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.

Background

Blepharophimosis, ptosis, and epicanthus inversus syndrome (BPES, OMIM 110100) is a rare and autosomal dominant disease, with an estimated prevalence of 1 per 50,000 births in the general population [1]. Eyelid malformation of BPES is characterized by four major cardinal features, including a narrow horizontal aperture of the eyelids, congenital ptosis, epicanthus inversus, and telecanthus. Two main phenotypic subtypes of BPES harbor the same craniofacial disorders: BPES type I, which is also associated with premature ovarian failure (POF), whereas BPES type II is associated with eyelid malformations only without systemic features [2, 3]. Other common ocular symptoms include squint, refractive error, amblyopia, and stenosis of the lacrimal canaliculi [4].

Currently, the role of the forkhead box L2 (FOXL2, OMIM 605597) gene in the pathogenesis of BPES is well established, approximately 80% of BPES cases are attributed to heterozygous variants of the FOXL2 gene, as reported previously [5, 6]. Foxl2 knockout mice also exhibit both craniofacial malformations and female infertility, which recapitulates the BPES phenotype [7, 8]. FOXL2 is a single-exon gene of 2.7 kb encoding a 376 amino acid protein that contains a 110 amino acid DNA-binding forkhead domain (FHD) and a polyalanine (poly-Ala) tract of 14 residues [8]. FOXL2 is localized in the nucleus as a transcriptional regulator and is selectively expressed in developing eyelid mesenchymal cells, granulosa cells of the ovary, and gonadotropin cells of the anterior pituitary gland [8, 9]. Moreover, FOXL2 affects granulosa cell proliferation, differentiation, and steroidogenesis by regulating the expression of multiple downstream genes, such as steroidogenic acute regulatory (StAR) [8, 9] and odd-skipped related 2 transcription factor (OSR2) [10], which may be involved in the synthesis and function of hormones that are associated with the development of POF.

Prompt diagnosis is crucial for female patients with BPES type I, but it remains a major challenge for clinicians. It is difficult to make a prompt diagnosis because these patients are usually children when first seen at the hospital, meaning that the symptoms that would enable a diagnosis of POF and variation of ovarian reserve function are not yet apparent [11]. Thus, researching genotype‒phenotype correlations is highly valuable for predicting the classification of BPES. More than 200 mutations in the FOXL2 gene have been reported in patients with BPES [12]. These variants include missense changes, frameshift and nonsense mutations, in-frame deletions, and duplications [13]. Several researchers have proposed that there is a genotype‒phenotype correlation between FOXL2 mutations and the phenotype of BPES. Mutations resulting in a predicted truncated protein or complete loss of function of the FOXL2 gene usually lead to BPES with POF, whereas mutations leading to an extended protein result in BPES type II [14,15,16]. However, clinical heterogeneity and genetic differences still exist among BPES patients. For example, Yang et al. [17] reported that a duplicate mutation (c.844_860dup17, p.His291Argfs*71) in FOXL2 was present in a Chinese family with both types of BPES. Therefore, additional evidence is needed to confirm the correlation between FOXL2 mutations and the phenotype of BPES. In this study, we identified a FOXL2 heterozygous mutation in a Chinese family with BPES type II, and conducted functional validation. By systematically reviewing prior studies that have elucidated the clinical classification of BPES and functionally characterized disease-associated mutations, we comprehensively summarize the genotype-phenotype correlations of FOXL2 mutations in BPES and propose evidence-based guidelines for genetic evaluation.

Methods

Patients

A Chinese family with BPES comprised a total of seventeen individuals, of which 6 affected individuals and 5 unaffected individuals were recruited from the Eye Hospital Affiliated with Wenzhou Medical University. All patients presented with typical BPES manifestations, including small palpebral fissures, ptosis, epicanthus inversus, and telecanthus (Fig. 1a). Written informed consents were obtained from all participants or their legal guardians (for participants under 16 years of age). This study adhered to the tenets of the Declaration of Helsinki and was approved by the Ethics Committee of the Eye Hospital of Wenzhou Medical University (2022-046-K-31-01).

