Pathogenic missense mutation pattern of forkhead box genes in neurodevelopmental disorders.

Background: Forkhead box (FOX) proteins are a family of transcription factors. Mutations of three FOX genes, including FOXP1, FOXP2, and FOXG1, have been reported in neurodevelopmental disorders (NDDs). However, due to the lack of site-specific statistical significance, the pathogenicity of missense mutations of these genes is difficult to determine.

Methods: DNA and RNA were extracted from peripheral blood lymphocytes. The mutation was detected by single-molecule molecular inversion probe-based targeted sequencing, and the variant was validated by Sanger sequencing. Real-time quantitative PCR and western blot were performed to assay the expression of the mRNA and protein. To assess the pattern of disorder-related missense mutations of NDD-related FOX genes, we manually curated de novo and inherited missense or inframeshift variants within FOXP1, FOXP2, and FOXG1 that co-segregated with phenotypes in NDDs. All variants were annotated by ANNOVAR.

Results: We detected a novel de novo missense mutation (NM_001244815: c.G1444A, p.E482K) of FOXP1 in a patient with intellectual disability and severe speech delay. Real-time PCR and western blot revealed a dramatic reduction of mRNA and protein expression in patient-derived lymphocytes, indicating a loss-of-function mechanism. We observed that the majority of the de novo or transmitted missense variants were located in the FOX domains, and 95% were classified as pathogenic mutations. However, 10 variants were located outside of the FOX domain and were classified as likely pathogenic or variants of uncertain significance.

Conclusion: Our study shows the pathogenicity of missense and inframeshift variants of NDD-related FOX genes, which is important for clinical diagnosis and genetic counseling. Functional analysis is needed to determine the pathogenicity of the variants with uncertain clinical significance.