GBP1影响甲状腺细胞迁移导致先天性甲减发生的分子机制研究

先天性甲减是一种常见的严重影响儿童智力发育的先天性内分泌代谢病。课题组已收集552例先天性甲减样本，前期研究发现约一半的先天性甲减致病基因不明。对未明确致病基因的患者进行全外显子组测序及生信分析，识别了23个候选的先天性甲减致病基因；利用实验室建立的斑马鱼快速筛选体系，发现GBP1基因敲低引起甲减及影响甲状腺细胞迁移和甲状腺发育。在此基础上，本项目将通过先天性甲减人群遗传筛查和临床随访，明确GBP1导致先天性甲减的遗传模式和临床特点及转归；通过过表达或敲低的甲状腺细胞系结合斑马鱼和小鼠等模式生物功能研究，明确GBP1基因突变对其功能的影响，明确GBP1是否通过影响细胞粘附连接和β-catenin通路，调控甲状腺细胞迁移和甲状腺发育，导致先天性甲减发生的分子机制，并识别参与甲状腺细胞迁移的GBP1的上游调节因子，证明GBP1是否是先天性甲减的新的致病基因，为先天性甲减的精准治疗提供科学依据。

Congenital hypothyroidism is a common human congenital endocrine disorder. Untreated congenital hypothyroidism inevitably leads to cretinism, causing irreversible brain dysfunction and dwarfism. Congenital hypothyroidism are primarily caused by thyroid dysgenesis, such as athyreosis, thyroid ectopy, hypoplasia, and hemiagenesis, and thyroid dyshormonogenesis. The pathogenesis of congenital hypothyroidism is still a conundrum, although several candidate genes have been associated with congenital hypothyroidism, comprehensive screening of causative genes has been limited. Among a total of 552 patients with congenital hypothyroidism collected by our study consortium, 192 samples has been screened by the panels with 21 previously reported congenital hypothyroidism causative genes. And we detected the biallelic mutations of 15 reported genes in 94 patients with congenital hypothyroidism, suggesting nearly half of congenital hypothyroidism patients with unknown causative genes. Firstly, we achieved the whole exome sequencing and bioinformatic analysis in the 98 congenital hypothyroidism patients with unknown causative genes and identified 23 candidate causative genes for congenital hypothyroidism, including 4 pathogenic GBP1 mutations from 3 congenital hypothyroidism patients in 3 pedigrees. Next, when we used zebrafish morpholino knockdown models to screen the 23 candidate causative genes with recurrent or compound heterozygous mutations, we found that gbp1 knockdown zebrafish exhibited the phenotypes with increased tsh expression level compared with controls, “bell-like” thyroid morphology, the thyroid cells remained piled up together, and appeared to lose their ability to move along the anterior-posterior axis and the ability to move along endothelial cells simultaneously, suggesting that GBP1 may play an important role in the aetiology of congenital hypothyroidism by affecting thyroid cell migration and thyroid development. Secondly, we will recruit additional patients with congenital hypothyroidism and perform the genetic screening for the 23 candidate causative genes of congenital hypothyroidism to verify these new congenital hypothyroidism causative genes. At the same time, we will presume the possible inheritance pattern of GBP1 and undertake the epidemiological follow-up study for the clinical characteristics, treatment, and prognosis in the congenital hypothyroidism patients harboring GBP1 mutations, providing theoretical basis for the precision medicine of congenital hypothyroidism. Finally, we are going to establish wild-type or mutated GBP1 overexpression or knockdown or knock-out models by CRISPR/Cas9 system in human papillary thyroid carcinoma TPC1 cell lines, zebrafish, and mice, in order to investigate the mechanism how GBP1 can regulate embryonic thyrocyte migration along the pharyngeal midline by affecting adherens junctions and Wnt/β-catenin signaling pathway, cause thyroid dysgenesis and induce the pathogenesis of congenital hypothyroidism. Taken together, this study can confirm that GBP1 is a causative gene of congenital hypothyroidism, thus uncovering fresh insights into the prevention and treatment of congenital hypothyroidism.