L26/O-324 The novel G42C point mutation in the neuropeptide s receptor - role in endometriosis

Abstract Study question Does the G42C point mutation in NPSR1 alter Neuropeptide S (NPS) signalling and contribute to inflammatory and invasive mechanisms in endometriosis? Summary answer The G42C NPSR1 mutation alters NPS signalling, enhances cytoskeletal remodelling, and dysregulates sphingolipid-related genes, potentially contributing to endometriosis pathogenesis. What is known already Endometriosis is a chronic, estrogen-dependent inflammatory disease characterized by ectopic endometrial growth, pelvic pain, and infertility. Genetic susceptibility contributes to its development, with NPSR1 variants linked to advanced disease. Neuropeptide S (NPS) signalling promotes epithelial invasion, while sphingosine-1-phosphate (S1P) pathways support lesion survival and inflammation. Tapmeier et al. 2021 identified rare and common NPSR1 variants in humans and rhesus macaques, highlighting a 5-Mb candidate region flanked by D7S683–D7S510. Prisinzano et al. 2024 showed that 100 nM NPS significantly increases endometriotic epithelial cell invasion, providing functional evidence that NPSR1 variation may influence disease progression. Study design, size, duration An in vitro experimental study was conducted using human endometrial epithelial cell lines and human SH-SY5Y wild-type or G42C-mutant NPSR1 SH-SY5Y clone cells (c.7_3469A>G, p.Gly42Cys). Gene expression and cytoskeletal remodeling responses to NPS stimulation were assessed in a concentration response curve and across multiple time points. Participants/materials, setting, methods SH-SY5Y clones were stimulated with 100 nM neuropeptide S (NPS) for 45 minutes, in parallel to ECC-1 and 12Z endometrial cell lines. Expression of sphingosine-related genes (CIB1, SPHK1, SPHK2, SPGL1) was assessed by qRT-PCR (two repeats). Cytoskeletal remodelling was analysed after treatment with 100, 300, or 900 nM NPS for 45 minutes using TRITC-phalloidin/Hoechst 33258 staining. Experiments were performed in duplicate, with imaging by confocal microscopy (Olympus FV1000) and analysis by ANOVA, GraphPad Prism v10. Main results and the role of chance Gene expression was quantified using the 2−ΔΔCt method, normalising to GAPDH and using untreated cells as controls. Sphingosine pathway genes were upregulated mainly in ECC-1 cells, showing over two-fold increases (p < 0.05). SPHK1 and SPHK2 were highly expressed in ECC-1, with SPHK2 particularly elevated, while SPHK1 showed moderate expression in ECC-1 and 12Z. SGPL1 was detected only in ECC-1. No gene expression was detected in the G42C clone after NPS [TT1] stimulation (100 nM). Actin cytoskeletal staining revealed no differences between treated and untreated cells. qRT-PCR findings underscore the relevance of genetic background in modulating cellular responses to NPS, indicating that NPSR1 variants, including the G42C-mutant SH-SY5Y cells, can markedly affect gene regulatory mechanisms by attenuating downstream transcriptional activation, potentially contributing to endometriosis pathophysiology. Limitations, reasons for caution We have investigated the sphingosine-related signalling pathways but not inflammatory ones yet. These in vitro data are interesting and will need to be translated into in vivo experiments to see any systemic effects of NPSR1 mutations on inflammatory phenotypes. Wider implications of the findings These findings highlight mutations in NPSR1 and aberrant signalling as potentially contributing to endometriosis pathogenesis and could go some way to explain the genetic connection identified earlier. Trial registration number No