12419 Single-nuclei Sequencing Of Normal Donor Cadaveric Parathyroid Glands Reveals Distinct Cellular Subpopulations With Key Differences In Gland Anatomical Location And Suppressed Vs Non-suppressed Physiological Status

Abstract Disclosure: T. Glinin: None. E. Khanafshar: None. J.A. Sosa: Advisory Board Member; Self; Novo Nordisk, AstraZeneca, Eli Lilly & Company. Grant Recipient; Self; Exelixis, Inc., Eli Lilly & Company. J. Koh: None. Despite the significant clinical burden of parathyroid disorders, little is known at the molecular level about the cellular composition of normal parathyroid glands (PTGs). We performed a single-nuclei RNAseq study of six cadaveric PTGs (three from the superior anatomical location, three inferior) from four donors (two female). A board-certified pathologist confirmed that the sequenced PTGs were histologically normal. Using the split-pool method, we sequenced 83,693 nuclei, generating 1.7B reads. Data integration was performed with the Harmony algorithm in Seurat 5.0.1. Cluster analysis revealed 14 discrete populations of PTG origin. Gene set enrichment identified clusters corresponding to endothelial cells, neurons, microglia, mast cells, dendritic cells, stromal fibroblasts, macrophages, and smooth muscle. The remaining six clusters were closely related but distinct populations sharing markers including PTH, CaSR, SPOCK3, PRKCE, and PLCB1 (p<2e-300 for group specificity) but with differentially expressed gene (DEG) patterns between clusters suggestive of specialized functions in VEGF signaling (KIAA1549L), GPCR downstream signaling (GRM7, ADGRL3), and subcellular spatial organization (SYNE1) (p<2e-24 for cluster specificity). Superior and inferior PTGs were distinguished by 8 superior-specific DEGs (p<2e-245) including HOXB3, expressed in 71% of superior PTG cells vs 6% of inferior PTG cells. Enhanced expression of HOXB3, a homeobox transcription factor found to inhibit tumorigenesis in multiple tissues, could explain in part why adenomas are three times less likely to arise in superior PTGs. During procurement, one of the presumptive normal donors was found to have a single grossly enlarged, hypercellular PTG, elevated PTH (140 pg/ml), and hypercalcemia (11.2 mg/dL), consistent with primary hyperparathyroidism (PHPT). In PHPT patients with a single tumor, contralateral PTGs are functionally suppressed by the dominant adenoma. We compared the histologically normal suppressed and unsuppressed PTGs. Fifteen genes were more highly expressed in suppressed PTGs, including LRP2, a multi-ligand receptor known to bind PTH (p = 2e-300). Fourteen genes were more highly expressed in unsuppressed PTGs (p<2.14e-296), including PDE11A, NRG2, NRXN3, and PRLR, which are all associated with GABA receptor signaling (q = 2.8e-5). These data independently support our group’s prior work proposing a role for the GABA receptor in PHPT. Differential expression in endothelial cells, neurons, and fibroblasts from suppressed PTGs suggests that non-endocrine cells may also be responsive to changes in PTG activity. Finally, the different proportions of cells with suppressed or unsuppressed gene expression signatures observed between clusters suggests that PTGs incorporate distinct populations of cells reflective of physiological activity or functional suppression. Presentation: 6/2/2024