Fig. 1

Functional characterization of rare FOXP2 variants. a Schematic representation of the FOXP2 protein showing rare variants found in individuals with neurodevelopmental disorders. Stop-gain and frameshift variants are shown in black and missense variants in red. Known domains are labelled: glutamine-rich (Q-rich) region (hatched shading) including polyglutamine tracts (solid shading), zinc finger (ZF), leucine zipper (LZ) and forkhead domain (FOX). Nuclear localization signals are indicated with red bars. b Fluorescence-based measurement of FOXP2 expression level. HEK293 cells were transfected with YFP-FOXP2, together with mCherry for normalization. Fluorescence intensity was measured 48 h post-transfection. Values are mean YFP/mCherry fluorescence ratios ± S.D. (n = 3), expressed relative to the value for wild-type (WT) FOXP2. c Fluorescence micrographs of HEK293 cells transfected with YFP-FOXP2. Nuclei were stained with Hoechst 33342. d, e Luciferase reporter assays for transcriptional regulatory activity of FOXP2. HEK293 cells were transfected with a firefly luciferase reporter vector containing the SV40 promoter (d) or the human SRPX2 promoter (e), together with a Renilla luciferase normalization plasmid and YFP-FOXP2, or YFP alone (control). Values are mean relative luciferase activity ± S.D. (n = 3), expressed relative to the control. Asterisks indicate significant differences compared to wild-type (WT) FOXP2 (p < 0.05, one-way ANOVA followed by Bonferroni post hoc correction). NS not significant. Exact p values for both the SV40 and the SRPX2 assays are <0.0001 for the control and the R553H, R328*, and Q390Vfs*7 variants, and >0.9999 for all other variants