Mutational screening of FOXO3A and FOXO1A in women with premature ovarian failure

FOXO3A and FOXO1A are excellent candidate genes for the development of premature ovarian failure and have not been analyzed previously in POF patients. Potentially causal mutations in FOXO3A (2/90; 2.2%) and FOXO1A (1/90; 1.1%) were identified in POF patients; however, the pathological role of these mutations will be determined only by screening increased numbers of patients and controls, or by functional studies. FOXO3A and FOXO1A are excellent candidate genes for the development of premature ovarian failure and have not been analyzed previously in POF patients. Potentially causal mutations in FOXO3A (2/90; 2.2%) and FOXO1A (1/90; 1.1%) were identified in POF patients; however, the pathological role of these mutations will be determined only by screening increased numbers of patients and controls, or by functional studies. Premature ovarian failure (POF) or premature menopause is defined as ovarian failure before the age of 40 years and affects approximately 1% of women, although this varies depending on ethnicity (1Coulam C.B. Adamson S.C. Annegers J.F. Incidence of premature ovarian failure.Obstet Gynecol. 1986; 67: 604-606PubMed Google Scholar, 2Luborsky J.L. Meyer P. Sowers M.F. Gold E.B. Santoro N. Premature menopause in a multi-ethnic population study of the menopause transition.Hum Reprod. 2003; 18: 199-206Crossref PubMed Scopus (281) Google Scholar). The diagnosis of POF is confirmed by two blood tests, at least a month apart, to measure FSH. The causes of POF are diverse and largely idiopathic. It has been estimated that up to 30% of POF cases may result from genetic causes (3Vegetti W. Grazia Tibiletti M. Testa G. de Lauretis Y. Alagna F. Castoldi E. et al.Inheritance in idiopathic premature ovarian failure: analysis of 71 cases.Hum Reprod. 1998; 13: 1796-1800Crossref PubMed Scopus (118) Google Scholar, 4van Kasteren Y.M. Hundscheid R.D. Smits A.P. Cremers F.P. van Zonneveld P. Braat D.D. Familial idiopathic premature ovarian failure: an overrated and underestimated genetic disease?.Hum Reprod. 1999; 14: 2455-2459Crossref PubMed Scopus (119) Google Scholar); however, few gene targets have been positively correlated with POF, which necessitates further screening of candidate genes. With increased understanding of the genes and pathways involved in POF, screening will enable early detection or prediction of those who are at risk for developing POF. Future management and treatments may restore fertility to these women or at least provide the information to empower life choices that maximize fertile years. FOXL2 was investigated after the discovery that mutations in this gene cause a rare syndrome called blepharophimosis-ptosis-epicanthus-inversus syndrome (BPES). This syndrome is characterized by a distinctive eyelid phenotype that is associated with POF in type I BPES, whereas the eyelid phenotype occurs in isolation in type II BPES (5De Baere E. Dixon M.J. Small K.W. Jabs E.W. Leroy B.P. Devriendt K. et al.Spectrum of FOXL2 gene mutations in blepharophimosis-ptosis-epicanthus inversus (BPES) families demonstrates a genotype–phenotype correlation.Hum Mol Genet. 2001; 10: 1591-1600Crossref PubMed Scopus (230) Google Scholar). We since have found evidence for FOXL2 involvement in isolated POF (6Harris S.E. Chand A.L. Winship I.M. Gersak K. Aittomaki K. Shelling A.N. Identification of novel mutations in FOXL2 associated with premature ovarian failure.Mol Hum Reprod. 2002; 8: 729-733Crossref PubMed Scopus (153) Google Scholar). Two other members of the forkhead transcription factor family, FOXO1A and FOXO3A, both are expressed in the ovary and are thought to play roles in ovarian development and function and therefore were chosen for mutation screening in POF patients. The Foxo3a-knockout mouse exhibited a POF phenotype that is characterized by early depletion of functional ovarian follicles, preceded by global follicular activation leading to oocyte death (7Castrillon D.H. Miao L. Kollipara R. Horner J.W. DePinho R.A. Suppression of ovarian follicle activation in mice by the transcription factor Foxo3a.Science. 