Publications by authors named "Brian C Schutte"

54 Publications

Secondary Palate Development in the Dog ().

Cleft Palate Craniofac J 2021 Feb 24;58(2):230-236. Epub 2020 Jul 24.

Microbiology & Molecular Genetics, 3078Michigan State University, East Lansing, MI, USA.

Objective: To investigate the gestational timing of morphologic events in normal canine secondary palate development as a baseline for studies in dog models of isolated cleft palate (CP).

Methods: Beagle and beagle/cocker spaniel-hybrid fetal dogs were obtained by cesarean-section on various days of gestation, timed from the initial rise of serum progesterone concentration. Morphology of fetal heads was determined by examining serial coronal sections.

Results: On gestational day 35 (d35), the palatal shelves pointed ventrally alongside the tongue. On d36, palatal shelves were elongated and elevated to a horizontal position above the tongue but were not touching. On d37, palatine shelves and vomer were touching, but the medial epithelial seam (MES) between the apposed shelves remained. Immunostaining with epithelial protein markers showed that the MES gradually dissolved and was replaced by mesenchyme during d37-d44, and palate fusion was complete by d44. Examination of remnant MES suggested that fusion of palatal shelves began in mid-palate and moved rostrally and caudally.

Conclusion: Palate development occurs in dogs in the steps described in humans and mice, but palate closure occurs at an intermediate time in gestation. These normative data will form the basis of future studies to determine pathophysiologic mechanisms in dog models of CP. Added clinical significance is the enhancement of dogs as a large animal model to test new approaches for palate repair, with the obvious advantage of achieving full maturity within 2 years rather than 2 decades.
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http://dx.doi.org/10.1177/1055665620943771DOI Listing
February 2021

SPECC1L regulates palate development downstream of IRF6.

Hum Mol Genet 2020 03;29(5):845-858

Department of Anatomy and Cell Biology, University of Kansas Medical Center, Kansas City, KS 66160, USA.

SPECC1L mutations have been identified in patients with rare atypical orofacial clefts and with syndromic cleft lip and/or palate (CL/P). These mutations cluster in the second coiled-coil and calponin homology domains of SPECC1L and severely affect the ability of SPECC1L to associate with microtubules. We previously showed that gene-trap knockout of Specc1l in mouse results in early embryonic lethality. We now present a truncation mutant mouse allele, Specc1lΔC510, that results in perinatal lethality. Specc1lΔC510/ΔC510 homozygotes showed abnormal palate rugae but did not show cleft palate. However, when crossed with a gene-trap allele, Specc1lcGT/ΔC510 compound heterozygotes showed a palate elevation delay with incompletely penetrant cleft palate. Specc1lcGT/ΔC510 embryos exhibit transient oral epithelial adhesions at E13.5, which may delay shelf elevation. Consistent with oral adhesions, we show periderm layer abnormalities, including ectopic apical expression of adherens junction markers, similar to Irf6 hypomorphic mutants and Arhgap29 heterozygotes. Indeed, SPECC1L expression is drastically reduced in Irf6 mutant palatal shelves. Finally, we wanted to determine if SPECC1L deficiency also contributed to non-syndromic (ns) CL/P. We sequenced 62 Caucasian, 89 Filipino, 90 Ethiopian, 90 Nigerian and 95 Japanese patients with nsCL/P and identified three rare coding variants (p.Ala86Thr, p.Met91Iso and p.Arg546Gln) in six individuals. These variants reside outside of SPECC1L coiled-coil domains and result in milder functional defects than variants associated with syndromic clefting. Together, our data indicate that palate elevation is sensitive to deficiency of SPECC1L dosage and function and that SPECC1L cytoskeletal protein functions downstream of IRF6 in palatogenesis.
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http://dx.doi.org/10.1093/hmg/ddaa002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7104672PMC
March 2020

Endogenous Avian Leukosis Virus in Combination with Serotype 2 Marek's Disease Virus Significantly Boosted the Incidence of Lymphoid Leukosis-Like Bursal Lymphomas in Susceptible Chickens.