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

Peripheral blood DNA extraction and screening for gene mutations via WES and Sanger sequencing

Peripheral blood (5 ml) samples were collected from all participants in EDTA-K2 anticoagulation tubes. Genomic DNA was extracted from leukocytes using a Qiagen DNA Blood Midi/Mini Kit (Qiagen GmbH, Hilden, Germany). Whole-exome sequencing (WES) was performed in 4 affected individuals (II-4, II-6, II-7, III-4) and 3 unaffected individuals (I-1, III-5, III-7) by Berry Genomics Co. Ltd. Beijing, China. After the raw sequencing reads were obtained, the data were aligned to the human reference genome (hg38/GRCh38) using the Burrows–Wheeler Aligner tool. Variant annotation and interpretation were conducted by ANNOVAR [18] and the Enliven® Variants Annotation Interpretation System provided by Berry Genomics.

After genomic DNA was extracted from peripheral blood, polymerase chain reaction (PCR) was performed in 50 μl reactions following the manufacturer’s protocol for the 2x Taq PCR Master Mix kit (APE x BIO, Catalog No. K1034, USA), and primers were designed to amplify the region of FOXL2 through PCR: FOXL2-F: GCACAGTCAAGGAGCCAGA; FOXL2-R: GCCCTTCTCGAACATGTCTT. The PCR products were separated on a 2% agarose gel and purified using a QIAquick Gel Extraction Kit (Qiagen, Valencia, CA). To validate the identified suspected pathogenic variant, Sanger sequencing was performed at the Sangon Biotech DNA sequencing core facility, and multiple sequence alignments were analyzed with SnapGene software (Dotmatics, Boston, Massachusetts, United States). Segregation analysis was performed on the proband and other family members.

Prediction of the pathogenicity of shared mutations and protein models

The shared mutations in all affected members were identified. All variants were classified according to the American College of Medical Genetics and Genomics (ACMG) standards and guidelines [19] for sequence variant interpretation. Furthermore, the variants were identified and evaluated using Protein Variation Effect Analyzer (PROVEAN), Polymorphism Phenotyping v2 (PolyPhen-2), and Sorting Intolerant from Tolerant (SIFT). Combined Annotation Dependent Depletion (CADD) was also used to score the deleteriousness of single nucleotide variants. The FOXL2 gene coding sequence (NM_023067) was entered into Missense 3D (Department of Life Sciences, Imperial College London, UK) to construct the protein model, and the stability was assessed by DUET. Moreover, the residues around 105 of the FOXL2 protein sequence were evaluated for conservation across multiple species via UniProt software.

Plasmid construction and transfection

The coding sequence of FOXL2 (NM_023067) was cloned and inserted into the pEGFP-N1 and pcDNA3.1-3xflag-N vectors, which were designated pEGFP-WT and pcDNA-WT, respectively. Mutant plasmids carrying c.313 A > C were generated using targeted mutagenesis PCR, with the wild-type (WT) FOXL2 expression vector used as a template, and the two mutant-types (MT) were designated pEGFP-MT and pcDNA-MT1, respectively. Human embryonic kidney (HEK) 293T cells were obtained from our laboratory stock. The cells were cultured in complete medium consisting of Dulbecco’s modified Eagle’s medium (DMEM; Gibco™, United States) supplemented with 10% fetal bovine serum (Gibco™) and 1% penicillin‒streptomycin solution (Gibco™) at 37 °C and 5% CO₂. Lipofectamine 3000 (Invitrogen, Carlsbad, CA, United States) was used for transfection. The pEGFP-N1 and pcDNA3.1-3xflag-N plasmids were gently mixed in 250 μl of Opti-MEM™ I Reduced Serum Medium (Gibco™), and 10 μl Lipo-3000 was gently mixed into 250 μl of Opti-MEM™ I Reduced Serum Medium. The diluted DNA mixture was combined with the Lipo-3000 mixture and incubated for 10–15 min at room temperature. After 6 h, the medium was replaced with complete medium.

