Rapid detection of VHL exon deletions using real-time quantitative PCR

Lab Invest. 2005 Jan;85(1):24-33. doi: 10.1038/labinvest.3700209.


Various types of mutations exist that exert an effect on the normal function of a gene. Among these, exon/gene deletions often remain unnoticed in initial mutation screening. Until recently, no fast and efficient methods were available to detect this type of mutation. Molecular detection methods for gene copy number changes included Southern blot (SB) and fluorescence in situ hybridisation, both with their own intrinsic limitations. In this paper, we report the development and application of a fast, sensitive and high-resolution method for the detection of single exon or larger deletions in the VHL gene based on real-time quantitative PCR (Q-PCR). These deletions account for approximately one-fifth of all patients with the von Hippel-Lindau syndrome, a dominantly inherited highly penetrant familial cancer syndrome predisposing to specific malignancies including phaeochromocytomas and haemangioblastomas. Our VHL exon quantification strategy is based on SYBR Green I detection and normalisation using two reference genes with a normal copy number, that is, ZNF80 (3q13.31) and GPR15 (3q12.1). Choice of primer sequences and the use of two reference genes appears to be critical for accurate discrimination between 1 and 2 exon copies. In a blind Q-PCR study of 29 samples, all 14 deletions were detected, which is in perfect agreement with previously determined SB results. We propose Q-PCR as the method of choice for fast (within 3.5 h), accurate and sensitive (ng amount of input DNA) exon deletion screening in routine DNA diagnosis of VHL disease. Similar assays can be designed for deletion screening in other genetic disorders.

Publication types

  • Evaluation Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Benzothiazoles
  • Diamines
  • Exons / genetics*
  • Gene Deletion*
  • Genetic Testing / methods
  • Humans
  • Organic Chemicals
  • Point Mutation
  • Quinolines
  • Reproducibility of Results
  • Reverse Transcriptase Polymerase Chain Reaction / methods*
  • Tumor Suppressor Proteins / genetics*
  • Ubiquitin-Protein Ligases / genetics*
  • Von Hippel-Lindau Tumor Suppressor Protein
  • von Hippel-Lindau Disease / diagnosis
  • von Hippel-Lindau Disease / genetics*


  • Benzothiazoles
  • Diamines
  • Organic Chemicals
  • Quinolines
  • Tumor Suppressor Proteins
  • SYBR Green I
  • Ubiquitin-Protein Ligases
  • Von Hippel-Lindau Tumor Suppressor Protein
  • VHL protein, human