OR08-4 A Novel Mutation in the Follicle-Stimulating Hormone Receptor (Ile423Thr) in a Woman with Normal Pubertal Development and Primary Amenorrhea: In Vivo, In Vitro, and In Silico Studies

Abstract

Women with inactivating mutations in the FSHR may exhibit an array of phenotypes, from lack of pubertal development, primary amenorrhea, and complete insensitivity to FSH, to secondary amenorrhea and premature ovarian failure. In contrast to the LHCGR, only 19 inactivating mutations in the FSHR have been described to date. We report herein the case of a 22 year old women who presented with normal pubertal development but primary amenorrhea, associated with elevated serum FSH and LH levels (27-30 IU/L), low serum estradiol (E2) (21-40 pg/ml) and normal serum AMH (2.2-2.5 ng/ml) levels. After administration of recombinant FSH (total dose 1425 IU/9 days) serum E2 levels rose from 20 to 65 pg/ml with a parallel increase in number (from 5 to 17 follicles) and size (from 3 to 10 mm diameter) of follicles, and ovarian volumes (from 4 to 10 cm3). Sequencing of the FSHR revealed a new homozygous mutation in codon 423, where ATC was changed to ACC, generating the Ile423Thr substitution at the junction of the transmembrane domain (TM) 2 and the exo-loop (EL) 1 of the FSHR. The mother and two half-brothers were heterozygous for the same mutation. The molecular physiopathogenesis of the novel Ile423Thr mutation was analyzed by a series of in silico and in vitro biochemical studies in HEK293 cells transiently expressing either the wild-type (Wt) FSHR or the Ile423Thr mutant cloned into the pSG5 vector, and the results were compared with those obtained in cells transfected with the expression-deficient Asp408Tyr mutant, which leads to a more severe form of hypergonadotropic hypogonadism (1). Compared with the Wt FSHR, the plasma membrane expression of the mature (~80 kDa) form of the Ile423Thr mutant was slightly reduced, while that of the Asp408Tyr mutant was remarkably low, as disclosed by immunoblotting. A dissociation in the responses to FSH-stimulated signaling between the mutant receptors was found: compared with the Wt FSHR, FSH-stimulated cAMP production and ERK phosphorylation were reduced by 67% and 27% in the Ile423Thr mutant and by 52% and 43% in the Asp408Tyr FSHR, respectively. In silico molecular dynamics simulations of the FSHRs in an explicit membrane environment unveiled important differences in the behavior of both mutants vs the Wt receptor, particularly in the interhelical interactions of Thr423 with TM7 and EL2, and Tyr408 with TMs 5 and 7. Concurrently, these data suggest that conformational differences during the inactive (particularly during trafficking of the receptor proteins from the endoplasmic reticulum to the plasma membrane) and active (agonist-occupied receptor) states, account for the distinct expression levels and differential signaling exhibited by the Ile423Thr and Asp408Tyr mutant FSHRs. (1) Bramble MS, et al. A novel follicle-stimulating hormone receptor mutation causing primary ovarian failure: a fertility application of whole exome sequencing. Hum. Reprod. 2016; 31;905-914.