J Virol 2019 12 13;93(23). Epub 2019 Nov 13.

United States Department of Agriculture, Agricultural Research Service, Avian Disease and Oncology Laboratory, East Lansing, Michigan, USA

In 2010, sporadic cases of avian leukosis virus (ALV)-like bursal lymphoma, also known as spontaneous lymphoid leukosis (LL)-like tumors, were identified in two commercial broiler breeder flocks in the absence of exogenous ALV infection. Two individual ALV subgroup E (ALV-E) field strains, designated AF227 and AF229, were isolated from two different breeder farms. The role of these ALV-E field isolates in development of and the potential joint impact in conjunction with a Marek's disease virus (MDV) vaccine (SB-1) were further characterized in chickens of an experimental line and commercial broiler breeders. The experimental line 0.TVB*S1, commonly known as the rapid feathering-susceptible (RFS) line, of chickens lacks all endogenous ALV and is fully susceptible to all subgroups of ALV, including ALV-E. Spontaneous LL-like tumors occurred following infection with AF227, AF229, and a reference ALV-E strain, RAV60, in RFS chickens. Vaccination with serotype 2 MDV, SB-1, in addition to AF227 or AF229 inoculation, significantly enhanced the spontaneous LL-like tumor incidence in the RFS chickens. The spontaneous LL-like tumor incidence jumped from 14% by AF227 alone to 42 to 43% by AF227 in combination with SB-1 in the RFS chickens under controlled conditions. RNA-sequencing analysis of the LL-like lymphomas and nonmalignant bursa tissues of the RFS line of birds identified hundreds of differentially expressed genes that are reportedly involved in key biological processes and pathways, including signaling and signal transduction pathways. The data from this study suggested that both ALV-E and MDV-2 play an important role in enhancement of the spontaneous LL-like tumors in susceptible chickens. The underlying mechanism may be complex and involved in many chicken genes and pathways, including signal transduction pathways and immune system processes, in addition to reported viral genes. Lymphoid leukosis (LL)-like lymphoma is a low-incidence yet costly and poorly understood disease of domestic chickens. The observed unique characteristics of LL-like lymphomas are that the incidence of the disease is chicken line dependent; pathologically, it appeared to mimic avian leukosis but is free of exogenous ALV infection; inoculation of the nonpathogenic ALV-E or MDV-2 (SB-1) boosts the incidence of the disease; and inoculation of both the nonpathogenic ALV-E and SB-1 escalates it to much higher levels. This study was designed to test the impact of two new ALV-E isolates, recently derived from commercial broiler breeder flocks, in combination with the nonpathogenic SB-1 on LL-like lymphoma incidences in both an experimental egg layer line of chickens and a commercial broiler breeder line of chickens under a controlled condition. Data from this study provided an additional piece of experimental evidence on the potency of nonpathogenic ALV-E, MDV-2, and ALV-E plus MDV-2 in boosting the incidence of LL-like lymphomas in susceptible chickens. This study also generated the first piece of genomic evidence that suggests host transcriptomic variation plays an important role in modulating LL-like lymphoma formation.
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http://dx.doi.org/10.1128/JVI.00861-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6854487PMC
December 2019

The TFAP2A-IRF6-GRHL3 genetic pathway is conserved in neurulation.

Hum Mol Genet 2019 05;28(10):1726-1737

Departments of Biochemistry and Molecular Biology.

Mutations in IRF6, TFAP2A and GRHL3 cause orofacial clefting syndromes in humans. However, Tfap2a and Grhl3 are also required for neurulation in mice. Here, we found that homeostasis of Irf6 is also required for development of the neural tube and associated structures. Over-expression of Irf6 caused exencephaly, a rostral neural tube defect, through suppression of Tfap2a and Grhl3 expression. Conversely, loss of Irf6 function caused a curly tail and coincided with a reduction of Tfap2a and Grhl3 expression in tail tissues. To test whether Irf6 function in neurulation was conserved, we sequenced samples obtained from human cases of spina bifida and anencephaly. We found two likely disease-causing variants in two samples from patients with spina bifida. Overall, these data suggest that the Tfap2a-Irf6-Grhl3 genetic pathway is shared by two embryologically distinct morphogenetic events that previously were considered independent during mammalian development. In addition, these data suggest new candidates to delineate the genetic architecture of neural tube defects and new therapeutic targets to prevent this common birth defect.
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http://dx.doi.org/10.1093/hmg/ddz010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6494790PMC
May 2019

IRF6 and AP2A Interaction Regulates Epidermal Development.

