Molecular Vision 2013; 19:2298-2305
Received 23 August 2013 | Accepted 14 November 2013 | Published 16 November 2013
The first three authors contributed equally to this work.
1Henan Eye Institute, Henan Eye Hospital, Henan, People’s Republic of China; 2Tianjin Key Lab of Ophthalmology and Visual Science, Tianjin Eye Institute, Tianjin, People’s Republic of China; 3Tianjin Eye Hospital, Clinical College of Ophthalmology, Tianjin Medical University, Tianjin, People’s Republic of China; 4Red Cross Jinan Bright Hospital, Shandong, People’s Republic of China; 5Affiliated Hospital of Jiyuan Medical College, JiYuan, Henan, People’s Republic of China
Correspondence to: Ningdong Li, Tianjin Eye Hospital, Tianjin, 300070, People’s Republic of China; Phone: 86+13001326471; FAX: 86+22+27313133; email: firstname.lastname@example.org
Purpose: To determine the genetic origin of disease in four Chinese families with blepharophimosis syndrome.
Methods: Four Han Chinese families with blepharophimosis syndrome were ascertained and patients underwent complete physical and ophthalmic examinations. Blood samples were collected and genomic DNA was extracted. Sequence analysis of the forkhead transcriptional factor 2 (FOXL2) gene was performed by direct sequencing and mutations were analyzed.
Results: Three mutations in FOXL2 were found in four families, including c.672_701dup30 (p.Ala224_Ala234dup10), c.313C>A (p.N105H), and c.430G>T (p.R144W). The c.672_701dup30 (p.Ala224_Ala234dup10) mutation was reported previously and predicted to result in expansions of the polyalanine tract. The mutations of c.313C>A (p. N105H) and c.430G>T (p.R144W) are two novel missense mutations.
Conclusions: Our study further supports the view that the expansion of the polyalanine tract is the hotspot of mutations within FOXL2. The two novel missense mutations detected in this study will expand the mutation spectrum of the FOXL2 gene and contribute to the research on the molecular pathogenesis of FOXL2.
Blepharophimosis syndrome (BPES, MIM #110100) is a rare autosomal dominant inheritable disorder with an estimated incidence of 1 in 50,000 births . It is a complex eyelid disease characterized by blepharophimosis, ptosis, epicanthus inversus, and telecanthus. Other ocular abnormities with BPES are strabismus, refractive error, and amblyopia in one or both eyes . There are two forms of BPES distinguished by the eyelid defects with (type I) or without (type II) premature ovarian failure . However, both types of BPES are caused by mutations of the forkhead transcriptional factor 2 (FOXL2) gene spanning approximately 2.7 kb on chromosome 3q23.
FOXL2 (OMIM 605597) is a member of the superfamily of Forkhead Box (Fox) transcription factors. It is a single-exon gene and encodes a protein with 376 residues containing a 100 amino acid DNA-binding forkhead domain and a polyalanine (poly-Ala) tract that is highly conserved in mammals. Currently, more than 100 different mutations have been identified in FOXL2, including frameshifts, insertions, nonsense mutations, and missense mutations [4,5]. Although some authors have found that mutations resulting in a predicted truncated protein before the poly-Ala tract are associated with BPES type I, whereas mutations resulting in an extended protein might lead to BPES type II, no clear genotype-phenotype correlation has been confirmed between mutations and BPES types because the genetic and clinical heterogeneity are found in and between families with BPES [6,7].
Here, we report three mutations identified in the FOXL2 gene from four Chinese families with BPES. Two of the mutations are missense, and one of them is indel.
Four Han Chinese families with BPES (Figure 1) were recruited through the Ophthalmic Genetics Clinic of the Tianjin Eye Hospital, Tianjin, China. Patients with BPES were diagnosed depending on the following clinical criteria: blepharophimosis, ptosis, epicanthus inversus, and telecanthus. Premature ovarian failure was defined as cessation of menses for a duration of ≥6 months before the age of 40 and a concentration of follicle-stimulating hormone of >40 IU/l.
After informed consent was received, 3 ml blood samples were taken from the blood vessels of 27 affected and 21 unaffected individuals from the four unrelated families, and DNA was extracted from blood lymphocytes according to the standard methods of protocol (Roche Biochemical, Inc. Penzberg, Germany). This study obtained institutional review board (IRB) approval from the Tianjin Eye Hospital and conformed to the tenets of the Declaration of Helsinki.
