A range of testing methodologies on germline or tumour samples are needed to identify pathogenic changes in the RB1 gene including:
- sequencing (deep sequencing method is recommended for better detection of mosaic pathogenic variants)
- copy number analysis (e.g. MLPA)
- methylation analysis (tumour loss of heterozygosity and methylation studies)
- analysis for structural rearrangements (conventional karyotype) and deletions of the 13q locus (array)
Approximately 6-8% of individuals with retinoblastoma have a large deletion of 13q14 locus involving deletion of multiple genes which are often associated with developmental delay, failure to thrive and congenital anomalies.
When no somatic RB1 pathogenic variants are identified in unilateral retinoblastoma in infants, particularly under the age of 12 months, analysis of amplification of MYCN gene in tumour tissue is recommended. Approximately 1.5% of children with sporadic unilateral retinoblastoma have high-level MYCN amplification on tumour tissue. MYCN amplification and somatic RB1 pathogenic variants are mutually exclusive.r
Information about DNA tests and testing laboratories is available from:
If a decision is made to test this gene as part of a cancer gene panel, care should be taken to select a panel where the individual genes tested have both clinical validity and clinical utility.
Genetic testing of tumour tissue is often needed to determine heritability and relative recurrence risks. Tumour DNA can be extracted from either fresh frozen tumour or micro-dissected tumour from paraffin blocks. The decision to start analyses on either peripheral blood or tumour DNA is based on disease extent, tissue availability and family history:
- In a proband with apparently sporadic (no family history), unilateral, unifocal retinoblastoma initial testing should ideally occur in tumour tissue seeking to identify two RB1 tumour pathogenic variants. Ideally peripheral blood should then be tested to differentiate sporadic unilateral from heritable unilateral disease. If tumour tissue is not available, then sequence and copy number analysis of RB1 are performed on peripheral blood DNA. (See RB1 gene – probability of a cryptic heritable RB1 pathogenic variant in an isolated case of unilateral retinoblastoma)
- In a proband with bilateral or multi-focal retinoblastoma, or a family history of retinoblastoma, initial testing should occur in blood seeking to identify a heritable (germline) RB1 pathogenic variant. If no pathogenic variant is identified testing of tumour tissue should occur to differentiate low-level somatic mosaicism from a presumed cryptic heritable RB1 pathogenic variant. (See RB1 gene – interpretation of results in an isolated case of bilateral retinoblastoma)
NOTE: Blood mosaicism down to approximately 20% can usually be detected by conventional analyses such as Sanger sequencing, and can be detected down to approximately 5% mosaicism with high depth massively parallel sequencing analyses. The failure to detect a RB1 pathogenic variant in blood reduces but cannot eliminate the probability that the individual has a RB1 pathogenic variant that involves the germline (mosaic or non-mosaic).