Disruption of FOXF2 as a Likely Cause of Absent Uvula in an Egyptian Family

Abstract

This study investigated the genetic basis of an unusual autosomal dominant phenotype characterized by familial absent uvula, with a short posterior border of the soft palate, abnormal tonsillar pillars, and velopharyngeal insufficiency. Cytogenetic analysis and single-nucleotide polymorphism-based linkage analysis were investigated in a 4-generation family with 8 affected individuals. Whole exome sequencing data were overlaid, and segregation analysis identified a single missense variant, p.Q433P in the FOXF2 transcription factor, that fully segregated with the phenotype. This was found to be in linkage disequilibrium with a small 6p25.3 tandem duplication affecting FOXC1 and GMDS. Notably, the copy number imbalances of this region are commonly associated with pathologies that are not present in this family. Bioinformatic predictions with luciferase reporter studies of the FOXF2 missense variant indicated a negative impact, affecting both protein stability and transcriptional activation. Foxf 2 is expressed in the posterior mouse palate, and knockout animals develop an overt cleft palate. Since mice naturally lack the structural equivalent of the uvula, we demonstrated FOXF2 expression in the developing human uvula. Decipher also records 2 individuals with hypoplastic or bifid uvulae with copy number variants affecting FOXF2. Nevertheless, given cosegregation with the 6p25.3 duplications, we cannot rule out a combined effect of these gains and the missense variant on FOXF2 function, which may account for the rare palate phenotype observed.

Keywords: cleft palate; craniofacial biology/genetics; gene expression; oral pathology; speech pathology; transcription factor(s).

Figure 1.

Egyptian family with absent uvula. (A) Absent uvula palate in the proband (IV.5). Note the short posterior border of the soft palate where the uvula should be and the unusual structure of the pillar of fauces. ATP, anterior tonsillar pillar; SP, soft palate; T, tonsil. (B) Family pedigree showing autosomal dominant inheritance of hypernasality and absent uvula. Eleven family members were genotyped for linkage analysis (+). Exome sequencing analysis was performed on 3 individuals (‡), and Sanger sequencing (*) was used to assess segregation of candidate gene variants in additional individuals (↑).

Figure 2.

The functional domains of FOXF2. (A) The locations of nonsynonymous missense variants p.A25G, p.A41S, and p.Q433P are given. Note that the 2 N-terminal variants p.A25G and p.A41S were classified bioinformatically as benign. (B) The evolutionary conservation of each variant is indicated in the boxed area.

Figure 3.

Functional analysis of the FOXF2 p.Q433P variant. (A) Transcriptional activation of FOXF2 WT and Q433P constructs in HeLa cells. Data represent the mean ± SD of triplicate samples at 24 h posttransfection. (B) Quantitative expression difference between the WT and p.Q433P mutation constructs measured by RTqPCR in HeLa cells. (C) Western blot shows FOXF2 protein expression in HeLa cells transfected with FOXF2 WT, Q433P mutation, and pCMV6-XL “empty” vector constructs. (D) Transcriptional activity normalized to protein level. (E) FGF18 and (F) SHOX2 transcript levels assessed by RTqPCR and normalized to ACTB in HeLa cells at 24 h following transfection of WT, Q433P constructs, and empty vector. All data presented are representative of at least 3 replicate experiments. *P < 0.05. **P < 0.01. RTqPCR, quantitative reverse transcription polymerase chain reaction; WT, wild type.

Figure 4.

Expression of FOXF2 in the oral cavity of human embryos. Coronal sections anterior to posterior (left to right) of human embryo heads at CS22, CS23, and L8pcw (top, middle, and bottom panels, respectively), showing maxilla, tongue, and palatal shelves. The second column is approximately midpalate; the third column is toward the back of the palatal shelves; and the fourth column is from among the last sections in each embryo, which show the rudimentary palatal shelves (arrowheads) that will become the uvula upon fusion. Expression of FOXF2 is seen in the tongue and in the palatal shelves. In the developing palate, expression is mostly on the oral half of anterior regions but becomes more widely expressed throughout the posterior palatal shelf mesenchyme and bordering oral tissues. At later stages, expression includes the more posterior oral epithelial surfaces. ES, epithelial seam; NS, nasal septum; PS, palatal shelf; T, tongue.