Molecular Vision 2007; 13:108-113 <http://www.molvis.org/molvis/v13/a13/> Received 8 December 2006 | Accepted 25 January 2007 | Published 26 January 2007

Download Reprint

FOXL2 mutations in Chinese patients with blepharophimosis-ptosis-epicanthus inversus syndrome

Juan Wang,¹ Jinling Liu,² Qingjiong Zhang¹
 
(The first two authors contributed equally to this publication)
 
 

¹State Key Laboratory of Ophthalmology, and ²Eye Hospital, Zhongshan Ophthalmic Center, Sun Yat-Sen University, Guangzhou, China

³Contribute equally.

Correspondence to: Qingjiong Zhang, MD, PhD, Ophthalmic Genetics & Molecular Biology, Zhongshan Ophthalmic Center, Sun Yat-Sen University, 54 Xianlie Road, Guangzhou 510060, China; Phone: (+86)-20-87330422; FAX: (+86)-20-87333271; email: qingjiongzhang@yahoo.com

Abstract

Purpose: Blepharophimosis-ptosis-epicanthus inversus syndrome (BPES) is an autosomal dominant disorder where eyelid malformation associated with (type I) or without (type II) premature ovarian failure (POF). It is ascribed to mutations in the forkhead transcriptional factor2 (FOXL2) gene. The purpose of this study is to identify mutations in FOXL2 of Chinese patients with BPES.

Methods: Genomic DNA was prepared from leucocytes of peripheral venous blood. The coding regions and nearby intron sequences of FOXL2 were analyzed by cycle and cloning sequencing.

Results: Four mutations in FOXL2 were identified in six families, including c.241T>C, c.650C>G, c.804dupC, and c.672_701dup. Of the four, the c.241T>C and c.650C>G were novel and would result in missense changes of the encoded proteins, i.e., p.Tyr81His and p.Ser217Cys, respectively. The c.672_701dup (p.Ala224_Ala234dup) was detected in three families, indicating a mutation hotspot. The c.804dupC (p.Gly269ArgfsX265) mutation was found in one family.

Conclusions: Our results expand the spectrum of FOXL2 mutations and confirm the mutation hotspot in FOXL2.

Introduction

Blepharophimosis-ptosis-epicanthus inversus syndrome (BPES, OMIM 110100) is a rare autosomal dominant disease with a prevalence of about 1 in 50,000 [1]. Clinically, BPES has been divided into two subsets depending on the association of ocular malformation with (type I) or without (type II) premature ovarian failure (POF) [2]. Genetically, however, both types are caused by mutations in FOXL2, and a genotype-phenotype correlation has been described in some cases [3,4].

The human FOXL2 gene (OMIM 605597), located at 3q23, is a member of winged/forkhead transcription factor gene family [5]. This single-exon gene codes a protein with 376 residues, which consists of a DNA-binding forkhead domain (resudes 52-152) and a polyalanine domain (residues 221-234) [3,6,7]. A number of mutations in FOXL2 have been identified [8], including six novel mutations in the Chinese population [9-11].

Here, we report four mutations identified in six Chinese families with BPES. Two novel missense mutations were associated with BPES type II.

Methods

Patients

Thirteen probands with BPES from unrelated families were collected from the Zhongshan Ophthalmic Center. Informed consent conforming to the tenets of the Declaration of Helsinki and following the Guidance of Sample Collection of Human Genetic Diseases (863-Plan) by the Ministry of Public Health of China was obtained from all participated individuals or their guardians prior to the study. The diagnosis of BPES was based on criteria previously established [12] with exclusion of microphthalmia.

