Clinical Diagnosis

The diagnosis of retinoblastoma (RB) is usually established by examination of the fundus of the eye using indirect ophthalmoscopy. CT, MRI, and ultrasonography are used to support the diagnosis and stage the tumor.

Retinoblastoma is:

• Unilateral if only one eye is affected by retinoblastoma. Usually, in individuals with unilateral retinoblastoma the tumor is also unifocal, i.e., only a single retinoblastoma tumor is present. However, in most persons with unilateral retinoblastoma the tumor is large and it is not possible to determine if the tumor represents only a single retinoblastoma.

• Bilateral if both eyes are affected by retinoblastoma. Usually, in individuals with bilateral retinoblastoma one or both eyes clearly show multifocal tumor growth, i.e., multiple retinoblastoma tumors are present. A few individuals have multifocal tumors in one eye (unilateral multifocal retinoblastoma). Intraocular seeding (metastasizing) may mimic true multifocal tumor growth.

• Trilateral when bilateral (or, rarely, unilateral) RB and a pinealoma co-occur.

Testing

Histopathology. Diagnosis of retinoblastoma can be confirmed by histopathologic investigation. Careful investigation of the optic nerve is required to identify possible invasion of tumor cells.

Chromosome analysis. Cytogenetic analysis of peripheral blood lymphocytes detects cytogenetically visible deletions or rearrangements involving 13q14.1-q14.2 in approximately 5% of individuals with unilateral RB and approximately 7.5% of individuals with bilateral RB. Cytogenetic resolution at the 600-650 band level is recommended and at least 30 metaphases should be analyzed in order to detect mosaic aberrations that are present in about 1% of individuals with RB.

Molecular Genetic Testing

Gene. RB1 is the only gene known to be associated with retinoblastoma.

Clinical testing

For an overview of current techniques and problems, see also the information provided by the European Molecular Genetics Quality Network (EMQN).

• Sequence analysis/mutation scanning is used to identify small deletions, insertions, and base substitutions in exons and splice site consensus regions which account for about 70% of oncogenic RB1 mutations [Lohmann et al 1996, Richter et al 2003, Houdayer et al 2004].

• Sequence analysis of RNA from blood is used to identify missplicing resulting from mutations in splice-site consensus regions and missplicing as a result of deep intronic changes not detected by conventional DNA sequencing.

• Gross deletion/duplication analysis

  • FISH. Deletions of all or parts of the RB1 gene have been identified by FISH analysis using probes derived from sequences of the RB1 gene (e.g., LSI 13 [RB1] 13q14 SpectrumOrange Probe, Vysis, Abbott laboratories). A specific role for FISH analysis is identification of individuals who have mosaicism for a deletion.

  • MLPA (multiplex ligation-dependent probe amplification), quantitative multiplex PCR with high-resolution fragment length analysis, and other methods are used to identify submicroscopic whole-exon and multiexon deletions, insertions, and rearrangements, which account for about 16% of oncogenic RB1 mutations [Richter et al 2003].

  • Genotyping of polymorphic loci

    • Heterozygosity testing. Comparison of the genotypes of RB1 polymorphic loci in DNA from peripheral blood between the individual and his/her parents can be used to show absence of a parental allele, a finding that may result from a de novo germline deletion.

    • Testing for loss of heterozygosity in tumors. Comparative genotyping of polymorphic loci within and flanking the RB1 gene in DNA from peripheral blood and tumor can reveal somatic mutations that result in allele loss.

• Targeted mutation analysis. Recurrent CpG transitions at 11 CGA codons that result in nonsense mutations account for about 25% of oncogenic RB1 alterations and can be detected by mutation-specific detection methods. These methods are particularly useful for detecting mosaic recurrent mutations in blood and can detect mutant DNA levels that are below the limit of conventional sequence analysis. Low levels of mutational mosaicism have been identified in bilateral probands and in unilateral patients with affected children who inherited the mutation and, therefore, are clinically relevant [Rushlow et al 2009].

• Methylation analysis. Hypermethylation of the RB1 gene promoter, which results in silencing gene expression, is observed in about 10%-12% of tumors from individuals with sporadic, unilateral retinoblastoma [Zeschnigk et al 2004]. In these individuals, analysis of the promoter methylation status in DNA from tumor is needed to identify the two inactive RB1 alleles that triggered tumor development.

