Free Access
Horm Res Paediatr 2012;77:2-11

FOXL2 Impairment in Human Disease

Verdin H. · De Baere E.
Center for Medical Genetics, Ghent University Hospital, Ghent, Belgium
email Corresponding Author


 goto top of outline Key Words

  • FOXL2 mutation
  • BPES
  • POF
  • Adult GCT
  • Anti-testis action

 goto top of outline Abstract

FOXL2 encodes a forkhead transcription factor that plays important roles in the ovary during development and in post-natal, adult life. Here, we focus on the clinical consequences of FOXL2 impairment in human disease. In line with other forkhead transcription factors, its constitutional genetic defects and a somatic mutation lead to developmental disease and cancer, respectively. More than 100 unique constitutional mutations and regulatory defects have been found in blepharophimosis syndrome (BPES), a complex eyelid malformation associated (type I) or not (type II) with premature ovarian failure (POF). In agreement with the BPES phenotype, FOXL2 is expressed in the developing eyelids and in fetal and adult ovaries. Two knock-out mice and at least one natural animal model, the Polled Intersex Syndrome goat, are known. They recapitulate the BPES phenotype and have provided many insights into the ovarian pathology. Only a few constitutional mutations have been described in nonsyndromic POF. Moreover, a recurrent somatic mutation p.C134W was found to be specific for adult ovarian granulo-sa cell tumors. Functional studies investigating the consequences of FOXL2 mutations or regulatory defects have shed light on the molecular pathogenesis of the aforementioned conditions, and contributed considerably to genotype-phenotype correlations. Recently, a conditional knock-out of Foxl2 in the mouse induced somatic transdifferentiation of ovary into testis in adult mice, suggesting that Foxl2 has an anti-testis function in the adult ovary. This changed our view on the ovary and testis as terminally differentiated organs in adult mammals. Finally, this might have potential implications for the understanding and treatment of frequent conditions such as POF and polycystic ovary syndrome.

Copyright © 2012 S. Karger AG, Basel

goto top of outline Introduction

FOXL2 is a single-exon gene of 2.7 kb encoding a highly conserved protein of 376 amino acids containing a 110-amino-acid DNA-binding forkhead domain, classifying FOXL2 in the family of forkhead transcription factors involved in a wide variety of biological processes during development and postnatal life. Besides the forkhead domain, FOXL2 also contains a polyalanine tract of 14 residues that is conserved in mammals; however, the exact function is unknown (fig. 1). As several forkhead transcription factors have a strict spatiotemporal expression pattern in early development, a mutation dysregulating this pattern could lead to developmental disease. Currently, 11 human forkhead genes have been shown to be mutated in human hereditary developmental disorders, four of which have an ocular phenotype [1]. One of them is FOXL2, leading to blepharophimosis-ptosis-epicanthus inversus syndrome with or without ovarian dysfunction when mutated (BPES, OMIM 110100) [2]. The expression pattern of FOXL2 is compatible with the BPES phenotype, as expression studies in human, mouse and goat demonstrated the presence of the nuclear protein in the mesenchyme of developing eyelids and in fetal and adult supporting granulosa cells but not in the oocytes. As FOXL2 expression in the ovary is observed before folliculogenesis, it is the earliest known marker of ovarian differentiation in mammals. Moreover, FOXL2 is strongly expressed in adult follicular cells, suggesting not only a role in ovarian somatic cell differentiation but also in adult female fertile life in follicular development and maintenance. FOXL2 expression has also been demonstrated in the developing pituitary, and in gonadotrope as well as thyrotrope cell types of the adult pituitary, suggesting an involvement in pituitary organogenesis [3,4,5,6].

Fig. 1. Schematic outline of the FOXL2 gene and protein. FOXL2 encodes a protein of 376 amino acids. The characteristic forkhead domain and the polyalanine tract are indicated by an arrowhead. Adapted from Beysen et al. [19].


goto top of outline FOXL2 Impairment in BPES

goto top of outline Clinical Features of BPES

Complex Eyelid Malformation
BPES is a rare developmental disorder of the eyelids and ovary essentially presenting with an autosomal dominant inheritance. Typically, four major characteristics of the eyelids are present at birth (fig. 2): (1) a reduction of the horizontal dimension of the palpebral fissures (blepharophimosis), (2) drooping of the upper eyelid leading to a narrowing of the vertical palpebral fissure (ptosis), (3) a small skin fold rising from the lower eyelid and running inwards and upwards (epicanthus inversus), and (4) a lateral displacement of the canthi with normal interpupillary distance (telecanthus). Besides this clinical tetrad, other ophthalmic features associate with BPES including nasolacrimal drainage problems caused by lateral displacement, duplication, or stenosis of the lacrimal puncta. Especially the lateral displacement of the inferior punctum is a relatively unknown and important anatomical hallmark for the clinical diagnosis of BPES [7]. Minor features observed in BPES are a broad nasal bridge, low-set ears and a short philtrum [8]. The eyelid malformation can be corrected with oculoplastic surgery for both aesthetic and functional reasons. Traditionally, a medial canthoplasty to correct the epicanthic folds is performed at the age of 3-5 years depending on the severity of the ptosis, followed by ptosis correction. Decock et al. [7] described two surgical procedures to correct ptosis, i.e. super-maximal resection and frontalis suspension. Indeed, super-maximal resection of the levator muscle is the preferred method as it leads to a good cosmetic outcome as well as to an improved muscle function.

Fig. 2. Complex eyelid malformation in BPES. The four major characteristics of the eyelids are: (1) blepharophimosis or a reduction of the horizontal dimension of the palpebral fissures, (2) ptosis or drooping of the upper eyelid, (3) epicanthus inversus or a skin fold rising from the lower eyelid and running inwards and upwards, and (4) telecanthus or a lateral dislocation of the canthi with normal inter-pupillary distance.

