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Paediatric Laboratory Medicine
Paediatric Laboratory Medicine
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Retinoblastoma (RB)

Background
Who should be tested?
Testing Methodology
Potential Outcomes & Interpretation of Test Results
Cautions
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Background

Retinoblastoma (RB) is an eye tumor that occurs in children, usually before the age of five. Approximately one in 20,000 children are affected by this condition. One or both eyes may be involved. Although the majority of cases of RB are treatable, RB often leads to blindness unless detected early. This rapidly growing tumor can spread along the optic nerve into the brain and in some cases causing death.

RB is caused by mutations in the RB1 gene located on chromosome 13 at 13q14. In 60% of people with RB, mutations in the RB1 gene are found only in the cells of the tumour, and the other body cells contain two copies of the normal RB1 gene. This is called non-hereditary RB. The relatives and children of individuals with non-hereditary RB do not have an elevated risk of developing RB, compared to the risk in the general population.

In the remaining 40% of people with RB, the mutation is found in all the cells of the body. This is called hereditary RB. In these cases, the mutation occurred spontaneously around the time of conception (90% of hereditary cases), or was inherited from a parent (10% of hereditary cases). If the mutation occurred at conception, only the children of the affected individual are at increased risk. If the mutation was inherited from one of the parents, the children and relatives of the affected individual may have an increased risk of developing tumours themselves. It is often helpful to first identify the mutation(s) in an affected family member or in the parent of the affected family member. If the familial mutation can be identified in this way, the molecular test is conducted only for the familial mutation.

In some cases (10-18%) individuals with retinoblastoma have a mutation in only a subset of their cells. This is called genetic mosaicism. These mutations may be difficult or impossible to detect by analysis of the blood unless the mutations are first identified in the tumour. Thus it is very important to analyze tumour material, if available, before analysis of the blood. Individuals who are mosaic have an increased risk of recurrent tumours and of passing the mutation to their offspring.

Approximately 90% of all RB1 mutations can be detected by molecular analysis. Therefore, genetic testing can be used to distinguish between cases of hereditary and non-hereditary RB, which in turn can help determine the risk of tumour development for relatives. However, disease penetrance is about 95-99%, therefore genetic testing cannot predict with certainty whether a child will develop eye tumours. Approximately 1 out of every 20 individuals who have an RB1 mutation are not affected with Retinoblastoma.

Who should be tested

  • individuals affected with bilateral or multifocal RB (analysis of blood sample DNA)
  • individuals affected with unilateral RB (analysis of both tumour and blood sample DNA is required)
  • parents, siblings and unaffected relatives of individuals with hereditary RB, to determine the carrier status of unaffected individuals
  • pregnancies at risk due to a family history of RB

Testing Methodology

Direct Mutation Analysis: The method of analysis depends on the family history and the availability of specimens. In families where an RB1 mutation has previously been identified, blood samples from relatives are analyzed using the method most appropriate for detecting the family mutation. In isolated bilateral or unilateral cases, where there is no family history of retinoblastoma and no previous genetic testing, it is important to test both blood and tumour samples, if available, to determine if the individual is mosaic for an RB1 mutation.
The testing methods employed by the laboratory include:

  • Karyotyping and FISH analysis
  • DNA deletion analysis by quantitative PCR and/or MLPA
  • Direct mutation identification by DNA sequencing
  • Promoter Methylation analysis: An allele specific methylation PCR assay is used to determine the methylation status of the promotor region of the RB1 gene in unilateral retinoblastoma patients with the sporadic form.

Test Sensitivity: Approximately 90% of all RB1 mutations can be detected by molecular analysis whereas 10% of the mutations causing retinoblastoma cannot be detected by current testing procedures in place in the Molecular Diagnostics Laboratory. For example, of 10 people diagnosed with retinoblastoma, molecular analysis will identify the RB1 mutations in approximately 9 of them, but will not detect any mutations in 1.

In approximately 10% of individuals with the sporadic form of unilateral retinoblastoma the promoter region of the RB1 gene in the tumour will have methylation alterations.

Potential Outcomes & Interpretation of Test Results

Reason
for referral

RB1 Gene Mutations

allele 1 / allele 2

Explanation
carrier testingnone detected /
none detected
  • This individual is unlikely to be a carrier of Retinoblastoma.
carrier testingmutation detected / none detected
  • This individual is a carrier of Retinoblastoma, and may transmit a mutation to offspring.
diagnosisnone detected /
none detected
  • This result does not support a diagnosis of hereditary Retinoblastoma.
diagnosismutation detected / none detected
  • This result supports a diagnosis of hereditary Retinoblastoma.
diagnosismutation detected / mutation detected
  • This result (in eye tumour) supports a diagnosis of Retinoblastoma. This individual may be a carrier of Retinoblastoma (in blood).

Cautions

  • In some cases of hereditary RB, the mutation is found only in the germ cells of the parents (germline mosaicism). The mutation is not detectable in DNA extracted from parental blood samples. In these families, siblings of the affected individual are at high risk of developing retinoblastoma, but more distant relative are at very low risk.
  • Genetic testing cannot determine whether an individual will develop eye tumours. Approximately 5 per cent of people who have an RB1 mutation (one out of every 20) are not affected with retinoblastoma (i.e. disease penetrance is ~95 per cent
  • Current molecular testing will not detect all possible mutations in this gene. A negative test, therefore, does not rule out the diagnosis of retinoblastoma, or eliminate the possibility the individual is a carrier.
  • This test was developed and its performance characteristics validated by the Molecular Genetics Laboratory at the Hospital for Sick Children. It has not been cleared or approved by U.S. Food and Drug Administration. The FDA has determined that such clearance or approval is not necessary. This test is used for clinical purposes.

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