Tong Yong-Qing, Liu Bei, Fu Chao-Hong, Zheng Hong-Yun, Gu Jian, Liu Hang, Luo Hong-Bo, Li Yan
Department of Clinical Laboratory, Renmin Hospital of Wuhan University, Wuhan, 430060, China.
Department of Pathology, Affiliated Tianyou Hospital of Wuhan University of Science and Technology, Wuhan, 430064, China.
J Huazhong Univ Sci Technolog Med Sci. 2016 Oct;36(5):758-766. doi: 10.1007/s11596-016-1658-8. Epub 2016 Oct 18.
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基因突变被发现与常染色体隐性多囊肾病（ARPKD）有关。然而，由于PKHD1的开放阅读框非常长，通过普通聚合酶链反应（PCR）检测这些突变并不方便。最近，长程（LR）PCR通过直接测序被证明是一种对PKHD1更敏感的突变筛查方法。在本研究中，通过29次反应扩增了PKHD1的整个编码区域，以避免对单个外显子进行特异性PCR扩增，通过LR PCR产生了大小为1至7 kb的产物。该方法与一家参考实验室对15例ARPKD患者采用对该基因的每个单个外显子进行标准直接测序的筛查方法进行了比较。结果显示，通过LR PCR测序共检测到37个基因变化，其中包括参考实验室采用标准直接测序法鉴定出的33个变异。LR PCR测序的灵敏度为100%，特异性为96%，准确率为97.0%，均高于标准直接测序法。总之，LR PCR测序是一种检测基因变异的可靠方法，具有高灵敏度、特异性和准确性。它还具有更多的内含子覆盖范围和更低的成本，是一种适用于复杂基因分析的临床方法。