Subcellular localization assays

To assess whether the mutations affected the localization of proteins, HEK293T cells were transfected with plasmids containing the empty vectors pEGFP-N1, pEGFP-FOXL2, pEGFP-FOXL2-MT, and Lipo-3000. At 24 h after transfection, the nuclei were visualized via Hoechst 33,342 staining (Beyotime Institute of Biotechnology, Jiangsu, China) for 5 min at 37 °C and 5% CO₂. The cells were subsequently observed under a Zeiss LSM880 (Carl Zeiss AG, Oberkochen, Germany) and DMi8 fluorescence microscope (Leica, Delaware, USA).

Quantitative real-time PCR

To assess whether the mutations affected the expression of the downstream target genes OSR2 and StAR, SYBR Green real-time quantitative (q) PCR was performed using Biosystems MicroAmp EnduraPlate plastic consumables (Thermo Fisher Scientific, Massachusetts, USA). The pcDNA3.1-3xflag expression vector (10 μg) containing WT or mutant FOXL2 cDNA or the empty pcDNA3.1-3xflag vector was transfected into HEK293T cells in 6-well plates using Lipo-3000. At 48 h after transfection, total mRNA was extracted from the cells using FastPure Cell/Tissue Total RNA Isolation Kit V2 (Vazyme, Nanjing, China), after which the mRNA (1 μg) was reverse transcribed into cDNA using HiScript III RT SuperMix (Vazyme) in 20 μl reactions. The cDNA (1 μg) was subjected to real-time PCR using Taq Pro Universal SYBR qPCR Master Mix (Vazyme). GAPDH was used as an endogenous control to standardize the data, and the primers used are shown in Table 1.

Table 1 List of primers used for SYBR green real-time quantitative PCR

Statistical analysis

All the statistical values conformed to a normal distribution. Thus, statistical analysis was performed via one-way ANOVA using GraphPad Prism software version 8.0 (GraphPad, San Diego, California, USA), with a P value < 0.05 considered statistically significant.

Results

Clinical manifestations

The clinical characteristics of the family members are summarized in Table 2. Typical clinical manifestations of BPES, including small palpebral fissures, ptosis of the eyelids, epicanthus inversus, and telecanthus, had been observed in most of the affected patients from birth (Fig. 1a). The majority of patients had symptoms involving both eyes, and only one patient (patient III-4) had symptoms involving a single eye (the affected eye presented with ptosis only). All patients except patient I-2, who was amblyopic, had best corrected visual acuity within the normal range. None of the patients had strabismus but had high astigmatism. The clinical form was classified as BPES type II because the affected females in this family did not have fertility problems. The autosomal dominant mode of inheritance of BPES was confirmed by the fact that the disease is inherited vertically from generation to generation, irrespective of sex, which is even more strongly illustrated by the fact that all patients were heterozygous mutation carriers.

Table 2 Clinical characteristics of family members

Gene mutation analysis

A heterozygous missense mutation, c.313 A > C, in the FOXL2 gene was identified by WES between the proband (II-7) and other affected patients (II-4, II-6, III-4) but was not detected in the unaffected individuals (I-1, III-5, III-7), which cosegregated with the disease phenotype in this family. These results were further confirmed by Sanger sequencing (Fig. 1b). The A to C change at nucleotide position 313 resulted in an asparagine at codon 105, which has a neutral side chain, substituted by a histidine (N105H), which has a positively charged side chain, making it hydrophilic (Fig. 2a). The mutation c.313 A > C (p.N105H) was previously reported to be a nonsynonymous SNV within the coding region of the FHD, and multiple sequence alignment of the FOXL2 protein revealed that the position was highly conserved among different species, including humans, mice, pigs, bovines, rabbits, and goats (Fig. 2b, UniProtKB). Therefore, it is likely essential to the function of the protein. According to ACMG guidelines, the FOXL2 variant c.313 A > C (p.N105H) was classified as Likely Pathogenic (LP), supported by the following evidence criteria: PS1, PM1, PM2, and PP3.The N105H amino acid changes were predicted by PROVEAN software with a score of − 4.94. This mutation was predicted to be probably damaging with a score of 1.0 by the POLYPHEN2 program and a score of 32.0 by the CADD software. These values indicate that the mutation c.313 A > C (p.N105H) is likely to have a deleterious effect on the FOXL2 protein. The DUET-predicted change in protein stability upon mutation revealed that the protein became destabilizing (ΔΔG =-0.973 kcal/mol), and the FOXL2 protein structure is shown in Fig. 2c.

