OR06-3 ESR1 Pathogenic Variant With Incomplete Estrogen Insensitivity

Abstract Introduction Estrogen is vital to human reproduction and acts primarily through two receptors: estrogen receptor alpha (ERα, encoded by ESR1) and estrogen receptor beta (ERβ, encoded by ESR2). Surprisingly, very few human ESR1 pathogenic variants have been recognized, despite the existence of >1000 different androgen receptor pathogenic variants causing androgen insensitivity syndrome. Functional analysis of an ESR1 homozygous missense variant (p.Glu375His) in the ERα ligand-binding domain (LBD) showed a 240-fold decrease in estrogen response in a patient with complete estrogen insensitivity and abnormal pubertal development. We predict that less severe ESR1 variants will present with unexplained infertility. Previously, we performed whole exome sequencing on 200 females with unexplained infertility and identified 4 likely pathogenic ESR1 heterozygous missense variants confirmed by Sanger sequencing, including one variant (p.Thr313Met) in the LBD. In this study, we performed in vitro functional analysis to evaluate if the p.Thr313Met likely pathogenic variant found in a patient with unexplained infertility causes incomplete estrogen insensitivity. Hypothesis We hypothesize that the p.Thr313Met likely pathogenic variant in the ERa LBD will result in incomplete estrogen insensitivity. Methods The p.Thr313Met variant plasmid was constructed using site-directed mutagenesis (SDM), and confirmed by Sanger sequencing. An in vitro cell model was used to evaluate estrogenic activity. HepG2 cells were transiently transfected with WT ESR1, p.Thr313Met, or p.Glu375His plasmid, along with the Firefly estrogen response element (ERE) luciferase reporter (3X ERE TATA luc) and Renilla luciferase (pRL-TK luc) plasmids using the Effectene transfection protocol. Cells were treated with 17β-estradiol (0.01, 0.1, 1, 10, and 100 nM). A dual luciferase reporter assay was used to compare relative Firefly and Renilla luciferase activity between WT ESR1, p.Thr313Met, and p.Glu375His plasmids using a luminometer. Estrogen dose response curves were generated, and a two-way ANOVA with Tukey's test was performed to compare the relative luciferase activity across WT and variant plasmids using GraphPad Prism v.9. Differences were considered significant at p<0.05. Results Estrogen response curves were generated for WT, p.Thr313Met, and p.Glu375His plasmids. The p.Thr313Met variant showed lower overall activity with a right shifted dose response curve with a statistically significant decrease in relative luciferase activity in comparison to WT (p<0.05). The p.Thr313Met variant was more active at higher doses (1 and 10 nM) with a left shifted dose curve when compared to the p.Glu375His variant (p<0.05). Conclusion We have identified a human ESR1 variant that impairs estrogen signaling in vitro, suggesting incomplete estrogen insensitivity as a putative mechanism for unexplained infertility. The identification of ESR1 pathogenic variants and the evaluation of their functionality could provide improved diagnosis and genetic counseling for patients with unexplained infertility. Presentation: Saturday, June 11, 2022 12:00 p.m. - 12:15 p.m.