Ovarian Phenotype
Two types BPES can be clinically distinguished: both types have the clinical tetrad in common while in type I, premature ovarian failure (POF) manifests with the ocular phenotype [9]. POF is defined as amenorrhea for at least 4 months before the age of 40 years, combined with a decreased serum concentration of estradiol and an increased serum concentration of follicle-stimulating hormone (FSH >40 IU/l) [10]. Only little is known about the molecular mechanisms of FOXL2 dysregulation in the ovary of a BPES patient. In a first report by Fraser et al. [11], small hypoplastic uteri and streak ovaries were observed in two sisters with BPES type I (age difference of 8 years) using ultrasonography. On ovarian biopsy, primordial follicles progressing into scars were described. No genetic study was performed however. A second unique study by Meduri et al. [12] included an extensive clinical, genetic, hormonal and ovarian histological investigation in two BPES type I patients. Histological and immunohistological studies were performed on ovarian biopsies from these 2 patients, in which FOXL2 elongating frameshift mutations were found. The ovarian histological phenotype of the first patient was similar to that observed in the knock-out mice, while that of the second patient was apparently normal (fig. 3). In both patients, FOXL2 protein expression was observed in the granulosa cells. In patient 1, a predominant intracytoplasmatic expression was detected, while on the contrary a predominant nuclear expression was seen in patient 2. Taken together, different ovarian phenotypes, follicular defects and distribution of FOXL2 protein were observed in these two patients with molecularly proven defects [12].

Fig. 3. Histology of ovarian biopsy of BPES type 1 patient. a Numerous, crowded, small follicles are observed in the ovarian cortex. HE. b Double oocytes in two small follicles, a third follicle (lower left) is normal. HE. c Empty spaces in a small follicle suggesting deposits of cholesterol crystals which have dissolved during fixation leaving cholesterol clefts. Adapted from Meduri et al. [12].

In female type I BPES patients, both emotional and physical management of POF is imperative. A first issue in the management of POF is the necessity of hormone replacement therapy to reduce postmenopausal symptoms and prevent long-term health effects of estrogen deficiency, such as an increased risk of osteoporosis [13]. In general, POF reduces the chance of conceiving naturally to 5-10%. Of these pregnancies another 20% result in pregnancy loss [14]. Currently, three fertility-preserving strategies are available: embryo cryopreservation, oocyte cryopreservation and ovarian tissue cryopreservation. The only established method is embryo cryopreservation. This option, however, is restricted to patients who have a partner or are willing to use donor sperm. This also requires a cycle of ovarian stimulation. For those patients without a partner or not willing to use donor sperm, oocyte cryopreservation can be performed. This strategy also requires a cycle of ovarian stimulation. The success rate of oocyte cryopreservation is however still very low with pregnancy rates ranging from 1 to 5% [15,16]. The aforementioned strategies are not suitable in prepubertal patients however, because of the necessity of ovarian stimulation. Ovarian tissue cryopreservation is in theory possible before puberty. The aim of this technique is to freeze ovarian cortex tissue, and then to reimplant the tissue orthotopically (into the pelvic cavity) or heterotopically (e.g. the forearm or abdominal wall). Orthotopic reimplantation allows the possibility of pregnancy without further reproductive medical assistance. On the contrary, heterotopic reimplantation needs to be followed by in vitro fertilization (IVF). However, because the revascularization after the reimplantation needs time to develop, ischemia damages the follicles resulting in massive follicle loss. Therefore, further research is needed to enhance the freezing and revascularization processes. To date, only 15 live births have been reported, none of which were derived from cryopreserved prepubertal tissue [15,16,17]. Of note, most BPES type I patients are diagnosed in infancy. Two other options that need to be considered are adoption and oocyte donation. The latter option gives the patient the chance of experiencing a pregnancy herself. Traditionally, fresh oocyte donations were performed, but these are hampered by inefficiency, difficulties of synchronization between recipient and donor, very long waiting periods and lack of quarantine measures. The advances in oocyte cryopreservation techniques with fertilization, implantation and pregnancy rates comparable to fresh oocyte donation, diminishes the disadvantages of fresh oocyte donation and may become the new standard in the future [18]. The fact that young patients are most often not ready to decide on their reproductive future and that the age of onset of POF cannot be predicted, makes counseling on fertility preservation very challenging.In general, BPES type I patients require a thorough follow-up of ovarian function by both an endocrinologist and gynecologist.

goto top of outline Molecular Genetics of BPES

In 2001, FOXL2 was identified as the causal gene for BPES [2]. Using a combined mutation detection approach consisting of (1) sequencing of the FOXL2 open reading frame, (2) copy number screening of the FOXL2 gene, and (3) copy number screening of the regulatory domain of FOXL2, the underlying molecular defect can be identified in 88% of typical BPES patients. Of all genetic defects identified, intragenic mutations represent the largest group (71%). Deletions encompassing FOXL2 and located outside its transcription unit represent 12 and 5% of molecular defects, respectively [19].

Intragenic Mutations
Intragenic mutations occur along the total coding region of FOXL2 and all types of mutations have been identified, mostly in BPES. More than 100 unique FOXL2 mutations have been described ( [19]. The largest group (44%) contains frameshift mutations. Following are the in-frame changes (33%), the nonsense mutations (12%) and finally the missense mutations (11%). Notably, 93% of the in-frame mutations lead to polyalanine expansions, representing the most important mutational hotspot in FOXL2 [20].

Several genotype-phenotype correlations emerged after the identification of the first mutations in FOXL2. Before any functional studies were done, it was proposed that mutations predicted to result in proteins with truncation before the poly-Ala tract might by associated with BPES type I, whereas polyalanine expansions might rather lead to BPES type II. For missense mutations and mutations leading to a truncated or extended protein containing an intact forkhead domain and polyalanine tract, no correlation could be made [20,21].