Mutation Analysis

Genomic DNA was prepared from leucocytes of peripheral venous blood [13]. Amplification of the genomic fragments encompassing FOXL2 coding regions (NCBI human genome build 35.1, NC_000003 for gDNA, NM_023067 for mRNA, and NP_075555 for protein) was carried out by PCR using primers as follows: AF: 5'-CAG CGC CTG GAG CGG AGA G-3', AR: 5'-CTT GCC GGG CTG GAA GTG C-3', BF: 5'-GAC CCG GCC TGC GAA GAC A-3', BR: 5'-GGC CGC GTG CAG ATG GTG T-3', CF: 5'-CGC GGC CGC TGT GGT CAA G-3', CR: 5'-GCT GGC GGC GGC GTC GTC-3'. The sizes of the amplified DNA fragments are 545 bp, 517 bp, and 500 bp, respectively.

PCR amplification was carried out initially at 95 °C for 8 min, followed by 5 cycles at 94 °C for 40 s, at 68 °C for 40 s, at 72 °C for 40 s, then 5 cycles at 94 °C for 40 s, at 66 °C for 40 s, at 72 °C for 40 s, and a further 30 cycles at 94 °C for 40 s, at 64 °C for 40 s, at 72 °C for 40 s, and finally an elongation step at 72 °C for 5 min. Due to the high GC-rich nature of FOXL2, an additional 10% dimethylsulfoxide and 10% glycerol were added to the PCR mixture in order to successfully amplify the genomic fragments.

Direct sequencing of the PCR products was performed with an ABI BigDye Terminator Cycle Sequencing Kit v3.1 (ABI Applied Biosystem, Foster City, CA), using an ABI 3100 Genetic Analyzer. Sequencing results from patients as well as the FOXL2 consensus sequences from the NCBI Human Genome Database (NC_000003) were imported into the SeqManII program of the Lasergene package (DNAStar Inc., Madison, WI) and then aligned to identify variations. Each mutation was confirmed by bidirectional sequencing. Mutation description followed the nomenclature recommended by the Human Genomic Variation Society (HGVS).

Any variation detected in FOXL2 was further evaluated in available family members as well as in 100 normal controls by heteroduplex-SSCP analysis as described previously [14]. Two additional pairs of primers were used for heteroduplex-SSCP analysis. The sequence of these primers were: DF: 5'- CCG TAA GCG GAC TCG TGC-3', DR: 5'- AGT AGT TGC CCT TGC GCT C-3', EF: 5'- CGC ACT TCC AGC CCG GCA A-3', and ER: 5'- TGT GTA CGG CCC GTA CGA-3'.

In addition, one variation of insertions with multiple nucleotides that was found in three families was further analyzed by cloning sequencing. PCR products harboring this mutation were subcloned to pMD18-T Simple Vector (TaKaRa BIO, Japan) according to the manufacture's instructions. Clones with the mutant allele as well as the normal allele were selected by using heteroduplex-SSCP analysis. Sequence of the cloned fragment was identified by cycle sequencing as described above. Mutations were confirmed by sequencing three positive clones from each family. One mutation, c.241T>C, was further analyzed by PCR-RFLP analysis since the mutation creates a new FOKI site.

Results

All patients demonstrated typical features of BPES, including small palpebral fissures, ptosis of the eyelids, and epicanthus inversus (Figure 1). Upon complete sequencing analysis of FOXL2 for 13 probands with BPES, four heterozygous mutations were found in six probands, including c.241T>C, c.650C>G, c.804dupC, and c.672_701dup (Figure 2; Table 1). Of the four, c.241T>C and c.650C>G are novel. All four heterozygous mutations were further detected by heteroduplex-SSCP analysis, and one (c.241T>C) was further detected by FOKI digestion (Figure 3). These mutations were also present in affected patients from corresponding families but neither in unaffected individuals nor in 100 controls.

The c.241T>C (p.Tyr81His) mutation results in substitution of a charge-free tyrosine with a charge-positive basic hydrophilic histidine within the forkhead domain. The c.650C>G (p.Ser217Cys) mutation is located immediately upstream of the polyalanine domain. The tyrosine at position 81 and the serine at position 217 are well conserved in FOXL2 by ClustalW analysis of 11 orthologs from related vertebrate species (Figure 4).

