多重竞争性荧光PCR检测X连锁Alport综合征大片段缺失突变

发布时间：2018-05-30 22:30

本文选题：Alport综合征 + X连锁　；参考：《北京大学学报(医学版)》2017年05期

【摘要】：目的:探讨多重竞争性荧光PCR在X连锁Alport综合征分子诊断中的应用。方法:选择20例在北京大学第一医院确诊且未进行基因诊断的X连锁Alport综合征患者为研究对象,同时选择2例经多重连接依赖性探针扩增技术检测到COL4A5基因大片段缺失突变的患者作为阳性对照和1例经肾活检组织电子显微镜检查证实非Alport综合征的男性作为正常对照。首先应用多重竞争性荧光PCR技术扩增COL4A5基因53个外显子和4个参照基因,对于检测到COL4A5基因缺失第1外显子者,进而应用相同技术扩增COL4A5基因外显子1~4、COL4A6基因外显子1~4、两基因共用启动子以及3个参照基因;对于检测到拷贝数缺失者,应用琼脂糖凝胶电泳鉴定扩增后的PCR产物或直接测序。结果:两例阳性对照应用多重竞争性荧光PCR技术检测到的COL4A5基因缺失突变与应用多重连接依赖性探针扩增技术检测到的COL4A5基因缺失突变一致。20例患者中6例(30%)明确了基因型,其中2例患者具有累及COL4A5和COL4A6两个基因5'端的大片段缺失,2例患者具有累及COL4A5基因30个外显子以上的大片段缺失,1例患者具有累及COL4A5基因至少1个外显子的大片段缺失,1例患者具有COL4A5基因缺失13个碱基的小的缺失突变,未检测到重复突变。结论:多重竞争性荧光PCR技术可用于检测X连锁Alport综合征大片段缺失突变,是对该病分子诊断检测方法的重要补充。
[Abstract]:Objective: to investigate the application of multiple competitive fluorescent PCR in molecular diagnosis of X-linked Alport syndrome. Methods: twenty patients with X-linked Alport syndrome diagnosed in the first Hospital of Peking University without gene diagnosis were selected as subjects. At the same time, two patients with large deletion mutations of COL4A5 gene were selected as positive control and one male with non Alport syndrome confirmed by electron microscopy of renal biopsy tissue as normal control. At first, 53 exons and 4 reference genes of COL4A5 gene were amplified by multiplex competitive fluorescence PCR. For those with COL4A5 gene missing exon 1, Then the same technique was used to amplify exon 1n4COL4A6 of COL4A5 gene, the two genes shared promoter and three reference genes, and the amplified PCR products or direct sequencing were identified by agarose gel electrophoresis for those with copy number deletion detected. Results: the deletion mutations of COL4A5 gene detected by multiplex competitive fluorescent PCR in two positive controls were identical to those of COL4A5 gene deletion detected by multiplex conjugate dependent probe amplification. The genotypes were identified in 6 out of 20 patients. Of these, 2 patients had large deletions at the 5'end of the COL4A5 and COL4A6 genes and 2 patients had large deletions involving more than 30 exons of the COL4A5 gene. One patient had a large area involving at least one exon of the COL4A5 gene. One patient with segment deletion had a small deletion mutation with 13 bases of COL4A5 gene deletion. No repeated mutations were detected. Conclusion: multiplex competitive fluorescent PCR technique can be used to detect large fragment deletion mutations in X-linked Alport syndrome, which is an important supplement to the molecular diagnostic methods of X-linked Alport syndrome.
【作者单位】：北京大学第一医院儿科;
【基金】：国家十二五科技支撑计划(2012BAI03B02) 国家重点研发计划(2016YFC0901505) 国家自然科学基金(81070545) 北京市自然科学基金(7102148) 儿科遗传性疾病分子诊断与研究北京市重点实验室(Z141107004414036)资助~~
【分类号】：R440;R692

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