• Linkage analysis. Linkage analysis using highly informative microsatellite markers within and tightly linked to the RB1 gene can be used in two settings:

  • To track the mutant allele in families with more than two affected individuals
    
    Note: Indirect testing in a two-generation family with an affected parent and an affected child may be unreliable because of the possibility of germline mosaicism in the "founder" parent.

  • To determine if an individual at risk in a family with only one affected individual has inherited either RB1 allele present in the affected individual. If the individual at risk does not have either RB1 allele in common with the affected relative, the individual's risk of developing retinoblastoma decreases to that of the general population [Greger et al 1988, Wiggs et al 1988].

Table 1. Summary of Molecular Genetic Testing Used in Retinoblastoma

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Gene Symbol	Test Method		Mutations Detected	Mutation Detection Frequency by Test Method 1, 2	Test Availability
RB1	Gross deletion / duplication analysis 3	FISH	Submicroscopic deletions and translocations	>8%	Clinical
		Heterozygosity testing		8%
		MLPA, quantitative multiplex PCR, other methods 3	Submicroscopic whole exon(s) deletions, insertions, and rearrangements	16%
	Mutation scanning		Single-base substitutions, small length mutations	70%-75%
	Sequence analysis (genomic)
	Targeted mutation analysis		Specific panel of recurrent point mutations	25% 4
	Methylation analysis		Hypermethylation of the promoter region	10%-12% 5
	Sequence analysis of RNA from blood		(Deep intronic) splice mutations, gross rearrangements	<5% 6

Test Availability refers to availability in the GeneTests Laboratory Directory. GeneReviews designates a molecular genetic test as clinically available only if the test is listed in the GeneTests Laboratory Directory by either a US CLIA-licensed laboratory or a non-US clinical laboratory. GeneTests does not verify laboratory-submitted information or warrant any aspect of a laboratory's licensure or performance. Clinicians must communicate directly with the laboratories to verify information.

1. The ability of the test method used to detect a mutation that is present in the indicated gene

2. From Lohmann et al [2002]. In individuals with normal chromosome studies; refers to the ability to detect a germline mutation if one is present. Note: Table 2 lists the probability that a germline mutation would be present based on family history and tumor presentation.

3. Testing that identifies whole-exon deletions/duplications not readily detectable by sequence analysis of genomic DNA; a variety of methods including quantitative PCR, long-range PCR, multiplex ligation-dependent probe amplification (MLPA), or targeted array GH (gene/segment-specific) may be used. A full array GH analysis that detects deletions/duplications across the genome may also include this gene/segment. See array GH.

4. Rushlow et al [2009]

5. In retinoblastoma tumor tissue

6. In individuals without a mutation identified by DNA-based analyses [Zhang et al 2008]

Interpretation of test results For issues to consider in interpretation of sequence analysis results, click here.

• A combination of clinical presentation, family history, and molecular genetic testing is used to determine if a proband has a germline (heritable) mutation or two somatic (non-heritable) mutations. See Table 2.
  
  If a disease-causing RB1 mutation is found in the DNA of white blood cells of the affected individual, (s)he has a high probability of having a germline mutation.
  
  If neither disease-causing RB1 mutation identified in tumor tissue is found in the DNA of white blood cells, the affected individual has a low probability of having an RB1 germline mutation. Note: Because blood mosaicism as low as 20% can usually be detected by conventional molecular analysis such as sequencing, the failure to detect an RB1 disease-causing mutation in the DNA of white blood cells reduces but cannot eliminate the probability that the individual has an RB1 mutation in his/her germline.

Table 2. Probability of Germline Mutation Being Present in a Proband with Retinoblastoma Based on Family History and Tumor Presentation

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Family History	Retinoblastoma Presentation			Probability that an RB1 Germline Mutation is Present
	Unilateral		Bilateral
	Multifocal	Unifocal
Positive 1		+		100%
	+			100%
			+	100%
Negative 2			+	Close to 100% 3
	+			14%-95%
		+		~14%

1. Positive = more than one affected family member (10% of retinoblastoma)

2. Negative = only one affected individual in the family (90% of retinoblastoma)

3. RB1 mutations are identified by conventional molecular testing in 90%-95% of simplex cases with bilateral involvement; the remaining 5% may have translocations, deep intronic splice mutations, or low-level mosaic mutations which may or may not be in the germline.

