Abstract Primary failure of eruption (PFE) is a rare autosomal disorder provoked by heterozygous mutations in the parathyroid hormone receptor 1 ( PTH1R ) gene. PTH1R is a G protein-coupled receptor (GPCR) which regulates intracellular signaling molecules like cAMP. By using CRISPR/Cas9, the pathogenic PTH1R variant, c.1050-3C>G, was introduced into the periodontal ligament (PDL-hTERT) cell line to investigate molecular mechanisms in a PFE in vitro model. The PDL-hTERT immortal cell line, derived from human primary PDL cells, is a well-established model for dental diseases and expresses PTH1R . We performed different functional assays to compare the behavior of the PDL-hTERT WT versus PTH1R -mutated cells. cAMP synthesis and PKA activation were compared between different cell lines by live-cell imaging using Förster Resonance Energy Transfer (FRET)-based biosensors. Phosphorylation of VASP was measured to validate and compare the PKA activation between the cell lines. In summary, our experiments show that the mutated cell line has no major phenotypic changes, but the PTH1R downstream signaling cascade is impaired. Key messages A rare autosomal disorder linked to mutations in the PTH1R gene, which encodes a G protein-coupled receptor regulating intracellular signaling, including cAMP production. The pathogenic PTH1R mutation (c.1050-3C>G) was introduced into a periodontal ligament (PDL hTERT) cell line to model PFE and study molecular mechanisms in vitro. Functional assay revealed that while the mutated cell line displayed no major phenotypic changes, the PTH1R downstream signaling cascade, including cAMP synthesis and PKA activation, was disrupted. Techniques like FRET-based biosensors and VASP phosphorylation assays highlighted specific impairments in signaling pathways in cells with the PTH1R mutations
Marnet et al. (Mon,) studied this question.