From the first mutation studies, it was hypothesized that these mutations were loss-of-function alleles leading to haploinsufficiency of FOXL2 [2,21]. This was supported by the observation that FOXL2 deletions and intragenic mutations lead to the same phenotype [19]. However, the functional consequences were not clear for missense mutations.

Molecular Consequences of Intragenic Mutations
Most insights into the molecular effects of FOXL2 mutations contributing to genotype-phenotype correlations resulted from in vitro studies. The most frequent polyalanine expansion p.Ala224_Ala234dup was found to lead to intranuclear aggregation and cytoplasmic mislocalization of the protein, and to interfere with the availability of a co-expressed normal FOXL2 [22]. This was corroborated by a potential promoter-specific dominant-negative effect of this polyalanine expansion [23]. However, the fact that this mutation might keep partial transactivation capacity on high-affinity promoters might explain why its phenotypic expression is often mild (i.e. BPES without POF) [23].

Most missense mutations are located in the forkhead domain. A first study by Beysen et al. [24] suggested that missense mutations in the forkhead domain leading to mislocalization and aggregation, and thus severely impairing transactivation, would lead to a more severe ovarian phenotype than missense mutations not significantly affecting protein localization and function [24]. In addition, two mutations downstream of the forkhead domain (p.S217F and p.S217C) were found to lead to a mild BPES phenotype [24]. Dipietromaria et al. [25] developed a prediction tool for FOXL2 intragenic (missense and other) mutations, the validation of which was based on known phenotypic effects of a ‘training set' of mutations (BPES type I or type II). A clear correlation was found between the transcriptional activity of FOXL2 mutations on two different reporter promoters and the BPES type [25]. In a very recent study by Todeschini et al. [26], the amino acids of the helices of the forkhead domain of FOXL2 were systematically replaced by glycine residues to assess the impact of these artificial mutations. A number of mutations led to protein mislocalization, aggregation and to partial or complete loss of transactivation ability on a dozen of luciferase reporter systems. No clear-cut correlation was found between protein mislocalization or aggregation and the position of the mutation. However, the localization of the side chain of each amino acid was found to correlate very well with the impact of its mutation on FOXL2 transactivation capacity. Extrapolation of this analysis to natural mutations was in agreement with the findings obtained for the artificial mutations. This study brought important insights into the molecular effects of FOXL2 missense mutations located in the forkhead domain, and provided an apparently reliable in silico predictive tool for their phenotypic effects [26].


goto top of outline FOXL2 Encompassing Deletions

Deletions of the FOXL2 gene have been identified in 10% of the BPES patients. These patients are phenotypically indistinguishable from those carrying intragenic mutations, emphasizing that correct gene dosage of FOXL2 is critical for normal development. The observed rearrangements range from partial and total FOXL2 deletions to microdeletions encompassing neighboring genes. Not only are the sizes of the deletions diverse, also the breakpoint locations are heterogeneous, indicating a lack of rearrangements hotspots. Genotype-phenotype correlations might be especially helpful in providing a prognosis in newborns with BPES, mainly regarding associated features such as psychomotor retardation and POF. FOXL2 deletions were found to lead to BPES type I. Genetic counseling and endocrinologic follow-up is therefore of utmost importance in BPES females carrying a FOXL2 deletion [27,28]. Other potential correlations such as an ATR deletion and microcephaly, and a SOX14 deletion and limb anomalies, are still elusive [28].

goto top of outline Regulatory Defects

Before the identification of FOXL2 as the disease gene for BPES, three balanced translocations were found in BPES patients. Because the deletion breakpoints were located upstream of FOXL2, a position effect was assumed [27,29]. Recently, this hypothesis has been strengthened by the identification of several deletions outside the transcription unit of FOXL2 in typical BPES patients [27,30]. A very subtle deletion of only 7.4 kb defines the shortest region of overlap (SRO) encompassing 8 conserved noncoding sequences (CNCs) and a long noncoding RNA (lnc-RNA) named PISRT1 that is likely co-expressed with FOXL2 in human granulosa cells. This is in line with a presumed regulatory function of PISRT1, requiring a tissue and cell-type specific expression. The potential regulatory function of the CNCs was validated using in vitro luciferase assays in a FOXL2 expressing and nonexpressing cell line. Cell-type specific regulatory potential could be observed for the 3 CNCs located in the SRO. This supports that at least a fraction of the tested CNCs might be involved in the tissue-specific expression of FOXL2. Finally, chromosome conformation capture (3C) of a 625-kb region flanking FOXL2 was conducted in different cellular systems. In the FOXL2-expressing KGN cell line, the FOXL2 core promoter proved to come in close vicinity to 3 chromosomal fragments located upstream of FOXL2, of which 1 contains the 7.4-kb de-letion. Furthermore, all three distant sequences were found to mutually interact and contact the FOXL2 core promoter [30].

