北京大学学报（医学版）2017，Vol.49Issue(5)：760-767,8.DOI:10.3969/j.issn.1671-167X.2017.05.004
多重竞争性荧光PCR检测X连锁Alport综合征大片段缺失突变
Detection of large deletions in X linked Alport syndrome using competitive multiplex fluorescence polymerase chain reaction
摘要
Abstract
Objective:To evaluate the ability of multiplex competitive fluorescence polymerase chain reaction in detection of large deletion and duplication genotypes of X-linked Alport syndrome.Methods:Clinical diagnosis of X-linked Alport syndrome was based on either abnormal staining of type Ⅳ collagen α5 chain in the epidermal basement membrane alone or with abnormal staining of type Ⅳ collagen α5 chain in the glomerular basement membrane and Bowman's capsule/ultrastructural changes in the glomerular basement membrane typical of Alport syndrome.A total of 20 unrelated Chinese patients (13 males and 7 females) clinically diagnosed as X-linked Alport syndrome were included in the study.Their genotypes were unknown.Control subjects included a male patient with other renal disease and two patients who had large deletions in COL4A5 gene detected by multiplex ligation-dependent probe amplification.Genomic DNA was isolated from peripheral blood leukocytes in all the participants.Multiplex competitive fluorescence polymerase chain reaction was used to coamplify 53 exons of COL4A5 gene and four reference genes in a single reaction.When a deletion removed exon 1 of COL4A5 gene was identified,the same method was used to coamplify the first 4 exons of COL4A5 and COL4A6 genes,a promoter shared by COL4A5 and COL4A6 genes,and three reference genes in a single reaction.Any copy number loss suggested by this method was verified by electrophoresis of corresponding polymerase chain reaction amplified products or DNA sequencing to exclude possible DNA variations in the primer regions.Results:Genotypes of two positive controls identified by multiplex competitive fluorescence polymerase chain reaction were consistent with those detected by multiplex ligation-dependent probe amplification.Deletions were identified in 6 of the 20 patients,including two large deletions removing the 5'part of both COL4A5 and COL4A6 genes with the breakpoint located in the second intron of COL4A6,two large deletions removing more than 30 exons of COL4A5 gene,one large deletion removing at least 1 exon of COL4A5 gene,and one small deletion involving 13 bps.No duplication was found.Conclusion:Our results show that multiplex competitive fluorescence polymerase chain reaction is a good alternative to classical techniques for large deletion genotyping in X-linked Alport syndrome.关键词
Alport综合征,X连锁/大片段缺失突变/COL4A5基因/多重竞争性荧光聚合酶链反应Key words
Alport syndrome, X-linked/Large deletion/COL4A5 gene/Multiplex competitive fluorescence polymerase chain reaction分类
医药卫生引用本文复制引用
王芳,张琰琴,丁洁,俞礼霞..多重竞争性荧光PCR检测X连锁Alport综合征大片段缺失突变[J].北京大学学报（医学版）,2017,49(5):760-767,8.基金项目
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)和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 Supported by the National Twelfth Five-Year-Plan for Scientific and Technological Support Project (2012BAI03B02),the National Key Research and Development Program of China (2016YFC0901505),the National Natural Science Foundation of China (81070545),the Beijing Nature Science Foundation (7102148),and the Beijing Key Laboratory of Molecular Diagnosis and Study on Pediatric Genetic Diseases (Z141107004414036) （2012BAI03B02）
