- Email: [email protected]
Analysis of FOXO3 mutation in 114 Chinese women with premature ovarian failure
Reproductive BioMedicine Online (2010) 20, 499– 503
www.sciencedirect.com www.rbmonline.com
ARTICLE
Analysis of FOXO3 mutation in 114 Chinese women with premature ovarian failure Binbin Wang a,b,1, Yuan Mu a,b,1, Feng Ni c, Sirui Zhou Yunxia Cao c,*, Xu Ma a,b,d,*
a,b
, Jing Wang
a,b
,
a Graduate School, Peking Union Medical College, Beijing, PR China; b National Research Institute for Family Planning, 12, Dahuisi Road, Haidian, Beijing 100081, PR China; c Reproductive Medicine Center, The First Affiliated Hospital, Anhui Medical University, Hefei, PR China; d World Health Organization Collaborating Centre for Research in Human Reproduction, Beijing, PR China
* Corresponding authors. E-mail addresses: [email protected] (Y Cao), [email protected] (X Ma). 1 B.W. and Y.M. contributed equally to this work.
Professor Yunxia Cao obtained her MD degree in 1985, her MSc degree in 1988 and her PhD in 2000. In 1998, she established the Reproductive Medical Center in Anhui Province, China, and produced the first IVM baby and first baby derived from frozen-thawed oocytes in Anhui Province, China. Professor Yunxia Cao is a well-known infertility specialist in China, and she is a National Board Member for Reproductive Medicine. Her research interests include the mechanisms underlying polycystic ovary syndrome, fertility preservation for women and the simplification of IVF treatment.
Abstract This study screened FOXO3 coding regions for mutations in a sample of 114 Chinese women with premature ovarian failure
and discovered six novel single-nucleotide variants: c.71C>A (p.Pro24His), c.140C>T (p.Pro47Leu), c.184G>A (p.Asp62Asn), c.1652C>T (p.Ser551Phe), c.1697C>G (p.Gly566Ala) and c.1185G>A (silent). The nonsynonymous single-nucleotide variants, c.71C>A (p.Pro24His), c.140C>T (p.Pro47Leu), c.184G>A (p.Asp62Asn), c.1652C>T (p.Ser551Phe) and c.1697C>G (p.Gly566Ala), were not detected in any of 100 controls. These missense mutations might cause abnormal oocyte apoptosis and primordial follicle activation, thereby adversely affecting early follicle depletion in the ovary. RBMOnline ª 2010, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. KEYWORDS: female infertility, FOXO3, premature ovarian failure
Introduction Premature ovarian failure (POF) or premature menopause is defined as ovarian failure before the age of 40 and affects approximately 1% of women, although this varies depending on ethnicity (Coulam et al., 1986; Luborsky et al., 2003). POF results in amenorrhoea and infertility and has systemic
consequences such as reduced bone density caused by sex steroid deficiency (Kalataridou and Nelson, 2000). A number of physiological changes are associated with menopause: decline in the number of ovarian follicles, menstrual irregularities, ovarian hormonal deficiency, anovulation, decreased fertility and, finally, a complete and irreversible cessation of menses (McKinlay et al., 1972; Wyshak and
1472-6483/$ - see front matter ª 2010, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.rbmo.2010.01.008
500 Frisch, 1982). The diagnosis of POF is normally confirmed by two blood tests at least 1 month apart showing circulating serum FSH concentrations of >40 IU/l and is clinically recognized as amenorrhoea before the age of 40 with low concentrations of oestrogen (Dixit et al., 2006). POF has distinct aetiologies, including chemo/radiotherapy, galactosaemia, X-chromosome abnormalities and autoimmune oophoritis (Aittomaeki et al., 1995; Bione et al., 1998). The causes of POF are diverse and largely idiopathic. It has been estimated that up to 30% of women with POF might have a genetic cause (van Kasteren et al., 1999; Vegetti, 1998). However, few gene targets have been positively correlated with POF, which necessitates further screening of candidates. Previous studies suggested that intrinsic abnormalities of (or damage to) primordial follicles affecting their initial recruitment or survival are the unifying pathophysiology for POF (Board et al., 1979; Russell et al., 1982; Zarate et al., 1970). Defects in the normal physiological processes that deplete primordial follicles probably play a major role. Two mechanisms could explain this phenomenon: oocyte apoptosis and primordial follicle activation (PFA) (McGee and Hsueh, 2000; Tilly, 2001). Both oocyte apoptosis and PFA occur continually before menopause. Thus, even subtle defects in either process could have a significant cumulative impact on primordial follicle numbers over a woman’s lifetime and thereby influence the age at menopause. The forkhead transcription factor FOXO3 is a master regulator and potent suppressor of PFA. Activation of FOXO3a generally leads to either cell cycle arrest or cell death. A recent study clearly shows that FOXO3a activation induced by growth factor deprivation leads to up-regulation of Pink1 (Mei et al., 2009). The FOXO3 protein is expressed in the ovary and is thought to play roles in ovarian development and function. Therefore, it was chosen here for mutation screening in patients with POF. The FOXO3 knockout mouse exhibits a POF phenotype that is characterized by early depletion of functional ovarian follicles, preceded by global follicular activation leading to oocyte death (Castrillon
B Wang et al. et al., 2003) (Figure 1). These findings raised the possibility that mutations or naturally occurring polymorphisms of the human orthologue FOXO3 (95% sequence identity at the amino acid level) might contribute to POF resulting from primordial follicle depletion before menarche (Timmreck and Reindollar, 2003). Finally, heterozygosity for FOXO3 mutations might be biologically significant given that a number of hereditary disorders are caused by loss-of-function mutation of a single forkhead transcription factor allele (haploinsufficiency) (Watkins et al., 2006). This study’s goal was to determine whether perturbations in the FOXO3 gene occur in Chinese women with POF.
