Functional Analysis of PTH1R Variants Found in Primary Failure of Eruption

Abstract

Although many variants of the parathyroid hormone 1 receptor (PTH1R) gene are known to be associated with primary failure of eruption (PFE), the mechanisms underlying the link remains poorly understood. We here performed functional analyses of PTH1R variants reported in PFE patients—namely, 356C>T (P119L), 395C>T (P132L), 439C>T (R147C), and 1148G>A (R383Q)—using HeLa cells with a lentiviral vector-mediated genetic modification. Two particular variants, P119L and P132L, had severe reduction in a level of N-linked glycosylation when compared with wild-type PTH1R, whereas the other 2 showed modest alteration. PTH1R having P119L or P132L showed marked decrease in the affinity to PTH1-34, which likely led to severely impaired cAMP accumulation upon stimulation in cells expressing these mutants, highlighting the importance of these 2 amino acid residues for ligand-mediated proper functioning of PTH1R. To further gain insights into PTH1R functions, we established the induced pluripotent stem cell (iPSC) lines from a patient with PFE and the heterozygous P132L mutation. When differentiated into osteoblastic-lineage cells, PFE-iPSCs showed no abnormality in mineralization. The mRNA expression of RUNX2, SP7, and BGLAP, the osteoblastic differentiation-related genes, and that of PTH1R were augmented in both PFE-iPSC-derived cells and control iPSC-derived cells in the presence of bone morphogenetic protein 2. Also, active vitamin D3 induced the expression of RANKL, a major key factor for osteoclastogenesis, equally in osteoblastic cells derived from control and PFE-iPSCs. In sharp contrast, exposure to PTH1-34 resulted in no induction of RANKL mRNA expression in the cells expressing P132L variant PTH1R, consistent with the idea that a type of heterozygous PTH1R gene mutation would spoil PTH-dependent response in osteoblasts. Collectively, this study demonstrates a link between PFE-associated genetic alteration and causative functional impairment of PTH1R, as well as a utility of iPSC-based disease modeling for future elucidation of pathogenesis in genetic disorders, including PFE.

Validating clinical characteristics of primary failure of eruption (PFE) associated with PTH1R variants

“…However, a PTH1R variant was found only in 64% of PFE patients (30 of 47), and only 23% of these (11 out of 47) were carriers of pathogenic or likely pathogenic variants. The functional analysis of the effects of all PTH1R variants at the mRNA and protein level would be very useful, but it is challenging and has rarely been done [ 33 – 35 ]. We suspect that the phenotypic manifestation of PFE observed in the remaining patients might be due to variants in other genes, encoding other proteins involved in the complex process of tooth morphogenesis and eruption and not implicated in PFE pathogenesis yet [ 3 – 5 ].…”

Expanding genotypic and phenotypic spectrums of LTBP3 variants in dental anomalies and short stature syndrome

“…Failure of tooth eruption in the maxilla has not been reported in patients with DASS, however, decreased TGFβ signaling results in decreased expression of Parathyroid hormone 1 receptor gene (PTH1R; MIM 168468), 1 and mutations in PTH1R cause primary failure of tooth eruption. 9 Together, this suggests that failure of tooth eruption and osteosclerosis in the proband are related to a disruption in the LTBP3-TGFβ-PTH1R signaling pathway. Defects in root development, such as taurodontism and single-rooted molars observed in the proband, have not previously been reported in patients with LTBP3 mutations, but as TGFβ signaling has important roles in root development, 10 a role for LTBP3 in this process is likely (Figure 3).…”

Die Rolle des Parathormonrezeptors Typ 1 bei der primären Zahndurchbruchsstörung

“…Letztlich sind jedoch funktionelle Untersuchungen notwendig, um eine sichere Aussage über die Effekte der jeweiligen Mutante auf die Funktion des Wildtyp PTH1R in den betroffenen Zellen nachzuweisen. Hier wurden für ausgewählte Mutanten in den letzten Jahren unter Verwendung verschiedener in vitro Zellmodelle erste Fortschritte erzielt [21,22]. Es zeigten sich beispielsweise Unterschiede zwischen Wildtyp PTH1R und der c.1092delG p.(Val-365Cysfs*141) Mutante, welche stärker im Cytoplasma der Zelle vorhanden war, während Wildtyp mehr an Membranen und Transportvesikeln nachgewiesen werden konnte [23].…”