Amplification of the genomic fragments encompassing FOXL2 coding regions (NCBI human genome build 35.1, NC_000003 for gDNA, NM_023067 for messenger RNA, and NP_075555 for protein) was performed by PCR using the primers in Table 1. PCR was carried in 20 μl of standard PCR buffer containing 1.5 mM MgCl2, 0.2 mM of each dNTP, 0.5 μM of each primer, 1 U of Taq polymerase, and 50 ng of DNA. The amplification program was an initial 2 min denaturation at 98 °C, followed by 30 cycles of 30 s at 94 °C, 30 s at 55 °C, 1 min at 72 °C, and a final 7 min extension step at 72 °C. The PCR products were separated on a 2% agarose gel and purified with the QIAquick Gel Extraction Kit (Qiagen, Valencia, CA).
DNA sequencing analysis was performed using the BigDye Terminator Cycle Sequencing V3.1 kit on an ABI PRISM 3130 Genetic Analyzer (Applied Biosystems). Sequencing results were assembled and analyzed with the Seqman program of DNASTAR software (DNASTAR Inc., Madison, WI). Each heterozygous mutant was further validated with bidirectional sequencing in all relatives of the affected families and 100 normal healthy controls. Cloning sequencing was performed for the validation of multiple nucleotides insertions. A multiple sequence alignment was performed using the Clustal W algorithm in the software package. Mutation naming followed the nomenclature recommended by the Human Genomic Variation Society.
All patients in the four families were found to have typical features of BPES, including small palpebral fissures, ptosis of the eyelids, and epicanthus inversus (Figure 2). The clinical forms of families A and B were classified as BPES type II because the affected females in these two families did not have fertility problems. However, we were unable to evaluate the BPES types for families C and D because there were no female patients in family C, while the two affected females in family D were of pubertal age and had normal hormonal measurements and menstrual cycle (data not shown).
Sequence analysis of FOXL2 revealed three heterozygous mutations in four probands from the four families with BPES, including c.672_701dup30 (p.Ala224_Ala234dup10), c.313C>A (p. N105H), and c.430G>T (p.R144W; Figure 3). These mutations were also present in the affected patients from the corresponding families, but none of the unaffected family members or 100 normal control subjects examined carried these mutations.
The mutation of c.672_701dup30 (p.Ala224_Ala234dup10) was previously reported and was detected in both family A and B. It is a frameshift mutation within the coding region of the poly-Ala tract and was predicated to result in poly-Ala expansions. To examine the possibility of a common origin between family A and B, four single-nucleotide polymorphisms (rs56799713, rs1482976, rs12630172, and rs2291252) harboring the FOXL2 locus were genotyped in the probands of the families of A and B (Table 2). The haplotypes of these two probands were quite distinct. This suggested that the same mutation, c.672_701dup30 (p.Ala224_Ala234dup10), occurred independently in these two families rather than that they had descended from a common ancestor.
The heterozygous mutations of c.313C>A and c.430G>T were detected in family C and D, respectively. The C to T change at nucleotide position 313 would result in an asparagine at codon 105 substituted by a histidine (N105H), whereas the C to T change at the nucleotide position 430 would be predicted to result in an arginine at codon 144 replaced by a tryptophan residue (R144W).
The multiple sequence alignment of the FOXL2 protein shows that N105H and R144W were conserved among human, Pongo (chimpanzee), mouse, cow, chicken and zebrafish (Figure 4). The N105H and R144W amino acid changes produce PROVEAN scores of −4.943 and −6.766, respectively, as predicted by the PROVEAN tool. Both of them produce a position-specific independent counts ( PSIC) profile score of 1.0 calculated by the POLYPHEN2 program. These values indicate that the amino acid substitutions at N105 and R144 are likely to have a deleterious effect on the protein. These results suggest that the mutations of c.313C>A (p. N105H) and c.430G>T (p.R144W) are novel mutations in the FOXL2 gene.