Materials and methods A total of 114 patients with POF and 100 unrelated healthy controls were recruited from the First Affiliated Hospital, Anhui Medical University, China. The study was approved by the ethics committee of the National Research Institute for Family Planning, and informed consent was obtained from all participants. Diagnosing criteria were menopause occurring before the age of 40, with at least two serum FSH concentrations of >40 IU/l, and with an exclusion of chromosomal abnormalities. Patients with associated endocrinopathies or autoimmune disorders, iatrogenic agents, such as chemotherapy or radiotherapy, and infections, were also excluded. Controls were individuals under 40 years of age with proven fertility, normal menstrual cycles and ovary morphology, without a history of subfertility treatment. Genomic DNA was extracted from peripheral blood leukocytes using standard methods (Wang et al., 2008). The coding regions of FOXO3 were amplified using polymerase chain reaction (PCR) with two pairs of FOXO3 gene-specific primers. The first exon was amplified using primers FOXO3– 1F (50 -gcccgggataaccaactctccttct-30 ) and FOXO3–1R (50 -acctacctcctcggtgctg-30 ). The second exon was amplified using primers FOXO3–2F (50 -tatatcatctgggtgctcggtttt-30 ) and FOXO3–2R (50 -cccctcatccccatattgttatt-30 ). PCR products
Figure 1 Schematic illustrating the FOXO3 null phenotype. Colonization of the gonad by germline stem cells occurs normally during embryonic development (Castrillon et al., 2003), as do subsequent stages of female germline development up to cyst breakdown leading to the formation of primordial follicles (John et al., 2007b). Immediately following primordial follicle individualization (within a few days of birth), all primordial follicles in FOXO3/ ovaries undergo activation in a synchronized manner, resulting in dramatically increased ovarian size by post-natal day 14. Despite this global activation, maturation of some follicles proceeds normally and females have viable progeny until total follicle atresia occurs by 15 weeks. Since follicle growth is irreversible and activated, growing follicles have a finite lifespan (Elvin and Matzuk, 1998; McGee and Hsueh, 2000) and FOXO3 deficiency results in premature ovarian failure due to depletion of the follicle pool. PGC = primordial germ cell; PND = post-natal day.
FOXO3 mutations and premature ovarian failure were denatured and reannealed to form potential heteroduplexes (wild-type strand paired with mutant strand). The amplified samples were subjected to agarose gel electrophoresis to ensure single bands and then purified. Samples were sequenced using Big Dye Terminator 3.1 chemistry (Applied Biosystems, USA) with the above primers and run on a 3730xl ABI DNA analyser (Applied Biosystems). An additional in-silico analysis using PolyPhen (http://genetics. bwh.harvard.edu/pph, accessed 03 September 2009) was used to predict the effect of the variants discovered in sequencing.
Results Among the 114 subjects with POF, five novel nonsynonymous mutations were found: c.71C>A (p.Pro24His), c.140C>T (p.Pro47Leu), c.184G>A (p.Asp62Asn), c.1652C>T (p.Ser551 Phe) and c.1697C>G (p.Gly566Ala) and none of them were found in the controls (Figure 2). These mutations are located in a highly conserved domain seen in Mus musculus, Rattus norvegicus, Sus scrofa and Xenopus laevis (Figure 3). Predicted using Polyphen, the position-specific independent count (PSIC) score difference of the p.Pro24His variant was 2.025, the p.Pro47Leu variant 2.250, the p.Asp62Asn variant 1.125, the p.Ser551Phe variant 1.837 and the p.Gly566Ala variant 0.113. The clinical data of the 15 patients with POF who were heterozygous carriers of FOXO3 variants are given in Table 1. Three synonymous (silent) variants were also found in the POF group: c.105G>A (p.Leu35Leu), c.159C>T (p.Ala53Ala) and c.1185G>A (p.Thr395Thr). The c.159C>T variant was also present in the controls; c.105G>A and c.1185G>A were novel synonymous mutations.
