新的先天性甲减致病基因EIF4B的识别及其致病分子机制的研究

Congenital hypothyroidism (CH), the most common neonatal metabolic disorder, is characterized by impaired neurodevelopment. The pathogenesis of CH is still a conundrum, although several candidate genes have been associated with CH, comprehensive screening of causative genes has been limited. In this study, to discover the causal variant(s)/gene(s) of CH, firstly we carried out the targeted resequencing for all exons and exon–intron boundaries of 21 previously reported CH causative genes in the total of 192 CH patients, and detected the biallelic mutations of 15 reported genes in 94 patients with CH. Then, we achieved the whole exome sequencing in the 98 CH patients with unknown causative genes and identified 22 candidate causative genes for CH. Secondly, we used zebrafish morpholino knockdown models to screen the five candidate causative genes with recurrent or compound heterozygous mutations and found that eif4ba/eif4bb knockdown zebrafish exhibited the phenotypes with lower heart rate and shorter body length and showed the increased tsh, tg, nkx2-1, and tpo expression level compared with controls, suggesting that eif4ba/eif4bb may play an important role in the aetiology of CH. Next, we will recruit additional patients with CH and perform the genetic screening for the 22 candidate causative genes of CH to prove these new CH causative genes. Thirdly, we are going to establish EIF4B knock-out models by CRISPR/Cas9 system in Nthy ori 3.1 thyroid cell lines, zebrafish and mice, in order to investigate the mechanism how EIF4B can cause thyroid dysgenesis or thyroid dyshormonogenesis and induce the pathogenesis of CH. Finally, we will presume the possible genetic model of EIF4B and undertake the epidemiological follow-up study for the clinical presentation, treatment and prognosis in the CH patients harboring EIF4B and DUOX2 mutations, providing theoretical basis for the precision medicine of CH. Taken together, this study can confirm that EIF4B is a causal gene of CH, thus uncovering fresh insights into the prevention and treatment of CH.

先天性甲减（CH）是一种常见的严重影响儿童生长发育和智力的内分泌疾病。课题组收集192例CH样本，通过二代测序技术对已知的21个CH致病基因进行筛查，发现约一半的CH致病基因不明。对未知致病基因的患者进行全外显子组测序及信息生物学分析，识别了22个候选的CH致病基因；利用实验室建立的斑马鱼快速筛选体系，对其中的5个带有热点和复合杂合突变的候选基因进行筛查，发现EIF4B基因敲低引起甲减。在此基础上，本项目将通过CH人群遗传筛查，结合斑马鱼和小鼠等模式生物功能研究，证明EIF4B是否是CH的新的致病基因，并证明EIF4B导致CH的遗传模式；并通过体内外研究，明确EIF4B是否通过介导甲状腺发育不良或甲状腺激素合成障碍，进而引起CH的发生，阐明其导致甲减的分子机制；最后通过临床随访和对照研究，明确EIF4B基因突变导致的CH是否有其特殊的临床特点和转归，为CH的精准治疗提供科学依据。

该项目进展顺利，取得了一些新的成果，并产生了重要的可继续深入的研究的新的领域。通过对在未知基因致病的307例先天性甲减患者全外显子组测序数据的系列生物信息学分析，识别了一个新的先天性甲减的候选变异基因EIF4B，我们发现13例患者携带EIF4B的变异，其中1例携带EIF4B的复合杂合变异。接下来，我们通过构建敲除和敲低的斑马鱼模型均很好还原了甲减的表型，而小鼠的敲除模型表现为致死性的表型。进一步的分子生物学实验表明EIF4B可通过影响甲状腺激素合成和发育基因的表达进而参与甲减的发生。以上研究结果为这种基因变异的先天性甲减的治疗和预防提供精准和科学的依据。在该项目的资助下，已发表SCI收录论文15篇。