A novel PTH1R mutation causes primary failure of eruption via the cAMP-PI3K/AKT pathway

Abstract

Abstract Background Primary failure of eruption (PFE) is a rare disorder characterized by a posterior open bite. While mutations in the parathyroid hormone 1 receptor (PTH1R) gene have been demonstrated to cause PFE, the underlying mechanisms remain largely unknown. Methods Whole exome sequencing was conducted to identify PTH1R variants in a PFE family. MG63 cells that stably expressed the corresponding mutant PTH1R were established using lentiviruses. Next, osteogenesis was assessed by measuring cell alkaline phosphatase activity, conducting alizarin red staining, and evaluating osteoblast-specific gene expression. Then, computational analysis of binding affinity and RNA sequencing were carried out. Lastly, rescue experiments were performed to validate the mechanism underlying the pathogenesis of PFE. Results A novel PTH1R missense mutation (c.904G > A, p.E302K) was identified in a Chinese family affected by PFE. Moreover, the E302K mutation inhibited the expression of osteogenic-specific genes and proteins in MG63 cells. Computational analysis revealed the E302K mutation decreased the binding affinity of Gαs to the PTH1R protein. Consistently, cAMP accumulation assays demonstrated that the E302K mutation impaired the intracellular PTH1-34 -induced accumulation of cAMP. Further RNA sequencing analysis and validation experiments revealed that the PI3K-AKT signaling pathway was predominantly down-regulated in response to the E302K mutation. Finally, forskolin partially restored the effects of the E302K mutation on osteogenesis. Conclusions This study indicated that the E302K mutation in PTH1R decreased the binding affinity of PTH1R protein for Gαs, down-regulated the cAMP-PI3K/AKT signaling pathway, and inhibited osteogenesis, eventually leading to PFE. This study not only expands the genotypic spectrum of PTH1R mutations but also elucidates the underlying pathogenic mechanism of PTH1R-associated PFE.

Keywords

• Primary failure of eruption (PFE)
• Parathyroid hormone 1 receptor (PTH1R)
• Affinity
• Osteogenesis
• Cell signaling