Testing Strategy

To confirm/establish the diagnosis in a proband

• Individuals with familial or bilateral retinoblastoma. The goal is to identify the constitutional RB1 mutation that caused inactivation of one RB1 allele.

  • Molecular genetic testing is first performed on peripheral blood DNA to identify the constitutional RB1 mutation. About 90%-95% of individuals have a detectable RB1 mutation in blood.

  • In some individuals with bilateral retinoblastoma and no family history of retinoblastoma, an oncogenic RB1 mutation is not detected in peripheral blood. In such cases, tumor DNA should be investigated.

    • If tumor DNA demonstrates that each of the two tumor alleles have an RB1 mutation or hypermethylation of the RB1 promoter region, peripheral blood DNA can be tested for the presence of the RB1 mutations identified in the tumor.

    • If neither of the two RB1 mutations identified in the tumor is detected in DNA from peripheral blood, mutational mosaicism has to be assumed.

• Individuals with unilateral retinoblastoma and no family history of retinoblastoma (simplex cases). The goal is to identify the two RB1 mutations that caused inactivation of both RB1 alleles.

  • Molecular genetic testing is first performed on tumor tissue. If tumor DNA demonstrates that each of the two tumor alleles have an RB1 mutation or hypermethylation of the RB1 promoter region, peripheral blood DNA can be tested for the presence of the RB1 mutations identified in the tumor.

  • In about 14% of such individuals with unilateral retinoblastoma and no family history of retinoblastoma (see Table 2), one of the RB1 mutations identified in the tumor is also detected in peripheral blood, either as a heterozygous mutation (indicating the presence of a germline mutation) or in a mosaic state (indicating a mutation that occurred after conception).

Predictive testing for at-risk asymptomatic family members requires prior identification of the disease-causing mutation in the family.

Prenatal diagnosis and preimplantation genetic diagnosis (PGD) for at-risk pregnancies require prior identification of the disease-causing mutation in the family.

Note: It is the policy of GeneReviews to include clinical uses of testing available from laboratories listed in the GeneTests Laboratory Directory; inclusion does not necessarily reflect the endorsement of such uses by the author(s), editor(s), or reviewer(s).

Genetically Related (Allelic) Disorders

No phenotypes other than hereditary predisposition to retinoblastoma and second cancers (see Natural History, Related tumors) are known to be associated with mutation of RB1.

Natural History

Probands with retinoblastoma (RB) usually present in one of the following clinical settings:

• Chromosome deletion involving band 13q14. Up to 5% of all index cases with unifocal RB and 7.5% of all index cases with multifocal RB have a chromosomal deletion of 13q14. Such chromosomal abnormalities are often associated with developmental delay and birth defects [Baud et al 1999].

• Normal cytogenetic study and one of the following

  • Positive family history and unilateral or bilateral RB: ~10% of index cases

  • Negative family history and bilateral RB: 30% of index cases

  • Negative family history and unilateral RB: 60% of index cases

About 60% of individuals with RB have unilateral retinoblastoma with a mean age at diagnosis of 24 months. About 40% have bilateral retinoblastoma with a mean age at diagnosis of 15 months. In individuals with a positive family history (~10%) who undergo clinical surveillance via serial fundoscopic examinations, tumors are often identified in the first month of life.

The most common presenting sign of RB is a white pupillary reflex (leukocoria). Strabismus is the second most common presenting sign and may accompany or precede leukocoria [Abramson et al 2003]. Unusual presenting symptoms include glaucoma, orbital cellulitis, uveitis, hyphema, or vitreous hemorrhage. Most affected children are diagnosed before age five years. Atypical manifestations are more frequent in older children.

In most children with bilateral tumors, both eyes are affected at the time of initial diagnosis. Some children who are initially diagnosed with unilateral retinoblastoma later develop a tumor in the contralateral unaffected eye.

Retinoma and associated eye lesions. These lesions range from retinal scars to calcified phthisical eyes resulting from spontaneous regression of retinoblastoma, and include benign retinal tumors (called retinocytoma or retinoma) that have undergone spontaneous growth arrest.

Related tumors. Individuals with germline RB1 mutations are at increased risk of developing tumors outside the eye.