2003; 301: 215-218Crossref PubMed Scopus (705) Google Scholar). The FoxO1a protein was found to be a key regulator of the G1/S transition in granulosa cells, therefore influencing their growth during development of the ovarian follicle (8Cunningham M.A. Zhu Q. Hammond J.M. FoxO1a can alter cell cycle progression by regulating the nuclear localization of p27kip in granulosa cells.Mol Endocrinol. 2004; 18: 1756-1767Crossref PubMed Scopus (47) Google Scholar). A cohort of 30 POF patients from New Zealand and 60 from Slovenia were recruited by the Departments of Obstetrics and Gynaecology in Auckland, New Zealand (University of Auckland) and Ljubljana, Slovenia (University Medical Centre), after institutional review board approval. Premature ovarian failure was diagnosed as described elsewhere (9Shelling A.N. Burton K.A. Chand A.L. van Ee C.C. France J.T. Farquhar C.M. et al.Inhibin: a candidate gene for premature ovarian failure.Hum Reprod. 2000; 15: 2644-2649Crossref PubMed Scopus (147) Google Scholar). Thirty normal control samples were obtained from each of the general populations of New Zealand and Slovenia. The coding regions of FOXO3A (NM_001455) and FOXO1A (NM_002015) were amplified by polymerase chain reaction using genomic DNA extracted from blood (9Shelling A.N. Burton K.A. Chand A.L. van Ee C.C. France J.T. Farquhar C.M. et al.Inhibin: a candidate gene for premature ovarian failure.Hum Reprod. 2000; 15: 2644-2649Crossref PubMed Scopus (147) Google Scholar) and were screened by denaturing high-performance liquid chromatography (dHPLC) or DNA sequencing. The Primer Select module in the DNAStar computer program from Lasergene 1994 (DNAStar Inc., Madison, WI) was used to design primers. Standard or touchdown (10Don R.H. Cox P.T. Wainwright B.J. Baker K. Mattick J.S. “Touchdown” PCR to circumvent spurious priming during gene amplification.Nucleic Acids Res. 1991; 19: 4008Crossref PubMed Scopus (2234) Google Scholar) polymerase chain reaction was performed (details available on request from corresponding author). Denaturing HPLC analysis was performed on the dHPLC 2100 WAVE DNA Fragment Analysis System and DNASep column (Transgenomic, Omaha, NE). Polymerase chain reaction products to be sequenced were purified with the High Pure PCR Product Purification Kit (Roche Diagnostics GmbH, Mannheim, Germany). Sequencing reactions were performed using the ABI Prism Big Dye Terminator Sequencing Kit, version 3.1, under standard conditions, and then the products were separated on an ABI Prism 3100 Genetic Analyzer (PE Biosystems, Foster City, CA). Sequencing reactions were performed at the DNA Sequencing Facility, Centre for Genomics and Proteomics, University of Auckland. To confirm the sequencing data and screen the 120 control chromosomes for variant 3A.6 (1262C>T), variant 3A.7 (1517G>A), and variant 1A.3 (251C>T), the restriction enzymes Xcm1, BsaW1, and PstI (New England BioLabs Inc, Beverly, MA) were used, respectively, for restriction fragment length analysis (details available on request from corresponding author). The 95% confidence intervals (CIs) were calculated using the R commands “binom.test(2,90)” and “binom.test(1,90).” The CIs were constructed using the Clopper and Pearson procedure. Screening revealed eight variations in FOXO3A, which were denoted variants 3A.1–3A.8 (Table 1) and were numbered in accordance with current mutation nomenclature recommendations (11den Dunnen J.T. Antonarakis S.E. Mutation nomenclature extensions and suggestions to describe complex mutations: a discussion.Hum Mutat. 2000; 15: 7-12Crossref PubMed Scopus (1503) Google Scholar). The most significant of these were variants 3A.6 and 3A.7. The only FOXO3A variant previously found in the National Center for Biotechnology Information (NCBI) Single-Nucleotide Polymorphism (SNP) database was variant 3A.1 (rs11757217).TABLE 1Summary of mutation screening results.VariantExon {Intron}Mutation descriptionFrequency in POF patient chromosomes n (%)methodFrequency in controlchromosomes n (%)methodNucleotide changeAmino acid changeResultFOXO3A mutation screening results3A.11c.159C>T (hetero + homo)p.A53Ano change19/120 (15.8)asequencing18/120 (15.0)asequencing3A.21c.419C>T (hetero + homo)p.A140Vconservative5/120 (4.2)asequencing13/120 (10.8)asequencing3A.31 (forkhead domain)c.504C>Tp.R168Rno change2/120 (1.7)asequencing0/120 (0.0)asequencing3A.4{1}A>Gn/an/a1/120 (0.8)asequencingNS3A.52c.747G>Ap.R249Rno change1/120 (0.8)bdenaturing high performance liquid chromatographyNS3A.62c.1262C>Tp.S421Lnon conservative1/180 (0.6)asequencing0/120 (0.0)crestriction fragment length polymorphism.3A.72c.1517G>Ap.R506Hconservative1/180 (0.6)asequencing0/120 (0.0)crestriction fragment length polymorphism.3A.82c.1857C>Tp.S619Sno change2/120 (1.7)bdenaturing high performance liquid chromatographyNSFOXO1A mutation screening results1A.11c.