Mol. Hum. Reprod. Advance Access originally published online on June 4, 2004
Molecular Human Reproduction 2004 10(8):555-557; doi:10.1093/molehr/gah078
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Mutations in the coding region of the FOXL2 gene are not a major cause of idiopathic premature ovarian failure
1Department of Biology and Genetics, Medical Faculty and 2First Department of Obstetrics and Gynaecology, University of Milan, Italy
3 To whom correspondence should be addressed at: Department of Biology and Genetics for Medical Sciences, University of Milan, Via Viotti 3/5, 20133 Milan, Italy. Email: anna.marozzi{at}unimi.it
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Abstract |
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Premature ovarian failure (POF) is a heterogeneous disorder whose aetiology is still unknown. Recently, the autosomal FOXL2 gene, highly expressed in the adult ovary, has been correlated with the disorder. FOXL2 mutations, causing a truncation of the FOXL2 protein in the forkhead domain or in the poly-Ala tract lead to blepharophimosis–ptosis–epicanthus–inversus syndrome associated with POF (BPES I). Interestingly, in two out of 70 idiopathic POF patients, a 30 bp deletion (898–927del) and a missense mutation (1009T
A) were identified. To further evaluate the correlation between POF and FOXL2 mutations, 120 phenotypically normal women affected by POF were analysed by direct sequencing of the FOXL2 coding region. The analysis did not reveal any mutation in the 240 analysed chromosomes, indicating that mutations in the FOXL2 coding region are rarely associated with non-syndromic POF. Key words: FOXL2/mutation/ovarian failure/premature ovarian failure/sterility
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Introduction |
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Complex molecular events control fertility in women, starting in the fetus with organogenesis and primordial follicle formation, and after birth involving the highly coordinate interactions of the hypothalamic–pituitary system and the ovaries (Matzuk and Lamb, 2002
; Schmidt et al., 2004). Ovarian failure might manifest as primary amenorrhoea resulting from a complete failure in germ cell development, or secondary amenorrhoea due to a reduction in the number of germ cells. Premature ovarian failure (POF) is defined as secondary hypergonadotrophic amenorrhoea occurring before the age of 40 years. This condition affects 1% of females (Coulam et al., 1986) and in most cases its aetiology is still unknown. Families in which more than two women have experienced premature menopause (Vegetti et al., 1998; van Kasteren et al., 1999) suggest a genetic basis for POF. Although premature menopause is strongly associated with X chromosome defects (Marozzi et al., 2000; Prueitt et al., 2002; Schlessinger et al., 2002) several observations indicate the involvement of autosomal genes. The inactivating point mutations of FSH receptor are responsible for POF inherited as an autosomal recessive trait recognized in Finland (Aittomaki et al., 1995) and the 769GA mutation in exon 2 of the inhibin 
gene was found to be associated with POF (Shelling et al., 2000; Marozzi et al., 2002). Recently, a single autosomal locus was strongly involved in POF associated with blepharophimosis–ptosis–epicanthus–inversus syndrome (BPES) (MIM 110100). BPES type I is characterized by a complex eyelid malformation associated with premature ovarian failure, whereas in type II the eyelid defect occurs as an isolated entity. Mutations of the FOXL2 gene cause both BPES type I and type II (Crisponi et al., 2001; De Baere et al., 2003; Udar et al., 2003). FOXL2 is a single exon gene of 2.7 kb located at 3q23, belonging to the family of winged-helix/forkhead transcription factors, and containing a 100 amino acid DNA-binding forkhead domain followed by a polyalanine tract (Crisponi et al., 2001). Mutations leading to a truncation of the FOXL2 protein in the forkhead domain are at high risk for the development of POF (BPES type I). Conversely, mutations causing a truncated or extended protein with an intact forkhead and polyalanine tract cause both types of BPES, even in the same family (De Baere et al., 2003). FOXL2 transcript is highly expressed in the human and the mouse adult ovary and in the developing eyelid of the mouse (Crisponi et al., 2001). Moreover the expression of FOXL2 was detected in the early ovary of mice, during sex determination and associated with both somatic and germ cell population (Loffler et al., 2003). Since FOXL2 gene seems to be essential for follicular development and ovarian maintenance, it has been indicated as a candidate gene in non-syndromic POF (Harris et al., 2002; De Baere et al., 2003). Two mutations (898–927del and 1009TA) in the FOXL2 coding region were detected in two out of 70 idiopathic POF patients (Harris et al., 2002). The mutation 898–927del is a heterozygous 30 bp deletion, that is predicted to remove 10 of 14 alanines from the polyalanine tract (amino acids 221–230), whereas 1009TA is a heterozygous nucleotide substitution, that causes a non-conservative amino acid change from tyrosine to asparagine (Y258N). On the contrary, the screening of a different cohort of POF women (70 individuals) did not reveal the occurrence of such mutations, nor of other pathogenic mutations in the FOXL2 coding region (De Baere et al., 2002, 2003). To further evaluate the association between POF and FOXL2 mutations, 120 women affected by POF were molecularly analysed.