Pinealomas occur in "retinal-like" tissue in the pineal gland of the brain. Co-occurrence of pinealomas or primitive neuroectodermal tumors and retinoblastoma is referred to as trilateral retinoblastoma. Pinealoma is rare and, unlike retinoblastoma of the eye, which is generally curable, usually fatal [Kivela 1999].

The risk of other specific extraocular primary neoplasms (collectively called second primary tumors) is increased. Most of the second primary cancers are osteosarcomas, soft tissue sarcomas, or melanomas. These tumors usually manifest in adolescence or adulthood. The incidence of second primary tumors is increased to more than 50% in individuals with retinoblastoma who have received external beam radiation therapy (EBRT) [Wong et al 1997]. Survivors of hereditary retinoblastoma who are not exposed to high-dose radiotherapy have a high lifetime risk of developing a late-onset cancer [Fletcher et al 2004].

Genotype-Phenotype Correlations

In the majority of families with retinoblastoma, all members who have inherited a germline mutation develop multiple tumors in both eyes. It is not unusual to find, however, that the founder (i.e., the first person in the family to have retinoblastoma) has only unilateral retinoblastoma. Most of such families segregate RB1 null alleles that are altered by frameshift or nonsense mutations. With few specific exceptions, RB1 null alleles show nearly complete penetrance (<99%) [Lohmann et al 1996, Sippel et al 1998, unpublished data].

Fewer than 10% of families show a "low penetrance" phenotype with reduced expressivity (i.e., increased prevalence of unilateral retinoblastoma) and incomplete penetrance (i.e., 25% or lower). This low penetrance phenotype is usually associated with mutant RB1 alleles showing in-frame or missense changes, distinct splice mutations, or mutations in the promoter region.

A third category of families shows reduced penetrance but no reduced expressivity in family members with retinoblastoma [Klutz et al 2002].

Cytogenetically visible deletions involving 13q14 that also result in deletions of other genes in the same chromosomal region in addition to the RB1 gene may cause developmental delay and mild-to-moderate facial dysmorphism. As sizeable deletions of 13q14 show reduced expressivity, a considerable proportion of individuals with such deletions show unilateral retinoblastoma only; some of these children develop no tumors at all.

Penetrance

See Genotype-Phenotype Correlations.

Anticipation

Milder phenotypic expression in founders has been associated with mutational mosaicism. No multigenerational anticipation has been observed to date.

Nomenclature

Glioma retinae is another name for retinoblastoma.

Prevalence

The incidence of retinoblastoma is estimated at between 1:15,000 and 1:20,000 live births [Moll et al 1997, Seregard et al 2004].

Evaluations Following Initial Diagnosis

To establish the extent of disease in an individual diagnosed with retinoblastoma, the following evaluations are recommended:

• Prior to the planning of therapy, the extent of the tumor within and outside the eye should be determined. In the absence of family history, most commonly the affected eye(s) contain large tumors, directly visible through the pupil as a white pupillary reflex. Extent of tumor is then estimated by imaging techniques such as CT scan and MRI, particularly focusing on the tumor-optic nerve relationship.

• For very large tumors with risk factors for extraocular disease, bone marrow aspiration and examination of cerebrospinal fluid (CSF) may also be performed at diagnosis, and certainly performed if pathologic examination of an eye reveals optic nerve invasion or significant choroidal invasion.

• In those individuals with a family history of retinoblastoma (RB) and in uncommon circumstances in which the child presents with strabismus or poor vision, the retinal tumors may be small and can be seen on clinical examination not to affect the optic nerve or extend outside the retina. CT scan or MRI would be unnecessary in evaluation when there is no risk of extraocular extension.

Treatment of Manifestations

Goals of treatment are preservation first of life, and then of sight. As optimum treatment may be complex, specialists skilled in the treatment of retinoblastoma from various fields including ophthalmology, pediatric ophthalmology, radiation oncology, and oncology often are included.

In addition to tumor stage, choice of treatment depends on several factors, including the number of tumor foci (unifocal, unilateral multifocal, or bilateral disease), localization and size of the tumor(s) within the eye, presence of vitreous seeding, and the age of the child.

