Missense mutations in the forkhead domain of FOXL2 lead to subcellular mislocalization, protein aggregation and impaired transactivation

Center for Medical Genetics, Ghent University Hospital, 9000 Ghent, Belgium.
Human Molecular Genetics (Impact Factor: 6.39). 08/2008; 17(13):2030-8. DOI: 10.1093/hmg/ddn100
Source: PubMed


Mutations of the FOXL2 gene have been shown to cause blepharophimosis syndrome (BPES), characterized by an eyelid malformation associated with premature ovarian failure or not. Recently, polyalanine expansions and truncating FOXL2 mutations have been shown to lead to protein mislocalization, aggregation and altered transactivation. Here, we study the molecular consequences of 17 naturally occurring FOXL2 missense mutations. Most of them map to the conserved DNA-binding forkhead domain (FHD). The subcellular localization and aggregation pattern of the mutant FOXL2 proteins in COS-7 cells was variable and ranged from a diffuse nuclear distribution like the wild-type to extensive nuclear aggregation often in combination with cytoplasmic mislocalization and aggregation. We also studied the transactivation capacity of the mutants in FOXL2 expressing granulosa-like cells (KGN). Several mutants led to a loss-of-function, while others are suspected to induce a dominant negative effect. Interestingly, one mutant that is located outside the FHD (S217F), appeared to be hypermorphic and had no effect on intracellular protein distribution. This mutation gives rise to a mild BPES phenotype. In general, missense mutations located in the FHD lead to classical BPES and cannot be correlated with expression of the ovarian phenotype. However, a potential predictive value of localization and transactivation assays in the making of genotype-phenotype correlations is proposed. This is the first study to demonstrate that a significant number of missense mutations in the FHD of FOXL2 lead to mislocalization, protein aggregation and altered transactivation, and to provide insights into the pathogenesis associated with missense mutations of FOXL2 in human disease.

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    • "It is evident from some earlier results that over-expression of PABPN1, which lacks entire polyA in N-terminus, can also induce protein aggregation in vivo and in vitro (Chartier et al., 2006). Missense mutation in another polyA protein FOXL2 also results protein aggregation in vitro, which is completely independent of polyA expansion (Beysen et al., 2008). These observations definitely indicate the presence of some non-polyA residues in those proteins, which have intrinsic propensity towards aggregation. "
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    ABSTRACT: Presence of polyalanine (polyA) stretches in some proteins is found to be associated with their aggregation, which causes disorders in various developmental processes. In this work, inherent propensities towards aggregation of some residues, which are not part of the polyA stretches, have been identified by using the primary sequences of seven polyA proteins with the help of Betascan, PASTA and Tango programs and explored unambiguously. This provides a basis for proposing molecular mechanism of this type of aggregation. Reported suppression of aggregation of polyA proteins by chaperones like HSP40 and HSP70 is substantiated through molecular docking. The hydrophobic residues of identified aggregating region are found to be interacting with hydrophobic surface of chaperones. This suggests a crucial clue for possible way to inhibit the aggregation of such proteins. Copyright © 2014 Elsevier Ltd. All rights reserved.
    Preview · Article · Nov 2014 · Computational Biology and Chemistry
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    • "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. "
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    ABSTRACT: To determine the genetic origin of disease in four Chinese families with blepharophimosis syndrome. 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. 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. 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.
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    • "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. "
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    ABSTRACT: Blepharophimosis-ptosis-epicanthus inversus syndrome (BPES) is a developmental disease characterized by a complex eyelid malformation associated or not with premature ovarian failure (POF). BPES is essentially an autosomal dominant disease, due to mutations in the forkhead box L2 (FOXL2) gene, encoding a forkhead transcription factor. More than one hundred unique FOXL2 mutations have been described in BPES in different populations, many of which are missense mutations in the forkhead domain. Here, we report on a very severe form of BPES resulting from a missense mutation outside the forkhead domain. A clinical and molecular genetic investigation was performed in affected and unaffected members of an Iranian family with BPES. The FOXL2 coding region was sequenced in an index case. Targeted mutation testing was performed in 8 family members. We have identified a heterozygous FOXL2 missense mutation c.650C→G (p.Ser217Cys) co-segregating with disease in members of a three-generation family with BPES type II. Only few missense mutations have been reported outside the forkhead domain so far. They were all found in mild BPES, in line with in vitro studies demonstrating mostly normal localization and normal or increased transactivation properties of the mutant proteins. Unlike previous studies, affected members of the family studied here showed a severe BPES phenotype, with bilateral amblyopia due to uncorrected ptosis. This is the first study demonstrating a severe BPES phenotype resulting from a FOXL2 missense mutation outside the forkhead domain, expanding our knowledge about the phenotypic consequences of missense mutations outside the forkhead domain in BPES.
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