501
Discussion Heterozygous mutations of FOXO3 have been described in women with POF from New Zealand, Slovenia, Italy, North America, France, Germany and South Korea. (Gallardo et al., 2008; Watkins et al., 2006) Nonsynonymous single nucleotide polymorphisms (SNPs) in the New Zealand and Slovenia data sets included c.1262C>T and c.1517G>A. The c.280C>T, c.1021G>A and c.1156C>T polymorphisms were detected among 93 Italian, 164 North American, 25 French, 10 German and 10 South Korean women with POF. However, none of these were detected in the Chinese subjects. Different ethnicity might explain these conflicting results. The c. 159C>T SNP detected in nine Chinese subjects is a common SNP that has been detected in women with POF from New Zealand, Slovenia, Italy, North America, France, Germany and South Korea. This SNP was reported previously to SNP databases. The FOXO3 gene encodes the forkhead family of transcription factors, which are characterized by a distinct forkhead domain. This gene likely functions as a trigger for apoptosis through expression of genes necessary for cell death. Translocation of this gene with the MLL gene is associated with secondary acute leukaemia. Alternatively, spliced transcript variants encoding the same protein have been observed. Numerous studies prompted us to test whether FOXO3deficient ovaries might exhibit defects in oocyte apoptosis in addition to the observed defects in PFA from which FOXO3 may play a specific and essential role (Castrillon et al., 2003; Hosaka et al., 2004; John et al., 2007a). FOXO3 is also dispensable for embryonic development, but primordial follicles in human FOXO3 mutant ovaries undergo global
Figure 2 Sequencing figures illustrating the five novel nonsynonymous mutations: c.71C>A (p.Pro24His), c.140C>T (p.Pro47Leu), c.184G>A (p.Asp62Asn), c.1697C>G (p.Gly566Ala) and c.1652C>T (p.Ser551Phe) found among the 114 Chinese women with premature ovarian failure.
502
B Wang et al.
Figure 3 Domain structure of FOXO3a and sequence alignments. (a) The domain architecture of FOXO3a is shown with the wellfolded forkhead (FH) domain, and three other conserved regions, CR1–CR3, are indicated. (b) Conserved domain seen in Mus musculus, Rattus norvegicus, Sus scrofa and Xenopus laevis, with the regions of the mutant residues indicated.
Table 1
Clinical and biochemical characteristics of patients carrying potential disease-associated FOXO3 sequence variants.
Gene variation
Protein variation
Phenotype
Age (years)
Menarche age (years)
FSH (IU/l)
LH (IU/l)
Familial case
c.71C>A c.71C>A c.71C>A c.71C>A c.71C>A c.71C>A c.140C>T c.184G>A c.184G>A c.184G>A c.184G>A c.1652C>T c.1697C>G c.1697C>G c.1697C>G
p.Pro24His p.Pro24His p.Pro24His p.Pro24His p.Pro24His p.Pro24His p.Pro47Leu p.Asp62Asn p.Asp62Asn p.Asp62Asn p.Asp62Asn p.Ser551Phe p.Gly566Ala p.Gly566Ala p.Gly566Ala
SA SA SA SA SA SA SA SA SA SA SA SA PA SA SA
35 35 32 32 32 35 24 27 15 25 35 43 17 20 32
13 17 16 13 13 13 12 17 14 17 14 17 NA 15 14
60.94 67.52 55.72 88.75 42.00 78.70 77.77 91.43 42.40 63.14 88.91 68.12 112.31 121.64 74.40
31.57 42.42 29.89 23.42 17.70 30.40 30.29 21.54 20.80 14.10 36.64 23.56 55.80 51.61 25.00
No No No No No No No Yes No No No No No No No
NA = not applicable; PA = primary amenorrhoea; SA = secondary amenorrhoea.
activation within a few days of birth, resulting in a characteristic syndrome of ovarian hyperplasia and early follicle depletion with consequent premature ovarian failure and secondary infertility (Gallardo et al., 2008; Hosaka et al., 2004). These findings further support the placement of FOXO3 mutants in a unique phenotypic class among female sterile mutants and highlight the importance of this gene in the regulation of PFA. In summary, this study has discovered five novel nonsynonymous mutations in the FOXO3 gene among 114 Chinese women with POF. None were present in the control population. Variants of FOXO3 are considered to have adverse effects on the function of the FOXO3 protein. Moreover, in-silico analysis using the PolyPhen program was performed to determine the potential effect of the variants. The PISC score differences showed that the variants Pro24His, Pro47Leu and Ser551Phe might be deleterious to the FOXO3 protein structure. Proline tends to terminate the protein
folding structure, the distinctive cyclic structure of proline’s side chain locks its u backbone dihedral angle at approximately 75, giving proline an exceptional conformational rigidity compared with other amino acids. By replacing proline with histidine and leucine, the variants p.Pro24His and p.Pro47Leu may cause a change to the protein structure. Meanwhile the p.Ser551Phe indicates a substitution of the non-polar amino acid, phenylalanine, for polar serine, which may also affect the protein structure. Meanwhile the Asp62Asn variant and the Gly566Ala variant are predicted to be benign (in-silico analysis using PolyPhen. Thus, Pro24His, Pro47Leu, Asp62Asn, Ser551Phe, Gly566Ala substitutions might cause abnormal oocyte apoptosis and primordial follicle activation, thereby adversely affecting early follicle depletion in the ovary. These might therefore be potential disease-associated FOXO3 sequence variants. Further functional studies are necessary to determine the functional significance of this missense mutation.
FOXO3 mutations and premature ovarian failure
Acknowledgements The authors would like to thank the POF patients for their involvement in this study and also the clinicians who provided these patients. This work was supported by the National Basic Research Program of China (2010CB945102), National Natural Science Foundation of China (30973197), the National Science & Technology Pillar Program of China (2008BAH24B05), and National Infrastructure Program of Chinese Genetic Resources (2006DKA21300).
