The Clinical Treatment Analysis Of Blepharophimosis-ptosis-epicanthus Inversus Syndrome (BPES) And Novel Mutations Detected In BPES Patients With Functional Study

Objective1.To explore the choice of therapeutic strategy about one-stage and two-stage correction of blepharophimosis-ptosis-epicanthus inversus syndrome（BPES）,we analyzed the treatment of our BPES patients.2.The purpose of this study was to found novel FOXL2 mutations through screening our BPES patients by the way of collecting their peripheral blood samples.3.To verify the perniciousness of our novel FOXL2 mutations,we designed functional experiments through constructed plasmids contained novel mutations.Methods 1.This study is a clinical retrospective analysis of BPES surgeries including one-stage and two-stage correction cases from Jan 2008 to Apr 2016 in the 3rd department of Plastic Surgery Hospital,Chinese Academy of Medical Sciences and Peking Union Medical College.The preoperative and postoperative parameters were the horizontal palpebral fissure length（HPFL）,vertical palpebral fissure height（VPFH）,inner intercanthal distance（IICD）and the ratio of IICD to HPFL（IICD/HPFL）,and these parameters were compared using statistical methods.2.Genomic DNA was isolated from peripheral blood leucocytes from BPES patients,and DNA sequencing by Sanger method and Capillary Electrophoresis Fragment Analysis.Analysis of DNA sequencing used Chromas、DNASTAR and GeneMapper V3.5 softwares.3.The wild and mutant plasmids were constructed and they are wild plasmid pEGFP-N1-WT（WT）,contained mutant c.274G>T plasmid pEGFP-N1-M1（Ml）,contained mutant c.17₉3del plasmid pEGFP-N1-M2（M2）and contained mutant c.971C>T plasmid pEGFP-N1-M3（M3）,then we transferred pEGFP-N1-Control（Control）and above-mentioned plasmids into HEK293T cells,using fluorescence microscope and RT-PCR to detect the expression of FOXL2,OSR2 and a-SMA.Results1.This study involved a retrospective analysis of 45 patients aged from 10 months to 21 years of age,comprising 24 females and 21 males.Changes of HPFL,VPFH,IICD andⅡCD/HPFL between preoperative and postoperative values were statistically significant for both one-stage and two-stage correction（P<0.00）.The initial age received correction shows statistically-significant difference（P<0.00）between one-stage（mean,14 ± 5.48）and two-stage（mean,4.59 ±4.64）procedures.2.Twenty-four samples were enrolled in this study,including two Chinese families and eleven sporadic patients.All patients demonstrated the typical features of BPES.In family A and family B,we found that the c.672₇01dup30（p.Ala224_Ala234dup10）mutation existed in all patients.Five novel mutations of FOXL2 were found in sporadic patients,including c.274G>T（p.E92*）,c.17₉3del（p.E9Rfs*61）,c.971C>T（p.P324L）,c.931C>T（p.H311 Y）and c.655C>A（p.Q219K）.3.The experiment results show that the proteins encoded by M1（c.274G>T）.M2（c.17₉3del）and M3（c.971C>T）could result in the agglutinate phenomenon in the nuclear;M1（c.274G>T）、M2（c.17₉3del）could influence the expression of FOXL2 and OSR2;M3（c.971C>T）could only influence the expression of OSR2.Conclusion 1.In our study we found that one-stage and two-stage corrections all can achieve satisfactory outcomes.Patient’s initial age for treatment exert an influence on the choice of one-stage or two-stage correction.2.In our study we found five novel mutations including c.274G>T（p.E92*）,c.17₉3del（p.E9Rfs*61）,c.971C>T（p.P324L）,c.931C>T（p.H311 Y）and c.655C>A（p.Q219K）,our results expand the spectrum of FOXL2 mutations.3.The cellular function research shows that the mutations of c.274G>T（p.E92*）、c.17₉3del（p.E9Rfs*61）and C.971C>T（p.P324L）could be the pathogenic mutations for BPES.