Apart from regulatory deletions, potentially interesting sequence variations have been found in the 3′ UTR of the FOXL2 transcription unit [31,32]. Their functional significance has not been studied so far however.


goto top of outline Guidelines for Molecular Genetic Testing of BPES

We proposed a decision tree for molecular genetic testing of BPES [28] (summarized in fig. 4). A first step in the diagnostic work-up of patients with a tentative diagnosis of BPES is a clinical classification of the patients into two groups: (1) typical BPES, and (2) BPES-like. Patients can be classified within the first group if: (1) the presence of four diagnostic criteria of BPES can be assessed (on a facial picture), including blepharophimosis, ptosis, epicanthus inversus and telecanthus, or (2) at least three of the four diagnostic criteria of BPES are mentioned on a standardized clinical questionnaire. Second, a different molecular diagnostic approach is required depending on the clinical classification. In case of typical BPES, the first step is direct sequencing of the FOXL2 open reading frame (ORF) as intragenic mutations can be detected in 72% of this group [19]. If no mutations can be detected, FOXL2 deletion screening should be performed as a second step (e.g. using multiplex ligation-dependent probe amplification or MLPA), as gene deletions are present in 10% of this group [19]. If negative, copy number screening targeting the FOXL2 region (e.g. using quantitative polymerase chain reaction [qPCR] or microarray-based comparative genome hybridisation [array CGH]) is recommended as a third step to exclude the presence of regulatory copy number changes affecting the long-range genetic control of FOXL2 which have been reported in 4% of the cases [27,30]. In case of a BPES-like phenotype, genome-wide microarray-based copy number screening is recommended, as copy number changes can be detected in a relatively large proportion of these patients (33%) [33].

Fig. 4. Decision tree for molecular diagnostics of BPES. Adapted from D'haene et al. [28].


goto top of outline FOXL2 Impairment in Ovarian Dysfunction

POF is defined as cessation of menses before the age of 40 for a period of at least 4 months. It is characterized by amenorrhea, hypoestrogenism and elevated serum gonadotrophin concentrations. This condition affects 1% of all women [10]. Multiple causes can underlie POF, including iatrogenic factors, autoimmune disease, metabolic and infectious factors, genetic defects, such as X chromosome aberrations and mutations in autosomal genes. However, the cause remains unknown in the majority of POF patients. Because mutations in FOXL2 have been associated with a syndromic form of POF, namely BPES type I, it has been considered to be a candidate gene for non-syndromic POF. Mutation studies were conducted in several cohorts with non-syndromic POF [21,34,35,36,37,38,39,40]. However, only in 3 patients a variation with a presumed pathogenic effect was found. First, a heterozygous 30-bp deletion was identified in a Slovenian POF patient resulting in a polyalanine contraction of 10 alanines. Interestingly, polyalanine expansions are known to cause BPES with or without POF. The patient presented with primary amenorrhea and hypergonadotrophic hypogonadism, but was still able to conceive spontaneously and give birth to two healthy children [35]. Second, a heterozygous single nucleotide substitution (c.772T>A) leading to a nonconservative amino acid change, p.Y258N, was identified in a sporadic New Zealand POF patient. Both mutations were absent in 100 control samples [35]. Third, variant p.G187D was found in a woman with POF in the absence of BPES. While FOXL2 localization was normal, the transactivation capacity of the mutant protein on two reporter promoters potentially relevant in an ovarian context proved to be lower than that of normal FOXL2 [38]. These studies indicate that mutations in the FOXL2 coding region are not a frequent cause of isolated POF.


goto top of outline FOXL2 Impairment in Granulosa Cell Tumors

Granulosa cell tumors (GCTs) represent less than 5% of all ovarian cancers. GCTs can be classified into adult and juvenile types based on different clinical and histopathologic features. Juvenile GCTs account for only 5% of all GCTs and typically occur in prepubertal girls and women younger than 30 years. These patients usually present at an early stage and have a good prognosis. On the other hand, adult GCTs occur most frequently in premenopausal women (fig. 5). Although GCTs may also present at an early stage of disease, relapses tend to occur 10-30 years after diagnosis with an estimated relapse rate of 18.6% and a mean survival rate of 5 years. Because of the tendency of a late relapse, a lifelong follow-up is advised [41]. Because no effective treatment is available to date, patients with late relapses or advanced-stage tumors at diagnosis have a poor prognosis [42]. Until recently, only little was known about the molecular basis of GCTs. Through whole-transcriptome paired-end RNA sequencing of only 4 GCTs, a somatic FOXL2 missense mutation c.402C>G (p.C134W) was found in all of them [43]. The presence of this unique recurrent somatic mutation in more than 95% of adult GCTs has been confirmed by several follow-up studies [44,45,46,47,48,49,50,51,52]. Localization of the mutated protein was found to be normal [43,44]. Differences have been found between wild-type and mutant FOXL2 however, such as increased induction of the target aromatase by the mutation [53] and a lower capacity of the mutation to induce apoptosis, thereby compromising cell death [47,48,53]. A very recent study by Benayoun et al. [54] suggested that this mutation might interfere with the capacity of FOXL2 to modulate the cell cycle.

Fig. 5. Histopathological features of a GCT. The histopathological picture of a GCT shows uniform nuclei with variable grooves. HE: Adapted from Shah et al. [43].

In conclusion, the identification of this recurrent adult GCT-specific FOXL2 mutation is a step forward for the diagnosis of adult GCTs, and might help to develop more targeted therapies and to elucidate the molecular pathogenesis of adult GCTs.


goto top of outline Role of FOXL2 in the Ovary: Insights from Animal Models

Two Foxl2 knock-out mice have been reported [55,56]. The perinatal lethality observed in the Foxl2 knock-out mice is high [55]. The craniofacial phenotype of the surviving homozygous animals recapitulates the BPES phenotype, with a severe eyelid malformation, and open eyes at birth [55].

Female homozygous mice are sterile with small and disorganized ovaries and lack of primary follicles [55,56]. There are some differences between the two models however: in the first model by Schmidt et al. [55], a correct formation of primordial follicles was seen, with normal oocytes but granulosa cells that did not undergo the normal squamous to cuboidal transition; in the second model by Uda et al. [56], disorganized ovaries were seen instead of normal primordial follicles, suggesting an earlier defect. In both models, oocytes seem to be intact during the first stages of folliculogenesis and perinatally. Shortly thereafter, the follicular reserve is depleted by a massive follicular atresia leading to sterility [55,56].

