title: Genetic analysis of the PKHD1 gene with long-rang PCR sequencing.
abstract: 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.
authors.affiliation: Department of Clinical Laboratory, Renmin Hospital of Wuhan University, Wuhan, 430060, China.
authors.forename: Yong-Qing
authors.initials: YQ
authors.lastname: Tong
authors.affiliation: Department of Pathology, Affiliated Tianyou Hospital of Wuhan University of Science and Technology, Wuhan, 430064, China.
authors.forename: Bei
authors.initials: B
authors.lastname: Liu
authors.affiliation: Department of Clinical Laboratory, Dongfeng Hospital, Hubei University of Medicine, Shiyan, 442008, China.
authors.forename: Chao-Hong
authors.initials: CH
authors.lastname: Fu
authors.affiliation: Department of Clinical Laboratory, Renmin Hospital of Wuhan University, Wuhan, 430060, China.
authors.forename: Hong-Yun
authors.initials: HY
authors.lastname: Zheng
authors.affiliation: Department of Clinical Laboratory, Renmin Hospital of Wuhan University, Wuhan, 430060, China.
authors.forename: Jian
authors.initials: J
authors.lastname: Gu
authors.affiliation: Department of Clinical Laboratory, Renmin Hospital of Wuhan University, Wuhan, 430060, China.
authors.forename: Hang
authors.initials: H
authors.lastname: Liu
authors.affiliation: Department of Urology, Renmin Hospital of Wuhan University, Wuhan, 430060, China. drluohb@163.com.
authors.forename: Hong-Bo
authors.initials: HB
authors.lastname: Luo
authors.affiliation: Department of Clinical Laboratory, Renmin Hospital of Wuhan University, Wuhan, 430060, China. yanlitf@gmail.com.
authors.forename: Yan
authors.initials: Y
authors.lastname: Li
mesh_terms: D004252:DNA Mutational Analysis; D005091:Exons / Q000235:genetics; D005820:Genetic Testing; D005838:Genotype; D006801:Humans; D007438:Introns / Q000235:genetics; D009154:Mutation; D017044:Polycystic Kidney, Autosomal Recessive / Q000175:diagnosis / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011956:Receptors, Cell Surface / Q000235:genetics* / Q000302:isolation & purification; D017422:Sequence Analysis, DNA
chemical_list: C451750:PKHD1 protein, human; D011956:Receptors, Cell Surface
keywords: PKHD1; autosomal recessive polycystic kidney disease; genetic analysis; long-range PCR