A heterozygous missense mutation of FOXL2
, p.Ser217Cys, was found to underly the phenotype here. Unlike most missense mutations, this mutation is located outside the forkhead domain between the forkhead domain and the polyalanine tract. All missense mutations outside the forkhead domain found in BPES are summarized in , with their phenotypic consequences, in silico predictions and in vitro assays, if any. In this study, affected patients presented with a severe form of BPES, leading to amblyopia and poor BCVA in individuals who did not undergo oculoplastic surgery. In females no signs of ovarian dysfunction were present, suggesting occurrence of type II BPES. This mutation was previously reported by Nallathambi et al. [13
] in an Indian BPES family, with a mild eyelid phenotype. A different mutation affecting the same residue p.Ser217Phe was found in a Belgian BPES family, in which a father and two pre-pubertal daughters displayed a similarly very mild BPES phenotype [12
]. Apart from the ocular findings, one of the siblings presented with alopecia areata, and the other one with growth hormone deficiency [12
]. In addition, Kumar et al. [19
] reported on missense mutation p.Tyr215Cys in an Indian family, in which all affected individuals exhibited mild to typical BPES with a normal visual acuity and normal ocular examination including mobility. All the patients except individual III-2 had chin elevation and telecanthus. Levator function was decreased in all the patients [19
]. From these previous studies was concluded that missense mutations outside the forkhead domain might lead to a rather mild BPES phenotype. In comparison with these previous mild cases however, the BPES phenotype of the affected individuals reported here is very severe. In those members who did not undergo successful oculoplastic surgery (I-2 and II-5), this led to bilateral amblyopia and poor BVCA.
Insights into the molecular effects of FOXL2
missense mutations contributing to genotype-phenotype correlations resulted from in vitro studies. First, the missense change p.Ser217Phe was shown to have no effect on subcellular localization of the FOXL2 protein and to increase its transactivation capacity on the DK3
promoter, suggesting hypermorphism [12
]. This might be in agreement with previous observations in FOXC1
-related phenotypes, in which haploinsufficiency of FOXC1 and hypermorphism (due to gene duplications) lead to similar but not identical anterior segment phenotypes [20
]. Reasoning the same way for FOXL2, this might explain why hypermorphic mutations such as p.Ser217Phe would give rise to a somewhat different, mild BPES phenotype. An equally mild BPES phenotype resulted from a different mutation in the same residue (p.Ser217Cys) of which neither localization nor transactivation was impaired [12
]. As said, we identified the p.Ser217Cys mutation in a severe form of BPES here. As this severe ocular phenotype was observed in all affected individuals of different generations, this might be attributed for instance to a cis
-effect of regulatory variants within or outside the transcription unit modulating the expression of mutant FOXL2
. Notably, as the origins of the families described by Nallathambi et al. [13
] and here are different (i.e., Indian versus Iranian, respectively), different haplotypes and regulatory contexts might be expected. As we could not substantiate this hypothesis so far by means of sequencing of the UTRs and by copy number screening of previously described SRO of regulatory deletions this remains speculative. However, several alternative possibilities, such as subtle sequence changes in regulatory elements, or promoter variations cannot be ruled out.
In addition, the phenotypic effect seems to be tissue-specific, as no ovarian involvement was observed in any of the affected females here. Indeed, this is in line with the study by Dipietromaria et al. [14
], in which a classification tool was developed for FOXL2
intragenic (missense and other) mutations, correlating the transcriptional activity of FOXL2
mutations on two different reporter promoters and the BPES type [14
]. Following this classification system, both p.Ser217Cys and p.Ser217Phe can be categorized as type II BPES mutations.
In conclusion, this study has expanded our knowledge about the phenotypic consequences of missense mutations outside the forkhead domain of FOXL2 by the identification of p.Ser217Cys, for the first time, in a very severe form of BPES in a family of Iranian descent.