Gene Mutation Detection And Functional Research Of Congenital Blepharophimosis-ptosis-epicanthus Inversus Syndrome(BPES)Families

PurposeCongenital blepharophimosis-ptosis-epicanthus inversus syndrome((BPES), is a relatively rare autosomal dominant inherited disease. The typical condition is characterized by eyelid ptosis, horizontal shortening of the palpebral fissure size, telecanthus, and epicanthus inversus. There are two subtypes of this syndrome. Type I is characterized by female infertility and hypogonadism with100%penetrance. Type II is transmitted both in affected males and females with a96.5%penetrance. FOXL2gene is the first candidate gene of the disease. It is not yet clear but the current hotspot of the mechanism of that how the FOXL2gene mutations affect the transcription function and result in the BPES. The purpose of this study:To collect two BPES families and analysis the clinical phenotype and genetic laws. To detect the FOXL2gene mutations and to predict the function of FOXL2gene using bioinformatics.To observe the mutated protein expression and functional changes in cells in vitro and explore the possible mechanism of the mutant FOXL2genes which lead to BPES.Method1In this study two BPES families were recruited from the department of the first Affiliated Hospital of the Medicine School of Zhejiang University. All the family members were received the history records and eye examinations. The clinical phenotypes and genetic laws were analysed.2Genomic DNA samples were extracted from the peripheral blood of the family members and normal controls. FOXL2gene mutations were detected using polymerase chain reaction (PCR) and DNA sequencing techniques. We applied pyrophosphate sequencing technology to determine the heterozygote content quantitatively, and analyze its correlation with phenorype severity. The impact of FOXL2mutations on protein structure and function were predicted using bioinformatics methods.3The wild and mutant plasmids were constructed after the amplifications were subcloned into the vector pEGFP-Nl. Then the plasmids were transfected into COS7cells in order to observe the protein subcellular localization. Simultaneously the other wild and mutant plasmids were also constructed after the amplifications were subcloned into the vector pCDB. These plasmids were transfected into the HEK-293cells and the regulation on target gene OSR2were measured using RT-PCR technology.Result1In Family I, there were3male and3female cases with clinical diagnosis of BPES distributed in four generations.4male and3female cases were collected among the three generations in Family II. In addition to the patient III:2characterized by mild ptosis and epicanthus inversus, all the other patients have obvious eyelid ptosis, horizontal shortening of the palpebral fissure size, telecanthus, and epicanthus inversus.All the female patients were fertile except an unmarried female in Family II.The diagnosis of the patients in both families were according to BPES II. The inherited model was identified as autosomal dominant according to the genealogy.2In Family I, sequencing of the complete coding sequence of the FOXL2gene revealed a heterozygous mutation (c.162G>T) leading to the replacement of lysine(Lys) by asparagine(Asn) at amino acid position54(AAG/AAT) in the FKH DNA-binding domain. In Family II, sequencing of the complete coding sequence of the FOXL2gene revealed a heterozygous mutation (c.308G>A) leading to the replacement of Argnine(Arg) by Hlstidine(His) at amino acid position103(CGC/CAC) in the FKH DNA-binding domain. These two were both novel mutations.Pyrophosphate sequencing found that the content of the mutated bases were lower than the mutated bases in patient III:2of Family I,the others were on the contrast.Bioinformatics analysis predicted that FOXL2(c.162g> T) and FOXL2(c.308g> A),these two mutations had no obvious effect on protein structure, but is likely to cause abnormal protein subcellular localization and damage its functions.3The plasmid pEGFP-Nl-FOXL2WT carrying the wild type FOXL2gene, FOXL2pEGFP-Nl-FOXL2-MUT1carrying c.162g> T mutant gene, and pEGFP-Nl-FOXL2-MUT2carrying c.308g> A mutant gene were constructed successfully. Then these three plasmids werer transfected into COS7cells, we found the wild protein localized in the nucleus, and the mutation proteins were both distributed in the nuclear, and little were found in the cytoplasm. The plasmidpCDB-FOXL2WT carrying the wild type FOXL2gene, pCDB-FOXL2-MUTl carrying c.162g> T mutant gene, andpCDB-FOXL2-MUT carrying c.308g> A mutant gene were constructed successfully. After transfected into HEK-293cells, wild type FOXL2increased the OSR2mRNA content with a significant difference than the control.While c.162g> T mutation FOXL2and c.308g> A mutation FOXL2decreased the OSR2mRNA content and the difference between these two mutation and the wild was significantly.Conclusions1In this study,we detected two novel missense mutations of the FOXL2gene leading to BPESâ…¡:c.162G>T and c.308G>A.2Eye malformation was lighter in Patients111:2of Family I.It may be related to the contents of the mutations bases were lower than the normal bases.3There were no obvious differences in the structure and subcellular localization of C.162g> T mutant FOXL2and c.308g> A mutant FOXL2protein with the wild type. But the two mutations reduce OSR2gene expression. We speculated that two family FOXL2mutations maybe by lowering the target genes OSR2regulation function and resulted in BPES II finally.4Our study broadened the BPES gene mutation spectrum, and revealed the possible mechanism of FOXL2mutations causing BPES. It provided the reference for the further study of congenital blepharophimosis-ptosis-epicanthus inversus syndrome.