Primary failure of eruption in heterozygous PTH1 receptor mutations: generation and characterization of cell models of clinically proven mutants

Primary failure of eruption (PFE) is a rare autosomal, non-syndromic disorder driven by heterozygous mutations in the parathyroid hormone receptor 1 (PTH1R) gene. PFE is characterized by impaired tooth eruption, predominantly affecting posterior teeth and associated with deficient development of the alveolar process in the affected region. Despite bone resorption and a clear eruption pathway, the teeth fail to emerge, suggesting a disruption in the underlying molecular mechanisms. The PTH1R gene encodes a G protein-coupled receptor that activates multiple intracellular signaling cascades, notably increasing cyclic adenosine monophosphate (cAMP) levels, a ubiquitous second messenger for numerous G protein-coupled receptors. However, the precise molecular pathways disrupted in PFE patients remain poorly understood. The objective of this study was to establish in vitro models to elucidate the molecular mechanisms of PFE using CRISPR/Cas9-engineered cell lines. We employed CRISPR/Cas9 technology to introduce clinically relevant mutations, specifically the c.1050-3C>G and c.1016G>A variants of PTH1R, into the PDL-hTERT cell line. These cells, derived from periodontal ligament cells, are a robust model for dental pathologies due to their stable expression of PTH1R. Functional assays were conducted to compare the behavior of the PDL-hTERT WT cells to the PTH1R mutated cells. Given the absence of significant functional differences between WT and mutant cell lines in standard assays, we explored potential alterations in downstream PTH1R signaling pathways. We specifically assessed cAMP synthesis and protein kinase A (PKA) activation using live-cell Förster Resonance Energy Transfer (FRET)-based biosensors. Results demonstrated reduced cAMP levels and impaired PKA activation in the mutated cell line compared to the WT cell line. To corroborate these findings, we utilized Western blot analysis to evaluate the phosphorylation of Vasodilator Stimulated Phosphoprotein (VASP), a downstream target of PKA. Phosphorylation levels in mutant cell lines indicated a trend consistent with reduced PKA activity, supporting our FRET-based observations. In parallel, further experiments were conducted using PTH1R variants c.463G>T and c.1016G>A, cloned into the pcDNA3.1 vector along with a V5 tag and transfected into HEK293T cells to examine receptor localization and expression. Immunofluorescence imaging revealed that PTH1R localization remained cytoplasmic, with no significant differences in receptor expression levels between WT and mutant variants. These findings suggest that PFE pathology may arise from defective intracellular signaling. The novel in vitro models developed in this study provide a valuable platform for future investigations into PFE pathogenesis and the development of targeted therapeutic strategies.