Retinoblastoma (RB): Test Description

Genes tested:

  • RB1
  • MYCN

Copy number changes
Our lab uses MLPA multiplex ligation dependent probe amplification to look for whole-exon and multi-exon deletions and duplications in the RB1 gene; this method simultaneously screens for small intra-exon insertions and deletions in RB1. Our lab also performs a QM-PCR test to measure MYCN copy number in any tumor sample that does not have an RB1 mutation. This test detects a new sub-set of retinoblastoma with early age at diagnosis, distinct histology, no RB1 mutations, and huge amplification of the MYCN oncogene. [Rushlow, D. et al. 2013. Lancet Oncology.]

Sequence analysis
Our lab sequences the RB1 gene core promoter and exons 1 through 25, as well as nearby flanking intronic regions. Our sequence analysis is able to detect mosaic mutations at a level of 15% or greater. We consider reported polymorphisms when designing our sequencing assays to ensure the accuracy of our sequence results.

Splice site analysis
We sequence a minimum of 25 nucleotides flanking each exon of RB1 to detect changes in splice sites. We use in silico analysis and scoring to determine whether a particular change is likely to cause missplicing. In the case of an intronic variant of uncertain significance we perform RNA transcript analysis on a fresh blood sample at no added charge.

Allele-specific PCR (AS-PCR) for eleven recurrent RB1 mutations
Our lab uses multiplex AS-PCR screens for rapid detection of eleven recurrent RB1 mutations, which are then confirmed by sequence analysis. The highly sensitive AS-PCR can detect the eleven mutations at mosaic levels as low as 1% mutant DNA.

Testing for methylation of the RB1 Promoter
Aberrant methylation of the RB1 promoter leads to reduced transcription of RB1, and can initiate unilateral sporadic retinoblastoma in the absence of an RB1 mutation. Our lab identifies RB1 promoter methylation leading to retinoblastoma in approximately 12% of unilateral sporadic tumors.

Amino acid conservation analysis
For missense amino acid changes of uncertain significance we employ several in silico conservation analysis programs to predict whether a particular missense change is likely to be pathogenic. In addition, when appropriate we will test known affected and unaffected relatives at no charge to clarify variant classification.


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