS families that lack an etiologic mutation within IRF6 exons. A rare mutation (350dupA) was found in a conserved IRF6 enhancer element (MCS9.7) in a Brazilian family. The 350dupA mutation abrogated the binding of p63 and E47 transcription factors to cis-overlapping motifs, and significantly disrupted enhancer activity in human cell cultures. Moreover, using a transgenic assay in mice, the 350dupA mutation disrupted the activation of MCS9.7 enhancer element and led to failure of lacZ expression in all head and neck pharyngeal arches. Interestingly, disruption of the p63 Motif1 and/or E47 binding sites by nucleotide substitution did not fully recapitulate the effect of the 350dupA mutation. Rather, we recognized that the 350dupA created a CAAAGT motif, a binding site for Lef1 protein. We showed that Lef1 binds to the mutated site and that overexpression of Lef1/β-Catenin chimeric protein repressed MCS9.7-350dupA enhancer activity. In conclusion, our data strongly suggests that 350dupA variant is an etiologic mutation in VWS patients and disrupts enhancer activity by a loss- and gain-of-function mechanism, and thus support the rationale for additional screening for regulatory mutations in patients with cleft lip and palate.
Human Molecular Genetics 01/2014; 23(10). DOI:10.1093/hmg/ddt664 · 6.68 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
The American Journal of Human Genetics 12/2013; 94(1). DOI:10.1016/j.ajhg.2013.11.009 · 10.99 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The signaling lymphocytic activation molecule-associated adaptor Ewing's sarcoma's-activated transcript 2 (EAT-2) is primarily expressed in dendritic cells, macrophages and natural killer cells. Including EAT-2 in a vaccination regimen enhanced innate and adaptive immune responses toward pathogen-derived antigens, even in the face of pre-existing vaccine immunity. Herein, we investigate whether co-vaccinations with two recombinant Ad5 (rAd5) vectors, one expressing the carcinoembryonic antigen (CEA) and one expressing EAT-2, can induce more potent CEA-specific cytotoxic T lymphocyte (CTL) and antitumor activity in the therapeutic CEA-expressing MC-38 tumor model. Our results suggest that inclusion of EAT-2 significantly alters the kinetics of Th1-biasing proinflammatory cytokine and chemokine responses, and enhances anti-CEA-specific CTL responses. As a result, rAd5-EAT2-augmented rAd5-CEA vaccinations are more efficient in eliminating CEA-expressing target cells as measured by an in vivo CTL assay. Administration of rAd5-EAT2 vaccines also reduced the rate of growth of MC-38 tumor growth in vivo. Also, an increase in MC-38 tumor cell apoptosis (as measured by hematoxylin and eosin staining, active caspase-3 and granzyme B levels within the tumors) was observed. These data provide evidence that more efficient, CEA-specific effector T cells are generated by rAd5 vaccines expressing CEA, when augmented by rAd5 vaccines expressing EAT-2, and this regimen may be a promising approach for cancer immunotherapy in general.Cancer Gene Therapy advance online publication, 16 August 2013; doi:10.1038/cgt.2013.53.
Cancer gene therapy 08/2013; 20(10). DOI:10.1038/cgt.2013.53 · 2.55 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Mandible shape in the mouse is a complex trait that is influenced by many genetic factors. However, little is known about the action of single genes on adult mandible shape so far, since most developmentally relevant genes are already required during embryogenesis, i.e., knockouts lead to embryonic death or severe deformations, before the mandible is fully formed. We employ here a geometric morphometric approach to identify subtle phenotypic differences caused by dosage effects of candidate genes. We use mouse strains with specific gene modifications (knockouts and knockins) to compare heterozygous animals with controls from the same stock, which is expected to be equivalent to a change of gene expression of the respective locus. Such differences in expression level are also likely to occur as part of the natural variation. We focus on Bmp pathway genes (Bmp4, its antagonist Noggin, and combinations of Bmp5-7 genotypes), but include also two other developmental control genes suspected to affect mandible development in some way (Egfr and Irf6). In addition, we study the effects of Hoxd13, as well as an extracellular matrix constituent (Col2a1). We find that subtle but significant shape differences are caused by differences in gene dosage of several of these genes. The changes seen for Bmp4 and Noggin are partially compatible with the action of these genes known from birds and fish. We find significant shape changes also for Hoxd13, although this gene has so far only been implicated in skeletal patterning processes of the limbs. Comparing the effect sizes of gene dosage changes to the variation found in natural populations of mice as well as quantitative trait loci (QTL) effects on mandible shape, we find that the effect sizes caused by gene dosage changes are at the lower end of the spectrum of natural variation, but larger than the average additive effects found in QTL studies. We conclude that studying gene dosage effects have the potential to provide new insights into aspects of craniofacial development, variation, and evolution.