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.

c.313 A > C (p.N105H) mutation in the FOXL2 protein results in nuclear aggregation

To further investigate the effect of the mutation c.313 A > C (p.N105H) on the subcellular location of the FOXL2 protein, three constructs, pEGFP-WT, pEGFP-MT, and the empty vector, were transfected into HEK293T cells. As expected, the wild-type FOXL2 protein was located in the nucleus in a diffuse manner, which is consistent with its function as a transcription factor. However, the cells transfected with the empty vector presented significant cytoplasmic localization. We found that, similar to the cells transfected with the wild-type construct, the HEK293T cells transfected with the mutant construct localized exclusively in the nucleus. In contrast to wild-type FOXL2, which exclusively localized to the nucleus in a diffuse manner, the mutant protein displayed nuclear aggregation in some transfected cells (Fig. 3a and b).

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

Effects of the pathogenic variant on transcriptional activity

We further evaluated the effect of the FOXL2 c.313 A > C (p.N105H) variant on the transactivation capacity of its downstream regulators by measuring the expression of endogenous OSR2 and StAR mRNAs via real-time PCR. The FOXL2 gene was expressed at low levels in HEK293T cells. However, when wild-type or mutant FOXL2 genes were transfected into HEK293T cells, the expression level of endogenous FOXL2 mRNA was significantly greater than that in cells transfected with the empty vector. Interestingly, there were no significant differences in the endogenous expression of either OSR2 mRNA or StAR mRNA in HEK293T cells transcribed with wild-type FOXL2, mutant FOXL2, or empty vector. However, OSR2 expression in HEK293T cells after transfection with wild-type FOXL2 was slightly greater than that after transfection with mutant FOXL2 (P = 0.1371) or the empty vector (P = 0.0647) (Fig. 3c).

Discussion

In this study, we identified and characterized a heterozygous mutation, c.313 A > C (p.N105H), within the FOXL2 gene in a Chinese family with BPES type II, which was consistent with an autosomal dominant pattern of inheritance. The mutation c.313 A > C (p.N105H) is a nonsynonymous SNV within the coding region of the FHD, and the A to C change at nucleotide position 313 results in a substitution of an asparagine (N) at codon 105 to a histidine (H). This locus is highly conserved among species and may provide key sites for N-linked glycosylation when it occurs in an Asn-X-Ser/Thr form in the motif [20]. Since glycosylation is crucial for both protein structure and function, the mutation c.313 A > C (p.N105H) may affect the structure and function of the FOXL2 protein, which was also predicted to be deleterious by biometric analysis software. The mutation c.313 A > C (p.N105H) of FOXL2 has been previously reported [21, 22]. The function of this specific mutation has not been thoroughly investigated, and the correlation between the mutation and the clinical manifestations of BPES has yet to be examined. The present study is the first to provide functional validation of the c.313 A > C (p.N105H) variant of FOXL2. A previous study reported that mutations in the FHD of FOXL2 are more likely to lead to cytoplasmic mislocalization and aggregation of proteins [23]. Therefore, we further explored the subcellular localization of the mutation, and the results indicated that the mutation displayed a pattern similar to that of the WT FOXL2 protein located in the nucleus. However, the mutant protein displayed nuclear aggregation in some transfected HEK293T cells, in contrast to wild-type FOXL2, which exclusively localized to the nucleus in a diffuse manner, which may affect interactions with downstream genes.

