Virzì Grazia Maria, Bruson Alice, Corradi Valentina, Gastaldon Fiorella, de Cal Massimo, Donà Marta, Cruz Dinna N, Clementi Maurizio, Ronco Claudio
Department of Nephrology, Dialysis and Transplant, St. Bortolo Hospital, Vicenza, Italy; IRRIV-International Renal Research Institute, Vicenza, Italy; Clinical Genetics Unit, Department of Women's and Children's Health, University of Padua, Padua, Italy.
J Clin Lab Anal. 2014 Jul;28(4):328-34. doi: 10.1002/jcla.21689. Epub 2014 Mar 22.
Autosomal dominant polycystic kidney disease (ADPKD) is an inherited condition caused by PKD1 and PKD2 mutations. Complete analysis of both genes is typically required in each patient. In this study, we explored the utility of High-Resolution Melt (HRM) as a tool for mutation analysis of the PKD2 gene in ADPKD families.
HRM is a mismatch-detection method based on the difference of fluorescence absorbance behavior during the melting of the DNA double strand to denatured single strands in a mutant sample as compared to a reference control. Our families were previously screened by linkage analysis. Subsequently, HRM was used to characterize PKD2-linked families. Amplicons that produced an overlapping profile sample versus wild-type control were not further evaluated, while those amplicons with profile deviated from the control were consequently sequenced.
We analyzed 16 PKD2-linked families by HRM analysis. We observed ten different variations: six single-nucleotide polymorphisms and four mutations. The mutations detected by HRM and confirmed by sequencing were as follows: 1158T>A, 2159delA, 2224C>T, and 2533C>T. In particular, the same haplotype block and nonsense mutation 2533C>T was found in 8 of 16 families, so we suggested the presence of a founder effect in our province.
We have developed a strategy for rapid mutation analysis of the PKD2 gene in ADPKD families, which utilizes an HRM-based prescreening followed by direct sequencing of amplicons with abnormal profiles. This is a simple and good technique for PKD2 genotyping and may significantly reduce the time and cost for diagnosis in ADPKD.
常染色体显性遗传性多囊肾病（ADPKD）是一种由PKD1和PKD2基因突变引起的遗传性疾病。通常需要对每位患者的这两个基因进行全面分析。在本研究中，我们探索了高分辨率熔解曲线分析（HRM）作为一种工具，用于分析ADPKD家系中PKD2基因的突变情况。
HRM是一种错配检测方法，其基于突变样本与对照样本中DNA双链熔解为单链时荧光吸收行为的差异。我们的家系先前已通过连锁分析进行筛选。随后，利用HRM对与PKD2相关的家系进行特征分析。产生与野生型对照重叠谱型的扩增子不再进一步评估，而那些谱型与对照有偏差的扩增子则进行测序。
我们通过HRM分析了16个与PKD2相关的家系。我们观察到十种不同的变异：六个单核苷酸多态性和四个突变。通过HRM检测并经测序确认的突变如下：1158T>A、2159delA、2224C>T和2533C>T。特别地，在16个家系中的8个家系中发现了相同的单倍型块和无义突变2533C>T，因此我们认为在我们省内存在奠基者效应。
我们开发了一种用于ADPKD家系中PKD2基因快速突变分析的策略，该策略利用基于HRM的预筛选，随后对谱型异常的扩增子进行直接测序。这是一种用于PKD2基因分型的简单且良好的技术，可能会显著减少ADPKD诊断的时间和成本。