References Aittomaeki, K., Dieguez Lucena, J., Pakarinen, P., et al., 1995. Mutation in the follicle-stimulating hormone receptor gene causes hereditary hypergonadotropic ovarian failure. Cell 82, 958–959. Bione, S., Sala, C., Manzini, C., et al., 1998. A human homologue of the Drosophila melanogaster diaphanous gene is disrupted in a patient with premature ovarian failure: evidence for conserved function in oogenesis and implications for human sterility. Am. J. Hum. Genet. 62, 533–541. Board, J., Redwine, F., Moncure, C., Frable, W., Taylor, J., 1979. Identification of differing etiologies of clinically diagnosed premature menopause. Am. J. Obstet. Gynecol. 134, 936–944. Castrillon, D., Miao, L., Kollipara, R., Horner, J., DePinho, R., 2003. Suppression of ovarian follicle activation in mice by the transcription factor Foxo3a. Science 301, 215–218. Coulam, C., Adamson, S., Annegers, J., 1986. Incidence of premature ovarian failure. Obstet. Gynecol. 67, 604–606. Dixit, H., Rao, L.K., Padmalatha, V.V., et al., 2006. Missense mutations in the BMP15 gene are associated with ovarian failure. Hum. Genet. 119 (4), 408–415. Elvin, J.A., Matzuk, M.M., 1998. Mouse models of ovarian failure. Rev. Reprod. 3 (3), 183–195. Gallardo, T.D., John, G.B., Bradshaw, K., et al., 2008. Sequence variation at the human FOXO3 locus: a study of premature ovarian failure and primary amenorrhea. Hum. Reprod. 23, 216–221. Hosaka, T., Biggs, W., Tieu, D., et al., 2004. Disruption of forkhead transcription factor (FOXO) family members in mice reveals their functional diversification. Proc. Natl. Acad. Sci. USA 101, 2975–2980. John, G.B., Shirley, L.J., Gallardo, T.D., Castrillon, D.H., 2007a. Specificity of the requirement for Foxo3 in primordial follicle activation. Reproduction 133, 855–863.
503 John, G.B., Shirley, L.J., Gallardo, T.D., Castrillon, D.H., 2007b. Specificity of the requirement for Foxo3 in primordial follicle activation. Reproduction 133, 855–863. Kalataridou, S., Nelson, L., 2000. Premature ovarian failure is not premature menopause. Ann. NY Acad. Sci. 900, 393–402. Luborsky, J., Meyer, P., Sowers, M., Gold, E., Santoro, N., 2003. Premature menopause in a multi-ethnic population study of the menopause transition. Hum. Reprod. 18, 199–206. McGee, E., Hsueh, A., 2000. Initial and cyclic recruitment of ovarian follicles. Endocr. Rev. 21, 200–214. McKinlay, S., Jefferys, M., Thompson, B., 1972. An investigation of the age at menopause. J. Biosoc. Sci. 4, 161–173. Mei, Yang, Zhang, Yiru, Yamamoto, Kazuo, Xie, Wei, Mak, Tak W., You, Han, 2009. FOXO3a-dependent regulation of Pink1 (Park6) mediates survival signaling in response to cytokine deprivation. PNAS 106 (13), 5153–5158. Russell, P., Bannatyne, P., Shearman, R., Fraser, I., Corbett, P., 1982. Premature hypergonadotropic ovarian failure: clinicopathological study of 19 cases. Int. J. Gynecol. Pathol. 1, 185. Tilly, J., 2001. Commuting the death sentence: how oocytes strive to survive. Nat. Rev. Mol. Cell Biol. 2, 838. Timmreck, L., Reindollar, R., 2003. Contemporary issues in primary amenorrhea. Obstet. Gynecol. Clin. North Am. 30, 287–302. van Kasteren, Y., Hundscheid, R., Smits, A., Cremers, F., van Zonneveld, P., Braat, D., 1999. Familial idiopathic premature ovarian failure: an overrated and underestimated genetic disease? Hum. Reprod. 14, 2455–2459. Vegetti, W., 1998. Inheritance in idiopathic premature ovarian failure: analysis of 71 cases. Hum. Reprod. 13, 1796–1800. Wang, Binbin, Zhou, Sirui, Yang, Ze, et al., 2008. Genetic analysis of tumor necrosis factor-a (TNF-a) G-308A and Saitohin Q7R polymorphisms with Alzheimer’s disease. J. Neurol. Sci. 270 (1– 2), 148–151. Watkins, W., Umbers, A., Woad, K., et al., 2006. Mutational screening of FOXO3A and FOXO1A in women with premature ovarian failure. Fertil. Steril. 86, 1518–1521. Wyshak, G., Frisch, R., 1982. Evidence for a secular trend in age of menarche. N. Engl. J. Med. 306, 1033–1035. Zarate, A., Karchmer, S., Gomez, E., Castelazo-Ayala, L., 1970. Premature menopause. A clinical, histologic and cytogenetic study. Am. J. Obstet. Gynecol. 106, 110–114. Declaration: The authors report no financial or commercial conflicts of interest. Received 6 May 2009; refereed 23 June 2009; accepted 17 December 2009.
