Building a growing genomic data repository for maternal and fetal health through the PING Consortium.

Background: Prenatally transmitted viruses can cause severe damage to the developing brain. There is unexplained variability in prenatal brain injury and postnatal neurodevelopmental outcomes, suggesting disease modifiers. Discordant outcomes among dizygotic twins could be explained by genetic susceptibly or protection. Among several well-recognized threats to the developing brain, Zika is a mosquito-borne, positive-stranded RNA virus that was originally isolated in Uganda and spread to cause epidemics in Africa, Asia, and the Americas. In the Americas, the virus caused congenital Zika syndrome and a multitude of neurodevelopmental disorders. As of now, there is no preventative treatment or cure for the adverse outcomes caused by prenatal Zika infection. The Prenatal Infection and Neurodevelopmental Genetics (PING) Consortium was initiated in 2016 to identify factors modulating prenatal brain injury and postnatal neurodevelopmental outcomes for Zika and other prenatal viral infections. Methods: The Consortium has pooled information from eight multi-site studies conducted at 23 research centers in six countries to build a growing clinical and genomic data repository. This repository is being mined to search for modifiers of virally induced brain injury and developmental outcomes. Multilateral partnerships include commitments with Children's National Hospital (USA), Instituto Nacional de Salud (Colombia), the Natural History of Zika Virus Infection in Gestation program (Brazil), and Zika Instituto Fernandes Figueira (Brazil), in addition to the Centers for Disease Control and Prevention and the National Institutes of Health. Discussion: Our goal in bringing together these sets of patient data was to test the hypothesis that personal and populational genetic differences affect the severity of brain injury after a prenatal viral infection and modify neurodevelopmental outcomes. We have enrolled 4,102 mothers and 3,877 infants with 3,063 biological samples and clinical data covering over 80 phenotypic fields and 5,000 variables. There were several notable challenges in bringing together cohorts enrolled in different studies, including variability in the timepoints evaluated and the collected clinical data and biospecimens. Thus far, we have performed whole exome sequencing on 1,226 participants. Here, we present the Consortium's formation and the overarching study design. We began our investigation with prenatal Zika infection with the goal of applying this knowledge to other prenatal infections and exposures that can affect brain development.

Neonatal Botulism: A Case Series Suggesting Varied Presentations.

Infantile botulism is an uncommon diagnosis and rarer still in the neonatal period. We describe three cases of neonatal-onset botulism that presented with symptoms typically (hypotonia, constipation, facial diplegia) or atypically seen in older infants (encephalopathy, seizures, and hypothermia). Our series shows a wider spectrum of clinical presentations in patients with neonatal-onset botulism. Our report also suggests that neonatal-onset botulism should be considered more broadly in the hypotonic infant, especially as the condition is treatable with intravenous botulinum-specific immune globulin.

Causes of Phenotypic Variability and Disabilities after Prenatal Viral Infections.

Prenatal viral infection can lead to a spectrum of neurodevelopmental disabilities or fetal demise. These can include microencephaly, global developmental delay, intellectual disability, refractory epilepsy, deafness, retinal defects, and cortical-visual impairment. Each of these clinical conditions can occur on a semi-quantitative to continuous spectrum, from mild to severe disease, and often as a collective of phenotypes. Such serious outcomes result from viruses' overlapping neuropathology and hosts' common neuronal and gene regulatory response to infections. The etiology of variability in clinical outcomes is not yet clear, but it may be related to viral, host, vector, and/or environmental risk and protective factors that likely interact in multiple ways. In this perspective of the literature, we work toward understanding the causes of phenotypic variability after prenatal viral infections by highlighting key aspects of the viral lifecycle that can affect human disease, with special attention to the 2015 Zika pandemic. Therefore, this work offers important insights into how viral infections and environmental teratogens affect the prenatal brain, toward our ultimate goal of preventing neurodevelopmental disabilities.

Complexities of Zika Diagnosis and Evaluation in a U.S. Congenital Zika Program.

The objective of the study was to describe the complexity of diagnosis and evaluation of Zika-exposed pregnant women/fetuses and infants in a U.S. Congenital Zika Program. Pregnant women/fetuses and/or infants referred for clinical evaluation to the Congenital Zika Program at Children's National (Washington, DC) from January 2016 to June 2018 were included. We recorded the timing of maternal Zika-virus (ZIKV) exposure and ZIKV laboratory testing results. Based on laboratory testing, cases were either confirmed, possible, or unlikely ZIKV infection. Prenatal and postnatal imaging by ultrasound and/or magnetic resonance imaging (MRI) were categorized as normal, nonspecific, or as findings of congenital Zika syndrome (CZS). Of 81 women-fetus/infant pairs evaluated, 72 (89%) had confirmed ZIKV exposure; 18% of women were symptomatic; only a minority presented for evaluation within the time frame for laboratory detection. Zika virus could only be confirmed in 29 (40%) cases, was possible in 26 (36%) cases, and was excluded in 17 (24%) cases. Five cases (7%) had prenatal ultrasound and MRI findings of CZS, but in only three was ZIKV confirmed by laboratory testing. Because of timing of exposure to presentation, ZIKV infection could not be excluded in many cases. Neuroimaging found CZS in 7% of cases, and in many patients, there were nonspecific imaging findings that warrant long-term follow-up. Overall, adherence to postnatal recommended follow-up evaluations was modest, representing a barrier to care. These challenges may be instructive to future pediatric multidisciplinary clinics for congenital infectious/noninfectious threats to pregnant women and their infants.

SPECC1L regulates palate development downstream of IRF6.

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.

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

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.

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

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.

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

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.

IRF6 and AP2A Interaction Regulates Epidermal Development.

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.

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

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.

Prenatal diagnosis of holoprosencephaly.

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.

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

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.

Intercellular Genetic Interaction Between Irf6 and Twist1 during Craniofacial Development.

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.

IRF6 expression in basal epithelium partially rescues Irf6 knockout mice.

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.

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

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.

Shared molecular networks in orofacial and neural tube development.

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.

Toward an orofacial gene regulatory network.

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.

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.

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.

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

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/ß-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.

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

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