Development Genes and Evolution 04/2013; 223(5). DOI:10.1007/s00427-013-0443-y · 2.18 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Interferon regulatory factor 6 () encodes a highly conserved helix-turn-helix DNA binding protein and is a member of the interferon regulatory family of DNA transcription factors. Mutations in lead to isolated and syndromic forms of cleft lip and palate, most notably Van der Woude syndrome (VWS) and Popliteal Ptyerigium Syndrome (PPS). Mice lacking both copies of have severe limb, skin, palatal and esophageal abnormalities, due to significantly altered and delayed epithelial development. However, a recent report showed that , an enhancer near , is active in the tongue, suggesting that may also be expressed in the tongue. Indeed, we detected Irf6 staining in the mesoderm-derived muscle during development of the tongue. Dual labeling experiments demonstrated that Irf6 was expressed only in the + cell lineage, which originates from the segmental paraxial mesoderm and gives rise to the muscles of the tongue. Fate mapping of the segmental paraxial mesoderm cells revealed a cell-autonomous function with reduced and poorly organized + cell lineage in the tongue. Molecular analyses showed that the -/- embryos had aberrant cytoskeletal formation of the segmental paraxial mesoderm in the tongue. Fate mapping of the cranial neural crest cells revealed non-cell-autonomous function with the loss of the inter-molar eminence. Loss of function altered , , , and signaling suggesting that these genes are involved in Irf6 signaling. Based on these data, plays important cell-autonomous and non-cell-autonomous roles in muscular differentiation and cytoskeletal formation in the tongue.
PLoS ONE 02/2013; 8(2):e56270. DOI:10.1371/journal.pone.0056270 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Purpose:
Mutations in the transcription factor IRF6 cause allelic autosomal dominant clefting syndromes, Van der Woude syndrome, and popliteal pterygium syndrome. We compared the distribution of IRF6 coding and splice-site mutations from 549 families with Van der Woude syndrome or popliteal pterygium syndrome with that of variants from the 1000 Genomes and National Heart, Lung, and Blood Institute Exome Sequencing Projects.
We compiled all published pathogenic IRF6 mutations and performed direct sequencing of IRF6 in families with Van der Woude syndrome or popliteal pterygium syndrome.
Although mutations causing Van der Woude syndrome or popliteal pterygium syndrome were nonrandomly distributed with significantly increased frequencies in the DNA-binding domain (P = 0.0001), variants found in controls were rare and evenly distributed in IRF6. Of 194 different missense or nonsense variants described as potentially pathogenic, we identified only two in more than 6,000 controls. PolyPhen and SIFT (sorting intolerant from tolerant) reported 5.9% of missense mutations in patients as benign, suggesting that use of current in silico prediction models to determine function can have significant false negatives.
Mutation of IRF6 occurs infrequently in controls, suggesting that for IRF6 there is a high probability that disruption of the coding sequence, particularly the DNA-binding domain, will result in syndromic features. Prior associations of coding sequence variants in IRF6 with clefting syndromes have had few false positives.
