Detection of germline deletions using real-time quantitative polymerase chain reaction in Japanese patients with von Hippel-Lindau disease

Abstract

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.

Figure 1

VHL gene structure and the locations of the five primer/probe sets for RQ‐PCR assay.

Figure 2

VHL gene dosage values detected by RQ‐PCR assay for the five primer/probe sets in 30 normal controls, 10 VHL deletion controls, and 33 Japanese patients. DNA samples of 30 healthy controls and patients, each of whom retained two copies of the VHL gene, displayed gene dosage values of approximately 1.0 (∼0.8 to ∼1.2), whereas the samples from 10 deletion controls and patients who each had a single copy of VHL displayed values of approximately 0.5 (∼0.4 to ∼0.6).