Association Between FSHR Mutations And Polymorphisms With Premature Ovarian Insufficiency And Risk Of Cervical Cancer In Han Chinese Population

Chapter ?.Novel FSHR Mutations Identified in Han Chinese Population with Premature Ovarian InsufficiencyBackgroud:Premature ovarian insufficiency(POI),also termed as premature ovarian failure(POF),is one of the common endocrine conditions in women.It displays high heterogeneity both in clinical manifestation and etiology.The clinical characteristics are primary amenorrhea or secondary amenorrhea before the age of 40,accompanied by the hypergonostogonidemia and hypoestrogenemia as well as perimenopausal symptoms.The etiology of POI is complicated,including genetic factors,immunological factors,iatrogenic factors,metabolic factors and environmental factors.1%of women before 40 years of age and 0.1%before 30 years of age are affected by POI,and more and more women are affected by this disorder.Actually,there are neither reliable early diagnostic indicators nor effective intervention.Therefore,studies on the etiology of POI and the early diagnosis of high-risk groups are of great significance.Current studies have confirmed that genetic defect accounts for 20%25%of POI patients.In recent years,with the development of genomic techniques,several dozen POI genes have been identified by means of screening candidate genes and whole exon sequencing.The pituitary glycoprotein hormone FSH plays a pivotal role in mammalian reproduction through binding and activating its specific receptor located on target cells.The normal function of the FSH-FSHR is considered to be essential for follic?lar development and estradiol production in females.In the classical signaling pathway,FSH-FSHR interaction activates the cyclic adenosine monophosphate(cAMP)-protein kinase A(PKA)cascade and then increases the phosphorylation of ERK1/2 protein,leading to follicle maturation and steroidogenesis.It has been considered that FSHR was the first single gene to cause nonsyndromic POI.Diverse inactivating mutations and polymorphisms have been described in the FSHR gene,most of which lead to a POI phenotype.Actually,the inactivating mutations in the FSHR gene have been largely reported in Finnish patients with POI,but are extremely rare in other populations.This study determined the potential FSHR mutations both in a POI family and sporadic POI patients of Han Chinese descent.Part ?.A Novel FSHR Mutation Identified in a Han Chinese Family with Premature Ovarian InsufficiencyObjective:To identify the potential FSHR mutation in a Han Chinese family with premature ovarian insufficiency(POI).Methods:A POI patient,her family members,and another 192 control women with regular menstruation were recruited in this study.Ovarian biopsy was performed in the patient.Hematoxylin and eosin staining of the ovarian section was applied to observe the follicular development.Sanger sequencing and genotype analysis were carried out for the patient,her sister,and parents.The novel variant identified was further confirmed in control subjects.ClustalW2 website was used for the conservation analysis.Expression vectors of both wild type and mutant FSHR were constructed and transfected into HEK293T cells.Western blot and immunofluorescence were performed to detect FSHR expression;and cAMP assay was carried out to monitor FSH-induced signaling.Results:Mutational screening and genotype analysis of the FSHR gene identified a novel homozygous mutation c.175C>T(p.R59X)in exon 2,which was inherited in the autosomal recessive mode from her heterozygous parents but was absent in her sister and the 192 control women.The amino acid involved was highly conserved among species.Functional studies demonstrated that in vitro the nonsense mutation caused the loss of full-length FSHR expression and that p.R59X mutant showed no response to FSH stimulation in the cAMP level.Histologic examination of the ovaries in the patient revealed follicular development up to the early antral stage.Conclusion:A novel homozygous mutation c.175C>T(p.R59X)of the FSHR gene was identified in a POI family.The mutation is causative for POI by means of arresting folliculogenesis.Part ?.Novel FSHR Mutations Identified in Sporadic POI patients of Han Chinese descentObjective:To determine the potential FSHR mutations in sporadic POI patients of Han Chinese descent.Methods:A total of 192 patients with sporadic POI and 192 control women were recruited from Center for Reproductive Medicine of Shandong University.All the participants were of Han Chinese origin with a normal karyotype(46,XX).Sanger sequencing was carried out for 192 patients with sporadic POI and the potential pathogenic variants were verified in 192 matched controls.ClustalW2 website was used for the conservation analysis.Immunofluorescence and flow cytometry were performed to detect FSHR expression;and AMP assay was carried out to monitor FSH-induced signaling.Western blot was applied to detect the FSH-induced ERK phosphorylation.Results:Sequencing analysis revealed 6 known single-nucleotide polymorphisms(SNPs)of the FSHR gene,two of which,rs1394205(c.