Genetics in medicine: official journal of the American College of Medical Genetics 11/2012; 15(5). DOI:10.1038/gim.2012.141 · 6.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Wound healing is a complex process that relies on proper levels of cytokines and growth factors to successfully repair the tissue. Of particular interest are the members of the transforming growth factor family. There are three TGF-ß isoforms-TGF- ß 1, 2, and 3, each isoform showing a unique expression pattern, suggesting that they each play a distinct function during development and repair. Previous studies reported an exclusive role for TGF-ß 3 in orofacial development and a potent anti-scarring effect. However, the role of TGF- ß 3 in excisional wound healing and keratinocyte migration remains poorly understood. We tested the effect of TGF-ß 3 levels on excisional cutaneous wounds in the adult mouse by directly injecting recombinant TGF-ß 3 or neutralizing antibody against TGF-ß 3 (NAB) in the wounds. Our results demonstrate that TGF-ß 3 does not promote epithelialization. However, TGF-ß 3 is necessary for wound closure as wounds injected with neutralizing antibody against TGF-ß 3 showed increased epidermal volume and proliferation in conjunction with a delay in keratinocyte migration. Wild type keratinocytes treated with NAB and Tgfb3-deficient keratinocytes closed an in vitro scratch wound with no delay, suggesting that our in vivo observations likely result from a paracrine effect.
PLoS ONE 10/2012; 7(10):e48040. DOI:10.1371/journal.pone.0048040 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Previous evidence from tooth agenesis studies suggested IRF6 and TGFA interact. Since tooth agenesis is commonly found in individuals with cleft lip/palate (CL/P), we used four large cohorts to evaluate if IRF6 and TGFA interaction contributes to CL/P. Markers within and flanking IRF6 and TGFA genes were tested using Taqman or SYBR green chemistries for case-control analyses in 1,000 Brazilian individuals. We looked for evidence of gene-gene interaction between IRF6 and TGFA by testing if markers associated with CL/P were overtransmitted together in the case-control Brazilian dataset and in the additional family datasets. Genotypes for an additional 142 case-parent trios from South America drawn from the Latin American Collaborative Study of Congenital Malformations (ECLAMC), 154 cases from Latvia, and 8,717 individuals from several cohorts were available for replication of tests for interaction. Tgfa and Irf6 expression at critical stages during palatogenesis was analyzed in wild type and Irf6 knockout mice. Markers in and near IRF6 and TGFA were associated with CL/P in the Brazilian cohort (p<10(-6)). IRF6 was also associated with cleft palate (CP) with impaction of permanent teeth (p<10(-6)). Statistical evidence of interaction between IRF6 and TGFA was found in all data sets (p = 0.013 for Brazilians; p = 0.046 for ECLAMC; p = 10(-6) for Latvians, and p = 0.003 for the 8,717 individuals). Tgfa was not expressed in the palatal tissues of Irf6 knockout mice. IRF6 and TGFA contribute to subsets of CL/P with specific dental anomalies. Moreover, this potential IRF6-TGFA interaction may account for as much as 1% to 10% of CL/P cases. The Irf6-knockout model further supports the evidence of IRF6-TGFA interaction found in humans.
PLoS ONE 09/2012; 7(9):e45441. DOI:10.1371/journal.pone.0045441 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: IFN regulatory factor 6 (IRF6) is a transcription factor that, in mammals, is required for the differentiation of skin, breast epithelium, and oral epithelium. However, the transcriptional targets that mediate these effects are currently unknown. In zebrafish and frog embryos, Irf6 is necessary for differentiation of the embryonic superficial epithelium, or periderm. Here we use microarrays to identify genes that are expressed in the zebrafish periderm and whose expression is inhibited by a dominant-negative variant of Irf6 (dnIrf6). These methods identify Grainyhead-like 3 (Grhl3), an ancient regulator of the epidermal permeability barrier, as acting downstream of Irf6. In human keratinocytes, IRF6 binds conserved elements near the GRHL3 promoter. We show that one of these elements has enhancer activity in human keratinocytes and zebrafish periderm, suggesting that Irf6 directly stimulates Grhl3 expression in these tissues. Simultaneous inhibition of grhl1 and grhl3 disrupts periderm differentiation in zebrafish, and, intriguingly, forced grhl3 expression restores periderm markers in both zebrafish injected with dnIrf6 and frog embryos depleted of Irf6. Finally, in Irf6-deficient mouse embryos, Grhl3 expression in the periderm and oral epithelium is virtually absent. These results indicate that Grhl3 is a key effector of Irf6 in periderm differentiation.Journal of Investigative Dermatology advance online publication, 30 August 2012; doi:10.1038/jid.2012.269.