Treatment options include enucleation, cryotherapy, photocoagulation, photochemistry, external beam radiation therapy, and radiation therapy using episcleral plaques. Novel treatment options include systemic chemotherapy combined with or followed by local therapy using laser or freezing to physically destroy residual disease [Gallie et al 1996, Bornfeld et al 1997, Schueler et al 2003].

Prevention of Secondary Complications

If possible, high-dose radiotherapy should be avoided to reduce lifetime risk of developing late-onset secondary cancers.

Surveillance

Detection of second tumors in individuals with retinoblastoma. Following successful treatment, children require frequent follow-up examinations for early detection of new intraocular tumors.

• It is recommended that children known to have an RB1 germline mutation have an eye examination every three to four weeks until age one year and then less frequently until age three years. Young or uncooperative children usually require examination under anesthesia.

• Individuals who have unilateral retinoblastoma are at risk of developing tumors in their normal eye.

  • If the two RB1 mutant alleles are identified in the tumor and if the individual is shown to have one of those two mutations in leukocyte DNA (~14% of individuals), the children are followed as described above. Mosaicism involving more than 20% of blood cells is molecularly detectable by conventional methods.

  • If the RB1 mutant alleles identified in the tumor are not detected in leukocyte DNA, there is still a risk that the individual has low-level mosaicism (involving <20% of blood cells) for the mutant allele and will develop a tumor in the other eye. [Lohmann et al 1997, Sippel et al 1998]. Possibly, this risk is small enough that examination under anesthesia may not be justified and may be replaced with regular clinical examination of the eyes.

Detection of second non-ocular tumors in individuals with retinoblastoma. Because of the high risk of sarcomas, the physician and parents should promptly evaluate complaints of bone pain or lumps. No specific screening protocols exist.

Individuals at risk for retinoblastoma who warrant surveillance for early manifestations of RB include the following:

• Individuals with retinomas

• Children who have inherited an RB1 disease-causing mutation OR children at risk for RB who have not undergone molecular genetic testing:

  • Eye examinations by an ophthalmologist experienced in the treatment of retinoblastoma starting directly after birth as described in Surveillance, Detection of second tumors in individuals with retinoblastoma. Young or uncooperative children may require examination under anesthesia.

• At-risk children who have not inherited the cancer-predisposing mutation known to be present in the family as determined by RB1 mutation analysis or linkage analysis:

  • Examination by an ophthalmologist familiar with retinoblastoma shortly after birth. Subsequent eye examinations should be performed as needed for routine pediatric care.

Agents/Circumstances to Avoid

It has been suggested by Fletcher et al [2004] that most of the excess cancer risks in hereditary retinoblastoma survivors may be preventable by limiting exposures to DNA-damaging agents (radiotherapy, tobacco, and UV light).

Testing of Relatives at Risk

Asymptomatic at-risk children. Use of molecular genetic testing for early identification of at-risk family members improves diagnostic certainty and reduces the need for costly screening procedures in those at-risk family members who have not inherited the disease-causing mutation [Noorani et al 1996, Richter et al 2003]. The American Society of Clinical Oncologists (ASCO) identifies RB as a Group 1 disorder, i.e., a hereditary syndrome for which genetic testing is considered part of the standard management for at-risk family members [ASCO 2003].

See Genetic Counseling for issues related to testing of at-risk relatives for genetic counseling purposes.

Therapies Under Investigation

Search ClinicalTrials.gov for access to information on clinical studies for a wide range of diseases and conditions. Note: There may not be clinical trials for this disorder.

Other

Genetics clinics, staffed by genetics professionals, provide information for individuals and families regarding the natural history, treatment, mode of inheritance, and genetic risks to other family members as well as information about available consumer-oriented resources. See the GeneTests Clinic Directory.

See Consumer Resources for disease-specific and/or umbrella support organizations for this disorder. These organizations have been established for individuals and families to provide information, support, and contact with other affected individuals.

Mode of Inheritance

Hereditary retinoblastoma (RB), caused by germline RB1 mutations, is inherited in an autosomal dominant manner.

Risk to Family Members

Parents of a proband. Some individuals diagnosed with retinoblastoma have an affected parent or a parent who has an RB1 mutation but is not affected; the majority of individuals with retinoblastoma have the disorder as the result of a de novo gene mutation.

