A new FOXL2 gene mutation in a woman with


A new FOXL2 gene mutation in a woman with
A new FOXL2 gene mutation in a woman with
premature ovarian failure and sporadic
blepharophimosis-ptosis-epicanthus inversus
Frederico Jos!
e Silva Corr^
ea, M.D.,a Adriano Bueno Tavares, Ph.D.,b Rinaldo Wellerson Pereira, Ph.D.,b
and Mauricio Sim~
oes Abr~
ao, Ph.D.c
Objective: To describe a new FOXL2 gene mutation in a woman with sporadic blepharophimosis-ptosis-epicanthus
inversus syndrome (BPES) and hypergonadotropic hypogonadism.
Design: Case report.
Setting: University medical center.
Patient(s): A 28-year-old woman.
Intervention(s): Clinical evaluation, hormone assays, gene mutation research.
Main Outcome Measure(s): FOXL2 gene mutation.
Result(s): The patient with hypergonadotropic hypogonadism was diagnosed with BPES due to a new FOXL2 gene
Conclusion(s): Blepharophimosis-ptosis-epicanthus inversus syndrome is a rare disorder associated with premature ovarian failure (POF). The syndrome is an autosomal dominant trait that causes eyelid malformations and
POF in affected women. Mutations in FOXL2 gene, located in chromosome 3, are related to the development of
BPES with POF (BPES type I) or without POF (BPES type II). This report demonstrates a previously undescribed
de novo mutation in the FOXL2 gene—a thymidine deletion, c.627delT (g.864delT)—in a woman with a sporadic
case of BPES and POF. This mutation leads to truncated protein production that is related to a BPES type I phenotype. This report shows the importance of family history and genetic analysis in the evaluation of patients
with POF and corroborates the relationship between mutations on the FOXL2 gene and ovarian insufficiency. (Fertil
Steril! 2009;-:-–-. "2009 by American Society for Reproductive Medicine.)
Key Words: Blepharophimosis-ptosis-epicanthus inversus syndrome, hypergonadotropic hypogonadism, premature ovarian failure, FOXL2 gene, gene mutation
Premature ovarian failure (POF) is considered in woman aged <40
years with absence of menstruation (amenorrhea), hypoestrogenism,
and elevated serum gonadotropins (1). More than 4 months of amenorrhea and two serum FSH levels of >40 mIU/mL obtained >1
month apart in a woman aged <40 years are the suggested criteria
for diagnosing POF (2). This disorder affects 1 in 10,000 women
by age 20 years, 1 in 1,000 women by age 30 years, and 1 in 100
women by age 40 years (3). In women with primary amenorrhea,
the prevalence of POF is 10%–28%; in those with secondary amenorrhea, POF occurs in 4%–18% (2). Isolated and familial cases have
been described; however, studies demonstrate that familial POF can
vary between 4% and 35% (4–6).
Premature ovarian failure is a heterogeneous disorder determined
by many pathogenic mechanisms (4). The principal causes of POF
Received July 31, 2009; revised August 9, 2009; accepted August 10,
F.J.S.C. has nothing to disclose. A.B.T. has nothing to disclose. R.W.P.
has nothing to disclose. M.S.A. has nothing to disclose.
! Silva Corre
^a, M.D., SHIS QI 15, conjunto
Reprint requests: Frederico Jose
4, casa 9, Lago Sul, Bras!ılia, DF 71.635-240, Brazil (FAX:
55-61-33526822; E-mail: [email protected]).
are chromosomal, genetic, autoimmune, metabolic, infectious, and
iatrogenic. A large proportion of cases remain without a known
cause, and these are classified as idiopathic or karyotypically normal
spontaneous premature failure (7, 8).
According to some investigators, POF could be explained by two
basic mechanisms, ovarian follicular depletion or follicular dysfunction. Follicular depletion can be an initial deficiency of the number
of primordial follicles or an early and accelerated follicular atresia
of the initial follicular endowment (2, 9).