www.sciencedirect.com www.rbmonline.com
ARTICLE
Analysis of FOXO3 mutation in 114 Chinese women with premature ovarian failure Binbin Wang a,b,1, Yuan Mu a,b,1, Feng Ni c, Sirui Zhou Yunxia Cao c,*, Xu Ma a,b,d,*
a,b
, Jing Wang
a,b
,
a Graduate School, Peking Union Medical College, Beijing, PR China; b National Research Institute for Family Planning, 12, Dahuisi Road, Haidian, Beijing 100081, PR China; c Reproductive Medicine Center, The First Affiliated Hospital, Anhui Medical University, Hefei, PR China; d World Health Organization Collaborating Centre for Research in Human Reproduction, Beijing, PR China
* Corresponding authors. E-mail addresses: [email protected] (Y Cao), [email protected] (X Ma). 1 B.W. and Y.M. contributed equally to this work.
Professor Yunxia Cao obtained her MD degree in 1985, her MSc degree in 1988 and her PhD in 2000. In 1998, she established the Reproductive Medical Center in Anhui Province, China, and produced the first IVM baby and first baby derived from frozen-thawed oocytes in Anhui Province, China. Professor Yunxia Cao is a well-known infertility specialist in China, and she is a National Board Member for Reproductive Medicine. Her research interests include the mechanisms underlying polycystic ovary syndrome, fertility preservation for women and the simplification of IVF treatment.
Abstract This study screened FOXO3 coding regions for mutations in a sample of 114 Chinese women with premature ovarian failure
and discovered six novel single-nucleotide variants: c.71C>A (p.Pro24His), c.140C>T (p.Pro47Leu), c.184G>A (p.Asp62Asn), c.1652C>T (p.Ser551Phe), c.1697C>G (p.Gly566Ala) and c.1185G>A (silent). The nonsynonymous single-nucleotide variants, c.71C>A (p.Pro24His), c.140C>T (p.Pro47Leu), c.184G>A (p.Asp62Asn), c.1652C>T (p.Ser551Phe) and c.1697C>G (p.Gly566Ala), were not detected in any of 100 controls. These missense mutations might cause abnormal oocyte apoptosis and primordial follicle activation, thereby adversely affecting early follicle depletion in the ovary. RBMOnline ª 2010, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. KEYWORDS: female infertility, FOXO3, premature ovarian failure
Introduction Premature ovarian failure (POF) or premature menopause is defined as ovarian failure before the age of 40 and affects approximately 1% of women, although this varies depending on ethnicity (Coulam et al., 1986; Luborsky et al., 2003). POF results in amenorrhoea and infertility and has systemic
consequences such as reduced bone density caused by sex steroid deficiency (Kalataridou and Nelson, 2000). A number of physiological changes are associated with menopause: decline in the number of ovarian follicles, menstrual irregularities, ovarian hormonal deficiency, anovulation, decreased fertility and, finally, a complete and irreversible cessation of menses (McKinlay et al., 1972; Wyshak and
1472-6483/$ - see front matter ª 2010, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.rbmo.2010.01.008
500 Frisch, 1982). The diagnosis of POF is normally confirmed by two blood tests at least 1 month apart showing circulating serum FSH concentrations of >40 IU/l and is clinically recognized as amenorrhoea before the age of 40 with low concentrations of oestrogen (Dixit et al., 2006). POF has distinct aetiologies, including chemo/radiotherapy, galactosaemia, X-chromosome abnormalities and autoimmune oophoritis (Aittomaeki et al., 1995; Bione et al., 1998). The causes of POF are diverse and largely idiopathic. It has been estimated that up to 30% of women with POF might have a genetic cause (van Kasteren et al., 1999; Vegetti, 1998). However, few gene targets have been positively correlated with POF, which necessitates further screening of candidates. Previous studies suggested that intrinsic abnormalities of (or damage to) primordial follicles affecting their initial recruitment or survival are the unifying pathophysiology for POF (Board et al., 1979; Russell et al., 1982; Zarate et al., 1970). Defects in the normal physiological processes that deplete primordial follicles probably play a major role. Two mechanisms could explain this phenomenon: oocyte apoptosis and primordial follicle activation (PFA) (McGee and Hsueh, 2000; Tilly, 2001). Both oocyte apoptosis and PFA occur continually before menopause. Thus, even subtle defects in either process could have a significant cumulative impact on primordial follicle numbers over a woman’s lifetime and thereby influence the age at menopause. The forkhead transcription factor FOXO3 is a master regulator and potent suppressor of PFA. Activation of FOXO3a generally leads to either cell cycle arrest or cell death. A recent study clearly shows that FOXO3a activation induced by growth factor deprivation leads to up-regulation of Pink1 (Mei et al., 2009). The FOXO3 protein is expressed in the ovary and is thought to play roles in ovarian development and function. Therefore, it was chosen here for mutation screening in patients with POF. The FOXO3 knockout mouse exhibits a POF phenotype that is characterized by early depletion of functional ovarian follicles, preceded by global follicular activation leading to oocyte death (Castrillon
B Wang et al. et al., 2003) (Figure 1). These findings raised the possibility that mutations or naturally occurring polymorphisms of the human orthologue FOXO3 (95% sequence identity at the amino acid level) might contribute to POF resulting from primordial follicle depletion before menarche (Timmreck and Reindollar, 2003). Finally, heterozygosity for FOXO3 mutations might be biologically significant given that a number of hereditary disorders are caused by loss-of-function mutation of a single forkhead transcription factor allele (haploinsufficiency) (Watkins et al., 2006). This study’s goal was to determine whether perturbations in the FOXO3 gene occur in Chinese women with POF.