FOXL2 is a transcriptional regulator that represses StAR transcription but upregulates OSR2 transcription. The StAR gene encodes a protein involved in the rate-limiting step of steroid hormone synthesis, which plays an essential role in folliculogenesis [24], and FOXL2 can preserve primordial follicles from premature follicular depletion by repressing the promoter of StAR. Thus, BPES type I could be induced if some FOXL2 mutations fail to bind to StAR, affecting its expression [24, 25]. In addition, FOXL2 has been proven to activate the OSR2 promoter directly. OSR2 is critical during craniofacial development [10], and OSR2 knockout mice exhibit eyelid malformation [26]. The regulatory interaction between FOXL2 and OSR2 could contribute to eyelid malformations in BPES patients. Thus, these two downstream genes were selected for functional validation of the FOXL2 variant in our study. The qPCR results revealed that, compared with the wild-type variant, the c.313 A > C (p.N105H) variant of FOXL2 did not affect the expression of the StAR gene. This gene slightly downregulated the expression of OSR2, although the difference was not significant, which may explain its generally mild phenotypic expression (i.e., BPES without POF) in this family.

Patients with BPES have diverse clinical phenotypes. In the present study, the majority of BPES patients were affected bilaterally, and they had four characteristic features of BPES in the eyelid malformation at birth, including narrow horizontal aperture of the eyelids, congenital ptosis, epicanthus inversus, and telecanthus. Interestingly, patient III-4 harbored the same variant of the FOXL2 gene as the other binocularly affected patients in the same family, but was only affected unilaterally. Almost no unilateral BPES patients have been previously reported. This patient presented with ptosis without other classic clinical signs of BPES. This suggests clinical heterogeneity of BPES in different patients, and we hypothesize that in addition to genotype, differential expression in tissues, epigenetic modifications, or the environment may also influence the clinical phenotype.

In addition to the four main eyelid symptoms, other ophthalmologic manifestations that may be associated with BPES or craniofacial features have been found in some BPES patients [1, 27]. Previous studies have shown that the incidence of strabismus, refractive error, and amblyopia is greater in patients with BPES than in the normal population [28]. The prevalence of refractive error and amblyopia is higher in patients with BPES at 34–94% and 40–60%, respectively, and the incidence of strabismus is approximately 20–40% [28, 29]. In the present study, BCVA was within the normal range in children as well as young and middle-aged patients with BPES, but one aged patient had amblyopia. The possible causes of amblyopia are deprivation amblyopia due to chronic ptosis or refractive amblyopia due to high astigmatism, and strabismic amblyopia was excluded because of the absence of strabismus in all patients. It is hypothesized that amblyopia that occurs in the late stages of BPES may be avoided or attenuated with regular monitoring and early intervention [29, 30].

Patients with BPES can be categorized into two subtypes depending on whether it co-occurs with POF (type I) or not (type II). Due to the absence of POF in the female patients, the patients in this family were categorized as having BPES type II. Identification of the typology of BPES is critical for female patients, which would be primarily a prediction of fertility, and recommended therapeutic considerations should be provided for patients with POF. However, there are significant challenges in the prompt diagnosis of BPES subtypes, which have not been assessed in many studies, mainly because of the prepubertal age and lack of endocrinological data in affected females or uninformative family history [22]. Thus, it is essential to clarify the relationship between the genotype and phenotype of BPES.

As reported previously, haploinsufficiency of FOXL2 remains the most common cause of BPES, and it is also the first autosomal gene associated with syndromic POF [9]. Approximately 80% of patients with BPES have heterozygous variants of the FOXL2 gene [5, 6], and more than 250 mutations have been reported. Intragenic variants within the FOXL2 gene, including frameshift, in-frame, nonsense, and missense variations, accounted for 81% of the cases. Whole-gene deletions and relatively large submicroscopic deletions in FOXL2 or neighboring genes accounted for 12% and 5% of the cases, respectively. In addition, some variants are located either upstream or downstream of the coding region [5]. Previous studies have generalized the correlation between genotype and clinical phenotype and concluded that FOXL2 variants resulting in a truncated protein, either lacking or containing the FHD or before the poly-A tract, lead to BPES type I, whereas variants leading to an expanded poly-Ala region are mostly associated with BPES type II [15]. However, FOXL2 gene variants would not have been expected on the basis of previous genotype‒phenotype correlations reported in previous studies. Yang et al. [17] reported that a duplicate mutation (c.844_860dup17, p.His291Argfs*71) in FOXL2 was identified in a Chinese family with both types of BPES. Other studies have reported that the same mutation leads to type I and type II BPES in different families [13, 31,32,33]. Therefore, we propose that classifying patients into type I or type II from the perspective of genotype alone is not comprehensive.