J Invest Dermatol 2018 12 18;138(12):2578-2588. Epub 2018 Jun 18.

Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, Michigan, USA. Electronic address:

Variants in IRF6 can lead to Van der Woude syndrome and popliteal pterygium syndrome. Furthermore, genes upstream and downstream of IRF6, including GRHL3 and TP63, are also associated with orofacial clefting. Additionally, a variant in an enhancer (MCS9.7) that regulates IRF6 is associated with risk for isolated orofacial clefting. This variant (rs642961) abrogates AP2A protein binding at MCS9.7. Here, we found that AP2A protein regulates MCS9.7 enhancer activity in vivo and IRF6 protein expression in epidermal development. In addition, loss of IRF6 leads to supra-basal expression of AP2A protein. Finally, using an IRF6 allelic series, we found that either increasing or decreasing IRF6 protein expression can destabilize AP2A protein expression in vivo. These data suggest that IRF6 regulates AP2A protein level in epidermal development. Therefore, we conclude that IRF6 and TFAP2A are part of a genetic regulatory network that is critical in epithelial development, with implications for both orofacial and cutaneous tissues. Our work provides in vivo, functional data to explain the relationship between AP2A protein binding and the MCS9.7 enhancer in orofacial clefting. This work is important because the MCS9.7 enhancer element contains a variant that abrogates AP2A protein binding and increases risk for orofacial clefting worldwide.
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http://dx.doi.org/10.1016/j.jid.2018.05.030DOI Listing
December 2018

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

PLoS Genet 2017 Sep 25;13(9):e1007009. Epub 2017 Sep 25.

Center for Regenerative Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, United States of America.

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.
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http://dx.doi.org/10.1371/journal.pgen.1007009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5628943PMC
September 2017

Altered thymocyte and T cell development in neonatal mice with hyperoxia-induced lung injury.

J Perinat Med 2018 May;46(4):441-449

Department of Pediatrics and Human Development College of Human Medicine, Division of Neonatology, Michigan State University, East Lansing, MI, USA.

Background: The adaptive immune system of neonates is relatively underdeveloped. The thymus is an essential organ for adaptive T cell development and might be affected during the natural course of oxygen induced lung injury. The effect of prolonged hyperoxia on the thymus, thymocyte and T cell development, and its proliferation has not been studied extensively.

Methods: Neonatal mice were exposed to 85% oxygen (hyperoxia) or room air (normoxia) up to 28 days. Flow cytometry using surface markers were used to assay for thymocyte development and proliferation.

Results: Mice exposed to prolonged hyperoxia had evidence of lung injury associated alveolar simplification, a significantly lower mean weight, smaller thymic size, lower mean thymocyte count and higher percentage of apoptotic thymocytes. T cells subpopulation in the thymus showed a significant reduction in the count and proliferation of double positive and double negative T cells. There was a significant reduction in the count and proliferation of single positive CD4+ and CD8+ T cells.

Conclusions: Prolonged hyperoxia in neonatal mice adversely affected thymic size, thymocyte count and altered the distribution of T cells sub-populations. These results are consistent with the hypothesis that prolonged hyperoxia causes defective development of T cells in the thymus.
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http://dx.doi.org/10.1515/jpm-2016-0234DOI Listing
May 2018

Intercellular Genetic Interaction Between Irf6 and Twist1 during Craniofacial Development.

Sci Rep 2017 08 2;7(1):7129. Epub 2017 Aug 2.

Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI, 48823, USA.

Interferon Regulatory Factor 6 (IRF6) and TWIST1 are transcription factors necessary for craniofacial development. Human genetic studies showed that mutations in IRF6 lead to cleft lip and palate and mandibular abnormalities. In the mouse, we found that loss of Irf6 causes craniosynostosis and mandibular hypoplasia. Similarly, mutations in TWIST1 cause craniosynostosis, mandibular hypoplasia and cleft palate. Based on this phenotypic overlap, we asked if Irf6 and Twist1 interact genetically during craniofacial formation. While single heterozygous mice are normal, double heterozygous embryos (Irf6 ; Twist1 ) can have severe mandibular hypoplasia that leads to agnathia and cleft palate at birth. Analysis of spatiotemporal expression showed that Irf6 and Twist1 are found in different cell types. Consistent with the intercellular interaction, we found reduced expression of Endothelin1 (EDN1) in mandible and transcription factors that are critical for mandibular patterning including DLX5, DLX6 and HAND2, were also reduced in mesenchymal cells. Treatment of mandibular explants with exogenous EDN1 peptides partially rescued abnormalities in Meckel's cartilage. In addition, partial rescue was observed when double heterozygous embryos also carried a null allele of p53. Considering that variants in IRF6 and TWIST1 contribute to human craniofacial defects, this gene-gene interaction may have implications on craniofacial disorders.
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http://dx.doi.org/10.1038/s41598-017-06310-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5540929PMC
August 2017

IRF6 expression in basal epithelium partially rescues Irf6 knockout mice.

Dev Dyn 2017 09 19;246(9):670-681. Epub 2017 Jul 19.

Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan.

Background: Mutations in IRF6, CHUK (IKKA), and RIPK4 can lead to a disease spectrum that includes cutaneous, limb, and craniofacial malformations. Loss of these alleles in the mouse leads to perinatal lethality and severe cutaneous, limb, and craniofacial defects also. Genetic rescue in the mouse has been shown for Ikka and Ripk4.

Results: Here, we show partial genetic rescue of Irf6 knockout embryos using the KRT14 promoter to drive Irf6 expression in the basal epithelium. In contrast to Irf6 knockout embryos, rescue embryos survive the immediate perinatal period. Macroscopic examination reveals rescue of skin adhesions between the axial and appendicular skeleton. Unexpectedly, KRT14-driven Irf6 expression does not completely rescue orofacial clefting and adhesions between the palate and tongue, suggesting the importance of cell-autonomous IRF6 expression in periderm. Like knockout embryos, Irf6 rescue embryos also have persistent esophageal adhesions, which likely contribute to postnatal demise.

Conclusions: Together, these data suggest that targeted expression of IRF6 can significantly reduce disease severity, but that a minimum level of Irf6 in both periderm and basal epithelial cells is necessary for orofacial development. Therefore, homologous human and mouse phenotypes are observed for IRF6, IKKA, and RIPK4. In this work, we show that altering the expression level of IRF6 dramatically modified this phenotype in utero. Developmental Dynamics 246:670-681, 2017. © 2017 Wiley Periodicals, Inc.
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http://dx.doi.org/10.1002/dvdy.24537DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5552062PMC
September 2017

Generation and characterization of a conditional allele of Interferon Regulatory Factor 6.

Genesis 2017 07 22;55(7). Epub 2017 Jun 22.

Genetics PhD Program, Michigan State University, East Lansing, Michigan.

