BRIEF REPORT

FOXL2 mutations in Chinese families with

Blepharophimosis syndrome (BPES)
JIA-YAN FAN, YE-FEI WANG, BING HAN, YONG-RONG JI, HUAI-DONG SONG,
and XIAN-QUN FAN
SHANGHAI, CHINA

Blepharophimosis syndrome (BPES) is a rare, autosomal dominant disease. Two clinical types of BPES have been distinguished. In BPES type I, an eyelid malformation is
associated with infertility in affected females as a result of premature ovarian failure.
In BPES type II, eyelid anomalies alone are observed. Mutations of FOXL2, which is
a gene encoding a forkhead transcription factor, have recently been shown to
cause both types of BPES. Here, we report 1 novel duplication mutation of the
FOXL2 gene identified in a large Chinese family affected by type II BPES and 1 less
recurrent 17-bp duplication in a large Chinese family affected by BPES of an undetermined type. These new cases give additional support to the previously reported genotypephenotype correlations and our findings have expanded the spectrum of
known mutations of the FOXL2 gene. (Translational Research 2011;157:4852)
Abbreviations: BPES Blepharophimosis syndrome; PCR polymerase chain reaction; POF
premature ovarian failure

lepharophimosis syndrome (BPES) is an autosomal dominant disease with a prevalence of

about 1 in 50,0001; it is characterized by blepharophimosis (shortened palpebral fissures), ptosis
(drooping eyelids), and epicanthus inversus (a vertical
skin fold arising from the lower eyelid). Two clinical

From the Department of Ophthalmology, Shanghai Ninth Peoples

Hospital; State Key Laboratory of Medical Genomics, Molecular
Medical Center, Shanghai Institute of Endocrinology, Ruijin
Hospital, Shanghai JiaoTong University School of Medicine,
Shanghai, China.
This work was supported by National Natural Science Foundation of
China [Grant number 30672273, 3097379] and Shanghai Leading
Academic Discipline Project [Grant number S30205].
Submitted for publication June 20, 2010; revision submitted August
12, 2010; accepted for publication August 13, 2010.
Reprint requests: Xianqun Fan, Department of Ophthalmology,
Shanghai Ninth Peoples Hospital, Shanghai JiaoTong University
School of Medicine, Shanghai, P.R. China; e-mail: fanxq@sh163.net.
1931-5244/$ - see front matter
2011 Mosby, Inc. All rights reserved.
doi:10.1016/j.trsl.2010.08.005

48

types of BPES have been distinguished. In type I, eyelid

abnormalities are associated with ovarian failure,
whereas in type II, only eyelid defects are found.2 Mutations in FOXL2, which is a gene encoding a forkhead
transcription factor containing a polyalanine domain of
14 alanines that is expressed in periocular tissues as well
as in the granulosa cells of fetal and adult ovaries3-5
have been shown recently to cause both types of BPES.3
To date, a total of 106 unique intragenic FOXL2 mutations has been identified in 206 unrelated families affected
by BPES from different ethnic origins6 Intragenic mutations are detected in 70% of BPES cases, and polyalanine
expansions represent a common type of in-frame mutation accounting for 30% of the mutations reported in the
FOXL2 open reading frame (ORF) and leading mainly
to BPES type II.6-8 The classification of the spectrum of
intragenic mutations in the FOXL2 gene according
to their effect on genotypephenotype correlations
indicates that mutations that result in a truncated protein
either lacking or containing the forkhead domain lead to
BPES type I, whereas polyalanine expansions lead to
BPES type II, in which only oculofacial dysmorphism
is present.8,9 However, the genotypephenotype

Translational Research
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AT A GLANCE COMMENTARY
Fan J-Y, et al.
Background

Blepharophimosis syndrome (BPES) is a rare, autosomal dominant disease in which an eyelid malformation is associated (type I) or not (type II) with
premature ovarian failure (POF). Mutations of
FOXL2, which is a gene encoding a forkhead transcription factor, have been shown to cause both
types of BPES.
Translational Significance

This work gives additional support to the previously reported genotypephenotype correlations,
and our findings have expanded the spectrum of
known mutations of the FOXL2 gene.

correlations for missense mutations and mutations

leading to a truncated or extended protein containing an
intact forkhead domain and polyalanine tract are still
elusive based on the data available.8,9 Large submicroscopic deletions might result in BPES associated
with mental retardation.10,11 Furthermore, a recent
article12 has revealed the smallest distant deletion (7.4
kb) involved of genetic changes of conserved non-coding
sequences in the cis-regulatory domain of FOXL2 in
BPES, which shows the importance of mutation screening
of cis-regulatory elements in human genetic disease.
In this study, we report 2 mutations in the FOXL2 gene
identified in 2 large Chinese families; 1 of these mutations
is novel. We demonstrate that our results agree with previously reported genotypephenotype correlations.
MATERIALS AND METHODS
Patients and clinical evaluation. The research was carried out according to the principles of the Declaration of
Helsinki; informed consent was obtained and Shanghai
Ninth Peoples Hospital Ethics Committee approved the
study. The patient photos, which are contained in this article, were taken by a hospital-based photographer at
Shanghai Ninth Peoples Hospital, Shanghai JiaoTong
University School of Medicine. Permission to use
these photos in this report has been obtained from all
the subjects who participated in this study. The 111
subjects involved in the study included 11 patients and
100 healthy individuals, including 5 relatives of the
affected families. The pedigrees of the 2 families are
shown in Figs 1, A and 2, A. One family was
a consanguineous, 4-generation Chinese family in