Materials and methods A total of 114 patients with POF and 100 unrelated healthy controls were recruited from the First Affiliated Hospital, Anhui Medical University, China. The study was approved by the ethics committee of the National Research Institute for Family Planning, and informed consent was obtained from all participants. Diagnosing criteria were menopause occurring before the age of 40, with at least two serum FSH concentrations of >40 IU/l, and with an exclusion of chromosomal abnormalities. Patients with associated endocrinopathies or autoimmune disorders, iatrogenic agents, such as chemotherapy or radiotherapy, and infections, were also excluded. Controls were individuals under 40 years of age with proven fertility, normal menstrual cycles and ovary morphology, without a history of subfertility treatment. Genomic DNA was extracted from peripheral blood leukocytes using standard methods (Wang et al., 2008). The coding regions of FOXO3 were amplified using polymerase chain reaction (PCR) with two pairs of FOXO3 gene-specific primers. The first exon was amplified using primers FOXO3– 1F (50 -gcccgggataaccaactctccttct-30 ) and FOXO3–1R (50 -acctacctcctcggtgctg-30 ). The second exon was amplified using primers FOXO3–2F (50 -tatatcatctgggtgctcggtttt-30 ) and FOXO3–2R (50 -cccctcatccccatattgttatt-30 ). PCR products
Figure 1 Schematic illustrating the FOXO3 null phenotype. Colonization of the gonad by germline stem cells occurs normally during embryonic development (Castrillon et al., 2003), as do subsequent stages of female germline development up to cyst breakdown leading to the formation of primordial follicles (John et al., 2007b). Immediately following primordial follicle individualization (within a few days of birth), all primordial follicles in FOXO3/ ovaries undergo activation in a synchronized manner, resulting in dramatically increased ovarian size by post-natal day 14. Despite this global activation, maturation of some follicles proceeds normally and females have viable progeny until total follicle atresia occurs by 15 weeks. Since follicle growth is irreversible and activated, growing follicles have a finite lifespan (Elvin and Matzuk, 1998; McGee and Hsueh, 2000) and FOXO3 deficiency results in premature ovarian failure due to depletion of the follicle pool. PGC = primordial germ cell; PND = post-natal day.
FOXO3 mutations and premature ovarian failure were denatured and reannealed to form potential heteroduplexes (wild-type strand paired with mutant strand). The amplified samples were subjected to agarose gel electrophoresis to ensure single bands and then purified. Samples were sequenced using Big Dye Terminator 3.1 chemistry (Applied Biosystems, USA) with the above primers and run on a 3730xl ABI DNA analyser (Applied Biosystems). An additional in-silico analysis using PolyPhen (http://genetics. bwh.harvard.edu/pph, accessed 03 September 2009) was used to predict the effect of the variants discovered in sequencing.
Results Among the 114 subjects with POF, five novel nonsynonymous mutations were found: c.71C>A (p.Pro24His), c.140C>T (p.Pro47Leu), c.184G>A (p.Asp62Asn), c.1652C>T (p.Ser551 Phe) and c.1697C>G (p.Gly566Ala) and none of them were found in the controls (Figure 2). These mutations are located in a highly conserved domain seen in Mus musculus, Rattus norvegicus, Sus scrofa and Xenopus laevis (Figure 3). Predicted using Polyphen, the position-specific independent count (PSIC) score difference of the p.Pro24His variant was 2.025, the p.Pro47Leu variant 2.250, the p.Asp62Asn variant 1.125, the p.Ser551Phe variant 1.837 and the p.Gly566Ala variant 0.113. The clinical data of the 15 patients with POF who were heterozygous carriers of FOXO3 variants are given in Table 1. Three synonymous (silent) variants were also found in the POF group: c.105G>A (p.Leu35Leu), c.159C>T (p.Ala53Ala) and c.1185G>A (p.Thr395Thr). The c.159C>T variant was also present in the controls; c.105G>A and c.1185G>A were novel synonymous mutations.