Interferon Regulatory Factor 6 (IRF6) is a critical regulator of differentiation, proliferation, and migration of keratinocytes. Mutations in IRF6 cause two autosomal dominant disorders characterized by cleft lip with or without cleft palate. In addition, DNA variation in IRF6 confers significant risk for non-syndromic cleft lip and palate. IRF6 is also implicated in adult onset development and disease processes, including mammary gland development and squamous cell carcinoma. Mice homozygous for a null allele of Irf6 die shortly after birth due to severe skin, limb, and craniofacial defects, thus impeding the study of gene function after birth. To circumvent this, a conditional allele of Irf6 was generated. To validate the functionality of the conditional allele, we used three "deleter" Cre strains: Gdf9-Cre, CAG-Cre, and Ella-Cre. When Cre expression was driven by the Gdf9-Cre or CAG-Cre transgenes, 100% recombination was observed as indicated by DNA genotyping and phenotyping. In contrast, use of the Ella-Cre transgenic line resulted in incomplete recombination, despite expression at the one-cell stage. In sum, we generated a novel tool to delete Irf6 in a tissue specific fashion, allowing for study of gene function past perinatal stages. However, recombination efficiency of this allele was dictated by the Cre-driver used.
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http://dx.doi.org/10.1002/dvg.23038DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5509482PMC
July 2017

Shared molecular networks in orofacial and neural tube development.

Birth Defects Res 2017 01;109(2):169-179

Department of Microbiology and Molecular Genetics and the Department of Pediatrics and Human Development, Michigan State University, East Lansing, Michigan.

Background: Single genetic variants can affect multiple tissues during development. Thus it is possible that disruption of shared gene regulatory networks might underlie syndromic presentations. In this study, we explore this idea through examination of two critical developmental programs that control orofacial and neural tube development and identify shared regulatory factors and networks. Identification of these networks has the potential to yield additional candidate genes for poorly understood developmental disorders and assist in modeling and perhaps managing risk factors to prevent morbidly and mortality.

Methods: We reviewed the literature to identify genes common between orofacial and neural tube defects and development. We then conducted a bioinformatic analysis to identify shared molecular targets and pathways in the development of these tissues. Finally, we examine publicly available RNA-Seq data to identify which of these genes are expressed in both tissues during development.

Results: We identify common regulatory factors in orofacial and neural tube development. Pathway enrichment analysis shows that folate, cancer and hedgehog signaling pathways are shared in neural tube and orofacial development. Developing neural tissues differentially express mouse exencephaly and cleft palate genes, whereas developing orofacial tissues were enriched for both clefting and neural tube defect genes.

Conclusion: These data suggest that key developmental factors and pathways are shared between orofacial and neural tube defects. We conclude that it might be most beneficial to focus on common regulatory factors and pathways to better understand pathology and develop preventative measures for these birth defects. Birth Defects Research 109:169-179, 2017. © 2016 Wiley Periodicals, Inc.
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http://dx.doi.org/10.1002/bdra.23598DOI Listing
January 2017

Toward an orofacial gene regulatory network.

Dev Dyn 2016 Mar 17;245(3):220-32. Epub 2015 Sep 17.

Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, Michigan.

Orofacial clefting is a common birth defect with significant morbidity. A panoply of candidate genes have been discovered through synergy of animal models and human genetics. Among these, variants in interferon regulatory factor 6 (IRF6) cause syndromic orofacial clefting and contribute risk toward isolated cleft lip and palate (1/700 live births). Rare variants in IRF6 can lead to Van der Woude syndrome (1/35,000 live births) and popliteal pterygium syndrome (1/300,000 live births). Furthermore, IRF6 regulates GRHL3 and rare variants in this downstream target can also lead to Van der Woude syndrome. In addition, a common variant (rs642961) in the IRF6 locus is found in 30% of the world's population and contributes risk for isolated orofacial clefting. Biochemical studies revealed that rs642961 abrogates one of four AP-2alpha binding sites. Like IRF6 and GRHL3, rare variants in TFAP2A can also lead to syndromic orofacial clefting with lip pits (branchio-oculo-facial syndrome). The literature suggests that AP-2alpha, IRF6 and GRHL3 are part of a pathway that is essential for lip and palate development. In addition to updating the pathways, players and pursuits, this review will highlight some of the current questions in the study of orofacial clefting.
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http://dx.doi.org/10.1002/dvdy.24341DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4755791PMC
March 2016

Identification of functional variants for cleft lip with or without cleft palate in or near PAX7, FGFR2, and NOG by targeted sequencing of GWAS loci.