Interestingly, several findings in animal models pointed to a testis-suppressing role of Foxl2 in the developing and adult ovary. First, this was suggested by observations in the Polled Intersex Syndrome goat. In the only natural animal model for BPES, a regulatory region upstream of goat FOXL2 is deleted, resulting in absence of horns and sex reversal in XX animals [57]. Second, similar findings in Foxl2-/- female mice further sustained an ‘anti-testis' role of Foxl2 in ovarian development [58,59]. Indeed, Foxl2-/- granulosa cells in mice acquire (male) Sertoli-like characteristics [59]. Third, a recent study dealing with a conditional knock-out of Foxl2 in mice provided evidence for an ‘anti-testis' role of Foxl2 in the adult ovary. Indeed, loss of Foxl2 in the adult ovary was shown to result in somatic transdifferentiation of the granulosa and thecal cells into testis-specific Sertoli- and Leydig-like cells, respectively. Moreover, this led to an immediate upregulation of testis-specific genes such as Sox9, assuming repression of Sox9 by Foxl2 [60]. The latter study profoundly changed our view on the ovary and testis as terminally differentiated organs in adult mammals. Finally, these studies might help to elucidate the molecular basis of SRY-negative XX sex reversal cases, although an initial study did not reveal any FOXL2 mu-tation [60]. Finally, these findings might have potential implications for the understanding of more frequent conditions such as POF and polycystic ovary syndrome (PCOS).


goto top of outline FOXL2 Impairment in Other Diseases

goto top of outline PCOS

In the previous study, the conditional deletion of Foxl2 in mice did not only transdifferentiate granulosa cells to testis-specific Sertoli-like cells, but also a change of thecal cells into Leydig-like cells, associated with an increase in androgen production. These findings are reminiscent of PCOS, the most common ovarian dysfunction in women in their reproductive life, as suggested by Murphy [61]. Although a link between FOXL2 dysregulation and PCOS has not yet been demonstrated clearly in humans, constitutional FOXL2 mutations found in syndromic POF (BPES type I) associated withandrogen-induced hirsutism [61], and in a BPES patient with reported PCOS [62] might represent first hints. However, it should be noted that PCOS is very common in the general population (6-10%) and can go up to 52% in the Indian subcontinent [63,64]. So it will be challenging to substantiate the connection between FOXL2 disruption and PCOS in future research.

goto top of outline Keloid

Keloid is a dermal fibroproliferative growth caused by pathologic wound healing following skin injury, and can be defined as a hypertrophic scar growing beyond the borders of the original wound [65]. A recent genomewide association study investigating 824 individuals with keloid (cases) and 3,205 unaffected controls in the Japanese population identified significant associations of keloid with four SNP loci in three chromosomal regions, one of which is 3q22.3-23 [66]. This region, with strong association with SNP rs1511412, included two genes: LOC389151, a hypothetical gene located 24 kb telomeric to rs1511412, and FOXL2, located 47 kb centromeric to rs1511412. Although speculative, a link between genetic variations influencing FOXL2 expression and keloid susceptibility might be found in potential effects on the levels of gonadotropin-releasing hormone (GnRH) and/or steroid hormones. On the one hand, FOXL2 is known to regulate the expression of GnRH and cholesterol metabolism and steroidogenesis, and on the other hand, it has been assumed that gonadal and steroid hormones might influence keloid formation [66].


goto top of outline Concluding Remarks

Mutations in FOXL2 illustrate the concept of pleiotropy and clinical heterogeneity. More than 100 unique constitutional FOXL2 mutations and multiple copy number changes of the FOXL2 region, and one unique recurrent somatic mutation have been described in human disease, varying from a rare developmental condition with manifestations in the ovary and eyelid to a specific tumor in adulthood.Functional studies investigating the con-sequences of FOXL2 mutations or regulatory defects considerably contributed to genotype-phenotype correlations, opening perspectives for fertility preservation. More importantly, an intact FOXL2 function is not only crucial during development but is also needed throughout the lifetime of a female to prevent ovarian dysfunction, somatic transdifferentiation and tumor formation. In this way, researchers might be challenged by further dissection of the molecular pathogenesis of SRY-negative XX sex reversal, and more frequent conditions such as POF and PCOS. In order to provide potential targets for therapy, further insights into the regulation of expression of FOXL2 and into potential ‘druggability' of its downstream targets are needed. This might ultimately lead to treatment perspectives for (one of) the conditions resulting from FOXL2 impairment.


goto top of outline Acknowledgements

H.V. is doctoral fellow from the Research Foundation Flanders (FWO). E.D.B. is a senior clinical investigator from the FWO. This study is supported by FWO grant G079711N.

 goto top of outline References
  1. Lehmann OJ, Sowden JC, Carlsson P, Jordan T, Bhattacharya SS: Fox's in development and disease. Trends Genet 2003;19:339-344.
  2. Crisponi L, Deiana M, Loi A, et al: The putative forkhead transcription factor FOXL2 is mutated in blepharophimosis/ptosis/epicanthus inversus syndrome. Nat Genet 2001;27:159-166.
  3. Cocquet J, Pailhoux E, Jaubert F, Servel N, Xia X, Pannetier M, De Baere E, Messiaen L, Cotinot C, Fellous M, Veitia RA: Evolution and expression of FOXL2. J Med Genet 2002;39:916-921.
  4. Cocquet J, De Baere E, Gareil M, Pannetier M, Xia X, Fellous M, Veitia RA: Structure, evolution and expression of the FOXL2 transcription unit. Cytogenet Genome Res 2003;101:206-211.
  5. Treier M, Gleiberman AS, O'Connell SM, Szeto DP, McMahon JA, McMahon AP, Rosenfeld MG: Multistep signaling requirements for pituitary organogenesis in vivo. Genes Dev 1998;12:1691-1704.
  6. Ellsworth BS, Egashira N, Haller JL, Butts DL, Cocquet J, Clay CM, Osamura RY, Camper SA: FOXL2 in the pituitary: molecular, genetic, and developmental analysis. Mol Endocrinol 2006;20:2796-2805.
  7. Decock CE, Claerhout I, Leroy BP, Kesteleyn P, Shah AD, De Baere E: Correction of the lower eyelid malpositioning in the blepharophimosis-ptosis-epicanthus inversus syndrome. Ophthal Plast Reconstr Surg 2011;27:368-370.