501
Discussion Heterozygous mutations of FOXO3 have been described in women with POF from New Zealand, Slovenia, Italy, North America, France, Germany and South Korea. (Gallardo et al., 2008; Watkins et al., 2006) Nonsynonymous single nucleotide polymorphisms (SNPs) in the New Zealand and Slovenia data sets included c.1262C>T and c.1517G>A. The c.280C>T, c.1021G>A and c.1156C>T polymorphisms were detected among 93 Italian, 164 North American, 25 French, 10 German and 10 South Korean women with POF. However, none of these were detected in the Chinese subjects. Different ethnicity might explain these conflicting results. The c. 159C>T SNP detected in nine Chinese subjects is a common SNP that has been detected in women with POF from New Zealand, Slovenia, Italy, North America, France, Germany and South Korea. This SNP was reported previously to SNP databases. The FOXO3 gene encodes the forkhead family of transcription factors, which are characterized by a distinct forkhead domain. This gene likely functions as a trigger for apoptosis through expression of genes necessary for cell death. Translocation of this gene with the MLL gene is associated with secondary acute leukaemia. Alternatively, spliced transcript variants encoding the same protein have been observed. Numerous studies prompted us to test whether FOXO3deficient ovaries might exhibit defects in oocyte apoptosis in addition to the observed defects in PFA from which FOXO3 may play a specific and essential role (Castrillon et al., 2003; Hosaka et al., 2004; John et al., 2007a). FOXO3 is also dispensable for embryonic development, but primordial follicles in human FOXO3 mutant ovaries undergo global
Figure 2 Sequencing figures illustrating the five novel nonsynonymous mutations: c.71C>A (p.Pro24His), c.140C>T (p.Pro47Leu), c.184G>A (p.Asp62Asn), c.1697C>G (p.Gly566Ala) and c.1652C>T (p.Ser551Phe) found among the 114 Chinese women with premature ovarian failure.
502
B Wang et al.
Figure 3 Domain structure of FOXO3a and sequence alignments. (a) The domain architecture of FOXO3a is shown with the wellfolded forkhead (FH) domain, and three other conserved regions, CR1–CR3, are indicated. (b) Conserved domain seen in Mus musculus, Rattus norvegicus, Sus scrofa and Xenopus laevis, with the regions of the mutant residues indicated.
Table 1
Clinical and biochemical characteristics of patients carrying potential disease-associated FOXO3 sequence variants.
Gene variation
Protein variation
Phenotype
Age (years)
Menarche age (years)
FSH (IU/l)
LH (IU/l)
Familial case
c.71C>A c.71C>A c.71C>A c.71C>A c.71C>A c.71C>A c.140C>T c.184G>A c.184G>A c.184G>A c.184G>A c.1652C>T c.1697C>G c.1697C>G c.1697C>G
p.Pro24His p.Pro24His p.Pro24His p.Pro24His p.Pro24His p.Pro24His p.Pro47Leu p.Asp62Asn p.Asp62Asn p.Asp62Asn p.Asp62Asn p.Ser551Phe p.Gly566Ala p.Gly566Ala p.Gly566Ala
SA SA SA SA SA SA SA SA SA SA SA SA PA SA SA
35 35 32 32 32 35 24 27 15 25 35 43 17 20 32
13 17 16 13 13 13 12 17 14 17 14 17 NA 15 14
60.94 67.52 55.72 88.75 42.00 78.70 77.77 91.43 42.40 63.14 88.91 68.12 112.31 121.64 74.40
31.57 42.42 29.89 23.42 17.70 30.40 30.29 21.54 20.80 14.10 36.64 23.56 55.80 51.61 25.00
No No No No No No No Yes No No No No No No No
NA = not applicable; PA = primary amenorrhoea; SA = secondary amenorrhoea.
activation within a few days of birth, resulting in a characteristic syndrome of ovarian hyperplasia and early follicle depletion with consequent premature ovarian failure and secondary infertility (Gallardo et al., 2008; Hosaka et al., 2004). These findings further support the placement of FOXO3 mutants in a unique phenotypic class among female sterile mutants and highlight the importance of this gene in the regulation of PFA. In summary, this study has discovered five novel nonsynonymous mutations in the FOXO3 gene among 114 Chinese women with POF. None were present in the control population. Variants of FOXO3 are considered to have adverse effects on the function of the FOXO3 protein. Moreover, in-silico analysis using the PolyPhen program was performed to determine the potential effect of the variants. The PISC score differences showed that the variants Pro24His, Pro47Leu and Ser551Phe might be deleterious to the FOXO3 protein structure. Proline tends to terminate the protein
folding structure, the distinctive cyclic structure of proline’s side chain locks its u backbone dihedral angle at approximately 75, giving proline an exceptional conformational rigidity compared with other amino acids. By replacing proline with histidine and leucine, the variants p.Pro24His and p.Pro47Leu may cause a change to the protein structure. Meanwhile the p.Ser551Phe indicates a substitution of the non-polar amino acid, phenylalanine, for polar serine, which may also affect the protein structure. Meanwhile the Asp62Asn variant and the Gly566Ala variant are predicted to be benign (in-silico analysis using PolyPhen. Thus, Pro24His, Pro47Leu, Asp62Asn, Ser551Phe, Gly566Ala substitutions might cause abnormal oocyte apoptosis and primordial follicle activation, thereby adversely affecting early follicle depletion in the ovary. These might therefore be potential disease-associated FOXO3 sequence variants. Further functional studies are necessary to determine the functional significance of this missense mutation.
FOXO3 mutations and premature ovarian failure
Acknowledgements The authors would like to thank the POF patients for their involvement in this study and also the clinicians who provided these patients. This work was supported by the National Basic Research Program of China (2010CB945102), National Natural Science Foundation of China (30973197), the National Science & Technology Pillar Program of China (2008BAH24B05), and National Infrastructure Program of Chinese Genetic Resources (2006DKA21300).