The recommendations for determining the genetic status of the parent of a proband with retinoblastoma or the conclusions about the genetic status of the parent depend on the following findings in the proband:

• Cytogenetically detectable chromosome 13 deletion or rearrangement

  • Recommendation. Parental cytogenetic studies to determine if either parent carries a balanced chromosome translocation or rearrangement

• Positive family history (i.e., the parent had retinoblastoma or a close relative of one parent had retinoblastoma)

  • Conclusion. The parent has an RB1 cancer-predisposing germline mutation.

• Negative family history

  • Recommendation. Examination of apparently unaffected parents by an ophthalmologist knowledgeable about retinoblastoma, retinoma, and retinoblastoma-associated eye lesions

  • Conclusion. If such a lesion is detected, the parent has an RB1 cancer-predisposing germline mutation.

• Presence of a germline RB1 cancer-predisposing mutation in the proband

  • Recommendation. Molecular genetic testing of a blood sample of both parents

  • Conclusions

    • If a heterozygous mutation is identified in either parent, the parent is at risk of developing non-ocular second primary tumors and is at 50% risk of transmitting the cancer-predisposing RB1 mutation to other offspring. In approximately 10% of simplex cases that have a heterozygous RB1 mutation detected in blood, one of the proband’s unaffected parents also has the mutation. In most cases this is a heterozygous “reduced penetrance” mutation, such as a missense mutation [Rushow et al 2009].

    • For approximately 1% of simplex cases that have a heterozygous RB1 mutation detected in blood, one of the proband’s unaffected parents has mutational mosaicism, , possibly at levels too low to be detectable by sequencing of DNA extracted from leukocytes, but detectable by more sensitive methods such as allele-specific PCR [Rushlow et al 2009]. Sibs of the proband are at risk of inheriting the mutation.

    • A parent may have germline mosaicism even in the absence of a detectable mutation in leukocyte DNA. Therefore, even if the RB1 mutation cannot be detected in leukocyte DNA from either parent, sibs of the proband should still be tested for the presence of the proband’s mutation.

• Presence of a mosaic RB1 cancer-predisposing mutation in the proband [Carlson & Desnick 1979]

  • Conclusion. The mutation occurred de novo as a post-zygotic event in the proband.

  • Recommendation. Molecular genetic testing of the parents is not necessary.

Sibs of a proband. The risk to sibs of a proband depends on the genetic status of the parents.

• If a parent is determined to have a germline RB1 cancer-predisposing mutation either by positive family history, by an eye examination that reveals a retinoblastoma-associated eye lesion, or by molecular genetic testing that reveals the presence of a cancer-predisposing RB1 mutation, the risk to each sib of the proband is 50% (or lower if the parent with the RB1 mutation has mosaicism) of inheriting the cancer-predisposing RB1 mutation. Given the approximately 99% penetrance of most RB1 cancer-predisposing mutations, the actual risk for retinoblastoma in these individuals is about 50% (or lower if the parent with the RB1 mutation has mosaicism). (In rare families with "familial low-penetrance retinoblastoma," the risk of tumor development in an individual with the mutation is reduced.)

• If neither parent shows the cancer-predisposing RB1 germline mutation that was identified in the proband, germline mosaicism in one parent is still possible. Thus, it is recommended that each sib be tested for the RB1 mutation identified in the proband.

• If the proband clearly shows mosaicism for an RB1 cancer-predisposing mutation in non-cancer cells such as leukocyte DNA, it is assumed that the mutation arose as a postzygotic event and that neither parent has an RB1 germline mutation. The risk to the sibs is not increased and thus the testing of sibs for the RB1 mutation identified in the proband is not warranted.

• If molecular genetic testing is not available or is uninformative, empiric risks based on tumor presentation (i.e., unifocal or multifocal) and family history can be used (Table 3). The low, but not negligible, risk to sibs of a proband with a negative family history presumably reflects the presence of either a germline RB1 mutation with reduced penetrance in one parent or somatic mosaicism (that includes the germline) for an RB1 mutation in one parent.

• If a parent has a cytogenetically detectable balanced chromosome 13 translocation or rearrangement, the sibs are at increased risk of inheriting an unbalanced chromosome rearrangement.

Offspring of a proband. The risk to the offspring of a proband depends on the following:

• If the proband has bilateral RB and no family history of RB, the presence of a germline RB1 cancer-predisposing mutation is assumed and the risk to each offspring of inheriting the mutation is 50%. Predictive DNA testing in offspring is possible if the cancer-predisposing RB1 mutation has been identified in the proband.

