[利用聚合酶链反应-高分辨率熔解曲线分析快速筛查MMACHC基因中的热点变异c.609G>A]
[Rapid screening of a hotspot variant c.609G>A in MMACHC gene by using PCR-high-resolution melting curve analysis].
作者信息
Lin Shuxiang, Wang Chao, Zhang Xinjie, Xu Xiaowei, Zou Qianqian, Cai Chunquan, Zhang Yuqin, Shu Jianbo
机构信息
Tianjin Pediatric Research Institute, Tianjin Children's Hospital, Tianjin 300134, China.
出版信息
Zhonghua Yi Xue Yi Chuan Xue Za Zhi. 2020 Jul 10;37(7):759-763. doi: 10.3760/cma.j.issn.1003-9406.2020.07.014.
OBJECTIVE
To carry out genetic testing for two families affected with cobalamin C (cblC) and establish a rapid method for the detection of a hotspot pathogenic variant c.609G>A of the MMACHC gene by using a PCR-high-resolution melting curve (PCR-HRM) method.
METHODS
Genomic DNA was extracted from peripheral blood samples of the probands and their parents. Potential variants of the MMACHC gene was analyzed by Sanger sequencing. The c.609G>A variant of the MMACHC gene was screened among 100 healthy children with the PCR-HRM method.
RESULTS
Sanger sequencing revealed that proband 1 carried compound heterozygous variants c.394C>T and c.609G>A of the MMACHC gene, while proband 2 carried compound heterozygous variants c.482G>A and c.609G>A of the same gene. PCR-HRM analysis of the two probands and the 100 healthy children were consistent with the Sanger sequencing.
CONCLUSION
c.609G>A is a hotspot pathogenic variant of the MMACHC gene. The diagnosis of cblC may be rapidly attained through detection by PCR-HRM.
目的
对两个患有钴胺素C（cblC）的家庭进行基因检测，并建立一种利用聚合酶链反应-高分辨率熔解曲线（PCR-HRM）方法检测MMACHC基因热点致病变异c.609G>A的快速方法。
方法
从先证者及其父母的外周血样本中提取基因组DNA。通过桑格测序分析MMACHC基因的潜在变异。采用PCR-HRM方法在100名健康儿童中筛查MMACHC基因的c.609G>A变异。
结果
桑格测序显示，先证者1携带MMACHC基因的复合杂合变异c.394C>T和c.609G>A，而先证者2携带同一基因的复合杂合变异c.482G>A和c.609G>A。对两名先证者和100名健康儿童的PCR-HRM分析结果与桑格测序一致。
结论
c.609G>A是MMACHC基因的热点致病变异。通过PCR-HRM检测可快速实现cblC的诊断。
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