Am J Hum Genet 2015 Mar 19;96(3):397-411. Epub 2015 Feb 19.

Department of Pediatrics, Carver College of Medicine, University of Iowa, Iowa City, IA 52242, USA.

Although genome-wide association studies (GWASs) for nonsyndromic orofacial clefts have identified multiple strongly associated regions, the causal variants are unknown. To address this, we selected 13 regions from GWASs and other studies, performed targeted sequencing in 1,409 Asian and European trios, and carried out a series of statistical and functional analyses. Within a cluster of strongly associated common variants near NOG, we found that one, rs227727, disrupts enhancer activity. We furthermore identified significant clusters of non-coding rare variants near NTN1 and NOG and found several rare coding variants likely to affect protein function, including four nonsense variants in ARHGAP29. We confirmed 48 de novo mutations and, based on best biological evidence available, chose two of these for functional assays. One mutation in PAX7 disrupted the DNA binding of the encoded transcription factor in an in vitro assay. The second, a non-coding mutation, disrupted the activity of a neural crest enhancer downstream of FGFR2 both in vitro and in vivo. This targeted sequencing study provides strong functional evidence implicating several specific variants as primary contributory risk alleles for nonsyndromic clefting in humans.
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http://dx.doi.org/10.1016/j.ajhg.2015.01.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4375420PMC
March 2015

Pedigree structure and kinship measurements of a mid-Michigan community: a new North American population isolate identified.

Hum Biol 2014 ;86(1):59-68

Department of Comparative Medicine and Integrative Biology, East Lansing, Michigan, USA; Department of Pediatrics and Human Development, East Lansing, Michigan, USA; Department of Microbiology and Molecular Genetics, East Lansing, Michigan, USA.

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 affected members from this community descend from a defined founder population. Using public records and personal interviews, we constructed a genealogical database that includes the affected 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 coefficient 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.
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http://dx.doi.org/10.3378/027.086.0103DOI Listing
November 2015

Interferon regulatory factor 6 regulates keratinocyte migration.

J Cell Sci 2014 Jul 28;127(Pt 13):2840-8. Epub 2014 Apr 28.

Department of Pediatrics, The University of Iowa, Iowa City, IA 52242, USA Interdisciplinary Graduate Program in Genetics, The University of Iowa, Iowa City, IA 52242, USA

Interferon regulatory factor 6 (Irf6) 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 with wild-type embryos. In vitro, Irf6-deficient murine embryonic keratinocytes were delayed in closing a scratch wound. Live imaging of the scratch showed 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-healing process. Interestingly, Irf6-deficient keratinocytes exhibited an increased network of stress fibers and active RhoA compared with that observed in wild-type keratinocytes. Blocking ROCK, a downstream effector of RhoA, rescued the delay in closing scratch wounds. The expression of Arhgap29, a Rho GTPase-activating protein, was reduced in Irf6-deficient keratinocytes. Taken together, these data suggest that Irf6 functions through the RhoA pathway to regulate cellular migration.
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http://dx.doi.org/10.1242/jcs.139246DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4075356PMC
July 2014

Community leader perceptions of the health needs of older adults.

West J Nurs Res 2015 May 22;37(5):599-618. Epub 2014 Apr 22.

Michigan State University, East Lansing, MI, USA.

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.
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http://dx.doi.org/10.1177/0193945914530046DOI Listing
May 2015

An etiologic regulatory mutation in IRF6 with loss- and gain-of-function effects.

Hum Mol Genet 2014 May 16;23(10):2711-20. Epub 2014 Jan 16.

Microbiology and Molecular Genetics.

DNA variation in Interferon Regulatory Factor 6 (IRF6) causes Van der Woude syndrome (VWS), the most common syndromic form of cleft lip and palate (CLP). 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 suggest 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 CLP.
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http://dx.doi.org/10.1093/hmg/ddt664DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3990169PMC
May 2014

Dominant mutations in GRHL3 cause Van der Woude Syndrome and disrupt oral periderm development.

