Search

In contrast to the progress that has been made toward understanding the genetic etiology of cleft lip with or without cleft palate, relatively little is known about the genetic etiology for cleft palate only (CPO). A common coding variant of grainyhead like transcription factor 3 ( GRHL3) was recently shown to be associated with risk for CPO in Europeans. Mutations in this gene were also reported in families with Van der Woude syndrome. To identify rare mutations in GRHL3 that might explain the missing heritability for CPO, we sequenced GRHL3 in cases of CPO from Africa. We recruited participants from Ghana, Ethiopia, and Nigeria. This cohort included case-parent trios, cases and other family members, as well as controls. We sequenced exons of this gene in DNA from a total of 134 nonsyndromic cases. When possible, we sequenced them in parents to identify de novo mutations. Five novel mutations were identified: 2 missense (c.497C>A; p.Pro166His and c.1229A>G; p.Asp410Gly), 1 splice site (c.1282A>C p.Ser428Arg), 1 frameshift (c.470delC; p.Gly158Alafster55), and 1 nonsense (c.1677C>A; p.Tyr559Ter). These mutations were absent from 270 sequenced controls and from all public exome and whole genome databases, including the 1000 Genomes database (which includes data from Africa). However, 4 of the 5 mutations were present in unaffected mothers, indicating that their penetrance is incomplete. Interestingly, 1 mutation damaged a predicted sumoylation site, and another disrupted a predicted CK1 phosphorylation site. Overexpression assays in zebrafish and reporter assays in vitro indicated that 4 variants were functionally null or hypomorphic, while 1 was dominant negative. This study provides evidence that, as in Caucasian populations, mutations in GRHL3 contribute to the risk of nonsyndromic CPO in the African population.

Rare mutations in IRF6 and GRHL3 cause Van der Woude syndrome, an autosomal dominant orofacial clefting disorder. Common variants in IRF6 and GRHL3 also contribute risk for isolated orofacial clefting. Similarly, variants within genes that encode receptor tyrosine kinase (RTK) signaling components, including members of the FGF pathway, EPHA3 and SPRY2, also contribute risk for isolated orofacial clefting. In the mouse, loss of Irf6 or perturbation of Fgf signaling leads to abnormal oral epithelial adhesions and cleft palate. Oral adhesions can result from a disruption of periderm formation. Here, we find that IRF6 and SPRY4 signaling interact in periderm function. We crossed Irf6 heterozygous ( Irf6+/–) mice with transgenic mice that express Spry4 in the basal epithelial layer ( TgKRT14::Spry4). While embryos with either of these mutations can have abnormal oral adhesions, using a new quantitative assay, we observed a nonadditive effect of abnormal oral epithelial adhesions in the most severely affected double mutant embryos ( Irf6+/–;TgKRT14::Spry4). At the molecular level, the sites of abnormal oral adhesions maintained periderm-like cells that express keratin 6, but we observed abnormal expression of GRHL3. Together, these data suggest that Irf6 and RTK signaling interact in regulating periderm differentiation and function, as well as provide a rationale to screen for epistatic interactions between variants in IRF6 and RTK signaling pathway genes in human orofacial clefting populations.

Craniofacial development in vertebrates involves the coordinated growth, migration, and fusion of several facial prominences during embryogenesis, processes governed by strict genetic and molecular controls. A failure in any of the precise spatiotemporal sequences of events leading to prominence fusion often leads to anomalous facial, skull, and jaw formation—conditions termed craniofacial defects (CFDs). Affecting approximately 0.1% to 0.3% of live births, CFDs are a highly heterogeneous class of developmental anomalies, which are often underpinned by genetic mutations. Therefore, identifying novel disease-causing mutations in genes that regulate craniofacial development is a critical prerequisite to develop new preventive or therapeutic measures. The Grainyhead-like ( GRHL) transcription factors are one such gene family, performing evolutionarily conserved roles in craniofacial patterning. The antecedent member of this family, Drosophila grainyhead ( grh), is required for head skeleton development in fruit flies, loss or mutation of Grhl family members in mouse and zebrafish models leads to defects of both maxilla and mandible, and recently, mutations in human GRHL3 have been shown to cause or contribute to both syndromic (Van Der Woude syndrome) and nonsyndromic palatal clefts. In this review, we summarize the current knowledge regarding the craniofacial-specific function of the Grainyhead-like family in multiple model species, identify some of the major target genes regulated by the Grhl transcription factors in craniofacial patterning, and, by examining animal models, draw inferences as to how these data will inform the likely roles of GRHL factors in human CFDs comprising palatal clefting. By understanding the molecular networks regulated by Grhl2 and Grhl3 target genes in other systems, we can propose likely pathways that mediate the effects of these transcription factors in human palatogenesis.

Van der Woude syndrome (VWS) is an autosomal-dominant oral facial disorder. To find a gene mutation in a Japanese family using fingernail DNA samples, we performed this study. We hypothesized that a gene mutation in IRF6 might be involved in VWS, and that fingernail DNA samples may be valuable for detecting such mutations. Linkage and haplotype analyses of the family mapped the disease locus to the 1q32-q41 region. Mutation analysis with an improved extraction method for fingernail DNA detected a novel missense mutation (1046A>T, E349V) in exon 7 of IRF6 in all the affected members of the family. Since the E349V change may disturb the hydrophobic core and affect regulatory activity of IRF6, it is most likely that the mutation is causative for VWS in this family. Fingernail DNA is thus useful for linkage and mutation analyses, since the fingernail can be easily obtained non-invasively, sent through the mail, and stored for a long period. We emphasize here the usefulness of fingernail DNA for the genetic analysis of a disease.

Van der Woude syndrome (VWS) (OMIM 119300) is a dominantly inherited, developmental disorder that is characterized by pits and/or sinuses of the lower lip and a cleft lip and/or cleft palate. Mutations in the interferon regulatory factor 6 gene ( IRF6) have been recently identified in patients with VWS, with more than 60 mutations reported. However, the VWS phenotype, IRF6 mutation genotypes, and their interrelationships in Chinese VWS patients have not been studied. Here, we report 11 Chinese families with variable clinical phenotypes of VWS and identified mutations in all patients. Of the 11 mutations, 8 appeared to be novel: CC5.6GT, T342A, 566delA, C748T, C756A, C989A, C1209G, and 1316delT. Seven mutations caused a change or loss of the IRF6 domain. The marked phenotypic variation may be caused by the action of certain modifier genes on IRF6 function. Abbreviations: VWS, Van der Woude syndrome; IRF6, interferon regulatory factor 6; CL/P, cleft lip and/or cleft palate; DBD, DNA-binding domain; SMIR, Smad-interferon regulatory factor-binding domain; Kb, kilobase; PCR, polymerase chain-reaction.

Genetic defects responsible for tooth agenesis are only now beginning to be uncovered. MSX1 and PAX9 have been associated with tooth agenesis in mice and humans, but interestingly for humans, these genes are associated with specific missing teeth. Mouse models also show that specific genes contribute to the development of specific types of teeth. A precise description of the phenotype specifying which teeth are missing has become fundamental. Mendelian segregation can be identified in families with tooth agenesis, but heterogenous or multiple genes may be responsible for the development of specific types of teeth agenesis in humans. Data from animal models are still very complex, and the human embryology is still poorly understood. Oral clefts and syndromic forms of tooth agenesis may be the best models for isolated tooth agenesis. In the future, a precise description of the missing teeth in syndromes involving tooth agenesis may be useful.