DUOX2基因突变导致先天性甲状腺功能减退症的遗传模式及其临床特征研究

Congenital hypothyroidism (CH) is a common genetic endocrinological diseases in newborns. According to the clinical outcome, it can be divided into permanent and temporary hypothyroidism. Mutations in DUOX2 gene cause disorder of thyroid hormone synthesis, which ultimately leads to CH. Previous studies found that genotype and clinical phenotype was not completely consistent in DUOX2 mutations. So the model of inheritance is not clear. In addition, temporary CH patients had normal thyroid function after the brain development. Whether these patients need long-term hormone replacement was also inconclusive. We collected 72 CH patients. After genetic scanning, 46 patients were found to have DUOX2 gene mutations. DUOX2 gene mutation was able to explain 41.7% of CH. We hypothesized that CH caused by DUOX2 gene mutation was in an autosomal recessive model and that CH patients with normal thyroid function after brain development require long-term hormone replacement therapy. Based on this hypothesis, zebrafish with compound heterozygous mutations of DUOX gene was constructed to study the relationship between genotype and phenotype. Population study was also carried out to clarify whether discontinue of hormone replacement(temporary CH patients carrying DUOX2 gene mutations have normal thyroid function) will influence the long-term growth and development. In the end, our work will have positive impact on promotion of population quality.

先天性甲状腺功能减退症(CH)是新生儿常见的内分泌遗传疾病，根据临床转归，可分为永久性和暂时性甲减。DUOX2基因突变会引起甲状腺激素合成障碍，最终导致CH。既往的研究发现DUOX2突变的基因型和临床表型不完全一致，其遗传模式尚不明确；另外，脑发育完成后甲功正常的暂时性CH患者是否需要长期激素替代治疗也尚无定论。我们收集了72例CH患者，通过基因筛查，发现46例患者携带有DUOX2基因突变，其中DUOX2基因突变能够解释41.7%的CH。我们推测DUOX2基因突变导致的CH呈常染色体隐性遗传模式，并且在脑发育完成后甲功正常的暂时性CH患者仍需要长期激素替代治疗。基于这一假设，通过构建DUOX基因复合杂合突变的斑马鱼，研究基因型与表型的关系；开展人群研究，明确携带DUOX2基因突变的暂时性CH患者甲功正常后停药是否会影响长期的生长发育。最终，通过我们的研究将会对人口素质的提升产生积极的影响。

采用靶向二代测序技术对273例先天性甲减患者进行了21个基因的测序，发现有217个患者在其中18个基因(DUOX2,TG,DUOXA1, TPO, DUOXA2, DUOX1,TSHR,SLC26A4,NKX2.1,GNAS,FOXE1,PAX8,NKX2.5,THRA,IYD,DIO1,SLC5A5,HHEX）上测到了突变，其中DUOX2是突变频率最高的基因，有160个患者在基因上检测到了突变，突变频率高达（73.7%）,其次是TG(13.55%,37/273), DUOXA1(11%,30/273)，绘制了中国人群中特异的基因突变谱。由于DUOX2是最主要的先天性甲减的致病基因，我们构建了野生型DUOX2质粒及64个突变型质粒，与DUOXA2共同转染293T细胞，检测野生型及突变型DUOX2产生H202的能力。我们发现5个突变位点的酶活性完全丧失(<10% of wt):R56W、N232K、R347T、A366V、R1110Q，53个突变位点酶活性下降（10-80% of wt），6个突变位点不影响酶活性：S363G、S906P、R1021X、R1211H、L1343F、D1537N。分别采用morpholino 和CRISPR Cas9技术分别敲低和敲除斑马鱼的DUOX基因，发现斑马鱼出现甲状腺激素合成障碍导致的甲状腺功能减退。随后通过体外实验得出的DUOX2基因突变残留的酶活性，选择DUOX2残留酶活性小于50%且两个等位基因的的酶活性均下降的DUOX2双突变患者76例，我们认为这76例患者是真正由于DUOX2突变导致的先天性甲减，称为DUOX2阳性突变患者。比较这些患者与其他107例不携带DUOX2突变的患者，发现携带DUOX2阳性突变的患者的FT4水平明显低于不携带DUOX2阴性突变患者的FT4水平。以DUOX2酶活性临界值把DUOX2双突变患者分为轻度或重度甲减两个亚组，通过随访这些患者发现酶活性临界点与两亚组之间TSH差异的p值呈”V”型曲线，发现22%的酶活性大小是一个最佳临界值，可以反映DUOX2阳性突变患者的先天性甲减的严重程度。此外，通过二代测序与靶向测序相结合，研究230名先天性甲减病人中的TPO突变。通过对于TPO突变病人随访，研究TPO突变类型和病人临床表现之间的关系，可以为指导病人用药剂量的调整以及疾病预后提供参考。

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