Wang F, Zhang Y Q, Ding J, Yu L X
Department of Pediatrics, Peking University First Hospital, Beijing 100034, China.
Beijing Da Xue Xue Bao Yi Xue Ban. 2017 Oct 18;49(5):760-767.
To evaluate the ability of multiplex competitive fluorescence polymerase chain reaction in detection of large deletion and duplication genotypes of X-linked Alport syndrome.
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.
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.
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.
评估多重竞争性荧光聚合酶链反应检测X连锁Alport综合征大缺失和重复基因型的能力。
X连锁Alport综合征的临床诊断基于仅表皮基底膜中IV型胶原α5链染色异常，或肾小球基底膜和鲍曼囊/肾小球基底膜超微结构改变伴IV型胶原α5链染色异常，这些改变是Alport综合征的典型表现。本研究共纳入20例临床诊断为X连锁Alport综合征的非亲属中国患者（13例男性和7例女性），其基因型未知。对照受试者包括1例患有其他肾脏疾病的男性患者和2例通过多重连接依赖探针扩增检测到COL4A5基因大缺失的患者。从所有参与者的外周血白细胞中分离基因组DNA。采用多重竞争性荧光聚合酶链反应在单一反应中共同扩增COL4A5基因的53个外显子和4个参考基因。当鉴定出缺失COL4A5基因外显子1时，采用相同方法在单一反应中共同扩增COL4A5和COL4A6基因的前4个外显子、COL4A5和COL4A6基因共享的一个启动子以及3个参考基因。该方法提示的任何拷贝数缺失均通过相应聚合酶链反应扩增产物的电泳或DNA测序进行验证，以排除引物区域可能的DNA变异。
多重竞争性荧光聚合酶链反应鉴定的两个阳性对照的基因型与多重连接依赖探针扩增检测的结果一致。20例患者中有6例检测到缺失，包括2例大缺失，缺失COL4A5和COL4A6基因5'部分，断点位于COL4A6的第二个内含子；2例大缺失，缺失COL4A5基因30多个外显子；1例大缺失，缺失COL4A5基因至少1个外显子；1例小缺失，涉及13个碱基对。未发现重复。
我们的结果表明，多重竞争性荧光聚合酶链反应是X连锁Alport综合征大缺失基因分型经典技术的良好替代方法。