    External Resources

  8. Oley C, Baraitser M: Blepharophimosis, ptosis, epicanthus inversus syndrome (BPES syndrome). J Med Genet 1988;25:47-51.
  9. Zlotogora J, Sagi M, Cohen T: The blepharophimosis, ptosis, and epicanthus inversus syndrome: delineation of two types. Am J Hum Genet 1983;35:1020-1027.
  10. De Vos M, Devroey P, Fauser BC: Primary ovarian insufficiency. Lancet 2010;376:911-921.

    External Resources

  11. Fraser IS, Shearman RP, Smith A, Russell P: An association among blepharophimosis, resistant ovary syndrome, and true premature menopause. Fertil Steril 1988;50:747-751.
  12. Meduri G, Bachelot A, Duflos C, Bstandig B, Poirot C, Genestie C, Veitia R, De Baere E, Touraine P: FOXL2 mutations lead to different ovarian phenotypes in BPES patients: case report. Hum Reprod 2010;25:235-243.
  13. Goswami D, Conway GS: Premature ovarian failure. Hum Reprod Update 2005;11:391-410.
  14. Kalantaridou SN, Davis SR, Nelson LM: Premature ovarian failure. Endocrinol Metab Clin North Am 1998;27:989-1006.
  15. Donnez J, Dolmans MM: Preservation of fertility in females with haematological malignancy. Br J Haematol 2011;154:175-184.

    External Resources

  16. Donnez J, Martinez-Madrid B, Jadoul P, Van Langendonckt A, Demylle D, Dolmans MM: Ovarian tissue cryopreservation and transplantation: a review. Hum Reprod Update 2006;12:519-535.
  17. Tao T, Del Valle A: Human oocyte and ovarian tissue cryopreservation and its application. J Assist Reprod Genet 2008;25:287-296.

    External Resources

  18. Cobo A, Remohi J, Chang CC, Nagy ZP: Oocyte cryopreservation for donor egg banking. Reprod Biomed Online 2011;23:341-346.
  19. Beysen D, De Paepe A, Baere ED: FOXL2 mutations and genomic rearrangements in BPES. Hum Mutat 2009;30:158-169.
  20. De Baere E, Beysen D, Oley C, et al: FOXL2 and BPES: mutational hotspots, phenotypic variability, and revision of the genotype-phenotype correlation. Am J Hum Genet 2003;72:478-487.
  21. De Baere E, Dixon MJ, Small KW, et al: Spectrum of FOXL2 gene mutations in blepharophimosis-ptosis-epicanthus inversus (BPES) families demonstrate a genotype-phenotype correlation. Hum Mol Genet 2001;10:1591-1600.
  22. Caburet S, Demarez A, Moumne L, Fellous M, De Baere E, Veitia RA: A recurrent polyalanine expansion in the transcription factor FOXL2 induces extensive nuclear and cytoplasmic protein aggregation. J Med Genet 2004;41:932-936.
  23. Moumne L, Dipietromaria A, Batista F, Kocer A, Fellous M, Pailhoux E, Veitia RA: Differential aggregation and functional impairment induced by polyalanine expansions in FOXL2, a transcription factor involved in cranio-facial and ovarian development. Hum Mol Genet 2008;17:1010-1019.
  24. Beysen D, Moumne L, Veitia R, Peters H, Leroy BP, De Paepe A, De Baere E: Missense mutations in the forkhead domain of FOXL2 lead to subcellular mislocalization, protein aggregation and impaired transactivation. Hum Mol Genet 2008;17:2030-2038.
  25. Dipietromaria A, Benayoun BA, Todeschini AL, Rivals I, Bazin C, Veitia RA: Towards a functional classification of pathogenic FOXL2 mutations using transactivation reporter systems. Hum Mol Genet 2009;18:3324-3333.
  26. Todeschini AL, Dipietromaria A, L'Hote D, Boucham FZ, Georges AB, Pandaranayaka PJ, Krishnaswamy S, Rivals I, Bazin C, Veitia RA: Mutational probing of the forkhead domain of the transcription factor FOXL2 provides insights into the pathogenicity of naturally occurring mutations. Hum Mol Genet 2011;20:3376-3385.
  27. Beysen D, Raes J, Leroy BP, et al: Deletions involving long-range conserved nongenic sequences upstream and downstream of FOXL2 as a novel disease-causing mechanism in blepharophimosis syndrome. Am J Hum Genet 2005;77:205-218.
  28. D'haene B, Nevado J, Pugeat M, et al: FOXL2 copy number changes in the molecular pathogenesis of BPES: unique cohort of 17 deletions. Hum Mutat 2010;31:E1332-E1347.

    External Resources

  29. Crisponi L, Uda M, Deiana M, Loi A, Nagaraja R, Chiappe F, Schlessinger D, Cao A, Pilia G: FOXL2 inactivation by a translocation 171 kb away: analysis of 500 kb of chromosome 3 for candidate long-range regulatory sequences. Genomics 2004;83:757-764.
  30. D'Haene B, Attanasio C, Beysen D, Dostie J, Lemire E, Bouchard P, Field M, Jones K, Lorenz B, Menten B, Buysse K, Pattyn F, Friedli M, Ucla C, Rossier C, Wyss C, Speleman F, De Paepe A, Dekker J, Antonarakis SE, De Baere E: Disease-causing 7.4 kb cis-regulatory deletion disrupting conserved non-coding sequences and their interaction with the FOXL2 promotor: implications for mutation screening. PLoS Genet 2009;5:e1000522.

