The Role And Mechanism Of TSC2 Gene Variants In The Pathogenesis Of Premature Ovarian Insufficiency

Objective:Premature ovarian insufficiency(POI)is defined as the loss of ovarian function in women before the age of 40,with clinical manifestation of abnormal menstruation accompanied by elevated gonadotropin levels(FSH>25IU/L)and decrease in estrogen levels.The prevalence of POI is about 3.7%and shows an increasing trend.POI patients not only suffer from infertility and menopausal symtoms,but also have a higher risk of developing aging diseases such as osteoporosis,cardiovascular disease,and Alzheimer’s disease.Therefore,POI affects women’s physical and mental health and the quality of life.And currently there are no effective measures to restore ovarian function.Etiological studies are important for the early prevention,diagnosis and intervention of POI.The etiology of POI is highly heterogeneous,including genetic,autoimmune and medical factors.Recently,studies have found that genetic factors account for approximately 20-25%of POI patients.However,known POI candidate genes explain only a small proportion of POI patients.With the development of next sequencing technology,the contribution of genetic factors in the etiology can be further investigated.mTOR pathway plays an important role in primordial follicle activation and follicle development.Tuberous Sclerosis Complex(TSC),composed of TSC1 and TSC2,is an important negative regulator of mTOR.Conditional knockout of Tscl or Tsc2 in mouse oocytes or pregranulosa cells resulted in premature awakening of primordial follicle and accelerated follicle depletion due to hyperactivation of mTOR.However,the pathogenicity of TSC1 and TSC2 gene defects in POI occurrence is unlear.Therefore,in this study we screened for TSC1 and TSC2 variants in POI-Whole Exome Sequencing(WES)database to investigate the role of TSC1 and TSC2 genes in the pathogenesis of POI.Methods:Variants in TSC1 and TSC2 were screened and analyzed through our own POIWES database.The pathogenic effects of TSC2 candidate variants and potential mechanism were further explored by functional studies in HEK293 cells.Ovarian cultured in vitro assay was conducted to further explore the pathogenic TSC2 variants on primordial follicles activation.Results:Through the variant screening of TSC1 and TSC2 in the POI-WES database,five rare heterozygous missense variants in TSC2 were firstly identified:p.R98Q,p.R901C,p.N919S,p.S1568C,p.E1756K.Next,in vitro experimental validation was conducted on TSC2 variants of uncertain significance(VUS).Overexpression of wild-type and mutant TSC2 vectors in HEK293 cells revealed that TSC2 p.R98Q caused reduced protein stability and lower expression in protein level,expression significantly lower than that of wild-type or other variants,affected the protein stability.Four VUS variants(p.R98Q,p.R901C,p.N919S and p.E1756K)exhibited increased levels of phosphorylation of S6K and 4EBP1,indicating that they compromised the repressive effect of TSC2 on mTOR kinase activity.Further studies revealed that p.R98Q,p.R901C,p.N919S affected the interaction of TSC1 and TSC2,while p.E1756 affected the TSC2 GTPase activity(GAP).In ovarian cultured in vitro assay,ovaries overexpressing Tsc2 p.R98Q showed hyperactivation of the mTOR pathway and a higher proportion of growing follicles.Conclusions:In this study,we screened for TSC1 and TSC2 variants in POI-WES database and identified five pathogenic variants of TSC2 in patients with sporadic POI.Combined with in vitro functional experiments,we found that the TSC2 pathogenic vairants impaired the role of TSC complex in negative regulation of mTOR activity and triggered the primordial follicle activation.This study firstly suggested defective TSC/mTOR pathway mediated hyperactivation of primordial follicle participating in the pathogenesis of POI.