A novel follicle-stimulating hormone receptor mutation causing primary ovarian failure: a fertility application of whole exome sequencing

Abstract

Study question: Can whole exome sequencing (WES) and in vitro validation studies be used to find the causative genetic etiology in a patient with primary ovarian failure and infertility?

Summary answer: A novel follicle-stimulating hormone receptor (FSHR) mutation was found by WES and shown, via in vitro flow cytometry studies, to affect membrane trafficking.

What is known already: WES may diagnose up to 25-35% of patients with suspected disorders of sex development (DSD). FSHR mutations are an extremely rare cause of 46, XX gonadal dysgenesis with primary amenorrhea due to hypergonadotropic ovarian failure.

Study design, size, duration: A WES study was followed by flow cytometry studies of mutant protein function.

Participants/materials, setting, methods: The study subjects were two Turkish sisters with hypergonadotropic primary amenorrhea, their parents and two unaffected sisters. The affected siblings and both parents were sequenced (trio-WES). Transient transfection of HEK 293T cells was performed with a vector containing wild-type FSHR as well as the novel FSHR variant that was discovered by WES. Cellular localization of FSHR protein as well as FSH-stimulated cyclic AMP (cAMP) production was evaluated using flow cytometry.

Main results and the role of chance: Both affected sisters were homozygous for a previously unreported missense mutation (c.1222G>T, p.Asp408Tyr) in the second transmembrane domain of FSHR. Modeling predicted disrupted secondary structure. Flow cytometry demonstrated an average of 48% reduction in cell-surface signal detection (P < 0.01). The mean fluorescent signal for cAMP (second messenger of FSHR), stimulated by FSH, was reduced by 50% in the mutant-transfected cells (P < 0.01).

Limitations, reasons for caution: This is an in vitro validation. All novel purported genetic variants can be clinically reported only as 'variants of uncertain significance' until more patients with a similar phenotype are discovered with the same variant.

Wider implications of the findings: We report the first WES-discovered FSHR mutation, validated by quantitative flow cytometry. WES is a valuable tool for diagnosis of rare genetic diseases, and flow cytometry allows for quantitative characterization of purported variants. WES-assisted diagnosis allows for treatments aimed at the underlying molecular etiology of disease. Future studies should focus on pharmacological and assisted reproductive treatments aimed at the disrupted FSHR, so that patients with FSH resistance can be treated by personalized medicine.

Study funding/competing interests: E.V. is partially funded by the DSD Translational Research Network (NICHD 1R01HD068138). M.S.B. is funded by the Neuroendocrinology, Sex Differences and Reproduction training grant (NICHD 5T32HD007228). The authors have no competing interests to disclose.

Keywords: follicle-stimulating hormone receptor; premature ovarian failure; primary amenorrhea; resistant ovary syndrome; whole exome sequencing.

Figure 1

FSHR mutations. (A) Table of previously reported FSHR mutations and patient phenotypes. (B) FSHR is a 695 amino acid GPCR with a ligand-binding extracellular domain at the amino tail, a transmembrane domain and an intracellular domain at the carboxyl tail. The locations of the novel mutation and the previously reported 11 inactivating missense mutations are demonstrated here.

Figure 2

Inheritance of the FSHR mutation. (A) A pedigree was obtained demonstrating consanguinity between the parents and no other cases of primary amenorrhea. The proband was patient III-6, and her affected sister was III-3. * indicates WES performed. (B) Sanger sequencing confirmed the homozygous variant status of the affected sisters as well as the parents' heterozygous status. One unaffected sister was found to be heterozygous for the variant and the other was WT.

Figure 3

Modeling of the FSHR mutation. The RaptorX protein structure and functional prediction software predicted that FSHR containing the substituted residue showed significant disordering of secondary structure when compared with the predicted model of WT FSHR. This region of disorder lies upstream of the actual mutation at residue 408.

Figure 4

In vitro analysis of FSHR function. (A) Flow cytometry demonstrated an average 48.05% reduction of surface signal detection in cells expressing the mutant p.Asp408Tyr FSHR construct relative to cells expressing a WT construct (**P < 0.01). Permeabilized cells from the same experimental groups showed an intracellular FSHR signal of 100% for both the mutant and the WT-transfected cells. (B) After FSH stimulation, cells transfected with p.Asp408Tyr were capable of generating a 22.58% (**P < 0.001) increase in the mean cAMP fluorescent signal compared with baseline mock-transfected cells. This was 50.22% lower than the WT FSHR-transfected cells, which produced a 44.96% (***P < 0.0001) higher fluorescent mean signal for cAMP relative to the mock transfection (**P < 0.01 for difference of means between WT and mutant by ANOVA/Tukey's HSD).