目的对山东地区有甲状腺大的先天性甲状腺功能减退症(congenital hypothyroidism,CH)患儿双氧化酶成熟因子1(DUOXA1)基因进行突变筛查,阐明DUOXA1基因突变类型及特点,为CH的基因诊断及治疗提供理论依据。方法通过山东省新生儿筛查系统选取山东省52例有甲状腺大的CH患儿及100例健康对照者,抽取外周静脉血,提取白细胞全基因组DNA,采用8对序列特异性引物,运用PCR扩增与直接测序法对DUOXA1基因的全部编码序列(codingsequence,CDS)进行突变筛查。测序结果与DUOXA1基因的美国国立生物技术信息中心参考序列:NG_033105.1进行比对,并对发现的单核苷酸多态性(single nucleotide polymor—phisms,SNP)位点的基因频率进行,检验。结果在52例有甲状腺大的CH患儿及100例健康对照CDS区序列中均未发现突变,但在9例患儿及11例健康对照中的第7外显子发现1个SNP位点(rs75981505,c.398G〉T)为错义突变,导致密码子由CGC突变为CTC,相应133位氨基酸由精氨酸变为组氨酸(p.Arg133His)。2组SNP基因频率比较,差异无统计学意义(17.3%比11.0%,x^2=1.24,P〉0.05)。结论DUOXA1基因在山东地区有甲状腺大CH患儿突变率低,可能不是该地区伴甲状腺大CH患儿的主要病因。
Objective To screen the dual oxidase maturation factor 1 (DUOXA1) gene mutations in children with congenital hypothyroidism (CH) and thyroid goiter from Shandong Province, China, and to identify the gene mutation type and characteristics of DUOXA1 gene mutations in order to provide some evidence for gene diagnosis and therapy of CH. Methods A cohort of 52 cases of CH with thyroid goiter and 100 normal controls were selected according to neonatal screening system in Shandong Province whose genomic DNA was isolated from peripheral blood leukocytes with a standard phenol chloroform method. The whole coding sequence (CDS) of DUOXA1 gene was amplified with 8 pairs of sequence specific primers by using PCR. The PCR products were directly sequenced with Sanger sequencing to detect new mutations types of DUOXA1 gene. The sequencing data were compared to the DUOXA1 gene reference sequence ( National Center for Biotechnology Information:RefSeq:NG_033105.1 ) to see if there was any mutation. A x^2 test was done for the gene frequency of discovered single nucleotide polymorphisms (SNP). Results There was no mutation in CDS of 52 CH patients with thyroid goiter and 100 normal controls. However,a SNP (rs75981505 ,c. 398G 〉 T) which was an missense mutation and could lead to a change of the codon from CGC to CTC, was found in 9 CH patients with thyroid goiter and 11 normal controls in the exon 7. The corresponding amino acid arginine was replaced by histidine( p. Arg133His). There was no significant difference in the SNP rate between CH patients with thyroid goiter and normal controls (17.3% vs 11.0% ,x^2 = 1.24 ,P 〉 0.05 ). Conclusion DUOXA1 gene mutation rate is very low which may not be the main cause of CH patients with thyroid goiter in the population of Shandong Province.