先天性甲状腺功能减退症患儿TSHR基因突变筛查及DUOX2基因突变致病机理研究

发布时间：2018-04-23 10:58

  本文选题：先天性甲状腺功能减退症 + 促甲状腺素受体　； 参考：《青岛大学》2014年硕士论文

【摘要】：先天性甲状腺功能减退症(CH)是婴幼儿最常见的内分泌疾病之一,主要表现为甲状腺功能部分或完全丧失导致的生长发育落后和智力发育迟缓。80%-85%的CH患者继发于甲状腺发育异常,与促甲状腺激素受体(TSHR)等基因突变有关；其余15%-20%的患者则是由甲状腺激素合成缺陷导致,其中,双氧化酶2(DUOX2)基因突变后因不能氧化NADPH提供H202,致使碘的有机化障碍,最终导致CH。 本实验第一部分以89例山东地区甲状腺异位导致CH的患儿为研究对象,采集外周血提取DNA, PCR扩增第10外显子,采用直接测序的方法对TSHR基因进行突变筛查。在89例研究对象第10外显子测序结果中,发现1个错义突变c.1269GA(p.V424I),1个SNP位点(rs1991517,c.2181GC,变异频率18.5%)。对小鼠、大鼠、猫、猪、牛、斑马鱼和人的TSHR蛋白进行同源性比较,发现第424密码子编码的氨基酸位于TSHR的保守区,该位点的缬氨酸被异亮氨酸取代(p.V4241),表明山东地区甲状腺异位导致CH的患者中,TSHR基因第10外显子突变率较低。 在前期的研究中,本课题组对10例甲状腺功能减退伴甲状腺肿大的患儿进行DUOX2基因突变筛查,共发现2个DUOX2基因新的错义杂合突变：c.106OCT (p. R354W)和c.G3616A(p.A1206T)。本实验第二部分对新发现的2个突变进行功能学研究,以阐明DUOX2基因突变导致甲减的致病机理。构建DUOX2野生型或突变型以及DUOXA2野生型真核表达载体,转染293T细胞,转染后48h加入50uM Amplex Red reagent和0.2U/ml辣根过氧化物酶,37℃避光孵育2h,利用多功能酶标仪监测595nm荧光强度,计算H202浓度；以DUOX2野生型或突变型与DUOXA2野生型真核表达载体共转染Hela细胞,36h后加入终浓度为100μg/ml的放线菌酮(CHX)阻断细胞内蛋白质合成,之后0-24h内取5个时间点分别裂解细胞,提取总蛋白,用Western blot技术对DUOX2进行定量检测,研究DUOX2突变后蛋白半衰期的改变。结果表明：无论野生型还是突变型DUOX2质粒单独转染293T细胞后都不能生成H202,而与DUOXA2共转染293T细胞后,野生型产生H202能力显著提高,而突变型生成H202能力则表现为部分缺陷型；DUOX2基因突变后表达的蛋白半衰期缩短,可能与突变蛋白更易降解有关。
[Abstract]:Congenital hypothyroidism (CHS) is one of the most common endocrine diseases in infants and young children. It is mainly characterized by growth retardation caused by partial or complete loss of thyroid function and mental retardation. 80-85% of Ch patients are secondary to thyroid dysplasia. The other 15% to 20% of the patients were caused by the deficiency of thyroid hormone synthesis. After the mutation of the dioxygenase 2DUOX2 gene, the NADPH could not be oxidized to provide H2022, which resulted in the organic disorder of iodine, which resulted in CH2. In the first part of this experiment, 89 children with Ch caused by thyroid heterotopia in Shandong province were studied. The peripheral blood samples were extracted, the exon 10 was amplified by PCR, and the mutation of TSHR gene was screened by direct sequencing. One missense mutation c.1269 GAp.V424Ihe and one SNP locus, rs1991517, c.2181GC, were found in 89 subjects. The mutation frequency was 18.5g. The homology of TSHR protein in mice, rats, cats, pigs, cattle, zebrafish and human was compared. It was found that the amino acid encoded at codon 424 was located in the conserved region of TSHR. The valine of this locus was replaced by isoleucine, indicating that the mutation rate of exon 10 of TSHR gene was lower in patients with Ch caused by thyroid ectopic in Shandong area. In the previous study, 10 children with hypothyroidism and goiter were screened for DUOX2 gene mutation. Two new missense heterozygosity mutations of DUOX2 gene were found in 10 children with hypothyroidism and goiter. R354W) and c. G3616A, p. A1206T _ _ _ In the second part of this experiment, the two new mutations were studied in order to elucidate the pathogenesis of hypothyroidism caused by DUOX2 gene mutation. DUOX2 wild-type, mutant and DUOXA2 wild-type eukaryotic expression vectors were constructed and transfected into 293T cells. After 48 h transfection, 50uM Amplex Red reagent and 0.2U/ml horseradish peroxidase were incubated at 37 鈩，

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