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Materials and methods |
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Patient population
A total of 120 women affected by POF were recruited by the Reproductive Endocrinology Services of the Departments of Obstetrics and Gynaecology in Milan. The POF status was defined as the cessation of menses for a period >6 months, before or at the age of 40 years, associated with a high gonadotrophin level (FSH
40 IU/l), detected on two different occasions. A complete medical and gynaecological history, including age at menarche and previous menses, was taken from all patients following a pre-established common protocol for homogeneous patient selection (Vegetti et al., 1998). All the patients included in this study were phenotypically normal, and were of average height and considered idiopathic because they did not show any POF-related conditions. Fifty-eight POF patients had a family history of premature menopause, with at least one relative experiencing POF and the remaining 62 patients were classified as having sporadic POF. Chromosome analysis based on high-resolution banding technique did not reveal any structural or numerical anomalies in any of the selected patients. All the women included in the study gave informed consent for their medical history to be reviewed and for collection of a peripheral blood sample suitable for further molecular analysis.
PCR and sequencing
Genomic DNA was obtained from peripheral blood by standard methods (Marozzi et al., 1999). The full coding region of the FOXL2 gene (1323 bp) was amplified by PCR with primers F and R (Crisponi et al., 2001). PCR reactions were carried out using 100 ng of genomic DNA as a template in the presence of 25 pmol of both primers and dimethylsulphoxide 10%, in a final volume of 50 µl. After denaturation at 95°C for 10 min, the samples underwent 33 cycles of amplification (95°C denaturation for 1 min, 65°C annealing for 1 min, 72°C extension for 2 min); the last cycle was followed by 10 min extension at 72°C. A nil DNA reaction was used as a negative control for all PCR reactions. The PCR products were then sequenced using primers F and R, and internal primers C and E (Crisponi et al., 2001). Sequencing reactions were performed following the Big Dye terminator protocol (Applied Biosystems, USA), and all fluorescent traces were analysed using the Applied Biosystems model 3100 DNA Sequencing System. All the sequencing reactions were performed two times.
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Results and discussion |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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A total of 120 POF patients were screened for mutations in the FOXL2 gene by direct sequencing. No pathogenic mutations were found in the 240 analysed chromosomes. In six patients we simultaneously detected a heterozygous silent substitution at nucleotide 738C
T and a 773CG transition causing a conservative change (A179G). Electropherograms comparing the two identified FOXL2 variants and wild-type sequences are shown in Figure 1A and B respectively. The derived frequency of the two variants is 4.2%, in agreement with a previous observation showing a frequency of 3.6 and 4.5% respectively in POF patients and controls (Harris et al., 2002). Thus, the two variants may be considered normal polymorphisms with no functional consequences.
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The FOXL2 gene shows a preferential expression in the human adult ovary, and thus it is difficult to exclude it as a candidate gene for ovarian failure. In fact, Foxl2lacZ homozygous mutant female mice showed no differentiation of granulosa cells, leading to the absence of secondary follicles and finally to progressive depletion of the primordial follicle pool and ovarian atresia (Schmidt et al., 2004
). Thus, FOXL2 mutation observed in BPES type I causing a truncated protein might be responsible for granulosa cell differentiation failure and consequently for POF. However, our results clearly indicate that structural alterations of the FOXL2 protein play a minor role in the pathogenesis of non-syndromic POF. To reconcile the results, it may be proposed that a modulation of the ovarian FOXL2 gene expression, due to specific mutations in the regulatory region, may be one of the causes of POF. Moreover, the obtained results strengthen the notion that POF may be considered a heterogeneous disorder whose pathogenesis is based on alteration of the structure and/or expression of different genes. In conclusion this study indicates that mutations in the FOXL2 coding region are very rarely associated with idiopathic POF, and thus the mutational analysis of the FOXL2 gene is not strictly recommended in POF families.
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References |
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Submitted on April 19, 2004; resubmitted on May 17, 2004; accepted on May 19, 2004.
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