Tan Ying-Cai, Blumenfeld Jon D, Anghel Raluca, Donahue Stephanie, Belenkaya Rimma, Balina Marina, Parker Thomas, Levine Daniel, Leonard Debra G B, Rennert Hanna
Department of Pathology and Laboratory Medicine, Weill Cornell Medical College, New York, New York, USA.
Hum Mutat. 2009 Feb;30(2):264-73. doi: 10.1002/humu.20842.
Genetic testing of PKD1 and PKD2 is useful for diagnosis and prognosis of autosomal dominant polycystic kidney disease (ADPKD), particularly in asymptomatic individuals or those without a family history. PKD1 testing is complicated by the large transcript size, complexity of the gene region, and the extent of gene variations. A molecular assay was developed using Transgenomic's SURVEYOR Nuclease and WAVE Nucleic Acid High Sensitivity Fragment Analysis System to screen for PKD1 and PKD2 variants, followed by sequencing of variant gene segments, thereby reducing the sequencing reactions by 80%. This method was compared to complete DNA sequencing performed by a reference laboratory for 25 ADPKD patients from 22 families. The pathogenic potential of gene variations of unknown significance was examined by evolutionary comparison, effects of amino acid substitutions on protein structure, and effects of splice-site alterations. A total of 90 variations were identified, including all 82 reported by the reference laboratory (100% sensitivity). A total of 76 variations (84.4%) were in PKD1 and 14 (15.6%) in PKD2. Definite pathogenic mutations (seven nonsense, four truncation, and three splicing defects) were detected in 64% (14/22) of families. The remaining 76 variants included 26 missense, 33 silent, and 17 intronic changes. Two heterozygous nonsense mutations were incorrectly determined by the reference laboratory as homozygous. "Probably pathogenic" mutations were identified in an additional five families (overall detection rate 86%). In conclusion, the SURVEYOR nuclease method was comparable to direct sequencing for detecting ADPKD mutations, achieving high sensitivity with lower cost, providing an important tool for genetic analysis of complex genes.
对多囊肾病1基因（PKD1）和多囊肾病2基因（PKD2）进行基因检测，对常染色体显性多囊肾病（ADPKD）的诊断和预后评估很有用，尤其适用于无症状个体或无家族病史者。PKD1检测因转录本大、基因区域复杂以及基因变异程度高而变得复杂。利用Transgenomic公司的SURVEYOR核酸酶和WAVE核酸高灵敏度片段分析系统开发了一种分子检测方法，用于筛查PKD1和PKD2变异，随后对变异基因片段进行测序，从而减少了80%的测序反应。将该方法与一家参考实验室对来自22个家庭的25例ADPKD患者进行的全DNA测序进行了比较。通过进化比较、氨基酸替换对蛋白质结构的影响以及剪接位点改变的影响，研究了意义不明的基因变异的致病潜力。共鉴定出90种变异，包括参考实验室报告的所有82种变异（灵敏度100%）。总共76种变异（84.4%）位于PKD1，14种（15.6%）位于PKD2。在64%（14/22）的家庭中检测到明确的致病突变（7个无义突变、4个截短突变和3个剪接缺陷）。其余76种变异包括26个错义突变、33个沉默突变和17个内含子变化。参考实验室将两个杂合无义突变错误地判定为纯合突变。在另外五个家庭中鉴定出“可能致病”突变（总体检测率86%）。总之，SURVEYOR核酸酶方法在检测ADPKD突变方面与直接测序相当，以较低成本实现了高灵敏度，为复杂基因的遗传分析提供了重要工具。
