Genetic analysis of the PKHD1 gene with long-rang PCR sequencing.
- Author:
Yong-Qing TONG
1
;
Bei LIU
2
;
Chao-Hong FU
3
;
Hong-Yun ZHENG
1
;
Jian GU
1
;
Hang LIU
1
;
Hong-Bo LUO
4
;
Yan LI
5
Author Information
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. drluohb@163.com.
5. Department of Clinical Laboratory, Renmin Hospital of Wuhan University, Wuhan, 430060, China. yanlitf@gmail.com.
- Publication Type:Journal Article
- Keywords:
PKHD1;
autosomal recessive polycystic kidney disease;
genetic analysis;
long-range PCR
- MeSH:
DNA Mutational Analysis;
Exons;
genetics;
Genetic Testing;
Genotype;
Humans;
Introns;
genetics;
Mutation;
Polycystic Kidney, Autosomal Recessive;
diagnosis;
genetics;
Polymerase Chain Reaction;
Receptors, Cell Surface;
genetics;
isolation & purification;
Sequence Analysis, DNA
- From:
Journal of Huazhong University of Science and Technology (Medical Sciences)
2016;36(5):758-766
- CountryChina
- Language:English
-
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.
