Publications by authors named "Youssef A Kousa"

20 Publications

  • Page 1 of 1

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

Acute Pontine Ischemic Stroke in a Healthy Child With Intracranial Vasculopathy.

J Child Neurol 2019 11 16;34(13):820-823. Epub 2019 Jul 16.

Division of Neurology, Children's National Health System, Washington, DC, USA.

Here we report the case of a previously healthy 8-year-old boy who presented with altered mental status, right facial droop and right-sided hemiplegia the day after playing in an inflatable bouncer. No head trauma was reported by the patient nor witnessed by the parents. Urgent magnetic resonance imaging (MRI) demonstrated acute ischemic infarction in the left pons; computed tomographic angiography excluded arterial dissection but identified a small hyperdense filling defect in the basilar artery, later confirmed to be a calcification at the origin of a perforating artery. Pediatric National Institutes of Health (PedNIH) Stroke Scale score was 15. Infectious, inflammatory, hypercoagulable and additional vascular causes were excluded. Although the cause of the calcification remains obscure, we speculate that, similarly to mineralizing microangiopathy, a minor trauma led to stroke in this child. To our knowledge, mineralizing microangiopathy, the well-described entity affecting perforating arteries of the anterior circulation in young children leading to basal ganglia stroke following minor head traumas has not been described in the posterior circulation or in previously healthy school-age children.
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http://dx.doi.org/10.1177/0883073819861851DOI Listing
November 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

Sequential Neuroimaging of the Fetus and Newborn With In Utero Zika Virus Exposure.

JAMA Pediatr 2019 01;173(1):52-59

Division of Fetal and Transitional Medicine, Children's National Health System, Washington, DC.

Importance: The evolution of fetal brain injury by Zika virus (ZIKV) infection is not well described.

Objectives: To perform longitudinal neuroimaging of fetuses and infants exposed to in utero maternal ZIKV infection using concomitant magnetic resonance imaging (MRI) and ultrasonography (US), as well as to determine the duration of viremia in pregnant women with ZIKV infection and whether the duration of viremia correlated with fetal and/or infant brain abnormalities.

Design, Setting, And Participants: A cohort of 82 pregnant women with clinical criteria for probable ZIKV infection in Barranquilla, Colombia, and Washington, DC, were enrolled from June 15, 2016, through June 27, 2017, with Colombian women identified by community recruitment and physician referral and travel-related cases of American women recruited from a Congenital Zika Program.

Interventions And Exposures: Women received 1 or more MRI and US examinations during the second and/or third trimesters. Postnatally, infants underwent brain MRI and cranial US. Blood samples were tested for ZIKV.

Main Outcomes And Measures: The neuroimaging studies were evaluated for brain injury and cerebral biometry.

Results: Of the 82 women, 80 were from Colombia and 2 were from the United States. In 3 of 82 cases (4%), fetal MRI demonstrated abnormalities consistent with congenital ZIKV infection. Two cases had heterotopias and malformations in cortical development and 1 case had a parietal encephalocele, Chiari II malformation, and microcephaly. In 1 case, US results remained normal despite fetal abnormalities detected on MRI. Prolonged maternal polymerase chain reaction positivity was present in 1 case. Of the remaining 79 cases with normal results of prenatal imaging, postnatal brain MRI was acquired in 53 infants and demonstrated mild abnormalities in 7 (13%). Fifty-seven infants underwent postnatal cranial US, which detected changes of lenticulostriate vasculopathy, choroid plexus cysts, germinolytic/subependymal cysts, and/or calcification in 21 infants (37%).

Conclusions And Relevance: In a cohort of pregnant women with ZIKV infection, prenatal US examination appeared to detect all but 1 abnormal fetal case. Postnatal neuroimaging in infants who had normal prenatal imaging revealed new mild abnormalities. For most patients, prenatal and postnatal US may identify ZIKV-related brain injury.
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http://dx.doi.org/10.1001/jamapediatrics.2018.4138DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6583436PMC
January 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

Admission EEG findings in diverse paediatric cerebral malaria populations predict outcomes.

