RB1 – genetic testing

• All individuals with a personal history of retinoblastoma.
• All first degree relatives (children and adults) of an individual with a known RB1 germline pathogenic variant.*
• Where a specific pathogenic variant has been identified on somatic testing in a tumour within RB1 spectrum.*

*In these settings a variant-specific test (rather than sequencing a single gene or gene panel) may be more appropriate and cost effective. 

Genetic testing is important for good clinical care of individuals who are suspected of having a heritable pathogenic variant in this gene.

The results of the following investigations may significantly influence the likelihood of detecting a heritable pathogenic variant in the RB1 gene:

• Fundoscopy*
  • in a retinoblastoma-affected child for multifocal or bilateral disease
  • in parents and siblings for regressed retinoblastoma or retinoma
• CNS imaging for a midline brain tumour**

The interpretation of RB1 gene testing in blood may be influenced by the result of RB1 gene testing in tumour tissue. It is recommended if enucleation is performed for diagnostic or treatment purposes, tumour tissue should be stored.

*Retinal examination with full pupillary dilation by a paediatric ophthalmologist
**The concurrence of a midline brain tumour in patients with bilateral retinoblastoma is referred to as trilateral retinoblastoma

Retinoblastoma has both heritable and sporadic forms. Most unilateral retinoblastoma is caused by biallelic somatic RB1 pathogenic variants and is non-hereditary, but 10-15% of children with unilateral retinoblastoma have a germline RB1 pathogenic variant.^rr

Nearly all bilateral/multifocal retinoblastoma is caused by a detectable heritable germline pathogenic variant in the RB1 gene.^rrr Approximately 90% of detected germline RB1 pathogenic variants are de novo. In families with heritable retinoblastoma families the initial RB1 pathogenic variant is mosaic, either in the proband or in one of the proband’s parents, in up to 10% of cases.^r

The frequency of germline RB1 pathogenic variants in individuals with retinoblastoma depends on:

• whether the disease is unilateral or bilateral/multifocal
• whether there is or is not a family history of retinoblastoma.^r

Factors	Probability of detecting a heritable pathogenic variant
Affected parent and offspring pair#	90-95%rr
Bilateral/multifocal retinoblastoma	90-95%rr
Apparently sporadic unilateral retinoblastoma (unifocal)	10-15%rr
Patient has a first or second degree relative with a germline RB1 pathogenic variant	Up to 50%

^#The likelihood of a germline RB1 pathogenic variant being present in families with multiple affected relatives is assumed to be 100% even when genetic testing is non-diagnostic for the affected proband – see Result Interpretation section. 

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)
• exon copy number analysis 
• 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 (MYCN^A) gene in tumour tissue is recommended. Approximately 1.5% of children with sporadic unilateral retinoblastoma and intact RB1 expression have high-level MYCN amplification on tumour tissue (MYCN^ARB1^+/+) representing a unique tumour subset. Focal MYCN amplification and somatic RB1 pathogenic variants can also coexist.^r

Information about DNA tests and testing laboratories is available from:

• RCPA catalogue of genetic tests and laboratories  
• GeneReviews^® 
• Genetic Testing Registry 
• NHS National Genomic Test Directory

A variant-specific test (rather than sequencing a single gene or gene panel) may be more appropriate and cost effective where a relative is known to have a germline pathogenic variant or a pathogenic variant has been identified in the patient’s tumour. 

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 and tumour tissue is available, 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).

Clinical scenario	Reference databases	Considerations and advice
Unilateral, bilateral or multifocal tumour. Pathogenic variant in blood	ClinVar RB1 LOVD database	Heritable retinoblastoma
Tumour unilateral or bilateral or multifocal and there is a family history. No pathogenic variant in blood		Heritable retinoblastoma due to cryptic (undetectable) heritable pathogenic variant
Tumour bilateral or multifocal and no family history. No pathogenic variant in blood		May have: cryptic heritable pathogenic variant or somatic mosaicism, tumour testing should be considered
Tumour unilateral and no family history. Two tumour pathogenic variants identified, both events excluded in blood		Heritable pathogenic variant essentially excluded
Tumour unilateral and no family history. Tumour testing not possible or testing fails to identify both tumour pathogenic variants. No pathogenic variant in blood		Small chance of a heritable, cryptic (undetectable) pathogenic variant or low-level somatic mosaicism Consider MYCN amplification studies in tumour
Predictive testing
Family pathogenic variant identified		For management of retinoblastoma see Skalet el al. 2018r
Family pathogenic variant not found		Population risk for retinoblastoma

Where there is no family history of retinoblastoma, and testing in blood does not identify an RB1 pathogenic variant, the probability of a heritable cryptic (undetectable) RB1 pathogenic variant or somatic mosaicism for a cryptic (including mobile element insertions) RB1 pathogenic variant that might involve the germline is dependent on whether DNA testing in tumour tissue is:

• fully informative (two RB1 pathogenic variants detected)
• partially informative (only one RB1 pathogenic variant detected) 
• uninformative (no RB1 pathogenic variants detected).