Am J Hum Genet 2014 Jan 19;94(1):23-32. Epub 2013 Dec 19.

Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI 48824, USA.

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.
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http://dx.doi.org/10.1016/j.ajhg.2013.11.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3882735PMC
January 2014

Haploinsufficiency of interferon regulatory factor 6 alters brain morphology in the mouse.

Am J Med Genet A 2014 Mar 19;164A(3):655-60. Epub 2013 Dec 19.

Department of Psychiatry, University of Iowa, Iowa City, Iowa.

Orofacial clefts are among the commonest birth defects. Among many genetic contributors to orofacial clefting, Interferon Regulatory Factor 6 (IRF6) is unique since mutations in this gene cause Van der Woude (VWS), the most common clefting syndrome. Furthermore, variants in IRF6 contribute to increased risk for non-syndromic cleft lip and/or palate (NSCL/P). Our previous work shows that individuals with either VWS or NSCL/P may have cerebral anomalies (larger anterior, smaller posterior regions), and a smaller cerebellum. The objective of this study was to test the hypothesis that disrupting Irf6 in the mouse will result in quantitative brain changes similar to those reported for humans with VWS and NSCL/P. Male mice heterozygous for Irf6 (Irf6(gt1/+); n = 9) and wild-type (Irf6(+/+) ; n = 6) mice at comparable age underwent a 4.7-T MRI scan to obtain quantitative measures of cortical and subcortical brain structures. There was no difference in total brain volume between groups. However, the frontal cortex was enlarged in the Irf6(gt1/+) mice compared to that of wild types (P = 0.028) while the posterior cortex did not differ. In addition, the volume of the cerebellum of Irf6(gt1/+) mice was decreased (P = 0.004). Mice that were heterozygous for Irf6 showed a similar pattern of brain anomalies previously reported in humans with VWS and NSCL/P. These structural differences were present in the absence of overt oral clefts. These results support a role for IRF6 in brain morphometry and provide evidence for a potential genetic link to abnormal brain development in orofacial clefting.
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http://dx.doi.org/10.1002/ajmg.a.36333DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4100789PMC
March 2014

Search for genetic modifiers of IRF6 and genotype-phenotype correlations in Van der Woude and popliteal pterygium syndromes.

Am J Med Genet A 2013 Oct 15;161A(10):2535-2544. Epub 2013 Aug 15.

Department of Pediatrics, University of Iowa, Iowa City, IA.

Van der Woude syndrome is the most common form of syndromic orofacial clefting, accounting for 1-2% of all patients with cleft lip and/or cleft palate. Van der Woude and popliteal pterygium syndromes are caused by mutations in IRF6, but phenotypic variability within and among families with either syndrome suggests that other genetic factors contribute to the phenotypes. The aim of this study was to identify common variants acting as genetic modifiers of IRF6 as well as genotype-phenotype correlations based on mutation type and location. We identified an association between mutations in the DNA-binding domain of IRF6 and limb defects (including pterygia). Although we did not detect formally significant associations with the genes tested, borderline associations suggest several genes that could modify the VWS phenotype, including FOXE1, TGFB3, and TFAP2A. Some of these genes are hypothesized to be part of the IRF6 gene regulatory network and may suggest additional genes for future study when larger sample sizes are also available. We also show that families with the Van de Woude phenotype but in whom no mutations have been identified have a lower frequency of cleft lip, suggesting there may be locus and/or mutation class differences in Van de Woude syndrome.
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http://dx.doi.org/10.1002/ajmg.a.36133DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3898350PMC
October 2013

Exploring the effects of gene dosage on mandible shape in mice as a model for studying the genetic basis of natural variation.

Dev Genes Evol 2013 Sep 6;223(5):279-87. Epub 2013 Apr 6.

Max-Planck-Institut für Evolutionsbiologie, August-Thienemann-str. 2, 24306 Plön, Germany.

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.
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http://dx.doi.org/10.1007/s00427-013-0443-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4013528PMC
September 2013

Cell-autonomous and non-cell-autonomous roles for IRF6 during development of the tongue.

