Fig. 2: In silico analyses of heterozygous FOXP4 variants.

(a) Linear representation of the FOXP4 protein (Q8IVH2–1) with the identified variants and functional domains annotated: FOX forkhead box domain, LZ leucine zipper, ZF zinc finger. (b) Conservation of FOXP4 across different species, with the amino acids affected by missense variants indicated. Species include Homo sapiens (UniProt sequence Q8IVH2), Pan troglodytes (A0A2J8NZN5), Mus musculus (Q9DBY0), Gallus gallus (A0A3Q2U1E5), Xenopus laevis (Q4VYR7), and Danio rerio (B3DJK9). Regions shown span amino acids 269–277, 425–433, and 501–525 of FOXP4 isoform 1 (Q8IVH2). (c) Visualization of missense variants in the FOX domain in a three-dimensional structure. A homology model for the FOX domain of FOXP4 (amino acids 456–542) was built based on template structure 2kiu.1.A (FOXP1 monomer), using the SWISS-MODEL Homology Modeling online tool.²⁰ (d) Alignment of missense variants in a subset of the FOX domain with pathogenic missense variants in other FOX proteins. An alignment was made of the Pfam Forkhead domain (PF00250) using Clustal Omega Multiple Sequence Alignment⁴⁴ of all FOX proteins with missense variants present in HGMD database 2019.3. Only missense variants labeled as pathogenic were included for this analysis. (e) Tolerance landscape of FOXP4 protein visualized via the MetaDome web server.²³ A tolerance landscape is computed based on single-nucleotide variants in the gnomAD database, and shows per amino acid position the missense over synonymous ratio in a sliding window of 21 residues. Green and blue peaks represent regions tolerant to missense variation; red valleys show intolerant regions. The missense variants in the FOX domain are located in extremely intolerant regions of FOXP4, while the two remaining missense variants are located in extremely tolerant regions.