Discussion

FOXL2 encodes a forkhead transcription factor containing a forkhead domain for DNA-binding and a polyalanine domain of uncertain function. Strong expression of FOXL2 has been found in eyelids [3,15], developing periocular muscles, and surrounding tissues [16,17]. Of the four mutations identified in this study, the c.241T>C affected the forkhead domain, while the other three (c.650C>G, c.804dupC, and c.672_701dup) were located upstream, within, and downstream of the polyalanine domain, respectively.

Missense mutations in FOXL2 reported so far usually occurred at the forkhead domain [9,17-19], except two, such as c.650C>T in a Belgian family [4] and c.644A>G in a five-generation family from south-India [20]. The clinical subtypes of the patients with the c.650C>T and c.644A>G mutations were unknown. The novel c.650C>G (p. Ser217Cys) mutation identified in Chinese family B occurred at the same site as that found in the Belgian family, which is located immediately upstream of the polyalanine domain. The serine at position 217 is well conserved in 11 orthologs (Figure 4). It has been shown that mutations affecting the polyalanine domain induce extensive nuclear and cytoplasmic protein aggregation [21,22]. Missense changes have been suggested to act as null allele leading to BPES phenotype due to haploinsufficiency [4] or dominant-negative effect [20,23].

It has been suggested that FOXL2 mutations truncating the protein led to BPES type I while those extending the mutant protein were associated with type II [3,4]. However, intra- and inter-family phenotypic variations have been found [3,4,19,24,25] so that this genotype-phenotype correlation might not be general [18,19,26]. The c.804dupC mutation has been shown to cause both types of BPES [4,19,25], and the c.672_701dup causing polyalanine expansion most likely leads to BPES type II [19]. Missense mutations have been associated with both BPES type I [17] and II [3,19]. The patients from families A and B in this study, with novel c.241T>C and c.650C>G mutations, respectively, had type II BPES. The c.650C>G mutation is the first mutation described that occurs immediately upstream of the polyalanine domain and associated with type II BPES. This may raise a possibility that the region containing the c.650C>G mutation is of importance for FOXL2 function.

The c.672_701dup (p.Ala224_Ala234dup) was found in families D, E, and F (Table 1), consistent with a mutation hotspot. To check the origin of the c.672_701dup mutation in three families (families D, E, and F in Figure 3), six SNPs (including rs13325788, rs2291252, rs28937885, rs7432551, rs28937884, and rs11924939) were analyzed (Table 2). The SNP at rs2291252 is different between patient II:1 from family D and patient III:1 from family E, which may suggest a different origin of the mutant allele. The mutation in family F is most likely a de novo event as BPES was not present in the patients' parents although the SNPs in the patient II:1 in family F were the same as that of II:1 in family D. It has been reported that 30% of the FOXL2 mutations lead to polyalanine expansion [19]. The c.672_701dup has been found in BPES families of Caucasian [4,19,27,28] and Asian origin [10,29].

In summary, we identified two novel and two known mutations in FOXL2 of six Chinese families with BPES. The two novel mutations are the first reported instances that were associated with BPES type II. Our results expanded the spectrum of FOXL2 mutations and confirmed the mutation hotspot in FOXL2.

Acknowledgements

The authors thank all patients and family members for their participation. This study was supported in part by the National 863 Plan of China (Z19-01-04-02 to QZ), National Natural Science Foundation of China (30572006 to QZ), and Foundation from the Ministry of Education of China (20050558073 to QZ).

References

1. Oley C, Baraitser M. Blepharophimosis, ptosis, epicanthus inversus syndrome (BPES syndrome). J Med Genet 1988; 25:47-51.

2. Zlotogora J, Sagi M, Cohen T. The blepharophimosis, ptosis, and epicanthus inversus syndrome: delineation of two types. Am J Hum Genet 1983; 35:1020-7.