• If the proband has had unilateral multifocal RB and no family history of RB, recurrence risk for offspring is lower [Sippel et al 1998].

• The risk to offspring of a proband with unilateral unifocal disease and a negative family history is 6%, reflecting the possibility that the proband has mosaicism for a germline mutation or a germline RB1 mutation associated with milder phenotypic expression. In families with "familial low-penetrance retinoblastoma," the risk of tumor development in persons with the low-penetrance RB1 allele is lower than the 95% observed with highly penetrant RB1 “null” alleles.

Molecular genetic testing of tumor DNA from probands who are simplex cases (i.e., a single occurrence in a family) with a unilateral tumor

• If both mutant RB1 alleles are identified and if one of the mutant alleles is heterozygous in DNA from leukocytes of the proband, the risk to offspring of a proband of inheriting the mutant alleles is 50%.

• If neither mutant allele is detected in DNA from leukocytes of the proband, there is an estimated 1.2% chance that the proband has germline mosaicism for the mutant allele, giving the offspring a 0.6% risk of inheriting a germline mutation [Richter et al 2003]. Molecular genetic testing in offspring must check for the two mutant alleles identified in the tumor of the proband.

• If one of the mutations identified in the tumor is mosaic in DNA from leukocytes of the proband, the level of germline involvement is uncertain. Quantitative mutation analysis of sperm may be helpful. All offspring should be checked for the mutation identified in leukocyte DNA.

Other family members of a proband. The risk to other family members depends on the status of the proband's parents. If a parent has a disease-causing mutation, his or her family members are at risk.

Related Genetic Counseling Issues

See Testing of Relatives at Risk for information on testing at-risk relatives for the purpose of early diagnosis and treatment.

Family planning

• The optimal time for determination of genetic risk and discussion of the availability of prenatal testing is before pregnancy.

• It is appropriate to offer genetic counseling (including discussion of potential risks to offspring and reproductive options) to young adults who are affected or at risk.

Genetic cancer risk assessment and counseling. For comprehensive descriptions of the medical, psychosocial, and ethical ramifications of identifying at-risk individuals through cancer risk assessment with or without molecular genetic testing, see Elements of Cancer Genetics Risk Assessment and Counseling (part of PDQ®, National Cancer Institute)

DNA banking is the storage of DNA (typically extracted from white blood cells) for possible future use. Because it is likely that testing methodology and our understanding of genes, mutations, and diseases will improve in the future, consideration should be given to banking DNA of affected individuals. See for a list of laboratories offering DNA banking.

Prenatal Testing

Molecular genetic testing. Prenatal diagnosis for pregnancies at increased risk is possible by analysis of DNA extracted from fetal cells obtained by amniocentesis usually performed at about 15 to 18 weeks' gestation or chorionic villus sampling (CVS) at about ten to 12 weeks' gestation. The disease-causing allele of an affected family member must be identified or linkage established in the family before prenatal testing can be performed.

Cytogenetic testing. Prenatal diagnosis for pregnancies at increased risk of having an unbalanced chromosome rearrangement is possible by chromosome analysis of fetal cells obtained by amniocentesis usually performed at about 15 to 18 weeks' gestation or chorionic villus sampling (CVS) at about ten to 12 weeks' gestation.

Note: Gestational age is expressed as menstrual weeks calculated either from the first day of the last normal menstrual period or by ultrasound measurements.

Ultrasound examination. If a disease-causing RB1 mutation is identified in the fetus, ultrasound examination may be used to identify intraocular tumors. If tumors are present, preterm delivery to enable early treatment may be considered [Gallie et al 1999].

Requests for prenatal testing for conditions (such as retinoblastoma) that do not affect intellect and have treatment available are not common. Differences in perspective may exist among medical professionals and within families regarding the use of prenatal testing, particularly if the testing is being considered for the purpose of pregnancy termination rather than early diagnosis. Although most centers would consider decisions about prenatal testing to be the choice of the parents, discussion of these issues is appropriate.

Preimplantation genetic diagnosis (PGD) may be available for families in which the disease-causing mutations have been identified. For laboratories offering PGD, see .