[Show abstract][Hide abstract] ABSTRACT: Thickening and the subsequent invagination of the epithelium are an important initial step in ectodermal organ development. Ikkα has been shown to play a critical role in controlling epithelial growth, since Ikkα mutant mice show protrusions (evaginations) of incisor tooth, whisker and hair follicle epithelium rather than invagination. We show here that mutation of the Interferon regulatory factor (Irf) family, Irf6 also results in evagination of incisor epithelium. In common with Ikkα mutants, Irf6 mutant evagination occurs in a NF-κB-independent manner and shows the same molecular changes as those in Ikkα mutants. Irf6 thus also plays a critical role in regulating epithelial invagination. In addition, we also found that canonical Wnt signaling is upregulated in evaginated incisor epithelium of both Ikkα and Irf6 mutant embryos.
[Show abstract][Hide abstract] ABSTRACT: DNA variation in Interferon Regulatory Factor 6 (IRF6) contributes risk for orofacial clefting, including a common DNA variant rs642961. This DNA variant is located in a multi-species conserved sequence that is 9.7 kb upstream from the IRF6 transcriptional start site (MCS9.7). The MCS9.7 element was shown to possess enhancer activity that mimicked the expression of endogenous Irf6 at embryonic day 11.5 in transient transgenic embryos, and also contains a p63 binding site that transactivates IRF6 expression. To analyze whether the MCS9.7 enhancer is sufficient to drive IRF6 expression, we generated stable transgenic murine lines that carry a MCS9.7-lacZ transgene. We hypothesized that MCS9.7 was sufficient to recapitulate the endogenous expression of Irf6 at other time-points during embryonic development.
We observed that MCS9.7 activity recapitulated endogenous Irf6 expression in most tissues, but not in the medial edge epithelium (MEE) at E14.5, when Irf6 expression was high during secondary palatal fusion. Also, while MCS9.7 activity and Irf6 expression were associated with p63 expression, we observed MCS9.7 activity and Irf6 expression in periderm, although p63 was absent.
These data suggest that MCS9.7 enhancer activity is not sufficient to recapitulate IRF6 expression, and that p63 expression is not always necessary nor sufficient for transactivation of IRF6.
[Show abstract][Hide abstract] ABSTRACT: Pterygium syndromes are complex congenital disorders that encompass several distinct clinical conditions characterized by multiple skin webs affecting the flexural surfaces often accompanied by craniofacial anomalies. In severe forms, such as in the autosomal-recessive Bartsocas-Papas syndrome, early lethality is common, complicating the identification of causative mutations. Using exome sequencing in a consanguineous family, we identified the homozygous mutation c.1127C>A in exon 7 of RIPK4 that resulted in the introduction of the nonsense mutation p.Ser376X into the encoded ankyrin repeat-containing kinase, a protein that is essential for keratinocyte differentiation. Subsequently, we identified a second mutation in exon 2 of RIPK4 (c.242T>A) that resulted in the missense variant p.Ile81Asn in the kinase domain of the protein. We have further demonstrated that RIPK4 is a direct transcriptional target of the protein p63, a master regulator of stratified epithelial development, which acts as a nodal point in the cascade of molecular events that prevent pterygium syndromes.
The American Journal of Human Genetics 12/2011; 90(1):69-75. DOI:10.1016/j.ajhg.2011.11.013 · 10.99 Impact Factor