Comprehensive screening for constitutional RB1 mutations by DHPLC and QMPSF

Hum Mutat. 2004 Feb;23(2):193-202. doi: 10.1002/humu.10303.


Constitutional mutations of the RB1 gene are associated with a predisposition to retinoblastoma. It is essential to identify these mutations to provide appropriate genetic counseling in retinoblastoma patients, but this represents an extremely challenging task, as the vast majority of mutations are unique and spread over the entire coding sequence. Since 2001, we have implemented RB1 testing on a routine basis as part of the clinical management of retinoblastoma. As most screening techniques do not meet the requirements for efficient RB1 testing, we have devised a semi-automated denaturing high-performance liquid chromatography (DHPLC) method for point mutation detection combined with a quantitative multiplex PCR of short fluorescent fragments (QMPSF) approach to screen for gene rearrangements. We report the results of this comprehensive screening of all exons and promoter of RB1 in 192 unrelated patients, mostly of French origin. Among 102 bilateral and/or familial cases and 90 unilateral sporadic probands, mutations were identified in 83 (81.5%) and 5 (5.5%) cases, respectively. A total of 43 mutations have not been previously reported. The mutational spectrum was found to be significantly different from previous published series, displaying a surprising amount of splice mutations and large deletions. This study demonstrates the reliability of DHPLC for RB1 analysis, but also illustrates the need for a deletion scanning approach. Finally, considering the benefits to retinoblastoma patients, RB1 testing should be widely implemented in routine healthcare because our study clearly illustrates its feasibility.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Child, Preschool
  • Chromatography, High Pressure Liquid / methods*
  • Chromatography, High Pressure Liquid / standards
  • Chromosome Deletion
  • DNA Mutational Analysis / methods
  • DNA, Neoplasm / genetics
  • Exons / genetics
  • Female
  • Fluorescent Dyes / metabolism
  • Genes, Retinoblastoma / genetics*
  • Genetic Testing / methods*
  • Humans
  • Male
  • Mutation / genetics*
  • Nucleic Acid Amplification Techniques / methods
  • Nucleic Acid Amplification Techniques / standards
  • Nucleic Acid Denaturation / genetics*
  • Polymerase Chain Reaction / methods*
  • Polymerase Chain Reaction / standards
  • RNA Splice Sites / genetics
  • Retinal Neoplasms / genetics
  • Retinoblastoma / genetics
  • Retinoblastoma Protein / genetics


  • DNA, Neoplasm
  • Fluorescent Dyes
  • RNA Splice Sites
  • Retinoblastoma Protein

Associated data

  • OMIM/180200