    External Resources

  31. Li WX, Wang XK, Sun Y, Wang YL, Lin LX, Tang SJ: A novel mutation in the FOXL2 gene in a Chinese family with blepharophimosis, ptosis, and epicanthus inversus syndrome. Zhonghua Yi Xue Yi Chuan Xue Za Zhi 2005;22:372-375.
  32. Qian X, Shu A, Qin W, Xing Q, Gao J, Yang J, Feng G, He L: A novel insertion mutation in the FOXL2 gene is detected in a big Chinese family with blepharophimosis-ptosis-epicanthus inversus. Mutat Res 2004;554:19-22.
  33. Gijsbers AC, D'Haene B, Hilhorst-Hofstee Y, Mannens M, Albrecht B, Seidel J, Witt DR, Maisenbacher MK, Loeys B, van Essen T, Bakker E, Hennekam R, Breuning MH, De Baere E, Ruivenkamp CA: Identification of copy number variants associated with BPES-like phenotypes. Hum Genet 2008;124:489-498.

    External Resources

  34. De Baere E, Lemercier B, Christin-Maitre S, Durval D, Messiaen L, Fellous M, Veitia R: FOXL2 mutation screening in a large panel of POF patients and XX males. J Med Genet 2002;39:e43.
  35. Harris SE, Chand AL, Winship IM, Gersak K, Aittomaki K, Shelling AN: Identification of novel mutations in FOXL2 associated with premature ovarian failure. Mol Hum Reprod 2002;8:729-733.
  36. Bodega B, Porta C, Crosignani PG, Ginelli E, Marozzi A: Mutations in the coding region of the FOXL2 gene are not a major cause of idiopathic premature ovarian failure. Mol Hum Reprod 2004;10:555-557.
  37. Ni F, Wen Q, Wang B, Zhou S, Wang J, Mu Y, Ma X, Cao Y: Mutation analysis of FOXL2 gene in Chinese patients with premature ovarian failure. Gynecol Endocrinol 2010;26:246-249.
  38. Laissue P, Lakhal B, Benayoun BA, Dipietromaria A, Braham R, Elghezal H, Philibert P, Saad A, Sultan C, Fellous M, Veitia RA: Functional evidence implicating FOXL2 in non-syndromic premature ovarian failure and in the regulation of the transcription factor OSR2. J Med Genet 2009;46:455-457.
  39. Gersak K, Harris SE, Smale WJ, Shelling AN: A novel 30 bp deletion in the foxl2 gene in a phenotypically normal woman with primary amenorrhoea: case report. Hum Reprod 2004;19:2767-2770.
  40. Chatterjee S, Modi D, Maitra A, Kadam S, Patel Z, Gokrall J, Meherji P: Screening for FOXL2 gene mutations in women with premature ovarian failure: an Indian experience. Reprod Biomed Online 2007;15:554-560.
  41. Geetha P, Nair MK: Granulosa cell tumours of the ovary. Aust NZ J Obstet Gynaecol 2010;50:216-220.

    External Resources

  42. Schumer ST, Cannistra SA: Granulosa cell tumor of the ovary. J Clin Oncol 2003;21:1180-1189.

    External Resources

  43. Shah SP, Kobel M, Senz J, et al: Mutation of FOXL2 in granulosa-cell tumors of the ovary. N Engl J Med 2009;360:2719-2729.
  44. Benayoun BA, Caburet S, Dipietromaria A, Georges A, D'Haene B, Pandaranayaka PJ, L'Hote D, Todeschini AL, Krishnaswamy S, Fellous M, De Baere E, Veitia RA: Functional exploration of the adult ovarian granulosa cell tumor-associated somatic FOXL2 mutation p.Cys134trp (c.402c>g). PLoS One 2010;5:e8789.

    External Resources

  45. Schrader KA, Gorbatcheva B, Senz J, Heravi-Moussavi A, Melnyk N, Salamanca C, Maines-Bandiera S, Cooke SL, Leung P, Brenton JD, Gilks CB, Monahan J, Huntsman DG: The specificity of the FOXL2 c.402c>g somatic mutation: a survey of solid tumors. PLoS One 2009;4:e7988.

    External Resources

  46. Jamieson S, Butzow R, Andersson N, Alexiadis M, Unkila-Kallio L, Heikinheimo M, Fuller PJ, Anttonen M: The FOXL2 c134w mutation is characteristic of adult granulosa cell tumors of the ovary. Mod Pathol 2010;23:1477-1485.
  47. Kim JH, Yoon S, Park M, Park HO, Ko JJ, Lee K, Bae J: Differential apoptotic activities of wild-type FOXL2 and the adult-type granulosa cell tumor-associated mutant FOXL2 (c134w). Oncogene 2011;30:1653-1663.
  48. Kim MS, Hur SY, Yoo NJ, Lee SH: Mutational analysis of FOXL2 codon 134 in granulosa cell tumour of ovary and other human cancers. J Pathol 2010;221:147-152.
  49. Hes O, Vanecek T, Petersson F, Grossmann P, Hora M, Perez Montiel DM, Steiner P, Dvorak M, Michal M: Mutational analysis (c.402c>g) of the FOXL2 gene and immunohistochemical expression of the FOXL2 protein in testicular adult type granulosa cell tumors and incompletely differentiated sex cord stromal tumors. Appl Immunohistochem Mol Morphol 2011;19:347-351.
  50. Al-Agha OM, Huwait HF, Chow C, Yang W, Senz J, Kalloger SE, Huntsman DG, Young RH, Gilks CB: FOXL2 is a sensitive and specific marker for sex cord-stromal tumors of the ovary. Am J Surg Pathol 2011;35:484-494.

