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目的研究山东地区先天性甲状腺功能减退症(CH)伴甲状腺肿大患儿双氧化酶1(DUOX1)基因突变类型及特点,为CH的诊断及基因治疗提供理论依据。方法选取43例经新生儿筛查确诊为CH伴甲状腺肿大患儿及100例正常对照新生儿,提取CH患儿及正常新生儿外周血基因组DNA,采用聚合酶链式反应(PCR)扩增及一代测序法(Sanger测序法),对DUOX1基因全部外显子进行基因突变检测,结合测序结果及生物信息学数据,分析DUOX1基因是否存在突变,并对发现的单核苷酸多态性(SNP)位点的基因频率进行χ2检验。结果在43例CH伴甲状腺肿大患儿及100例正常对照新生儿中,均未发现DUOX1基因突变。但在外显子区发现了2个SNP位点(rs16939752,c.3076T>C;rs1706804,c.3228A>G),前者为错义突变(Cys→Arg),后者为同义突变(Thr→Thr);内含子区发现了1个SNP位点(rs2020216,IVS8+129C>T)。经统计,本文实验组患儿与正常对照组新生儿SNP的基因频率比较,差异无统计学意义(P>0.05)。结论山东地区CH伴甲状腺肿大患儿中,DUOX1基因突变率低,可能不是该地区CH伴甲状腺肿大的主要病因。
Objective To investigate the types and characteristics of DUOX1 gene mutations in children with congenital hypothyroidism (CH) and thyroid enlargement in Shandong Province, and to provide a theoretical basis for the diagnosis and gene therapy of CH. Methods Forty-three neonates diagnosed as CH with thyroid enlargement and 100 normal controls were enrolled in the study. Genomic DNA was extracted from peripheral blood of children with CH and normal newborns and amplified by polymerase chain reaction (PCR) And generation sequencing (Sanger sequencing) to detect the gene mutations in all exons of DUOX1 gene. Combined with sequencing results and bioinformatics data, the DUOX1 gene was analyzed for the presence of mutations and the single nucleotide polymorphism SNP) locus gene frequency χ2 test. Results No DUOX1 gene mutation was found in 43 CH patients with thyroid enlargement and 100 normal control neonates. However, two SNPs were found in the exon region (rs16939752, c.3076T> C; rs1706804, c.3228A> G). The former was a missense mutation (Cys → Arg) and the latter was a synonymous mutation (Thr → Thr). One SNP site was found in the intron region (rs2020216, IVS8 + 129C> T). According to the statistics, there was no significant difference in the SNP gene frequencies between the experimental group and the normal control group (P> 0.05). Conclusions The prevalence of DUOX1 gene mutation in children with CH with thyroid enlargement in Shandong Province is low, which may not be the major cause of CH with thyroid enlargement in this area.