−30C>Tn/a (5′ UTR)n/a1/120 (0.8)bdenaturing high performance liquid chromatography0/120 (0.0)bdenaturing high performance liquid chromatography1A.21c.244G>Ap.D82Nconservative5/120 (4.2)bdenaturing high performance liquid chromatography4/120 (3.3)bdenaturing high performance liquid chromatography1A.31c.251C>Tp.P84Lconservative1/180 (0.6)bdenaturing high performance liquid chromatography0/120 (0.0)bdenaturing high performance liquid chromatographycrestriction fragment length polymorphism.1A.41 (forkhead domain)c.507C>Tp.I169Ino change1/120 (0.8)bdenaturing high performance liquid chromatographyNS1A.52c.921T>Cp.F307Fno change1/120 (0.8)bdenaturing high performance liquid chromatographyNS1A.62c.999C>Tp.T333Tno change1/120 (0.8)bdenaturing high performance liquid chromatographyNSNote: UTR = untranslated region; n/a = not applicable; NS = not screened. methodMutation detection method;Watkins. FOXO3A and FOXO1A screening in POF. Fertil Steril 2006.a sequencingb denaturing high performance liquid chromatographyc restriction fragment length polymorphism. Open table in a new tab Note: UTR = untranslated region; n/a = not applicable; NS = not screened. methodMutation detection method; Watkins. FOXO3A and FOXO1A screening in POF. Fertil Steril 2006. Variant 3A.6 involves a nonconservative amino-acid change from serine to leucine at position 421, resulting from the heterozygous nucleotide substitution 1262C>T. This was found in one Slovenian POF patient and was not present in any of the 120 control chromosomes. The Slovenian patient had primary amenorrhea and a high FSH level of 123 IU/L. The parents of this Slovenian patient were not screened for this mutation (they were deceased), but because the patient’s mother did not undergo POF, it is predicted to be a de novo mutation or to be inherited from the father, although nonpenetrance of mutations has been observed in some POF families (6Harris S.E. Chand A.L. Winship I.M. Gersak K. Aittomaki K. Shelling A.N. Identification of novel mutations in FOXL2 associated with premature ovarian failure.Mol Hum Reprod. 2002; 8: 729-733Crossref PubMed Scopus (153) Google Scholar, 9Shelling A.N. Burton K.A. Chand A.L. van Ee C.C. France J.T. Farquhar C.M. et al.Inhibin: a candidate gene for premature ovarian failure.Hum Reprod. 2000; 15: 2644-2649Crossref PubMed Scopus (147) Google Scholar). Serine residues are known to be sites of phosphorylation in the FOXO proteins, although phosphorylation of serine at position 421 (variant 3A.6) has not been reported (12Brunet A. Bonni A. Zigmond M.J. Lin M.Z. Juo P. Hu L.S. et al.Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor.Cell. 1999; 96: 857-868Abstract Full Text Full Text PDF PubMed Scopus (5333) Google Scholar). However, the change to a leucine residue is likely to induce a conformational change in the protein, which could have considerable repercussions, including effects on cellular events such as DNA binding and nuclear localization. Nuclear-cytoplasmic shuttling defects and/or defective DNA binding of FOXO3A would affect its ability to bind to promoter regions and to control the expression of target genes. Most of these target genes are thought to be proapoptotic, such as p27 (kip1), fas, and bim (12Brunet A. Bonni A. Zigmond M.J. Lin M.Z. Juo P. Hu L.S. et al.Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor.Cell. 1999; 96: 857-868Abstract Full Text Full Text PDF PubMed Scopus (5333) Google Scholar, 13Gilley J. Coffer P.J. Ham J. FOXO transcription factors directly activate bim gene expression and promote apoptosis in sympathetic neurons.J Cell Biol. 2003; 162: 613-622Crossref PubMed Scopus (542) Google Scholar, 14Chandramohan V. Jeay S. Pianetti S. Sonenshein G.E. Reciprocal control of Forkhead box O 3a and c-Myc via the phosphatidylinositol 3-kinase pathway coordinately regulates p27Kip1 levels.J Immunol. 