In the last 2 decades, many studies using molecular biology technology aimed to discover a relationship between POF and genetic
disorders (10–16). Among genetic causes, X-linked alterations
like Turner’s syndrome, X trisomy, and X mosaicism have been reported to be responsible for a large proportion of POF cases (4). The
most frequent POF-related genes in the X chromosome are the fragile site mental retardation 1 gene (FMR1) and the bone morphogenetic protein 15 gene (BMP15). However, mutations in autosomal
chromosomes have been identified in many cases of POF. Mutations
in genes of FSH receptor (FSHR), luteinizing hormone receptor
(LHR), galactose-1-phosphato uridyl transferase (GALT), guanine
nucleotide-binding protein, a-stimulating activity polypeptide 1
Fertility and Sterility# Vol. -, No. -, - 2009
Copyright ª2009 American Society for Reproductive Medicine, Published by Elsevier Inc.
(GANS), cytochrome P450c17a (CYP17), aromatase (CYP19), carbohydrate-deficient glycoprotein (CDG), and forkhead transcription
factor L2 (FOXL2) have been associated with POF (4).
Mutations in FOXL2 gene lead to blepharophimosis-ptosis-epicanthus inversus syndrome (BPES), which can be associated with
eyelid malformations and POF in the same woman, according to
the type of gene mutation.
This case report describes a new mutation on the FOXL2 gene in
a woman with POF and sporadic BPES.
A 28-year-old nulligravida white woman was referred to a tertiary
care unit for investigation of 8-year infertility. At that time the patient complained of amenorrhea since menarche at age 16 years,
only having menstrual bleeding with irregular use of oral combined
Her gynecologic history showed normal sexual development,
with breast and pubic hair compatible with Tanner stage 5, an active
sex life with the same partner for 8 years, and amenorrhea for 1 year.
Her medical history was positive for BPES. The family medical history for three sisters and three brothers, all phenotypically normal,
was unremarkable. At the time of clinical evaluation, her physical
examination showed diminished horizontal eye aperture, blepharoptosis, and epicanthus inversus. Her gynecologic examination results
were within normal limits. According to a hypothesis of primary
amenorrhea and primary infertility, she was submitted to a hormonal
profile, transvaginal ultrasound scan, and P withdrawal test.
Her laboratory tests results were as follows: FSH 31 mIU/mL, LH
24 mIU/mL, PRL 13.9 ng/mL, TSH 2.5 mIU/mL, free T4 1.0, mUI/
mL, and E2 28 pg/mL. Ultrasound scan revealed a uterine volume of
26 cm3, endometrial thickness of 4 mm, right ovarian volume of 1.1
cm3, and left ovarian volume of 1.7 cm3. The P withdrawal test results were negative. The association of hypergonadotropic hypogonadism and BPES is compatible with a diagnosis of BPES type I.
Her karyotype examination showed 46,XX. The patient is currently
under estrogen–progesterone replacement therapy and in a program
of oocyte reception. The proband and her parents accepted an invitation to undergo genetic investigation for FOXL2 gene mutation.
This study was approved by the internal review board, Bras!ılia, Brazil. Peripheral blood cells were collected from the patient and her
parents in an appropriate recipient with ethylenediaminetetraacetic
Extraction of DNA was performed according to the ‘‘salting out’’
protocol. The DNA quantification was realized by electrophoresis
on a 1% agarose gel with ethidium bromide. Amplification of the region of the FOXL2 gene was performed by polymerase chain reaction with four pairs of primers, as previously described by Crisponi
et al. (13) and De Baere et al. (17).
The PCR products were purified and submitted to direct sequencing by the dideoxy method using the ABI Prism BigDye Terminator
Cycle Sequencing Kit (Applied Biosystems, Foster City, CA) in the
ABI Prism 377 machine (Applied Biosystems).
The obtained DNA sequences were submitted to analysis with
help of software and tools like SeqScape, CodonCode Aligner, ClustalW (http://www.ebi.ac.uk/clustalw/), and Blastn (20). The sequences of the proband and her parents were compared with each
other and with the reference normal sequence of FOXL2 gene.
Corr^ea et al.
Throughout this analysis we detected that the proband presented
a mutation in her DNA sequence of FOXL2 gene. However, her father and mother presented a normal sequence of this gene. Thus the
abnormality detected in the patient is a de novo mutation leading to
a sporadic case of BPES. The mutation discovered is a single-nucleotide deletion, of a thymidine base at position 864 of gene sequence
(g.864delT) and 627 of exon sequence (c.627delT). Results from
electropherograms suggest that this mutation is in heterozygous
(Fig. 1A). The deletion observed is localized between the FOXL2
forkhead domain and the polyalanine domain. This mutation leads
to a modification in reading frame and in the amino acid sequence
of the predicted protein. The abnormal predicted protein presents
two important characteristics: the absence of polyalanine domain
and the presence of a premature termination codon leading to
a 107-amino-acids-shorter protein, p.Pro209fsX61 (Fig. 1B).
