西北地区甲状腺功能减低症患者DUOX2基因突变筛查和研究

发布时间：2018-06-09 17:07

本文选题：先天性甲状腺功能低下(CH) + 双氧化物酶2(DUOX2)　；参考：《西北大学》2017年硕士论文

【摘要】：先天性甲状腺功能减低症(congenital hypothyroidism,CH)是一种常见的由于甲状腺发育不全或者甲状腺内分泌机能障碍而引起的内分泌疾病。研究发现由于甲状腺激素分泌不足导致的CH多数是遗传因素导致的,双氧化物酶2(DUOX2)基因作为甲状腺激素合成的关键基因,其突变对CH的形成有着重要的影响。本研究共招募到西北地区135例CH患者和100例健康个体,采用高通量测序的方法对患者DUOX2全外显子和外显子内含子剪接处进行测序,同时对筛选出来的候选致病位点进行一代测序验证,并在100例健康样本对照中进行频率验证。然后利用计算生物学的方法对这些潜在致病位点的功能和效应进行分析,同时分析DUOX2突变谱和CH临床表型的关系,并且根据美国医学遗传学与基因组学学会和美国分子病理学会(ACMG/AMP)序列变异注释标准对其进行致病等级的划分。结果发现:在135例CH患者中共检测到49个罕见突变和2个功能多态性位点。在49个罕见突变中,有35个是错义突变,6个插入缺失突变,6个无义突变和2个剪接突变,其中11个是首次发现的新突变,它们在健康对照中的频率均小于1%。在135例患者中DUOX2突变检出率为60.7%(82/135),并且有4个热点突变(p.R1110Q,p.S1067L,p.R885Q,p.K530X)在CH患者和健康对照组中存在显著差异。采用SIFT,PolyPhen-2,MutationTaster,FATHMM,M-CAP等5个蛋白质功能预测软件对所有错义和无义突变的分析发现21个突变可能会损害蛋白质的功能;利用MaxEntScan,NetGene2和BDGP 3个软件对剪接突变分析发现2个突变(IVS28+1GT,IVS17+IGT)都可能会损害蛋白质的功能。研究发现患者所携带DUOX2的突变类型和位点数与临床表型没有直接的关系,其基因型和临床表型之间存在非常复杂的关系。对新发现的疑似致病位点进行7个物种间序列保守性分析,结果发现4个(p.D137E,p.R434_S440del,p.F591S,p.M1093V)位于保守区,可能对蛋白质结构和功能产生影响。最后综合目前已知的所有证据,根据ACMG/AMP对疑似致病位点的致病等级的划分标准,发现 7 个突变位点(p.R1110Q,p.R885Q,IVS28+1GT,p.R842X,IVS17+1GT,p.K530X,p.Q481X)为致病的(pathogenic)、9 个可能致病的(likeyly pathogenic)、33 个意义不明的(variants of unknown significance)以及 2 个(p.S1067L,p.H678R)良性突变位点(benign)。另外,本研究发现的两个功能多态性位点中,p.S1067L与CH的发生风险增高有关(OR=2.2,P=0)。总之,本研究首次采用高通量测序技术对西北地区CH患者的DUOX2基因突变进行筛查研究,分析了西北地区甲低患者中DUOX2突变的类型、特点、以及与临床表型的关系,这对于扩大DUOX2基因突变谱,进一步了解CH致病机制具有重要的理论价值。
[Abstract]:Congenital hypothyroidism is a common endocrine disease caused by hypothyroidism or hypothyroidism. It is found that most of the Ch secreted by thyroid hormone is caused by genetic factors. As the key gene of thyroid hormone synthesis, the mutation of the dioxase 2DUOX2 gene has an important effect on the formation of Ch. In this study, 135 Ch patients and 100 healthy individuals were recruited in Northwest China. High throughput sequencing was used to sequence the splicing sites of the full exon and intron of DUOX2 in patients. At the same time, the candidate pathogenicity sites were sequenced for one generation and the frequencies were verified in 100 healthy controls. Then the function and effect of these potential pathogenic sites were analyzed by computational biology, and the relationship between the DUOX2 mutation spectrum and the clinical phenotype of Ch was also analyzed. According to the American Society of Medical Genetics and Genomics and the American Society of Molecular Pathology (ACMG / AMP) sequence variation annotation standard, the disease grade was classified. The results showed that 49 rare mutations and 2 functional polymorphic loci were detected in 135 patients with Ch. Of the 49 rare mutations, 35 were missense mutations, 6 insertion deletion mutations, 6 nonsense mutations and 2 splicing mutations, of which 11 were new mutations found for the first time, and their frequencies were less than 1 in healthy controls. In 135 patients, the detection rate of DUOX2 mutation was 60.7 / 82 / 135, and there were significant differences between Ch patients and healthy controls. Five protein function prediction softwares such as SIFTX PolyPhen-2 Mutation Tasterboard FATHMMMM-CAP were used to analyze all missense and nonsense mutations. It was found that 21 mutations might damage the function of protein. Using MaxEntScann NetGene2 and BDGP to analyze splicing mutations, it was found that two mutants, IVS281GTG / IVS17 IGT, might damage the function of protein. It was found that the mutation type and site number of DUOX2 were not directly related to the clinical phenotype, but there was a very complex relationship between the genotype and the clinical phenotype. The conserved sequences of 7 new suspected pathogenicity loci were analyzed. The results showed that the conserved region was 4 p. D137EN. R434E p. R440dellp. F591Sn. M1093V), which might have an effect on the structure and function of proteins. Finally, synthesizing all known evidence, according to ACMG / AMP's criteria for the classification of suspected pathogenicity sites, It was found that 7 mutation loci, p. R1110QN, p. R885QnIVS28, GTP. R842XnIVS17, GTP.K530X, p. Q481X) were 9 pathogenetic of unknown significance) of pathogenicity, 33 variants of unknown significance) with unknown significance, and 2 benign mutation loci of p. S1067LPH678R. In addition, two functional polymorphic loci were found in this study. S1067L was associated with increased risk of Ch. In conclusion, the DUOX2 gene mutations in Ch patients in Northwest China were screened by high-throughput sequencing for the first time. The types, characteristics and clinical phenotypes of DUOX2 mutations in patients with hypothyroidism were analyzed. It has important theoretical value for expanding the mutation spectrum of DUOX2 gene and further understanding the pathogenesis of Ch.
【学位授予单位】：西北大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R581.2

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