As mentioned above, FOXL2 is a member of the winged helix/forkhead transcription factor family, and is mainly expressed in the developing eyelid and ovarian follicular granulosa cells. It encodes a protein with 376 amino acid residues, which contains a 100 amino acid DNA-binding forkhead domain from amino acid position 52 to 152 and an alanine-rich domain from amino acid position 221 to 234 downstream of the forkhead domain. Currently, 46 missense mutations have been found in the FOXL2 gene. Thirty-two of these are located in the forkhead domain [6-18], 1 in the alanine-rich domain, 10 in the region flanked by the forkhead and alanine-rich domains [9,12,17,19-22], and 3 downstream of the alanine-rich domain [6,8]. These missense mutations might lead to the BPES phenotype either by haploinsufficiency or through the dominant-negative effect. However, the exact molecular pathogenesis mechanism is not yet clear, nor can genetic and phenotype correlations be established for these missense mutations.
The missense mutation of N105H was located in the DNA-binding forkhead domain. It was caused by a C to A change at the nucleotide position 313. The mutation at N105 was reported previously by Crisponi et al. , and comprises a substitution of a serine (S) to an asparagine (N) due to an A to T change at the nucleotide position 314. The asparagine (N) at codon 105 is highly conserved among vertebrates and lower animals, and may provide key sites for N-linked glycosylation when occurring in an Asn-X-Ser/Thr form in the motif . Because glycosylation is important for both protein structure and function, substitution of the asparagine either by a serine or a histidine would be deleterious to the structure and function of the protein.
The R144W mutation was also located in the DNA-binding forkhead domain. R144 is highly conserved in vertebrates and low animals, and localized at the first position in the arginine/lysine (RK)-rich region (R144R145R146R147R148M149K150R151) at the C-terminal of the forkhead domain. The RK-rich region is conserved in several forkhead domains and might function as a nuclear localization signal. Mutations in the RK-rich region have been demonstrated to lead to mislocalization of the proteins . The missense mutation of R144W would affect the protein structure and function, as predicted by the PROVEAN tool and POLYPHEN2 program.
Previous studies showed that missense mutations inside or outside the forkhead domain could determine the expressivity of BPES. Mutations inside the forkhead domain might produce a more severe phenotype, while mutations outside it might produce a mild phenotype, except in an Indian BPES family carrying a missense mutation outside the forkhead domain but with a severe phenotype [15,26]. In this study, BPES families carrying either the N105H or R144W mutation had a typical clinical BPES phenotype, which further supported the possibility that the affected BPES individuals with missense mutations inside the forkhead domain might have a severe or typical phenotype.
The c.672_701dup30 mutation was reported previously and localized in the alanine-rich domain. Thus far, the role of the alanine-rich domain in FOXL2 has not yet been elucidated. It might be related to the function and structure of the protein because the number of alanine residues is strictly conserved among mammals. It was reported that the alanine-rich domain was a mutational “hotspot,” since the majority of the in-frame mutations occurred in this region and resulted in poly-Ala expansions. The possible mechanism for the in-frame changes in the poly-Ala tract of FOXL2 is the DNA hairpin–induced polymerase slippage that leads to the poly-Ala expansions . The poly-Ala expansions might lead to protein mislocalization and aggregation, which results mainly in BPES type II . Our study further supports that the poly-Ala domain is a mutational hotspot for these two independent families with BPES type II carried the same mutation of c.672_701dup30 in this region.
In summary, we identified three mutations—c.672_701dup30 (p.Ala224_Ala234dup10), c.313C>A (p.N105H), and c.430G>T (p.R144W)—in the FOXL2 gene in four Han Chinese families with BPES. The c.672_701dup30 was detected in the two families diagnosed as BPES type II, while the two missense mutations of c.313C>A and c.430G>T are novel mutations detected in the other two families with unknown types of BPES. Our data further support that poly-Ala expansions lead to BPES type II. For missense mutations, no predictions can yet be made because of a lack of sufficient information from families. The two novel missense mutations would expand the mutation spectrum of the FOXL2 gene and contribute to the study of the molecular pathogenesis of BPES.
The authors thank all participants in the families for their enthusiastic participations. This work is partially supported by the National Natural Science Foundation of China (Grant No.30940081 and No. 81170884), and Technology Foundation of Tianjin Health Bureau (Grant No. 09KR09).