3. Crisponi L, Deiana M, Loi A, Chiappe F, Uda M, Amati P, Bisceglia L, Zelante L, Nagaraja R, Porcu S, Ristaldi MS, Marzella R, Rocchi M, Nicolino M, Lienhardt-Roussie A, Nivelon A, Verloes A, Schlessinger D, Gasparini P, Bonneau D, Cao A, Pilia G. The putative forkhead transcription factor FOXL2 is mutated in blepharophimosis/ptosis/epicanthus inversus syndrome. Nat Genet 2001; 27:159-66.

4. De Baere E, Dixon MJ, Small KW, Jabs EW, Leroy BP, Devriendt K, Gillerot Y, Mortier G, Meire F, Van Maldergem L, Courtens W, Hjalgrim H, Huang S, Liebaers I, Van Regemorter N, Touraine P, Praphanphoj V, Verloes A, Udar N, Yellore V, Chalukya M, Yelchits S, De Paepe A, Kuttenn F, Fellous M, Veitia R, Messiaen L. Spectrum of FOXL2 gene mutations in blepharophimosis-ptosis-epicanthus inversus (BPES) families demonstrates a genotype--phenotype correlation. Hum Mol Genet 2001; 10:1591-600.

5. Carlsson P, Mahlapuu M. Forkhead transcription factors: key players in development and metabolism. Dev Biol 2002; 250:1-23.

6. Sullivan SA, Akers L, Moody SA. foxD5a, a Xenopus winged helix gene, maintains an immature neural ectoderm via transcriptional repression that is dependent on the C-terminal domain. Dev Biol 2001; 232:439-57.

7. Sutton J, Costa R, Klug M, Field L, Xu D, Largaespada DA, Fletcher CF, Jenkins NA, Copeland NG, Klemsz M, Hromas R. Genesis, a winged helix transcriptional repressor with expression restricted to embryonic stem cells. J Biol Chem 1996; 271:23126-33.

8. Beysen D, Vandesompele J, Messiaen L, De Paepe A, De Baere E. The human FOXL2 mutation database. Hum Mutat 2004; 24:189-93.

9. Or SF, Tong MF, Lo FM, Lam TS. Three novel FOXL2 gene mutations in Chinese patients with blepharophimosis-ptosis-epicanthus inversus syndrome. Chin Med J (Engl) 2006; 119:49-52.

10. Tang S, Wang X, Lin L, Sun Y, Wang Y, Yu H. Mutation analysis of the FOXL2 gene in Chinese patients with blepharophimosis-ptosis-epicanthus inversus syndrome. Mutagenesis 2006; 21:35-9.

11. Qian X, Shu A, Qin W, Xing Q, Gao J, Yang J, Feng G, He L. A novel insertion mutation in the FOXL2 gene is detected in a big Chinese family with blepharophimosis-ptosis-epicanthus inversus. Mutat Res 2004; 554:19-22.

12. Smith DW. Recognizable patterns of human malformation: genetic, embryologic, and clinical aspects. Major Probl Clin Pediatr 1970; 7:1-368.

13. Smith RJ, Holcomb JD, Daiger SP, Caskey CT, Pelias MZ, Alford BR, Fontenot DD, Hejtmancik JF. Exclusion of Usher syndrome gene from much of chromosome 4. Cytogenet Cell Genet 1989; 50:102-6.

14. Zhang Q, Minoda K. Detection of congenital color vision defects using heteroduplex-SSCP analysis. Jpn J Ophthalmol 1996; 40:79-85.

15. Small KW, Stalvey M, Fisher L, Mullen L, Dickel C, Beadles K, Reimer R, Lessner A, Lewis K, Pericak-Vance MA. Blepharophimosis syndrome is linked to chromosome 3q. Hum Mol Genet 1995; 4:443-8.