Fan et al

49

which a classic BPES phenotype was observed in 9

affected members, 8 of whom were alive. The index
patient was a 3-year-old girl with typical BPES, who
was not cooperative for visual acuity testing. All the
affected adult female participants do not show any
sign of premature ovarian failure (POF), such as
oligomenorrhea and irregular menses. Moreover, they
are fertile. We classified this family as BPES type II.
The other family was a 4-generation, BPES family
with 6 affected members; this family included twins
around the age of 5 (probands) with a typical BPES.
No female patient existed in this BPES family, which
made it difficult to reach an early diagnosis of the
BPES type.
Clinical examinations of affected and unaffected individuals were carried out by ophthalmologists. The clinical data are shown in Table I. Eyelid photographs of
some patients were taken before and/or after surgery
(Figs 1, B and 2, B).
DNA extraction and sequencing. Genomic DNA was
extracted from the peripheral blood leukocytes of the
patients and 100 randomly selected healthy volunteers,
including 5 relatives of the affected families, using the
Automatic Nucleic Acid Isolation System (QuickGene
800; Fujifilm Life Science, Tokyo, Japan). For DNA
amplification, the genomic region encompassing the
FOXL2 coding sequence was divided into 3 fragments
using 3 pairs of primers. The primers used for amplifying the FOXL2 gene ORF were as follows: FOXL2-1
sense: 50 - TCCGCAGTCTCCAGAAGTTT-30 , and
FOXL2-1 antisense: 50 - CCCTTCTCGAACATGTCT
TC-30 FOXL2-2 sense: 50 - ACAACCTCAGCCTCA
ACGAG-30 , and FOXL2-2 antisense: 50 - CCAGGCC
ATTGTACGAGTTC-30 FOXL2-3 sense: 50 - GCTTC
CTCAACAACTCGTGGC-30 , and FOXL2-3 antisense: 50 - CTGCATCCTCGCATCCGTCT-30 . The resulting polymerase chain reaction (PCR) products
were purified using a gel extraction kit (Qiagen,
Hilden, Germany) and sequenced in both directions
using an ABI 3730 DNA sequencer (Applied
Biosystems PerkinElmer, Foster City, Calif).
Mutation analysis. The PCR products were also inserted into the multiple cloning site (EcoRI) of the
pGEM-T Easy vector (Promega, Madison, Wis). The
plasmids were isolated and purified using anion
exchange columns (Qiagen), and all constructs were
sequenced in both directions on an ABI 3730 DNA
sequencer (Applied Biosystems PerkinElmer) to
confirm the mutations.
RESULTS AND DISCUSSION

The findings of this study revealed 1 novel duplication mutation c. [693_695dup; 666_695dup] in the

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Fan et al

Fig 1. (A) Four-generation BPES type II pedigree. (B) Facial photographs of the proband before (a) and after (b)
surgery. (C) Genomic analysis of the cloned PCR products of FOXL2 gene, which was inserted into the multiple
cloning site (EcoRI) of the pGEM-T Easy vector showing the duplication FOXL2 mutation
c.[693_695dup;666_695dup] (p.A221_231dup) found in affected members of this BPES type II family. These variants were absent in 100 control individuals, including 3 relatives of the affected families. (Color version of figure
is available online.)

FOXL2 gene (NM_023067) in a 4-generation family affected by BPES type II. In this family, all the affected
adult females do not show any sign of POF, such as oligomenorrhea and irregular menses. Moreover, all females are fertile. This mutation was confirmed by
sequencing of the cloned PCR products, which led to
an in-frame polyalanine expansion in the FOXL2 protein (p.A221_231dup). This variant was absent in 100
control individuals (Fig 1, C).
Heterozygous expansions of the polyalanine tract from
14 to 24 residues in length represent more than 30% of the
intragenic FOXL2 mutations. This type of mutation is predicted to result in a hypomorphic allele and lead to BPES

type II. In our study, polyalanine expansions of 111 residues (FOXL2Ala25) were identified in the BPES type II
family. The pathogenic mechanism of polyalanine expansion in BPES patients has been suggested to be a result of
cytoplasmic aggregation of the FOXL2 protein and inclusion into nuclear aggregates.7,13,14 The findings in our
report provide additional support to the genotype
phenotype correlation for this type of mutation
(polyalanine expansion).
In addition, we identified a less recurrent 17-bp duplication (c.855871dup; p.His291fs) in the FOXL2 gene
(NM_023067), which led to a truncated protein in the
4-generation BPES family (Fig 2, C). Interestingly, the

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Fan et al

51

Fig 2. A, Pedigree of the family. B, Facial photographs of the 5 members in this family. Five preoperative eyelid
photographs (II:1, III:1, III:3, IV:2a, and IV:3a), and 3 postoperative eyelid photographs (III:3b, IV:2b, and IV:3b)
are provided. C, Genomic analysis of the cloned PCR products of FOXL2 gene, which was inserted into the multiple cloning site (EcoRI) of the pGEM-T easy vector showing the recurrent 17-bp duplication c.855-871dup
(p.His291fs) in the FOXL2 gene found in affected members of this family. This variant was absent in 100 control
individuals, including 2 relatives of the affected families. (Color version of figure is available online.)

