发育性髋关节发育不良致病基因鉴定及PTH1R基因突变在其发病中的作用机制研究

Developmental dysplasia of the hip (DDH) is one of the most common diseases in the department of pediatric orthopedics, the incidence of which is up to 5‰. Genetic factor is the basic of the onset of DDH, however, the pathogenic gene and the pathogenesis of DDH were so far unknown. . In our former work, whole exome analysis of 3 generations (2 of them were severely affected family members) of a 37-member DDH family was performed to identify the pathogenic gene of DDH. Data were analyzed by bioinformatics and further validation within the family. We found that the c.C629T mutation of the parathyroid hormone 1 receptor (PTH1R) gene is closely related to the onset of DDH. However, whether PTH1R is the very disease-causing gene for DDH and the molecular mechanism of which leading to the onset of DDH are not clear. . To this end, we propose the hypothesis: PTH1R may be the DDH-causing gene and it involves in the onset of DDH through influencing it’s ability to banding to its ligand (PTH or PTHrP) and influencing the activation of its downstream signaling pathways. . In order to test this hypothesis, we will validate the mutation of PTH1R in different DDH group by conventional Sanger sequencing method and make sure whether PTH1R is the DDH disease-causing gene; We will combined a variety of methods, such as RT-PCR, WB, plasmid transfection, cell proliferation and apoptosis detection and expression profile microarray, form various of angles and levels, such as molecules, cells, tissues, to explore the impact of PTH1R mutation on PTH1R mediated signaling pathways and on the cell biological characteristics. Clear the molecular mechanism of PTH1R mutation on the pathogenesis of DDH.

发育性髋关节发育不良(DDH)是儿童骨科最常见疾病之一，发病率达5‰。遗传因素是DDH的发病基础，但DDH的致病基因及发病机制迄今不明。. 我们前期实验对一个37人DDH家系的3代人进行全基因外显子测序筛选致病基因，数据经分析并初步验证后发现甲状旁腺素1受体（PTH1R）基因突变与DDH发病密切相关。但PTH1R是否为DDH致病基因、其在DDH发病中的作用机制无文献报道。. 为此，我们提出假说：PTH1R可能是DDH的致病基因并通过影响其与配体的结合和下游信号通路的激活参与DDH发病。. 为验证这一假说，我们拟采用常规测序法在不同DDH人群验证PTH1R突变位点，明确该基因是否为DDH致病基因；采用RT-PCR、WB、质粒转染、表达谱分析等手段，从分子、细胞、组织水平多层面探索PTH1R突变对其下游信号通路及细胞生物学特性的影响，明确PTH1R在DDH发病中的作用及分子调控机制。

背景：发育性髋关节发育不良（DDH）是儿童骨科最常见疾病之一，遗传因素在其发病中发挥重要作用，但DDH的致病基因和发病机制仍不明确。我们前期研究发现甲状旁腺素1受体（PTH1R）基因的突变可能与DDH发病相关。研究内容：本研究拟验证和鉴定PTH1R，检测PTH1R对细胞生物学特性的影响。结果、数据及意义：测序验证发现其他3个DDH家系和332例DDH散发病例中无PTH1R基因的突变，提示PTH1R突变可能与DDH发病无关。为进一步探索DDH的发病机制，我们对37人DDH家系的另2位成员进行全外显子测序，综合分析5例家系成员外显子测序结果，发现骨形成蛋白2诱导激酶基因（BMP2K）的CAG插入或缺失突变和前列腺素 F2α受体基因（PTGFR）的终止密码子突变可能与DDH发病相关。Sanger测序进一步在该家系中验证BMP2K和PTGFR突变，结果显示家系中的全部 4个DDH患者有突变（4/4，100%），12个有血缘关系的3个正常人有突变（3/12，25%），8个无血缘关系的正常人中无突变（0/8，0%）。接着，测序验证BMP2K和PTGFR在300例DDH散发病例和300例正常对照中的突变情况，结果显示25例DDH散发病例（25/300，8.33%）和6例正常对照（6/300，2%）有 BMP2K突变（P＜0.01）；21例DDH散发病例（21/300，7%）和0例正常对照（0/300，0%）有PTGFR终止密码子突变（P＜0.01）。生物信息学分析和文献检索显示，PTGFR在所有人群（包括各类疾病和正常人群）中的突变率为1.6/十万、突变所在区域从鱼类到人类的各个物种中高度保守、突变有高度蛋白功能损害性和致病性，PTGFR及其所在信号通路在软骨细胞增殖、分化和软骨结节形成中发挥关键调控作用，软骨的异常发育与DDH的发病和发展密切关联，因此本项目进一步研究聚焦PTGFR在DDH发病中的功能探索。进一步检测PTGFR的表达结果提示PTGFR mRNA和蛋白在DDH病例关节囊和圆韧带中较对照组明显低表达，提示PTGFR可能直接参与DDH的表型形成。我们进一步成功构建了携带野生型（-WT）和突变型（-MUT）PTGFR的慢病毒载体并成功建立稳定转染的原始软骨细胞株ATDC5-WT和ATDC5-MUT，PTGFR在DDH发病中的作用分子机制正在持续研究中。