| BackgroundBPES is a rare autosomal dominant genetic disease,and there are two clinical types of BPES.Type I BPES patient demonstrates eyelid abnormalities and accompanied with infertility in affected females,while in type Ⅱ BPES only with eyelid abnormalities.The affected women with infertility is the main difference between two types,but because the genotype and phenotype is not completely consistent,the reason of the affected female infertility pathogenesis is unclear.Till now,there has no effective method to treat infertility accompanied with type I BPES.Although the eye ptosis of BPES can be corrected by the eyelid surgery and their appearance can be improved,but type I BPES affected female patients with infertility suffered great pain from body and spirit.In addition,the clinical level of phenotypic variation in patients with BPES is bigger,to the diagnosis of clinical doctors also brought considerable difficulties.According to Mendelian inheritance law,the next generation of patients with recurrence risk is 50%,so it is very necessary to make genetic counseling for the BPES patients.The most common reasons to cause BPES are FOXL2 gene mutations.FOXL2 gene is considered the first important autosomal gene which play an important role in maintaining the follicle development and normal ovarian function.FOXL2 gene is mainly expressed in medium and small follicular phase of granulosa cells and other cells,regulating the promoter transcriptional activity of downstream target genesCYP11A1,CYP19A1,CCND2.These genes involve in follicular granulosa cell proliferation and differentiation and play an important role in the processof the formation of ovarian steroid hormones.ObjectiveThe aim of this study is to performgene mutation screening for a FOXL2 BPES infertility family,and the new mutation function prediction and the function experiment in vitro and to explore the correlation between gene mutations and infertility patients.Thus the infertility BPES patients can obtain clinical diagnosis and genetic counseling.MethodsMutation screening on the exon region of FOXL2 was performed in a Chinese family with type I BPES and 223 controls in reproductive hospital affilicated to Shandong university with PCR and DNA sequencing technology.Subsequently,protein function prediction was assessed with Polyphen-2,SIFT and PROVEN software.Promoter transcriptional activity of CYP11A1,CYP19A and CCND2 gene was detected with the dual luciferase report system by cotransfected HEK293 cells and the protein expression and cellular localization function experiment was detected by cell immunofluorescence technique.ResultsThe two patients in this pedigree were accord with the clinical diagnosis criteria of type I BPES and the affected females were both infertility.The inheritance pattern was in line with the single gene autosomal dominant.A novel mutation was found in the base of 188 appeared in the template strand which is a hybrid peak thymine mutations for adenine for new mutation c.188 T>A and codes from the ATC to AAC(p.I le63Asn,p.I63N).This mutation located in the upstream of FKH structure domain and the heterozygous mutation of isoleucine was highly conserved in all kinds of mammals.Protein functional prediction softwares reminder that the mutation has damage function.This novel mutation was reported for the first time in the world.In vitro cotransfection HEK293 cells function experiments confirmed that the missense mutation p.I63N FOXL2 protein affected promoter transcriptional activity of downstream target gene CYP11A1,CYP19A1 and CCND2.The mutant protein slightly diminished the promoter transcriptional activity of CYP11A1(P>0.05),significantly impaired the repression activity on CYP19A1 promoter(P<0.05)indicating retained minimal repressor activity on CYP19A1while significantly enhanced the CCND2 promoter activity(P<0.05)indicating that not only is p.I63N mutation FOXL2’s repressor activity lost but p.I63N mutation may function as an enhancer of CCND2 promoter activity.Because of the heterozygous status of the patient,the dominant negative effect of the mutant over WT protein was assessed.Co-transfection of mutant with WT had no dominant-negative effect on CYPl1A1、CYP19A1and CCND2 promoter.Conclusions1.An infertility family pedigree was analysed andtheir symptoms were in accordance with BPES clinical diagnosis.The patients who have typical eyelid abnormalities and accompanied with infertility belongs to type Ⅰ BPES.2.A novel mutation was found in the base of 188 appeared in the template strand which is a hybrid peak thymine mutations for adenine for new mutation c.188 T>A and codes changing from ATC to AAC(p.Ile63Asn,p.I63N).This mutation located in the upstream of FKH DNA structure domain.The novel FOXL2 mutation with functional deficiency was identified in a BPES Chinese family and amplified the spectrum of FOXL2 mutations.3.The protein functional prediction software indicated that p.I63N was deletious effect on the protein function.4.Functional experiment in vitro showed that I63N influenced the promoter transcriptional inhibition activity of the downstream target gene CYP19A1 and CCND2 by the mechanism of haploinsufficiency.The p.I63N mutation resulted in abnormal granular cell function and steroid hormone generated,which may reveal possible mechanisms of female patients with infertility the BPES genealogy and provide experiment basis for the further study of BPES.’... |
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