For further information refer to RB1 gene - interpretation of results in an isolated case of bilateral retinoblastoma and RB1 gene - probability of a cryptic heritable RB1 pathogenic variant in an isolated case of unilateral retinoblastoma.

Note: up to 10% of families have a low penetrance pathogenic variant associated with increased prevalence of unilateral retinoblastoma and incomplete penetrance (≤25%); in some of these families there is a parent of origin effect.^r

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Version 8

Date	Summary of changes
28/08/2024	Protocol reviewed at June 2024 eviQ paediatric cancer genetics reference committee meeting. Discussions continued via email and MS Teams. Minor wording changes throughout. Version increased to V.8. Review in 4 years.

Version 7

Date

Summary of changes

20/02/2023

The following sections of the document were updated to align with the new eviQ cancer genetics genetic testing template: Target population:  Added "Where a specific pathogenic variant has been identified on somatic tumour testing.*" Added "*In these settings a variant-specific test (rather than sequencing a single gene or gene panel) may be more appropriate and cost effective." Circumstances in which testing is not indicated:  Removed "A variant-specific test (rather than sequencing a single gene or gene panel) may be more cost effective and appropriate where:  a known pathogenic variant has been identified in a relative a specific pathogenic variant has been identified on somatic tumour testing." Testing methods: Added "A variant-specific test (rather than sequencing a single gene or gene panel) may be more appropriate and cost effective where a relative is known to have a germline pathogenic variant or a pathogenic variant has been identified in the patient’s tumour." Result interpretation: HGMD and DMuDB removed from table. Template sentence added. Version increased to V.7.

Version 6

Date	Summary of changes
01/04/2021	Protocol reviewed at February 2020 paediatric cancer genetics reference committee meeting. Discussion continued via email. Approved for publication with the following changes made: Related pages: added link to "Guide for health professionals ordering genetic testing".  Removed link to "Pre-test counselling" Target population: Leap link added from  "all first degree relatives (children and adults) of an individual with a known pathogenic RB1 pathogenic variant" to "Circumstances in which testing is not indicated" section Investigations before genetic testing: Fundoscopy asterisked and footnote added  "CNS imaging for pineoblastoma" changed to "CNS imaging for midline brain tumour" and footnote added Probability of a heritable pathogenic variant: minor wording changes Circumstances in which testing is not indicated: template wording updated Testing methods:  Third bullet: "methylation analysis" changed to "methylation analysis (tumour loss of heterozygosity and methylation studies)" Fourth bullet: "analysis for structural rearrangements/large deletions involving 13q" changed to "analysis for structural rearrangements (conventional karyotype) and deletions of the 13q locus (array)" Added below bullet points: "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" Added "Approximately 1.5% of children with sporadic unilateral retinoblastoma have high-level MYCN amplification on tumour tissue" Note added: "Blood mosaicism down to approximately 20% can usually be detected by conventional analyses such 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)." Result interpretation:  Reference databases added to table Predictive testing - Family pathogenic variant identified: "For management of retinoblastoma see Skalet el al. 2018" added Template sentences added (including link to "Guide for health professionals ordering genetic testing") Counselling: section deleted Version number changed to V.6. Review in 2 years.
10/11/2021	Removed link to "European Directory of DNA Diagnostic Laboratories"

Version 5

Date	Summary of changes
18/11/2019	"Mutation" changed to "pathogenic variant" and "unclassified variant" changed to "variant of uncertain significance" throughout document for consistency among eviQ cancer genetics protocols per agreement among the cancer genetics reference committees' chairs Definition of "pathogenic variant" added as a pop-up Version number increased to V.5.

Version 4

Date	Summary of changes
30/10/2012	Discussed at August 2012 reference committee meeting, discussion continued via email and approved for publication.
02/12/2013	For review second yearly.
26/03/2014	Following discussion at March 5 2014 reference committee meeting the following change has been made: Diagnostic testing - Link to Human Genetics Society of Australasia removed.
03/02/2015	Discussed at October 2014 reference committee meeting, protocol approved with the following amendments: target population: minor wording change factors which influence the pre-test probability of a heritable mutation: minor wording change For 2 yearly review.
30/09/2015	Sentence added to genetic testing protocol template: "if a decision is made to test this gene(s) 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".
31/05/2017	Transferred to new eviQ website.Version change to V.2.
17/08/2017	Following discussion from May 2017 reference committee, protocol approved with the following amendments:  probability of a heritable mutation: minor wording change  testing methods: minor wording change  reference list updated  version change to V.3. for 2 yearly review.
01/06/2018	'Gene Tests' link in Testing Methods section relabelled as GeneReviews.
28/08/2019	Protocol title changed from 'Genetic testing for heritable mutations in the retinoblastoma 1 (RB1) gene' to 'RB1 genetic testing' in accordance with Cancer Genetics Reference Committees' consensus. Version number increased to V.4.