Preliminary Study On The Mechanism Of Different Mutation Sites Of FOXL2 Gene Causing Primary Ovarian Insufficiency

BackgroundFOXL2 regulates downstream genes such as AMH,cyclin D2,steroidogenic acute regulator(St AR),and aromatase(Cyp19)to control the proliferation and differentiation of granulosa cells,thereby inhibiting the activation of primitive follicles to protect ovarian reserve function,and preventing the release of immature follicles.FOXL2 is also involved in regulating follicular statin(FST),gonadotropin releasing hormone receptor(Gn RHR),and follicle stimulating hormone β(Follitropin subunit beta,FSH β)Isohypophyseal gonadal axis related genes participate in the endocrine regulation of the pituitary gonadal axis.In the nucleus,FOXL2 is primarily expressed in the ovaries,eyelids,and pituitary gland,and is essential for the formation of follicles,the growth and upkeep of the ovary,the formation of muscles around the eyelids,and the growth of the pituitary gland.FOXL2 is characterized by narrow eyelid fissures,ptosis,and a transition to epicanthus syndrome(BPES).BPES can be divided into two types based on protein mutations: in patients with type I,in addition to ocular symptoms,women may develop POF,and men may be fertile and pass the gene on to the next generation;Type II patients usually have only eyelid symptoms,and both sexes can have children.At Guangzhou Medical University’s Reproductive Medicine Center,two ART patients were discovered to have BPES,an eyelid phenotype,and inadequate oocyte capture.Therefore,full exon sequencing revealed that they carried FOXL2 mutations: c.317T>C and c.855-871 dup.The patient had 2-3 oocytes captured during 2-3 ART cycles,with an increase in FSH(>10 IU/L)or a decrease in AMH(<1.2 ng/m L),and was not clinically diagnosed as POI.Existing studies have failed to correlate mutations with phenotypes.Therefore,based on the clinical manifestations of patients,it is proposed to increase everyone’s understanding of the clinical relevance of the two FOXL2 mutations and their impact on ovarian function by studying the changes in protein structure and location caused by this mutation type.Method1.Conduct clinical and biochemical data collection and genetic evaluation for all patients,and analyze genotype phenotype correlation.Evaluate the impact of ART on pregnancy outcomes.2.Extract the patient’s whole blood DNA,design primers,and locate the FOXL2 mutation site of the case through PCR product sequencing.3.The patient’s whole blood DNA was subjected to PCR,T-A cloning,and sequencing to verify the FOXL2 mutation type.4.Organize the basic clinical information of patients;Mutation Taster website database was used to predict protein structural changes and protein localization caused by FOXL2 mutations c.317T>C and c.855-871 dup.5.The expression plasmid of FOXL2 mutant gene was constructed and the effects of FOXL2 mutation on protein expression and localization in HEK-293 T and KGN cells were studied in vitro.Result1.Case A has a FOXL2 c.317T>C mutation.Clinically,due to high FSH and a small number of sinus follicles(2-3 in total on both sides),the IVF cycle is canceled.It is recommended to have spontaneous cohabitation,follow up without pregnancy,and wait for egg donation2.Case B has a FOXL2 c.855-871 dup mutation,clinically FSH is normal,and the number of sinus follicles is acceptable(AFC 15/12).It is suspected of ovulation disorders.3.After testing and artificial egg breakage,guide the patient to have a successful pregnancy after self cohabitation.There is no relevant information about this patient’s child.4.In vitro cell experiments,the protein expressed by the FOXL2 c.317T>C mutant gene was not truncated compared to the WT group,but there was a difference in protein localization.FOXL2 was originally a nuclear protein and was only expressed in the nucleus,but this mutant protein was also expressed in the cytoplasm.The mutant expression protein containing FOXL2 c.855-871 dup has been truncated,and there are also cases of abnormal nuclear localization.ConclusionCase A has a FOXL2 c.317T>C mutation that severely affects ovarian function and cannot be interfered with through ART.This mutation mainly results in abnormal protein localization without protein truncation;Case B has a FOXL2 c.855-871 dup mutation,and its ovarian function is acceptable.It is not seriously affected and can be intervened through medical means.This mutation mainly causes protein truncation and also incorporates protein localization abnormalities.From the different mutation types and different cell phenotypes of the two cases,it can be seen that the original conclusion of FOXL2 cannot be directly matched with clinical patients in a general manner,and a large amount of clinical data is still needed to supplement this gap and provide clinical treatment reference for relevant patients.