OR35-04 Anti-Müllerian Hormone Variants identified in Patients with Polycystic Ovary Syndrome Affect Secretion and Immunoactivity

Abstract

Abstract Disclosure: L. Meng: None. A. McLuskey: None. A.E. Dunaif: None. J.A. Visser: None. Recently rare heterozygous AMH variants were identified in women with PCOS, causing reduced AMH signaling. However, the exact functional mechanism remains unknown. Here, we analyzed the processing, secretion and signaling of these AMH variants. Six PCOS-specific AMH variants (V12G, P151S, P270S, P352S, P362S, H506Q) and one control-specific variant (A519V) were introduced in an AMH expression vector. Signaling activity was measured using the BRE-Luc reporter in the mouse granulosa cell-line KK-1. AMH cleavage and secretion was analyzed by Western Blot, confocal microscopy and ELISA. Serum AMH levels in PCOS cases harboring these variants were measured by ELISA. hAMH-151S and hAMH-506Q have ∼90% decreased AMH signaling (P<0.001). The other five variants were comparable to wild-type (wt)-AMH signaling. Coexpression of hAMH-151S or hAMH-506Q with wt-hAMH at equal amounts inhibited wt-hAMH signaling by ∼30% (P<0.001). Western blot analysis revealed that hAMH-151S and hAMH-506Q proteins were only detected in the cell lysate but not in the supernatant, while wt-hAMH and the hAMH-352S and hAMH-362S variants were detected in both the cell lysate and the supernatant. Confocal analysis showed that cells expressing hAMH-151S and hAMH-506Q had higher cellular AMH protein levels with ER retention compared to cells expressing wt-hAMH and hAMH-352S. In agreement, using two commercial AMH ELISA’s, hAMH-151S was readily detected in the cell lysate, while only very low levels were detected in the supernatant. Both hAMH-362S and hAMH-519V were detectable using the Fujirebio-AMH ELISA, but showed severely reduced immunoactivity measured by the pico-AMH ELISA. Surprisingly, hAMH-506Q was undetectable in both the cell lysate and supernatant by both ELISA’s. However, in PCOS cases, heterozygous carriers of the P151S and H506Q variants still had detectable AMH in both assays. In conclusion, our results show that P151S and H506Q disrupt normal processing and secretion of AMH, leading to hampered secretion of wt-AMH. Additionally, AMH variants impair the AMH immunoactivity with (H506Q) or without (P362S and A519V) influencing their bioactivities. Presentation Date: Sunday, June 18, 2023