PLoS One 2013 22;8(2):e56270. Epub 2013 Feb 22.

Department of Otolaryngology, Vanderbilt University, Nashville, Tennessee, United States of America.

Interferon regulatory factor 6 (IRF6) 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 IRF6 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 Irf6 have severe limb, skin, palatal and esophageal abnormalities, due to significantly altered and delayed epithelial development. However, a recent report showed that MCS9.7, an enhancer near Irf6, is active in the tongue, suggesting that Irf6 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 Myf5+ 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 Irf6 function with reduced and poorly organized Myf5+ cell lineage in the tongue. Molecular analyses showed that the Irf6-/- 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 Irf6 function with the loss of the inter-molar eminence. Loss of Irf6 function altered Bmp2, Bmp4, Shh, and Fgf10 signaling suggesting that these genes are involved in Irf6 signaling. Based on these data, Irf6 plays important cell-autonomous and non-cell-autonomous roles in muscular differentiation and cytoskeletal formation in the tongue.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0056270PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3579850PMC
September 2013

Comparative analysis of IRF6 variants in families with Van der Woude syndrome and popliteal pterygium syndrome using public whole-exome databases.

Genet Med 2013 May 15;15(5):338-44. Epub 2012 Nov 15.

Department of Pediatrics, University of Iowa, Iowa City, IA, USA.

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.

Methods: We compiled all published pathogenic IRF6 mutations and performed direct sequencing of IRF6 in families with Van der Woude syndrome or popliteal pterygium syndrome.

Results: 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.

Conclusion: 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.
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http://dx.doi.org/10.1038/gim.2012.141DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3723330PMC
May 2013

Transforming growth factor Beta 3 is required for excisional wound repair in vivo.

PLoS One 2012 26;7(10):e48040. Epub 2012 Oct 26.

Department of Pediatrics, Carver College of Medicine, The University of Iowa, Iowa City, Iowa, United States of America.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0048040PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3482237PMC
April 2013

Interaction between IRF6 and TGFA genes contribute to the risk of nonsyndromic cleft lip/palate.

PLoS One 2012 20;7(9):e45441. Epub 2012 Sep 20.

Department of Oral Biology, University of Pittsburgh, School of Dental Medicine, Pittsburgh, PA, USA.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0045441PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3447924PMC
February 2013

Interferon regulatory factor 6 promotes differentiation of the periderm by activating expression of Grainyhead-like 3.

J Invest Dermatol 2013 Jan 30;133(1):68-77. Epub 2012 Aug 30.

Department of Otolaryngology-Head and Neck Surgery, University of Iowa, Iowa City, IA, USA.

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 [corrected] 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.
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http://dx.doi.org/10.1038/jid.2012.269DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3541433PMC
January 2013

The role of Irf6 in tooth epithelial invagination.

Dev Biol 2012 May 14;365(1):61-70. Epub 2012 Feb 14.

Department of Craniofacial Development and Comprehensive Biomedical Research Centre, Dental Institute, King's College London, UK.

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.
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http://dx.doi.org/10.1016/j.ydbio.2012.02.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7462630PMC
May 2012

Exome sequence identifies RIPK4 as the Bartsocas-Papas syndrome locus.

Am J Hum Genet 2012 Jan 22;90(1):69-75. Epub 2011 Dec 22.

Faculty of Medical and Human Sciences, Manchester Academic Health Sciences Centre, University of Manchester, UK.

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.
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http://dx.doi.org/10.1016/j.ajhg.2011.11.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3257958PMC
January 2012

MCS9.7 enhancer activity is highly, but not completely, associated with expression of Irf6 and p63.

Dev Dyn 2012 Feb 23;241(2):340-9. Epub 2011 Nov 23.

Microbiology and Molecular Genetics, Michigan State University, East Lansing, Michigan 48823, USA.

Background: 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.

Results: 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.

Conclusion: 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.
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http://dx.doi.org/10.1002/dvdy.22786DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4936416PMC
February 2012