    External Resources

  51. D'Angelo E, Mozos A, Nakayama D, Espinosa I, Catasus L, Munoz J, Prat J: Prognostic significance of FOXL2 mutation and mRNA expression in adult and juvenile granulosa cell tumors of the ovary. Mod Pathol 2011;24:1360-1367.

    External Resources

  52. Gershon R, Aviel-Ronen S, Korach J, Daniel-Carmi V, Avivi C, Bar-Ilan D, Barshack I, Meirow D, Ben-Baruch G, Cohen Y: FOXL2 C402G mutation detection using MALDI-TOF-MS in DNA extracted from Israeli granulosa cell tumors. Gynecol Oncol 2011;122:580-584.
  53. Fleming NI, Knower KC, Lazarus KA, Fuller PJ, Simpson ER, Clyne CD: Aromatase is a direct target of FOXL2:C134W in granulosa cell tumors via a single highly conserved binding site in the ovarian specific promoter. PLoS One 2010;5:e14389.
  54. Benayoun BA, Georges AB, L'Hote D, Andersson N, Dipietromaria A, Todeschini AL, Caburet S, Bazin C, Anttonen M, Veitia RA: Transcription factor FOXL2 protects granulosa cells from stress and delays cell cycle: role of its regulation by the SIRT1 deacetylase. Hum Mol Genet 2011;20:1673-1686.
  55. Schmidt D, Ovitt CE, Anlag K, Fehsenfeld S, Gredsted L, Treier AC, Treier M: The murine winged-helix transcription factor FOXL2 is required for granulosa cell differentiation and ovary maintenance. Development 2004;131:933-942.
  56. Uda M, Ottolenghi C, Crisponi L, Garcia JE, Deiana M, Kimber W, Forabosco A, Cao A, Schlessinger D, Pilia G: FOXL2 disruption causes mouse ovarian failure by pervasive blockage of follicle development. Hum Mol Genet 2004;13:1171-1181.
  57. Pailhoux E, Vigier B, Chaffaux S, Servel N, Taourit S, Furet JP, Fellous M, Grosclaude F, Cribiu EP, Cotinot C, Vaiman D: A 11.7-kb deletion triggers intersexuality and polledness in goats. Nat Genet 2001;29:453-458.
  58. Ottolenghi C, Omari S, Garcia-Ortiz JE, Uda M, Crisponi L, Forabosco A, Pilia G, Schles-singer D: FOXL2 is required for commitment to ovary differentiation. Hum Mol Genet 2005;14:2053-2062.
  59. Ottolenghi C, Pelosi E, Tran J, Colombino M, Douglass E, Nedorezov T, Cao A, Forabosco A, Schlessinger D: Loss of WNT4 and FOXL2 leads to female-to-male sex reversal extending to germ cells. Hum Mol Genet 2007;16:2795-2804.
  60. Uhlenhaut NH, Jakob S, Anlag K, Eisen-berger T, Sekido R, Kress J, Treier AC, Klugmann C, Klasen C, Holter NI, Riethmacher D, Schutz G, Cooney AJ, Lovell-Badge R, Treier M: Somatic sex reprogramming of adult ovaries to testes by FOXL2 ablation. Cell 2009;139:1130-1142.
  61. Murphy BD: Revisiting reproduction: what a difference a gene makes. Nat Med 2010;16:527-529.
  62. Kaur I, Hussain A, Naik MN, Murthy R, Honavar SG: Mutation spectrum of fork-head transcriptional factor gene (FOXL2) in Indian blepharophimosis ptosis epicanthus inversus syndrome (BPES) patients. Br J Ophthalmol 2011;95:881-886.
  63. Goodarzi MO, Dumesic DA, Chazenbalk G, Azziz R: Polycystic ovary syndrome: etiology, pathogenesis and diagnosis. Nat Rev Endocrinol 2011;7:219-231.
  64. Rodin DA, Bano G, Bland JM, Taylor K, Nussey SS: Polycystic ovaries and associated metabolic abnormalities in Indian subcontinent Asian women. Clin Endocrinol (Oxf) 1998;49:91-99.
  65. Marneros AG, Krieg T: Keloids - clinical diagnosis, pathogenesis, and treatment options. J German Soc Dermatol JDDG 2004;2:905-913.

    External Resources

  66. Nakashima M, Chung S, Takahashi A, Kamatani N, Kawaguchi T, Tsunoda T, Hosono N, Kubo M, Nakamura Y, Zembutsu H: A genome-wide association study identifies four susceptibility loci for keloid in the Japanese population. Nat Genet 2010;42:768-771.

 goto top of outline Author Contacts

Elfride De Baere, MD, PhD
Center for Medical Genetics Ghent
Ghent University, De Pintelaan 185
BE-9000 Ghent (Belgium)
Tel. +32 9 332 5186, E-Mail

 goto top of outline Article Information

Received: August 3, 2011
Accepted: November 20, 2011
Published online: January 12, 2012
Number of Print Pages : 10
Number of Figures : 5, Number of Tables : 0, Number of References : 66

 goto top of outline Publication Details

Hormone Research in Paediatrics (From Developmental Endocrinology to Clinical Research)

Vol. 77, No. 1, Year 2012 (Cover Date: February 2012)

Journal Editor: Czernichow P. (Paris)
ISSN: 1663-2818 (Print), eISSN: 1663-2826 (Online)

For additional information:

Copyright / Drug Dosage / Disclaimer

Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher or, in the case of photocopying, direct payment of a specified fee to the Copyright Clearance Center.
Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in goverment regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug.
Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.