2004; 172: 5522-5527Crossref PubMed Scopus (70) Google Scholar, 15Ghosh Choudhury G. Lenin M. Calhaun C. Zhang J.H. Abboud H.E. PDGF inactivates forkhead family transcription factor by activation of Akt in glomerular mesangial cells.Cell Signal. 2003; 15: 161-170Crossref PubMed Scopus (24) Google Scholar). Variant 3A.7 consisted of a heterozygous 1517G>A transition that resulted in a conservative change at amino-acid position 506, from arginine to histidine. It was found in one New Zealand POF patient and in none of the controls. This patient experienced POF at age 36 years (although menstruation was erratic from age 32 years), and her mother also had POF at age 39 years. Family members were not screened, because the patient was lost for follow-up. Six variants were identified in FOXO1A and were denoted variants 1A.1–1A.6 (Table 1), with the most significant of these being variant 1A.3. None of the FOXO1A variants described (Table 1) were found in the NCBI SNP database. Variant 1A.3, which consisted of a heterozygous nucleotide change (251C>T), is predicted to cause a conservative amino-acid change from proline to leucine at amino-acid position 84. This was found in one Slovenian POF patient and none of the controls. The Slovenian patient had normal puberty, with menarche at age 13 years, but became amenorrheic at age 18 years, with an FSH level of 48.8 IU/L. The results that were obtained after screening the coding region of FOXO1A in this POF patient cohort and controls reveal weak evidence for the role of FOXO1A gene mutations in the etiology of POF. Although FOXO1A gene mutations are unlikely to be responsible for POF, this does not rule out the involvement of FOXO1A by other mechanisms. Regulation of FOXO1A by FSH has been suggested via phosphorylation by protein kinase B, and serum and glucocorticoid-inducible kinase, resulting in its nuclear exclusion (16Richards J.S. Sharma S.C. Falender A.E. Lo Y.H. Expression of FKHR, FKHRL1, and AFX genes in the rodent ovary: evidence for regulation by IGF-I, estrogen, and the gonadotropins.Mol Endocrinol. 2002; 16: 580-599Crossref PubMed Scopus (155) Google Scholar). It is interesting to note that women with POF have elevated levels of FSH (>40 IU/L). Perhaps this regulation is deficient in women with POF, and FSH fails to inhibit apoptosis and cell cycle arrest via nuclear exclusion of FOXO1A. In summary, 2 (2.22%; 95% CI, 0.27–7.8) of 90 POF patients screened have mutations in the coding region of FOXO3A, and 1 (1.11%; 95% CI, 0.03–6.04) of 90 has mutations in FOXO1A, which lead to amino-acid changes and are not present in the 60 controls screened. It must be noted that total gene deletions and translocations would not have been detected by the methods of direct DNA sequencing and dHPLC that were used in this study. Our cohort of POF patients screened in this study was relatively small at only 60, although 90 POF patients were screened for variant 3A.6, variant 3A.7, and variant 1A.3. Mutations in FOXO3A (2.2%) and FOXO1A (1.1%) were detected less commonly than were mutations in the related FOXL2 gene, with 8 (3.43%; 95% CI, 1.49–6.65) of 233 mutations found in our POF patient cohort (data not shown). The variants identified appear to be polymorphisms (because they are also found in controls); rare polymorphisms that do not change the amino acid (silent) and are therefore of limited significance unless they alter mRNA folding or change splicing; or rare substitution polymorphisms that change the amino acid, but occur at low frequency. In conclusion, although potentially causal mutations were identified in FOXO3A and FOXO1A, they were rare and of unknown significance. Their pathologic role only will be determined by screening increased numbers of patients and controls and/or by functional studies. We conclude that mutations that lead to amino-acid changes in the coding regions of FOXO3A and FOXO1A are not common mechanisms for POF.