Abstract: Objective To investigate the effect of p.Y225X mutation on FOXL2, and explore the potential mechanism of Blepharophimosis-ptosis-epicanthus-inversus syndrome (BPES). Design Experimental study. Participants The wild-type and p.Y225X mutant FOXL2 genes. Method Wild-type and mutant sequence of FOXL2 genes were amplified, and then cloned into green fluorescence protein expression vector (pEGFP-N1) to construct GFP labeled expression vectors, which were later transfected into COS7 cells.  The confocal microscope was used to locate FOXL2 in COS7 cells and compare the difference between them. Wild-type and mutant FOXL2 genes were cloned into pcDNA3.1-myc-his(-)B vector, then transfected into HEK-293T and KGN cells. Proteins were extracted to make sure that wild-type and mutant FOXL2 were expressed after transient transfection. And then total RNA and protein were extracted to analyze OSR2 and StAR expression on transcriptional and protein levels by RT-PCR and Western blotting. Main Outcome Measures GFP locations,  target RNA and protein expression. Results The expression plasmids of wild-type and mutant FOXL2 labeled by GFP and myc/his flags were constructed successfully. The expression of FOXL2 was identified by Western blot assay in transfected HEK-293T and KGN cells． Under the confocal microscope, wild type FOXL2 evenly located in the nucleus, while the mutant FOXL2 located in both cytoplasm and nucleus. Furthermore, wild-type FOXL2 inhibited StAR in transcriptional and protein levels in KGN cells, while this inhibition effect in p.Y225X mutant were dramatically attenuated. Besides, wild-type FOXL2 enhanced OSR2 transcription and expression in HEK-293T cells, while the promotion of OSR2 expression in FOXL2 mutants was also reduced. Conclusion p.Y225X mutation, leading to FOXL2 dislocation in the cytoplasm, attenuates its regulation function, disturbs the expression of target genes, OSR2 and StAR, which contribute to impaired development of eyelid and ovary, and probably eventually lead to type I BPES.

Key words: Blepharophimosis-ptosis-epicanthus-inversus syndrome, FOXL2, mutation