A Novel FOXL2 Missense Mutation, c.1068G>C, In Chinese Families with Blepharophimosis-Ptosis-Epicanthus Inversus Syndrome Type II

Background Blepharophimosis-ptosis-epicanthus inversus syndrome (BPES) is a hereditary disease caused by a mutation in the forkhead box L2 ( FOXL2 ) gene. Female patients suffering from premature ovarian failure (POF) were classified as type I, and others were classified as type II. We aimed to clarify a novel FOXL2 indel mutation in Chinese families and to predict the POF risk in the affected patient. Methods Three generations of one Chinese family with BPES were enrolled in this study. Blood samples from patients of this family were collected and then analysed by whole-exome sequencing. Confocal microscopy was performed to observe the subcellular location. Transactivation studies were performed with real-time PCR. Results This novel mutation (c.1068G>C) is located in the downstream of DNA-binding forkhead(FHD) domain, and the mutant protein could also exhibit transactivation capacity of StAR , a key regulator of POF. Conclusively, we discovered a novel FOXL2 mutation and predicted that female patient in this family should be classified as type II BPES. Conclusions Our study revealed a novel missense mutation (c.1068G>C) and expanded the spectrum of FOXL2 gene mutations. Although we were not able to determine the classification from clinical manifestation, we discovered the patient developed type II BPES through subcellular distribution and transactivation analysis.


Background
Blepharophimosis-ptosis-epicanthus inversus syndrome (BPES; OMIM #110100) is clinically manifested in the form of eyelid malformation caused by autosomal dominant inheritance, and its incidence is approximately 1/50000 [1]. The characterization of this disease includes narrow horizontal palpebral fissures, congenital ptosis, telecanthus and epicanthus inversus [2]. BPES has been divided into two categories according to whether 3 the absence of infertility in female patients is caused by premature ovarian failure (POF): type II lacks POF compared with type I [3]. Currently, a mutation in the forkhead box L2 (FOXL2) gene (OMIM #605597) is thought to be the main reason for both types of BPES [4].
FOXL2 is a 2.7-kb gene with a single exon located at chromosome 3q23. FOXL2 encodes 376 amino acids, including a 100-amino acid DNA-binding forkhead domain (FHD) and a poly-Ala tract. FOXL2 is an important transcription factor expressed in ovary granulosa cells and the mesenchyme of the developing eyelid. Thus, mutant FOXL2 should be responsible for aberrant ovaries, embryogenesis and eyelids [5]. Some reports have indicated that steroid metabolism and reactive oxygen species (ROS) detoxification are regulated by the FOXL2 gene [6,7]. To date, studies have revealed that some diseases, such as polycystic ovary syndrome (PCOS), adult-type ovarian granulosa cell tumours (GCTs) and lacrimal gland underdevelopment, are caused by aberrant FOXL2 [8,9].
Until now, intragenic mutations, such as frameshift mutations (most frequent), in-frame mutations, nonsense mutations and missense mutations, have been responsible for 71% of BPES cases [10]. A previous report claimed that type I was caused by a truncation before poly-alanine, whereas type II was often caused by poly-Ala expansion mutations [11].
However, the definite relationship between BPES and such mutations needs to be further investigated. A recent report provided a better method to identify whether a mutation will cause POF through a transactivation reporter system, such as FLRE-luc and SIRT1-luc [12].
Steroidogenic acute regulatory protein (StAR) is an essential factor involved in the follicular differentiation of granulosa cells and the maturation of ovarian antral follicles [13]. Normally, the function of StAR is repressed by FOXL2 binding to its promoter, and any mutation that impacts this connection will give rise to POF [14,15].
Due to the patient inherited BPES syndrome from his father. We were not able to determine the classification from clinical manifestation. Here, we clarified a novel FOXL2 4 mutation, c.1068G>C, from a Chinese family with BPES. In addition, this mutation was in the downstream of FHD domain and didn't disturb subcellular location and transactivation ability, which indicated the affected patient developed type II BPES with limited POF risk.

DNA extraction and sequencing
We collected blood samples from patients of this family and applied an Automatic Nucleic Acid Isolation System to obtain genomic DNA from the leukocytes of peripheral venous blood. We applied whole-exome sequencing to analyse genomic DNA, and the consequences were compared with the reference from the GenBank database. To analyse this novel mutation, we adopted three kinds of overlapping primers to amplify the FOXL2 coding region as previous described [16]. The PCR amplification conditions were as follows: 5 95°C for 3 min, 40 cycles at 94°C for 60 s, 65°C for 30 s, and 72°C for 30 s; and a final elongation step at 72°C for 5 min. The mutation analysed by Finch TV software and the final consequences were compared to the reference sequence from the GenBank database.