References Aittomaeki, K., Dieguez Lucena, J., Pakarinen, P., et al., 1995. Mutation in the follicle-stimulating hormone receptor gene causes hereditary hypergonadotropic ovarian failure. Cell 82, 958–959. Bione, S., Sala, C., Manzini, C., et al., 1998. A human homologue of the Drosophila melanogaster diaphanous gene is disrupted in a patient with premature ovarian failure: evidence for conserved function in oogenesis and implications for human sterility. Am. J. Hum. Genet. 62, 533–541. Board, J., Redwine, F., Moncure, C., Frable, W., Taylor, J., 1979. Identification of differing etiologies of clinically diagnosed premature menopause. Am. J. Obstet. Gynecol. 134, 936–944. Castrillon, D., Miao, L., Kollipara, R., Horner, J., DePinho, R., 2003. Suppression of ovarian follicle activation in mice by the transcription factor Foxo3a. Science 301, 215–218. Coulam, C., Adamson, S., Annegers, J., 1986. Incidence of premature ovarian failure. Obstet. Gynecol. 67, 604–606. Dixit, H., Rao, L.K., Padmalatha, V.V., et al., 2006. Missense mutations in the BMP15 gene are associated with ovarian failure. Hum. Genet. 119 (4), 408–415. Elvin, J.A., Matzuk, M.M., 1998. Mouse models of ovarian failure. Rev. Reprod. 3 (3), 183–195. Gallardo, T.D., John, G.B., Bradshaw, K., et al., 2008. Sequence variation at the human FOXO3 locus: a study of premature ovarian failure and primary amenorrhea. Hum. Reprod. 23, 216–221. Hosaka, T., Biggs, W., Tieu, D., et al., 2004. Disruption of forkhead transcription factor (FOXO) family members in mice reveals their functional diversification. Proc. Natl. Acad. Sci. USA 101, 2975–2980. John, G.B., Shirley, L.J., Gallardo, T.D., Castrillon, D.H., 2007a. Specificity of the requirement for Foxo3 in primordial follicle activation. Reproduction 133, 855–863.
503 John, G.B., Shirley, L.J., Gallardo, T.D., Castrillon, D.H., 2007b. Specificity of the requirement for Foxo3 in primordial follicle activation. Reproduction 133, 855–863. Kalataridou, S., Nelson, L., 2000. Premature ovarian failure is not premature menopause. Ann. NY Acad. Sci. 900, 393–402. Luborsky, J., Meyer, P., Sowers, M., Gold, E., Santoro, N., 2003. Premature menopause in a multi-ethnic population study of the menopause transition. Hum. Reprod. 18, 199–206. McGee, E., Hsueh, A., 2000. Initial and cyclic recruitment of ovarian follicles. Endocr. Rev. 21, 200–214. McKinlay, S., Jefferys, M., Thompson, B., 1972. An investigation of the age at menopause. J. Biosoc. Sci. 4, 161–173. Mei, Yang, Zhang, Yiru, Yamamoto, Kazuo, Xie, Wei, Mak, Tak W., You, Han, 2009. FOXO3a-dependent regulation of Pink1 (Park6) mediates survival signaling in response to cytokine deprivation. PNAS 106 (13), 5153–5158. Russell, P., Bannatyne, P., Shearman, R., Fraser, I., Corbett, P., 1982. Premature hypergonadotropic ovarian failure: clinicopathological study of 19 cases. Int. J. Gynecol. Pathol. 1, 185. Tilly, J., 2001. Commuting the death sentence: how oocytes strive to survive. Nat. Rev. Mol. Cell Biol. 2, 838. Timmreck, L., Reindollar, R., 2003. Contemporary issues in primary amenorrhea. Obstet. Gynecol. Clin. North Am. 30, 287–302. van Kasteren, Y., Hundscheid, R., Smits, A., Cremers, F., van Zonneveld, P., Braat, D., 1999. Familial idiopathic premature ovarian failure: an overrated and underestimated genetic disease? Hum. Reprod. 14, 2455–2459. Vegetti, W., 1998. Inheritance in idiopathic premature ovarian failure: analysis of 71 cases. Hum. Reprod. 13, 1796–1800. Wang, Binbin, Zhou, Sirui, Yang, Ze, et al., 2008. Genetic analysis of tumor necrosis factor-a (TNF-a) G-308A and Saitohin Q7R polymorphisms with Alzheimer’s disease. J. Neurol. Sci. 270 (1– 2), 148–151. Watkins, W., Umbers, A., Woad, K., et al., 2006. Mutational screening of FOXO3A and FOXO1A in women with premature ovarian failure. Fertil. Steril. 86, 1518–1521. Wyshak, G., Frisch, R., 1982. Evidence for a secular trend in age of menarche. N. Engl. J. Med. 306, 1033–1035. Zarate, A., Karchmer, S., Gomez, E., Castelazo-Ayala, L., 1970. Premature menopause. A clinical, histologic and cytogenetic study. Am. J. Obstet. Gynecol. 106, 110–114. Declaration: The authors report no financial or commercial conflicts of interest. Received 6 May 2009; refereed 23 June 2009; accepted 17 December 2009.