-29G>A)and rs140106399(c.*111T>C)were identified in both groups with significantly different genotypic and allelic distributions.Importantly,three novel heterozygous variants,c.793A>G(p.M265V),c.1789C>A(p.L597I)and c.1821C>T(synonymous),were identified in three patients with POI,all of which were absent in the 192 controls.Leucine at site 597 was highly conserved across species,whereas methionine at site 265 was not.While both p.M265V and p.L5971 were confirmed to be surface-expressed,only p.L5971 mutant showed decreased membrane localization as compared to wild-type FSHR.Additionally,FSH-induced cAMP production and ERK1/2 phosphorylation were reduced in the cells transfected with p.L5971 mutant,but not in the cells transfected with p.M265V mutant.However,no dominant negative effect was observed for the p.L5971 mutant.Conclusion:Three novel mutations of the FSHR gene were identified in sporadic POI patients of Han Chinese descent,and the mutation frequency of the FSHR gene in POI patients was 1.6%(3/192).The novel mutation,c.1789C>A(p.L597I)in the FSHR gene resulted in decreased membrane localization of FSHR and an impaired FSH-induced signaling,revealing the contribution of the FSHR gene in the etiology of POI in Han Chinese population.Chapter ?.Association between FSHR Mutations and Polymorphisms with Risk of Cervical Cancer in Han Chinese PopulationBackground:Cervical cancer(CC)is the most common gynecologic tumor as well as the second common cancer in women in the world.Early diagnosis and treatment of CC are essential to reduce deaths from this disease.Through studies on association between genetic variation and susceptibility or resistance of CC,we can learn about the susceptibility or protective genotypes of CC,so as to obtain molecular tags for screening the susceptible or high-risk groups of CC.At present,studies about gene polymorphism and CC mainly includes 4 types:inflammatory-immune related genes,cytokine-related genes,metabolic enzyme genes and DNA damage repair genes.Follicle-stimulating hormone(FSH)plays its crucial role in ovarian steroid hormone formation by binding its specific receptor mainly expressed the granulosa cells in the ovary.Recently,the FSHR gene was demonstrated to be associated with several types of cancer,especially ovarian cancer.Our previous study identified a heterozygous FSHR mutation,C.175T(p.R59X),in a CC patient,which was also identified both in breast cancer and endometrial cancer.Recent studies have shown that the known SNP(rs1007541)of the FSHR gene is associated with CC risk in Tunisia.Considering FSH-FSHR interaction participates in estrogen biosynthesis,we suspect FSHR mutations might be associated with CC.All these suggest that the FSHR gene may be a candidate gene for CC.Objective:To investigate the association between FSHR mutations and polymorphisms with the risk of cervical cancer in Han Chinese population.Methods:A total of 130 Han Chinese CC patients and 200 aged-matched healthy controls were included in this retrospective case-control study,Sanger sequencing was performed for mutaion screening of the coding region of the FSHR gene and for genotying.Besides,Haploview software was used for Hardy-Weinberg equilibrium.Chi-square test was applied to compare the genotypic and allelic distriution of SNPs identified in CC patients and controls.The logistic regression analysis was used to analyze the risk of different genotypes and alleles in CC.Results:A heterozyous mutation was identified in the CC patients,but not in the controls.The allelic and genotypic distribution of rs 1394205(c.-29G>A)and rs140106399(c.*111T>C)was significantly different in both groups,while other tested FSHR SNPs were comparable between CC patients and control women.rs1394205 was analyzed to be associated with the CC risk.The Logistics regression analysis showed that rs1394205 was positively correlated with the incidence of CC:rs1394205 AA genotype increased the CC risk;the A allele was the risk factor for CC.Oppositely,rs140106399 was negatively correlated with the incidence of CC:rs140106399 CC genotype reduced the CC risk;the C allele was the protective factor for CC.However,Considering the small samples and its lower allele frequency(MAF),the correlation between rs140106399 and the CC risk remains to be further confirmed.Conclusions:One novel mutation of the FSHR gene were identified in three sporadic POI patients of Han Chinese descent(3/130).Two FSHR SNPs were identified with different distribution in CC patients and controls,one of which,rs1394205 showed a positive correlation with CC and AA genotype increased the CC risk.