Plasmid construction
Complementary DNA (cDNA) encoding wild-type FOXL2 and mutant FOXL2 were cloned with PCR and then inserted into the pcDNA3.1 and EGFP-N1 plasmids for constructing pcDNA3.1-FOXL2-wild-type and N1-FOXL2-EGFP-wild-type plasmids, respectively. All plasmids were isolated and purified with anion exchange columns (QIAGEN, Hilden, Germany). Plasmids were sequenced to confirm that there were no additional mutations.

Subcellular location of FOXL2-WT and FOXL2-MT
293T cells were seeded into 6 cm dishes and cultured to 50-60% confluence. Then, EGFP- Following the manufacturer's instructions, TRIzol (Invitrogen) was used to extract total mRNA from cells. Reverse transcription (RT)-PCR was performed with 2 µg of RNA using Super script II (Invitrogen). The housekeeping gene GAPDH was used as an endogenous control.

BPES patients with a novel mutation
We diagnosed 2 patients in a three-generation, 13-member Chinese family with BPES, as shown in Figure 1A. Patients were carefully examined by an ophthalmologist based on the following criteria: blepharophimosis, ptosis, epicanthus inversus and telecanthus (Table 1, Figure 1B).. Whole-genome sequencing analysis of genomic DNA extracted from patients' peripheral blood leukocytes revealed a novel missense mutation in guanine deoxynucleotides replacing cytosine deoxynucleotides at the 650 th base of the CDS of FOXL2 ( Figure 1C).. This novel missense mutation caused an aberrant amino acid sequence in which serine (Ser) at the 217 th amino acid position was replaced by cysteine (Cys), as shown in Figure 1D. The mutation was located downstream of the forkhead domain, upstream of the poly-Ala domain (Figure 2)..

Mutated protein is located in the nucleus.
To observe the subcellular localization of the FOXL2 mutant protein, we constructed wildtype and mutant FOXL2 plasmids with an EGFP tag and transfected them into 293T cells.

Mutated FOXL2 also represses downstream StAR
A previous study revealed thatFOXL2 strongly repressed the expression of StAR expression, a key regulator in the pathogenesis of POF, by binding to its promoter [11,17].
To further confirm whether this novel mutation abolished its transactivation capacity on StAR, wild-type FOXL2 and mutant FOXL2 were transfected into Chinese hamster ovarian (CHO) cells ( Figure 4A).. Notably, StAR expression was silenced after transfecting wildtype or mutant FOXL2 ( Figure 4B).. This result confirmed that this novel mutant FOXL2 could also regulate StAR expression as a transcription factor.

Discussion
FOXL2 is an evolutionarily conserved forkhead transcription factor that affects reproductive system development, sex determination, metabolism and tumourigenesis and has been shown to be associated with multiple diseases, such as polycystic ovary syndrome (PCOS) [18], pathologic scar formation [19] and reproductive system tumours [20]. FOXL2 is highly expressed in the ovary and eyelids, and its mutation causes BPES [21]. In addition, FOXL2 could direct bind to the promoter of its target gene and repress its expression, such as StAR and SIRT1 [22]. Because StAR and SIRT1 play a crucial role in the functional maturation of ovarian antral follicles, mutated FOXL2 could not only result in the malformation of the eyelid but also infernity [23].
It has been reported that BPES type I is closely associated with the truncation of the FOXL2 protein before the poly-Ala tract, whereas BPES type II is caused by poly-Ala expansions [24,25]. However, the situation is not exactly as it is described, and sometimes variation may occur. Some patients with type II BPES harbour a truncation in the FOXL2 protein before the poly-Ala tract, and type I BPES is caused by poly-Ala expansions [26]. It has also been reported that the same mutation in one family causes different types of BPES [26]. This phenotypic variability may result from the differences in the genetic background of each individual.
In our study, through whole-exome sequencing, we found a novel FOXL2 missense mutation (c.1068G>C) located in the downstream of the DNA-binding forkhead domain. It 8 should be noted that the two types of BPES were classified according to whether infertility was absent in female patients [27]. Here, we could not distinguish the clinical type of BPES in this patient from clinical information, because of the paternal inheritance. Here, we observed the subcellular distribution and tested the transactivation capacity of FOXL2 and predicted the limited risk of POF in the affected girl.

Availability of data and materials
The datasets used and analysed during the current study are available from the corresponding author on reasonable request.

Competing interests
The author declare that they have no competing interests.

Authors' contributions
In this report, SYW and FLH designed and performed the experiments and drafted the manuscript; RBJ were responsible for sample collection and data analysis; XS, RBJ and SFG discussed, revised and approved the manuscript. All authors approved this manuscript.