先天性甲减（CH）致病基因（TTF-1、TTF-2、NKX2.5、PAX8、TSHR及NIS）突变筛查研究

发布时间：2018-05-17 06:41

本文选题：先天性甲状腺功能减低症 + 甲状腺激素合成障碍　；参考：《青岛大学》2017年硕士论文

【摘要】：先天性甲状腺功能减低症(congenital hypothyroidism,CH)属于新生儿中最常见的内分泌疾病,主要通过新生儿筛查发现,若不及时诊断并治疗会造成患儿智力和身体发育的不可逆性损害。其主要分为两种:甲状腺发育异常(约占85%-90%),甲状腺激素合成障碍(约占10%-15%)。其中甲状腺发育异常包括缺如、异位、发育不良三种形式,病因不详,可能与某些基因(如TSHR)或转录因子(TTF-1、TTF-2、PAX8)的突变有关;而甲状腺激素合成障碍属于常染色体隐性遗传,可能与DUOXA2、DUOX2、NIS等基因的突变有关。本研究共分为两章,第一章CH伴甲状腺肿大患儿的NIS基因突变筛查。目的:NIS基因的突变与甲状腺激素合成障碍相关,在CH伴甲状腺肿大患者中进行NIS基因突变的筛查,以研究NIS基因突变类型、特点以及与临床表型的关系。方法:收集110例CH伴甲状腺肿大患儿(所选患儿都已经过DUOX2、DUOXA2等与甲状腺激素合成障碍相关基因的筛查)的血液样本,从外周血白细胞中提取全基因组DNA,通过聚合酶链反应扩增NIS基因所有外显子以及内含子与外显子交界部位,并进行测序分析。结果:在非血缘关系的家庭中,虽然未发现NIS基因的致病突变,共发现有2个错义突变(p.A514S、p.R569W),一个同义突变(p.L408=)和6个单核苷酸多态性(SNP)位点(IVS5-51、IVS6+11、IVS8+22、IVS14-8、IVS14+22、IVS14+28)。结论:在山东省内CH伴甲状腺肿大患儿中,NIS基因的突变率较低。在这项研究中虽发现两个罕见的变体(p.A514S、p.R569W),但需要进一步研究,以确定这些变体是否有功能的改变,或患儿是否有其他原因所致。第二章采用二代测序技术对CH伴甲状腺缺如患儿基因筛查研究。目的:转录因子(TTF-l、TTF-2、PAX8、NKX2.5)和促甲状腺激素受体因子(TSHR)的基因突变与甲状腺发育异常(TD)相关,而甲状腺缺如是TD的一种临床表现类型。通过对上述候选基因的筛查,在山东省内CH伴甲状腺缺如患儿中建立基因型与表型的关系。方法:利用二代测序技术对TTF-1、TTF-2、PAX8、NKX2.5、TSHR基因的全部外显子以及侧翼序列进行筛查,并对带有可疑基因突变的DNA样本进行sanger验证,以排除假阳性,并对发现变体的突变频率与数据库中的进行比较。结果:在TSHR基因中共发现有7个变体(p.P52T、p.R450H、p.C700E、p.G132R、p.M164K、p.A522V、p.R528S),TTF-2基因中发现有1个变体(p.P243T),NKX2.5基因中发现一个变体(p.N291I),TTF-1基因中发现有5个变体(p.G360V、p.R401Q、p.L418I、p.G44N、p.E453Q),PAX8基因中发现有2个变体(p.A355V、c.-26GA)。其中位于TSHR中p.G132R、p.M164K、p.A522V在已有的数据库资料中均未找到,为首次发现,并且p.G132R和p.M164L在同一患者中被发现。以及位于TSHR基因中的p.R450H和位于NKX2.5基因中的p.N291I在同一患者中被发现,位于TSHR基因中的p.R450H和位于PAX8基因中的p.A355V也在同一患者中被发现。结论:在山东地区CH伴甲状腺缺如患者中TSHR突变发生率较高约为7%,突变图谱的多样性。我们的研究进一步扩大了基因突变图谱,突变多位于高度保守的序列,且多位于重要结构域,TSHR基因突变可能为CH的致病基因,具体机制需要进一步的功能验证。与之相反,TTF-l、TTF-2、PAX8、NKX2.5等基因的突变率较低,不是CH伴甲状腺缺如患儿的主要致病突变。
[Abstract]:Congenital hypothyroidism (congenital hypothyroidism, CH) is the most common endocrine disease in newborns. It is found mainly through neonatal screening that untimely diagnosis and treatment can cause irreversible impairment of mental and physical development in children. It is divided into two types: thyroid dysplasia (about 85%-90%), thyroid stimulation The dysplasia of the hormone (about 10%-15%). Among them, abnormal thyroid development includes three forms of absence, ectopic, and dysplasia. The etiology is unknown and may be associated with mutations in some genes (such as TSHR) or transcription factors (TTF-1, TTF-2, PAX8); and thyroid hormone synthesis disorders are in autosomal recessive inheritance and may be associated with mutations in DUOXA2, DUOX2, NIS and other genes. This study is divided into two chapters. In this study, the first chapter is the screening of NIS gene mutations in CH with thyroid enlargement. Objective: the mutation of the NIS gene is related to the disorder of thyroid hormone synthesis. The NIS gene mutation is screened in the patients with CH with thyroid enlargement to study the type of mutation of the NIS gene, the characteristics and the relationship with the clinical