The additional functional validation of the mutation may provide more significant evidence for the typing of BPES. Therefore, investigating the clinical manifestations of patients, and the location and structural changes of the mutations combined with functional validation could provide additional evidence for the typing of BPES. We reviewed and summarized previous studies that have clarified the clinical typing of BPES and functionally validated the mutations (Table 3). According to criteria from De et al. [15], we also categorized the variants into A-I groups. It is widely believed that truncations before the poly-Ala tract, which induce loss of the C-terminal region that acts as a transcriptional inhibitory domain, are preferentially associated with BPES type I, whereas expansion of the poly-Ala tract leads to BPES type II [15, 34]. We also found that mutations predicted to result in truncated proteins without FHD (Group A) or partial deletion of FHD (Group B) are associated with BPES type I, and mutations that truncate proteins with complete FHD and without poly-Ala tract are also mostly associated with BPES type I (90.9%), except for the indel mutation c.675_690delinsT (p.Ala226_Ala230del) in the poly-Ala tract, indicating that either contraction or expansion of the poly-Ala tract may result in BPES type II. Interestingly, we observed that both subtypes of BPES could be induced by mutations that result in truncated or elongated proteins with complete forkhead and poly-Ala domains (Groups D, E). Moreover, the altered transactivation of StAR may assist in the identification of subtypes of BPES, and the mutation of FOXL2, which belongs to Group D or E, is more likely to be associated with BPES type I if the repression of the downstream transcription factor StAR is decreased compared with that of the wild type. Consistent with previous findings [15], mutations belonging to Group F were mostly associated with BPES type II. Surprisingly, almost all of these in-frame mutations were located downstream of the poly-Ala tract, which seems to be a region with high variant prevalence. Mutations belonging to Group G occur in families with BPES type I or type II, and further functional studies revealed that mutations that do not affect the transcript levels of their downstream regulator STAR are mostly associated with BPES type II, a conclusion that is also supported by the present study. However, previous studies have documented cases where specific Group G variants exhibiting altered STAR expression levels nevertheless manifested clinically as BPES type II [35, 36]. Furthermore, heterogeneity of clinical manifestations is reaffirmed by the fact that patients with the same mutation in the same or different pedigrees suffer from different subtypes of BPES, which also increases the difficulty of elucidating the correlation between genotypes and phenotypes. Therefore, more studies are needed to expand the spectrum of FOXL2 variants in BPES and to functionally validate mutations to provide broader insights into genotype–phenotype correlations.

Table 3 Summarization of the literatures on FOXL2 variants in BPES type I or type II patients

Limitations

While this study provides initial insights into the genotype-phenotype correlations in BPES, several limitations should be acknowledged. First, our conclusions are drawn from a single BPES type II pedigree, which may limit the generalizability of the findings. Second, the lack of access to affected tissues (e.g., eyelid and ovarian samples) precluded direct assessment of FOXL2 expression patterns, protein localization, and downstream transcriptional effects in disease-relevant cell types. Future studies should incorporate multi-center recruitment of diverse BPES pedigrees (types I and II) combined with functional validation using patient-derived tissues or appropriate cellular models. Addressing these gaps will strengthen the mechanistic understanding of FOXL2 variants and enhance the clinical translation of our findings.

Conclusions

We identified a FOXL2 mutation in a Chinese family and provided the first functional validation of this mutation. Our experimental findings suggest that the FOXL2 gene mutation is pathogenic and associated with BPES type II in this family. Furthermore, our results contribute to elucidating the genotype-phenotype correlation. Additionally, by reviewing previous literature, we analyzed the correlation between genotype and phenotype from multiple aspects, which could facilitate the early diagnosis and treatment of female BPES patients with POF.