Detection of germline deletions using real-time quantitative polymerase chain reaction in Japanese patients with von Hippel-Lindau disease
Detection of germline deletions using real-time quantitative polymerase chain reaction in Japanese patients with von Hippel-Lindau disease
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DOI：
10.1111/j.1349-7006.2006.00193.x
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发表时间：
2006-05-01
期刊：
影响因子：
5.7
通讯作者：
Yao, M
中科院分区：
文献类型：
--
作者：
Hattori, K;Teranishi, J;Yao, M
VHL基因的胚系突变是导致VHL的原因。大约70%的VHL家系表现出可通过测序检测到的微小基因内突变，而在其余大多数家系中已描述了部分或全部基因缺失。对于如此大的缺失，可能不得不使用测序以外的复杂基因技术。在这项研究中，我们描述了一种使用TaqMan荧光探针的RQ-PCR方法来检测胚系VHL缺失。针对VHL的3个编码外显子以及5‘启动子和3’非翻译区设计了RQ-PCR引物/探针组。对30例正常对照和10例已知VHL缺失对照标本的RQ-PCR检测具有较高的敏感性和特异性。然后，我们对19个典型VHL家系(16个1型，2个2A型，1个2B型)和1个Pheo家系的29名个体以及4例孤立的疑似病例进行了RQ-PCR检测，发现VHL基因缺失。我们在17个(89%)经典家系中检测到生殖系缺失，其中包括15个1型，1个2A型和1个2B型。我们还在我们的家族队列中发现了两个突变携带者和两个非携带者。1个Pheo家系和4个孤立病例未显示任何缺失模式。这些发现表明，基于TaqMan的RQ-PCR方法是一种简单而有效的技术，可用于VHL基因诊断的快速、敏感和特异性研究，可在更复杂的大片段缺失检测技术之前进行有益的应用。
Germline mutations of the VHL gene are responsible for VHL. Approximately 70% of VHL families display small intragenic mutations detectable by sequencing, whereas partial- or whole-gene deletions have been described in the majority of the remaining families. For such large deletions, complex genetic techniques other than sequencing might have to be used. In this study, we describe an RQ-PCR assay with TaqMan fluorescent probes to detect germline VHL deletions. The RQ-PCR primer/probe sets were designed for the three VHL coding exons as well as for the 5' promoter and 3' untranslated regions. The RQ-PCR assay for 30 normal and 10 known VHL deletion control samples demonstrated high sensitivity and specificity. We then screened 29 individuals from 19 classical VHL families (16 type 1, 2 type 2A, and one type 2B) and one PHEO family, as well as four solitary suspected cases, none displaying any sequence changes, for VHL deletions by the RQ-PCR assay. We detected germline deletions in 17 (89%) classical families including 15 type 1, one type 2A, and one type 2B. We also identified two mutation carriers and two non-carriers in our family cohort. The one PHEO family and four solitary cases did not display any deletion patterns. These findings indicated that the TaqMan-based RQ-PCR assay is a simple and potent technique for the rapid, sensitive, and specific investigation of VHL genetic diagnoses that could be used profitably before more complex large-deletion detection techniques.