Genetic Analysis of the PKHD1 Gene with Long-rang PCR Sequencing  

作　　者：童永清 刘蓓 付朝泓 郑红云 顾剑 刘航 罗洪波 李艳 

机构地区：[1]Department of Clinical Laboratory, Renmin Hospital of Wuhan University, Wuhan 430060, China [2]Department of Pathology, Affiliated Tianyou Hospital of Wuhan University of Science and Technology, Wuhan 430064, China [3]Department of Clinical Laboratory, Dongfeng Hospital, Hubei University of Medicine, Shiyan 442008, China [4]Department of Urology, Renmin Hospital of Wuhan University, Wuhan 430060, China

出　　处：《Journal of Huazhong University of Science and Technology(Medical Sciences)》2016年第5期758-766,共9页华中科技大学学报（医学英德文版）

基　　金：supported by grants from the Hubei Provincial Natural Science Foundation of China(No.2010CDB-06903);National Key Basic Research Program of China(“973”Program,No.2012CB526706);the National Natural Science Foundation of China(Nos.81000771 and 81271694)

摘　　要：PKHD1 gene mutations are found responsible for autosomal recessive polycystic kidney disease（ARPKD）. However, it is inconvenient to detect the mutations by common polymerase chain reaction（PCR） because the open reading frame of PKHD1 is very long. Recently, long-range（LR） PCR is demonstrated to be a more sensitive mutation screening method for PKHD1 by directly sequencing. In this study, the entire PKHD1 coding region was amplified by 29 reactions to avoid the specific PCR amplification of individual exons, which generated the size of 1 to 7 kb products by LR PCR. This method was compared to the screening method with standard direct sequencing of each individual exon of the gene by a reference laboratory in 15 patients with ARPKD. The results showed that a total of 37 genetic changes were detected with LR PCR sequencing, which included 33 variations identified by the reference laboratory with standard direct sequencing. LR PCR sequencing had 100% sensitivity, 96% specificity, and 97.0% accuracy, which were higher than those with standard direct sequencing method. In conclusion, LR PCR sequencing is a reliable method with high sensitivity, specificity and accuracy for detecting genetic variations. It also has more intronic coverage and lower cost, and is an applicable clinical method for complex genetic analyses.PKHD1 gene mutations are found responsible for autosomal recessive polycystic kidney disease（ARPKD）. However, it is inconvenient to detect the mutations by common polymerase chain reaction（PCR） because the open reading frame of PKHD1 is very long. Recently, long-range（LR） PCR is demonstrated to be a more sensitive mutation screening method for PKHD1 by directly sequencing. In this study, the entire PKHD1 coding region was amplified by 29 reactions to avoid the specific PCR amplification of individual exons, which generated the size of 1 to 7 kb products by LR PCR. This method was compared to the screening method with standard direct sequencing of each individual exon of the gene by a reference laboratory in 15 patients with ARPKD. The results showed that a total of 37 genetic changes were detected with LR PCR sequencing, which included 33 variations identified by the reference laboratory with standard direct sequencing. LR PCR sequencing had 100% sensitivity, 96% specificity, and 97.0% accuracy, which were higher than those with standard direct sequencing method. In conclusion, LR PCR sequencing is a reliable method with high sensitivity, specificity and accuracy for detecting genetic variations. It also has more intronic coverage and lower cost, and is an applicable clinical method for complex genetic analyses.

关 键 词：sequencing Sequencing recessive screening detecting conclusion applicable mutation genomic digestion 

分 类 号：R692[医药卫生—泌尿科学]