Note: It is the policy of GeneReviews to include clinical uses of testing available from laboratories listed in the GeneTests Laboratory Directory; inclusion does not necessarily reflect the endorsement of such uses by the author(s), editor(s), or reviewer(s).

Molecular Genetic Pathogenesis

Tumor development starts from cells that do not have a normal RB1 allele. See Figure 1.

Figure

Figure 1. Schematic of the molecular genetic mechanisms that result in non-hereditary and hereditary retinoblastoma (RB). The development of retinoblastoma is initiated if both alleles of the RB1 gene are mutated (rb rb).

In non-hereditary (more...)

Normal allelic variants. Twenty-seven exons are transcribed and spliced into a 4.7-kb mRNA. There is no indication of alternative splicing. No frequent polymorphic sites within the 2.7-kb open reading frame are known, but there are intronic variants and two highly polymorphic microsatellites (Rb1.20, Rbi2) and one minisatellite (RBD).

Pathologic allelic variants. More than 1000 distinct mutations have been observed in white blood-cell DNA of individuals with retinoblastoma or in tumors [Lohmann 1999, Richter et al 2003, Valverde et al 2005]. The majority of RB1 mutations result in a premature termination codon, usually through single base substitutions, frameshift mutations, or splice mutations. Mutations have been found scattered throughout exon 1 to exon 25 of the RB1 gene and its promoter region. Recurrent mutations are observed at 14 methylated CpG dinucleotides.

Normal gene product. The RB1 gene encodes a ubiquitously expressed nuclear protein that is involved in cell cycle regulation (G1 to S transition). The RB protein is phosphorylated by members of the cyclin-dependent kinase (cdk) system prior to the entry into S-phase. Upon phosphorylation, the binding activity of the pocket domain is lost, resulting in the release of cellular proteins. For a review see Goodrich [2006].

Abnormal gene product. The majority of mutant alleles, if expressed at all, code for proteins that have lost cell cycle-regulating functions. Retention of partial activities has been observed in proteins resulting from mutant alleles that are associated with low-penetrance retinoblastoma [Bremner et al 1997, Otterson et al 1997].

Literature Cited

1. Abramson DH, Beaverson K, Sangani P, Vora RA, Lee TC, Hochberg HM, Kirszrot J, Ranjithan M. Screening for retinoblastoma: presenting signs as prognosticators of patient and ocular survival. Pediatrics. 2003;112:1248–55. [PubMed: 14654593]
2. ASCO (2003) Statement on genetic testing for cancer susceptibility. American Society of Clinical Oncology.
3. Baud O, Cormier-Daire V, Lyonnet S, Desjardins L, Turleau C, Doz F. Dysmorphic phenotype and neurological impairment in 22 retinoblastoma patients with constitutional cytogenetic 13q deletion. Clin Genet. 1999;55:478–82. [PubMed: 10450867]
4. Bornfeld N, Schuler A, Bechrakis N, Henze G, Havers W. Preliminary results of primary chemotherapy in retinoblastoma. Klin Padiatr. 1997;209:216–21. [PubMed: 9293453]
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Published Statements and Policies Regarding Genetic Testing

1. American Society of Clinical Oncology. Statement on genetic testing for cancer susceptibility. Available at jco.ascopubs.org. 2003. Accessed 6-7-10.

Suggested Reading

1. Lohmann DR. Retinoblastoma. Atlas of Genetics and Cytogenetics in Oncology and Haematology. Available at atlasgenetics.oncology.org. 1998. Accessed 6-7-10.

Author History

Norbert Bornfeld, MD; University of Essen (2000-2004)
Brenda L Gallie, MD (2004-present)
Bernhard Horsthemke, PhD; University of Essen (2000-2004)
Dietmar R Lohmann, MD (2000-present)
Eberhard Passarge, MD; University of Essen (2000-2004)

Revision History

• 10 June 2010 (me) Comprehensive update posted live

• 7 May 2007 (me) Comprehensive update posted to live Web site

• 21 January 2005 (dl) Revision: Risk to offspring of a proband

• 28 December 2004 (me) Comprehensive update posted to live Web site

• 21 January 2003 (me) Comprehensive update posted to live Web site

• 18 July 2000 (me) Review posted to live Web site

• 21 January 1999 (dl) Original submission