Malar J 2018 May 22;17(1):208. Epub 2018 May 22.

Epilepsy Division, Department of Neurology, University of Rochester, 265 Crittenden Blvd, CU 420694, Rochester, NY, 14642, USA.

Background: Electroencephalography at hospital presentation may offer important insights regarding prognosis that can inform understanding of cerebral malaria (CM) pathophysiology and potentially guide patient selection and risk stratification for future clinical trials. Electroencephalogram (EEG) findings in children with CM in Uganda and Malawi were compared and associations between admission EEG findings and outcome across this diverse population were assessed. Demographic, clinical and admission EEG data from Ugandan and Malawian children admitted from 2009 to 2012 with CM were gathered, and survivors assessed for neurological abnormalities at discharge.

Results: 281 children were enrolled (Uganda n = 122, Malawi n = 159). The Malawian population was comprised only of retinopathy positive children (versus 72.5% retinopathy positive in Uganda) and were older (4.2 versus 3.7 years; p = 0.046), had a higher HIV prevalence (9.0 versus 2.8%; p = 0.042), and worse hyperlactataemia (7.4 versus 5.2 mmol/L; p < 0.001) on admission compared to the Ugandan children. EEG findings differed between the two groups in terms of average voltage and frequencies, reactivity, asymmetry, and the presence/absence of sleep architecture. In univariate analyses pooling EEG and outcomes data for both sites, higher average and maximum voltages, faster dominant frequencies, and retained reactivity were associated with survival (all p < 0.05). Focal slowing was associated with death (OR 2.93; 95% CI 1.77-7.30) and a lower average voltage was associated with neurological morbidity in survivors (p = 0.0032).

Conclusions: Despite substantial demographic and clinical heterogeneity between subjects in Malawi and Uganda as well as different EEG readers at each site, EEG findings on admission predicted mortality and morbidity. For CM clinical trials aimed at decreasing mortality or morbidity, EEG may be valuable for risk stratification and/or subject selection.
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http://dx.doi.org/10.1186/s12936-018-2355-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5963073PMC
May 2018

Prenatal diagnosis of holoprosencephaly.

Am J Med Genet C Semin Med Genet 2018 06 17;178(2):206-213. Epub 2018 May 17.

Division of Radiology, Children's National Health System, Washington, DC.

Holoprosencephaly is a spectrum of congenital defects of forebrain development characterized by incomplete separation of the cerebral hemispheres. In vivo diagnosis can be established with prenatal brain imaging and disease severity correlates with extent of abnormally developed brain tissue. Advances in magnetic resonance imaging (MRI) over the past 25 years and their application to the fetus have enabled diagnosis of holoprosencephaly in utero. Here, we report on the prenatal diagnosis of holoprosencephaly using MRI as part of a diagnostic and management evaluation at a tertiary and quaternary referral center. Using an advanced MRI protocol and a 1.5-Tesla magnet, we show radiographic data diagnostic for the holoprosencephaly spectrum, including alobar, semilobar, lobar, middle interhemispheric, and septopreoptic variant. Accurate prenatal evaluation is important because the severity of imaging findings correlates with postnatal morbidity and mortality in holoprosencephaly. Therefore, this work has implications for the evaluation, diagnosis, management, and genetic counseling that families can receive during a pregnancy.
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http://dx.doi.org/10.1002/ajmg.c.31618DOI Listing
June 2018

Management of intracranial hemorrhage in severe factor V deficiency and definitive treatment with liver transplantation.

Pediatr Transplant 2018 02 18;22(1). Epub 2017 Dec 18.

Hemophilia Treatment Center, Division of Hematology, Children's National Health System, Washington, DC, USA.

