Department of Biochemistry, School of Dentistry, Showa University, Tokyo, Japan.
Department of Orthodontics, School of Dentistry, Showa University, Tokyo, Japan.
J Dent Res. 2020 Apr;99(4):429-436. doi: 10.1177/0022034520901731. Epub 2020 Jan 27.
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 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 -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 PTH, 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 , and , the osteoblastic differentiation-related genes, and that of were augmented in both PFE-iPSC-derived cells and control iPSC-derived cells in the presence of bone morphogenetic protein 2. Also, active vitamin D induced the expression of , a major key factor for osteoclastogenesis, equally in osteoblastic cells derived from control and PFE-iPSCs. In sharp contrast, exposure to PTH resulted in no induction of mRNA expression in the cells expressing P132L variant PTH1R, consistent with the idea that a type of heterozygous 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.
尽管已知甲状旁腺激素 1 受体 (PTH1R) 基因的许多变体与原发性出牙失败 (PFE) 有关，但该关联的机制仍知之甚少。我们在这里使用携带慢病毒载体基因修饰的 HeLa 细胞对 PFE 患者报告的变体进行了功能分析，这些变体包括 356C>T (P119L)、395C>T (P132L)、439C>T (R147C) 和 1148G>A (R383Q)。与野生型 PTH1R 相比，两个特定的变体 P119L 和 P132L 的 -连接糖基化水平严重降低，而另外两个变体则显示出适度的改变。具有 P119L 或 P132L 的 PTH1R 对 PTH 的亲和力显著降低，这可能导致这些突变体表达的细胞中 cAMP 积累严重受损，突出了这两个氨基酸残基对配体介导的 PTH1R 正常功能的重要性。为了进一步深入了解 PTH1R 的功能，我们从一名 PFE 患者和杂合 P132L 突变建立了诱导多能干细胞 (iPSC) 系。当分化为成骨细胞谱系细胞时，PFE-iPSC 在矿化过程中没有异常。在骨形态发生蛋白 2 的存在下，PFE-iPSC 衍生细胞和对照 iPSC 衍生细胞中，成骨细胞分化相关基因和 的 mRNA 表达以及 的表达均增强。此外，活性维生素 D 同样在对照和 PFE-iPSC 衍生的成骨细胞中诱导了主要破骨细胞生成关键因子 的表达。相比之下，在表达 P132L 变体 PTH1R 的细胞中，PTH 的暴露没有诱导 mRNA 表达，这与一种杂合 基因突变会破坏成骨细胞中 PTH 依赖性反应的观点一致。总的来说，这项研究表明 PFE 相关遗传改变与 PTH1R 的功能损害之间存在关联，以及基于 iPSC 的疾病建模在包括 PFE 在内的遗传疾病发病机制阐明方面的应用。