Table I. Clinical features of Chinese families with BPES

BPES families

BPES type II family (Fig 1)

BPES type undetermined
family (Fig 2)

Affected
individuals

Age (years)

V:6
III:1
IV:2
IV:3

3
39
7
7

IPFH (mm)

HPFL (mm)

Levator function (mm)

IICD (mm)

RE

LE

RE

LE

RE

LE

Surgical stages
(preoperatively)

34
37
32
32

4
2
2
2

4
3
3
3

23
22
27
27

25
22
27
27

2
2
2
2

2
2
2
2

Stage 2
Stage 1
Stage 2
Stage 2

Abbreviations: HPFL, horizontal palpebral fissure length; IICD, inner intercanthal distance; IPFH, vertical interpalpebral fissure height; LE, left eye;
RE, right eye.

7 patients in this family were all male, which made it

difficult to reach an early diagnosis of the BPES type,
and this variant was absent in the female members of
this family. This mutation has been reported as a pathogenic change (Human FOXL2 Mutation Database;
http://mebgen.ugenp.be/foxl2). Recently, a 17-bp insertion (c.855_871dup) in the FOXL2 gene in Taiwanese
BPES patients has been reported to be associated with
POF.15 We, therefore, predict that if a female patient occurs in this family, there would be a risk of POF. A previous study emphasized that molecular testing alone is not
sufficient as a predictive marker for female infertility.16

We recommend that molecular genetic testing be

performed in the context of genetic counseling, and it
should be complemented by a multidisciplinary clinical
follow-up by an ophthalmologist and a reproductive
endocrinologist.6
Taken together, we have reported 1 novel duplication
mutation leading to a polyalanine expansion in a BPES
type II family and 1 less recurrent duplication mutation
in a BPES family, in which all members were of Chinese
origin. Our data provide additional support for the involvement of FOXL2 in the molecular etiology of
BPES, and genotypephenotype correlations exist in

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these Chinese BPES families. Our findings are in agreement with previous mutation studies of BPES.
We thank Professor Shengfang Ge for his helpful comments on the
manuscript. We are most grateful to the families and the volunteers
who participated in this study as well as to the clinicians and researchers who made this work possible. International Science Editing
reviewed the manuscript prior to submission.
REFERENCES

1. Oley C, Baraitser M. Blepharophimosis, ptosis, epicanthus inversus syndrome (BPES syndrome). J Med Genet 1988;25:4751.
2. Zlotogora J, Sagi M, Cohen T. The blepharophimosis, ptosis, and
epicanthus inversus syndrome: delineation of two types. Am J
Hum Genet 1983;35:10207.
3. 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;27:15966.
4. Cocquet J, Pailhoux E, Jaubert F, et al. Evolution and expression
of FOXL2. J Med Genet 2002;39:91621.
5. Cocquet J, De Baere E, Gareil M, et al. Structure, evolution and
expression of the FOXL2 transcription unit. Cytogenet Genome
Res 2003;101:20611.
6. De Baere E, De Paepe A, Beysen D. FOXL2 Mutations and genomic rearrangements in BPES. Hum Mutat 2009;30:15869.
7. De Baere E, Dixon MJ, Small KW, et al. Spectrum of FOXL2 gene
mutations in blepharophimosisptosisepicanthus inversus
(BPES) families demonstrates a genotype phenotype correlation.
Hum Mol Genet 2001;10:1591600.
8. De Baere E, Beysen D, Oley C, et al. FOXL2 and BPES: mutational hotspots, phenotypic variability, and revision of the

9.

10.

11.

12.

13.

14.

15.

16.

genotype-phenotype correlation. Am J Hum Gene 2003;72:

47887.
De Baere E, Copelli S, Caburet S, et al. Premature ovarian failure
and forkhead transcription factor FOXL2: blepharophimosisptosis-epicanthus inversus syndrome and ovarian dysfunction.
Pediatr Endocrinol Rev 2005;2:65360.
Dhaene 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;31:133247.
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;77:20518.
Dhaene B, Attanasio C, Beysen D, et al. 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.
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;41:9326.
Moumne L, Dipietromaria A, Batista F, et al. 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;17:10109.
Lin WD, Chou IC, Lee NC, et al. FOXL2 mutations in Taiwanese
patients with blepharophimosis, ptosis, epicanthus inversus syndrome. Clin Chem Lab Med 2010;48:4858.
Beysenl D, Jaegere SD, Amor D, et al. Identification of 34 novel
and 56 known FOXL2 mutations in patients with Blepharophimosis syndrome. Hum Mutat 2008;29:20519.