FOXL2 is a single-exon gene that belongs to a forkhead/winged helix transcription factor family. These classes of transcription factor
are involved in many of the developmental processes in humans
and other organisms. The FOXL2 gene, which has 2,745 bp and
its exon 1,131 bp, is extremely conserved between vertebrates
(12). This gene transcribes to a 376-amino-acid protein that has
a characteristic forkhead domain with 100 residues and a secondary
polyalanine domain with 14 residues of alanine (12).
Mutations in FOXL2 gene have been shown to cause BPES, a rare
genetic syndrome that leads to eyelid malformations with (BPES
type I) or without (BPES type II) POF. Mutations that leads to truncated protein production are often related to BPES type I phenotype.
However, mutations that lead to duplicated polyalanine sequence
and/or elongated protein are related to BPES type II, a hypomorphic
phenotype (18).
Blepharophimosis-ptosis-epicanthus inversus syndrome is an autosomal dominant inherited disease, but approximately 50% of
BPES cases are sporadic, caused by a de novo mutation in FOXL2
gene. Of all described mutations, 81% are intragenic. Genomic rearrangements comprising deletions encompassing FOXL2 and deletions outside the transcription unit represent 12% and 5%,
respectively. The proportion of each type of FOXL2 intragenic mutation is 11% missense mutations, 12% nonsense mutations, 44%
frameshift mutations, and 33% in-frame mutations (19).
As described by De Baere et al. (20), the mutations are classified into seven groups according to their effect on the predicted
protein. In groups A to D are present truncated proteins, without
forkhead domain (A), with partial forkhead domain (B), with
complete forkhead and without polyalanine domain (C), and
with complete forkhead and polyalanine domain (D). Group E
comprises frameshift mutations leading to elongated proteins
with complete forkhead and polyalanine domain. Group F mutations lead to in-frame changes, and group G contains missense
mutations (20).
In our case the found mutation led to a predicted protein with
complete forkhead domain and without polyalanine domain (group
C). The deletion of a thymidine at position 627 (c.627delT) of
FOXL2 gene, between the forkhead and polyalanine domain, led
to a predicted protein 107 residues shorter than the wide type and
to a truncated protein (Fig. 1B). Despite its involvement in phenotype BPES determination, the specific function of the polyalanine
tract is not clear. Studies with polyalanine tract expansion show an
intranuclear and cytoplasmic extensive protein aggregation. In this
Premature ovarian failure and sporadic BPES
Vol. -, No. -, - 2009
(A) Three electropherograms. The proband (top) demonstrates a diminished red wave (deletion of a thymidine base) into the dark blue circle
and a superposition of bases from this point. The middle and bottom electropherograms are the mother’s and the father’s sequences,
showing a normal DNA sequence. (B) Normal predicted protein with the domains forkhead (in orange) and polyalanine (in pink) and the
mutated predicted protein, with the forkhead domain but without polyalanine domain. The red arrow shows the point of mutation. The green
segment corresponds to an abnormal amino acid sequence from the mutation point to the stop codon, leading to an premature termination of
the protein.
Silva Corr^
ea. Premature ovarian failure and sporadic BPES. Fertil Steril 2009.
case the mutated protein promotes sequestration and aggregation of
wide type but stays active and compensates for the sequestered protein malfunction. Therefore the related phenotype is not so severe—
BPES type II (21). However, the truncated protein leads to retention
of the normal protein into intranuclear aggregations, impeding its
action (18). Thus the phenotype observed in this case is more expressive—BPES type I (21).
To date only 19 of the 106 described unique intragenic mutations
are located between the two domains of FOXL2 gene. Of these, only
three are deletions, and only two are deletions of a unique base pair
(c.500delT and c.576delC). Therefore, this is the first time that the
mutation c.627delT of FOXL2 gene is described in the world literature and in the FOXL2 mutation database (http://medgen.ugent.be/
Premature diagnosis of BPES syndrome is important to enable an
adequate approach and orientation of the women affected by the disease. Hormonal replacement therapy and IVF with donated oocytes
have to be offered to these patients.
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