Genetic and Functional Analyses of Two Missense Mutations in the Transcription Factor FOXL2 in Two Chinese Families with Blepharophimosis-Ptosis-Epicanthus Inversus Syndrome.

Abstract

Blepharophimosis-ptosis-epicanthus inversus syndrome (BPES) is a rare autosomal-dominant inherited disease. This study was carried out to investigate the genetic and functional changes within the FOXL2 gene in two Chinese families with BPES. DNA was extracted from the peripheral blood of 26 persons from two different Chinese BPES families (13 of which were affected), as well as 200 cataract patients to act as normal controls. FOXL2 gene mutations were detected using polymerase chain reaction (PCR) and DNA sequencing techniques. Bioinformatic analyses were performed to analyze the structures and functions of the mutant proteins. Wild-type and mutant FOXL2 genes were subcloned into pEGFP-N1 and pCDB vectors and then transfected into COS7 and HEK293T cell lines. We observed protein subcellular localization, and used quantitative real-time (qRT)-PCR and western blots to assess regulation of the target OSR2 gene. We detected two novel missense mutations, c.162G>T (p.Lys54Asn) and c.308G>A (p.Arg103His), in the FOXL2 gene; one in each of the study families. Bioinformatic analyses indicated no obvious differences between the wild-type and mutant protein structures. However, they did predict that the two mutations were likely damaging to protein function. We found that the two mutated proteins were both largely distributed within the nucleus and that there was little found in the cytoplasm. The OSR2 mRNA content decreased significantly when the plasmids carrying the c.162G>T and c.308G>A were transfected into COS7 and HEK293 cell lines, when compared to the empty and the wild-type FOXL2 carrier. Western blot analyses indicated, that after transfecting the c.162G>T mutation, the OSR2 protein level was relatively similar to the wild-type, but that the cells transfected with the c.308G>A mutation showed significantly decreased levels of the OSR2 protein. Our study broadens the BPES gene mutation spectrum and suggests a possible mechanism of action. It also provides reference data for the further studies of BPES.