phenotype. Methods: collect 1 10 children with CH with goiter (selected children have been screened for DUOX2, DUOXA2 and other genes related to thyroid hormone synthesis), the whole genome DNA was extracted from the peripheral blood white blood cells, and all the exons of the NIS gene and the junction of the introns and exons were amplified by polymerase chain reaction and sequenced. Results: in non related families, 2 missense mutations (p.A514S, p.R569W), a synonymous mutation (p.L408=) and 6 single nucleotide polymorphism (SNP) loci (IVS5-51, IVS6+11, IVS8+22, IVS14-8, IVS14+22, IVS14+28) were found in the unrelated family of the NIS gene. The mutation rate of the IS gene is low. Although two rare variants (p.A514S, p.R569W) are found in this study, further research is needed to determine whether these variants have functional changes or whether the children have other causes. The second chapter uses two generation sequencing techniques to screen for CH with hypothyroidism in children. Gene mutations of TTF-l, TTF-2, PAX8, NKX2.5) and thyroid stimulating hormone receptor factor (TSHR) are associated with thyroid dysplasia (TD), and thyroid deficiency is a clinical manifestation of TD. By screening these candidate genes, the relationship between genotype and phenotype is established in children with CH with thyroid deficiency in Shandong province. Methods: two Screening all exons and flanking sequences of TTF-1, TTF-2, PAX8, NKX2.5, TSHR genes and Sanger validation of DNA samples with suspicious gene mutations to eliminate false positive and compare the mutation frequency of the found variants to the database. Results: 7 variants (p.P52T, P) were found in TSHR gene. .R450H, p.C700E, p.G132R, p.M164K, p.A522V, p.R528S), there are 1 variants (p.P243T) found in the TTF-2 gene. The NKX2.5 gene is found in a variant (p.N291I), and there are 5 variants in the TTF-1 gene. 2 variants are found in the gene. V was not found in the existing database data for the first time, and p.G132R and p.M164L were found in the same patient. And the p.R450H in the TSHR gene and the p.N291I in the NKX2.5 gene were found in the same patient. The p.R450H in the TSHR gene and p.A355V in the PAX8 gene were also found in the same patient. Conclusion: the incidence of TSHR mutation in CH with thyroid deficiency in Shandong is about 7%, and the mutation map is varied. Our study further expands the gene mutation map. The mutation is mostly located in highly conserved sequences, and most of them are in the important domain. The mutation of TSHR gene may be the pathogenetic gene of CH, and the specific mechanism needs to be further studied. On the contrary, the mutation rates of TTF-l, TTF-2, PAX8, NKX2.5 and other genes are relatively low, and are not the main pathogenic mutations in children with CH and thyroid deficiency.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R725.8

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