Department of Prosthodontics, Peking University School and Hospital of Stomatology & National Center of Stomatology & National Clinical Research Center for Oral Diseases & National Engineering Research Center of Oral Biomaterials and Digital Medical Devices, Beijing 100081, China.
Int J Mol Sci. 2022 Jul 24;23(15):8142. doi: 10.3390/ijms23158142.
The purpose of this research was to investigate and identify PAX9 gene variants in four Chinese families with non-syndromic tooth agenesis. We identified pathogenic gene variants by whole-exome sequencing (WES) and Sanger sequencing and then studied the effects of these variants on function by bioinformatics analysis and in vitro experiments. Four novel PAX9 heterozygous variants were identified: two missense variants (c.191G > T (p.G64V) and c.350T > G (p.V117G)) and two frameshift variants (c.352delC (p.S119Pfs2) and c.648_649insC(p.Y217Lfs100)). The bioinformatics analysis showed that these variants might be pathogenic. The tertiary structure analysis showed that these four variants could cause structural damage to PAX9 proteins. In vitro functional studies demonstrated that (1) the p.Y217Lfs100 variant greatly affects mRNA stability, thereby affecting endogenous expression; (2) the p. S119Pfs 2 variant impairs the subcellular localization of the nuclear expression of the wild-type PAX9 protein; and (3) the four variants (p.G64V, p.V117G, p.S119Pfs2, and p.Y217Lfs100) all significantly affect the downstream transcriptional activity of the BMP4 gene. In addition, we summarized and analyzed tooth missing positions caused by PAX9 variants and found that the maxillary second molar (84.11%) and mandibular second molar (84.11%) were the most affected tooth positions by summarizing and analyzing the PAX9-related non-syndromic tooth agenesis positions. Our results broaden the variant spectrum of the PAX9 gene related to non-syndromic tooth agenesis and provide useful information for future genetic counseling.
本研究旨在调查和鉴定四个中国非综合征性牙缺失家系中 PAX9 基因的变异。我们通过全外显子组测序（WES）和 Sanger 测序鉴定了致病基因突变，然后通过生物信息学分析和体外实验研究了这些变异对功能的影响。共鉴定出 4 种 PAX9 杂合变异：2 种错义变异（c.191G>T(p.G64V)和 c.350T>G(p.V117G)）和 2 种移码变异（c.352delC(p.S119Pfs2)和 c.648_649insC(p.Y217Lfs100）。生物信息学分析表明这些变异可能具有致病性。三级结构分析表明，这 4 种变异可能导致 PAX9 蛋白结构损伤。体外功能研究表明：（1）p.Y217Lfs100 变异极大地影响了 mRNA 的稳定性，从而影响了内源性表达；（2）p.S119Pfs2 变异损害了野生型 PAX9 蛋白核表达的亚细胞定位；（3）四种变异（p.G64V、p.V117G、p.S119Pfs2 和 p.Y217Lfs100）均显著影响 BMP4 基因的下游转录活性。此外，我们总结和分析了 PAX9 变异引起的牙齿缺失位置，发现上颌第二磨牙（84.11%）和下颌第二磨牙（84.11%）是受 PAX9 相关非综合征性牙缺失位置影响最大的牙齿位置。我们的研究结果拓宽了与非综合征性牙缺失相关的 PAX9 基因突变谱，并为未来的遗传咨询提供了有用的信息。