16. Cocquet J, De Baere E, Gareil M, Pannetier M, Xia X, Fellous M, Veitia RA. Structure, evolution and expression of the FOXL2 transcription unit. Cytogenet Genome Res 2003; 101:206-11.

17. Dollfus H, Stoetzel C, Riehm S, Lahlou Boukoffa W, Bediard Boulaneb F, Quillet R, Abu-Eid M, Speeg-Schatz C, Francfort JJ, Flament J, Veillon F, Perrin-Schmitt F. Sporadic and familial blepharophimosis -ptosis-epicanthus inversus syndrome: FOXL2 mutation screen and MRI study of the superior levator eyelid muscle. Clin Genet 2003; 63:117-20.

18. Udar N, Yellore V, Chalukya M, Yelchits S, Silva-Garcia R, Small K, BPES Consortium. Comparative analysis of the FOXL2 gene and characterization of mutations in BPES patients. Hum Mutat 2003; 22:222-8.

19. De Baere E, Beysen D, Oley C, Lorenz B, Cocquet J, De Sutter P, Devriendt K, Dixon M, Fellous M, Fryns JP, Garza A, Jonsrud C, Koivisto PA, Krause A, Leroy BP, Meire F, Plomp A, Van Maldergem L, De Paepe A, Veitia R, Messiaen L. FOXL2 and BPES: mutational hotspots, phenotypic variability, and revision of the genotype-phenotype correlation. Am J Hum Genet 2003; 72:478-87.

20. Kumar A, Babu M, Raghunath A, Venkatesh CP. Genetic analysis of a five generation Indian family with BPES: a novel missense mutation (p.Y215C). Mol Vis 2004; 10:445-9 <http://www.molvis.org/molvis/v10/a56/>.

21. 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; 41:932-6.

22. Moumne L, Fellous M, Veitia RA. Deletions in the polyAlanine-containing transcription factor FOXL2 lead to intranuclear aggregation. Hum Mol Genet 2005; 14:3557-64.

23. Harris SE, Chand AL, Winship IM, Gersak K, Aittomaki K, Shelling AN. Identification of novel mutations in FOXL2 associated with premature ovarian failure. Mol Hum Reprod 2002; 8:729-33.

24. Fokstuen S, Antonarakis SE, Blouin JL. FOXL2-mutations in blepharophimosis-ptosis-epicanthus inversus syndrome (BPES); challenges for genetic counseling in female patients. Am J Med Genet A 2003; 117:143-6.

25. Kosaki K, Ogata T, Kosaki R, Sato S, Matsuo N. A novel mutation in the FOXL2 gene in a patient with blepharophimosis syndrome: differential role of the polyalanine tract in the development of the ovary and the eyelid. Ophthalmic Genet 2002; 23:43-7.

26. Bell R, Murday VA, Patton MA, Jeffery S. Two families with blepharophimosis/ptosis/epicanthus inversus syndrome have mutations in the putative forkhead transcription factor FOXL2. Genet Test 2001; 5:335-8.

27. Ramirez-Castro JL, Pineda-Trujillo N, Valencia AV, Muneton CM, Botero O, Trujillo O, Vasquez G, Mora BE, Durango N, Bedoya G, Ruiz-Linares A. Mutations in FOXL2 underlying BPES (types 1 and 2) in Colombian families. Am J Med Genet 2002; 113:47-51.

28. Vincent AL, Watkins WJ, Sloan BH, Shelling AN. Blepharophimosis and bilateral Duane syndrome associated with a FOXL2 mutation. Clin Genet 2005; 68:520-3.

29. Cha SC, Jang YS, Lee JH, Kim HK, Kim SC, Kim S, Baek SH, Jung WS, Kim JR. Mutational analysis of forkhead transcriptional factor 2 (FOXL2) in Korean patients with blepharophimosis-ptosis-epicanthus inversus syndrome. Clin Genet 2003; 64:485-90.