FV is primarily produced in the liver, and congenital FV deficiency is a disorder with an incidence of one in 1 million. Standard care is to treat severe bleeding phenotypes with FFP as there is no recombinant or plasma-derived FV concentrate. We present a case of a neonate with known severe FV deficiency diagnosed after prolonged bleeding after circumcision who represented at age 2 months with a large left intraparenchymal hemorrhage. His bleed was treated with FFP, platelet transfusion, recombinant VIIa, and emergent evacuation. He was maintained on plasma infusions but was unable to space his infusions beyond 48 hours. Liver transplantation was considered as a definitive treatment for this condition. While awaiting a suitable liver, his FV trough levels occasionally dropped below 5%, and he suffered from a second acute intracranial bleed. He received an orthotopic liver transplant at age 5 months, resulting in correction of his FV levels. He has not required any plasma infusions post-transplantation and has had no further bleeding episodes. Liver transplantation should be considered as definitive treatment early in the course for patients with severe FV deficiency and first time life-threatening bleed.
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http://dx.doi.org/10.1111/petr.13102DOI Listing
February 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

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

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

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

Immunogenicity when utilizing adenovirus serotype 4 and 5 vaccines expressing circumsporozoite protein in naïve and adenovirus (Ad5) immune mice.

Malar J 2012 Jun 21;11:209. Epub 2012 Jun 21.

Genetics Program, Michigan State University, 2240 E Biomedical and Physical Sciences Building, East Lansing, MI 48824, USA.

Background: Induction of potent long lasting effector T cell responses against liver stage malaria antigens strongly correlates with protection from malaria. While Adenovirus serotype 5 (Ad5) based malaria vaccine platforms have the ability to induce potent effector T cell responses against transgenes, high rates of pre-existing Ad5 immunity in malaria endemic regions has prompted study of alternative Ad serotype based malaria vaccines as replacements for Ad5 based malaria vaccines. The research described in this article examines the utility of alternative serotype adenovirus serotype 4 (Ad4) expressing a sporozoite surface protein (circumsporozoite protein (CSP)) (Ad4-CSP) to induce immune responses against CSP. The immunogenicity of Ad4-CSP was also tested in homologous and heterologous prime boost vaccinations in both Ad5 naïve and Ad5 immune backgrounds as compared to use of Ad5-CSP.

Results: In Ad5 naïve animals, use of Ad4-CSP priming vaccinations followed by boosting with Ad5-CSP (Ad4-CSP/Ad5-CSP) maximally increased the numbers of CSP specific cytokine secreting cytotoxic T cells relative to repeated use of Ad5-CSP. The Ad4-CSP/Ad5-CSP regimen also induced equivalent levels of CSP specific cell killing as did homologous prime-boost vaccinations with Ad5-CSP, despite stimulating lower numbers of CSP specific cytotoxic T cells. Priming with Ad4-CSP followed by a homologous boost resulted in significantly less CSP specific humoral responses than any other vaccination regimen tested in Ad naïve animals. In Ad5 immune animals, addition of Ad4-CSP in homologous or heterologous prime boost resulted in inductions of higher CSP specific responses than animals repeatedly vaccinated with Ad5-CSP alone. However, the observed responses were well below those observed in similarly treated Ad naïve mice.

Conclusions: While the Ad4-CSP/Ad5-CSP and Ad5-CSP/Ad5-CSP vaccination regimens resulted in equivalent CSP specific killing in Ad naïve animals, Ad4-CSP/Ad5-CSP achieved this result with a lower percentage of CSP specific CD8+ T cells and a higher number of IFNγ secreting cells, suggesting that the Ad4-CSP/Ad5-CSP vaccination regimen elicits more efficient cytotoxic T cells. In Ad5 immune animals use of Ad4-CSP improved CSP specific immune responses as compared to repeated use of Ad5-CSP, but could not achieve the levels of immunogenicity observed when the same vaccine regimens were used in Ad naïve animals. These data indicate the existence of some level of immunological cross-reactivity between these two adenovirus subgroups. Based on these results, it is suggested that future studies should undertake similarly stringent analyses of alternative Ad serotypes to establish their effectiveness as replacements for Ad5.
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http://dx.doi.org/10.1186/1475-2875-11-209DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3472263PMC
June 2012