Functional analysis of a novel FOXL2 mutation in blepharophimosis, ptosis, and epicanthus inversus syndrome type II and elucidation of the genotype-phenotype correlation

Abstract

Background: Blepharophimosis, ptosis, and epicanthus inversus syndrome (BPES) is a rare autosomal dominant disorder caused by genetic mutations. However, the genotype-phenotype correlation remains unclear. This study aimed to identify mutations in a Chinese family with BPES and elucidate the genotype-phenotype relationship.

Methods: A comprehensive clinical and molecular genetic analysis was conducted on a three-generation Chinese family with BPES, which was prospectively enrolled at the Eye Hospital of Wenzhou Medical University. Affected individuals underwent systematic phenotyping, including detailed physical and ophthalmic evaluations. Genomic DNA was isolated from peripheral blood samples and subjected to whole-exome sequencing, followed by targeted Sanger sequencing for variant validation. Candidate disease-associated variants were analyzed using in silico predictive algorithms to assess their potential structural and functional impact on encoded proteins. To further elucidate the pathogenicity of the identified mutation, functional studies were performed, including immunofluorescence-based subcellular localization assays and quantitative real-time PCR to evaluate transcriptional regulatory effects.

Results: Six affected individuals of this pedigree presented with canonical BPES features including small palpebral fissures, ptosis, epicanthus inversus, and telecanthus, without premature ovarian failure, consistent with a diagnosis of BPES type II. Whole-exome sequencing revealed a heterozygous missense mutation (c.313 A > C:p.N105H) in FOXL2, which was subsequently validated by Sanger sequencing. This variant demonstrated complete cosegregation with the BPES phenotype across all affected family members. According to ACMG guidelines, the variant was classified as Likely Pathogenic (PS1 + PM1 + PM2 + PP3). In silico pathogenicity prediction tools classified the p.N105H variant as deleterious. Immunofluorescence assays revealed aberrant nuclear aggregation of the mutant FOXL2 protein, and functional characterization via quantitative real-time PCR demonstrated no significant dysregulation (P > 0.05) of downstream targets (STAR, OSR2).

Conclusions: This study provides functional evidence of the pathogenic FOXL2 mutation (c.313 A > C, p.N105H) in BPES type II, demonstrating its disruptive effects on protein localization while maintaining normal transcriptional activity of downstream targets. These findings expand the mutational spectrum of FOXL2 related disorders and enhance our understanding of genotype-phenotype correlations in BPES.

Keywords: Blepharophimosis-ptosis-epicanthus inversus syndrome; FOXL2; Genotype‒phenotype correlation; Pathogenic variants; Whole-exome sequencing.

Fig. 1

Pedigrees, facial photographs and Sanger sequencing of the family members. (a) Affected individuals are indicated by filled darkened symbols, and the proband is indicated by upward arrows. Squares and circles indicate males and females, respectively. (b) Sanger sequencing revealed a heterozygous missense mutation in the FOXL2 gene (c.313 A > C: p. N105H) in affected members of this family. The top base sequence serves as the reference base, mutation loci are highlighted with red bounding boxes, and the black arrows point to the mutation sites

Fig. 2

Analysis of FOXL2 protein structural alterations at mutation sites. (a) Missense 3D prediction of molecular modeling of the wild-type and mutant variations in FOXL2 c.313 A > C, pN105H. The blue box indicates the location of the amino acid mutation, the structure of the wild-type FOXL2 is shown in red, and the mutant type is shown in blue. (b) The sequence alignments were compared among humans, mice, pigs, cows, rabbits, and goats via UniProt. The results indicated that the residues surrounding N105 of the FOXL2 gene are significantly conserved in all of these species. (c) The FOXL2 protein has a forkhead domain and a poly-Ala tract. The arrow points to the location of the mutation identified in this study.

Fig. 3

Subcellular localization and transcriptional activity of the FOXL2 variant. (a) Subcellular localization of the wild-type and mutant FOXL2 proteins. The first panel shows nuclear staining with Hoechst, the middle panel shows the subcellular localization of FOXL2 as a fusion protein with EGFP, and the third panel is a merged image. Yellow arrows indicate the representative cells shown in high-magnification views in boxed regions. Scale bar: 20 μM. (b) The first panel shows the cellular morphology captured via brightfield microscopy, the middle panel shows the subcellular localization of FOXL2 as a fusion protein with EGFP, and the third panel shows a merged image. Both wild-type and mutant FOXL2 (c.313 A > C, pN105H) localized exclusively in the nucleus. In contrast to wild-type FOXL2, which is exclusively localized in the nucleus in a diffuse manner, the mutant protein displayed nuclear aggregation in some transfected cells. Scale bar: 50 μM. (c) Real-time PCR revealed that the relative mRNA expression of FOXL2 was significantly elevated after transfection with FOXL2-WT/MT plasmids, and the mRNA expression of OSR2 or StAR was not different between the FOXL2-WT/MT and vector-transfected groups. However, OSR2 expression in HEK293T cells after transfection with FOXL2-WT was slightly greater than that in mutant FOXL2 cells. FOXL2, forkhead box L2; WT, wild type; MT, mutant type. **, P < 0.01; ****, P < 0.0001