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Hereditary Retinoblastoma (RB1) Sequencing and Deletion/Duplication

3005696

Hereditary Retinoblastoma (RB1) Sequencing and Deletion/Duplication 3005696

Method

Massively Parallel Sequencing

Recommended test for individuals with a suspected diagnosis or family history of heritable retinoblastoma

If a familial sequence variant has been previously identified, targeted sequencing for that variant may be appropriate; refer to the Laboratory Test Directory for additional information.

Retinoblastoma is a malignant tumor of the retina that typically occurs in children under 5 years of age. Hereditary retinoblastoma, caused by a single germline pathogenic variant in the RB1 gene, predisposes individuals to retinoblastoma and other nonocular tumors, including pinealoblastoma, osteosarcoma, soft tissue sarcoma, and melanoma.

Disease Overview

Retinoblastoma should be suspected in children with any of the following:

Leukocoria (white pupil)
Strabismus
Change in eye appearance
Reduced visual acuity

Hereditary retinoblastoma should be suspected in an individual with any of the following:

A diagnosis of retinoblastoma, including unilateral (unifocal and multifocal) and bilateral involvement
A retinoma
A family history of retinoblastoma

The diagnosis of hereditary retinoblastoma is established in an individual with both retinoblastoma/retinoma and a family history of retinoblastoma.

Approximately 6-8% of individuals with retinoblastoma have a chromosome deletion of 13q14, which can also be associated with developmental delay and birth defects.

Genetics

Gene

RB1 (NM_000321)

Etiology

Retinoblastoma affects approximately 1/15,000 to 1/20,000 live births. Hereditary retinoblastoma accounts for approximately 10% of retinoblastoma cases.

Inheritance

Autosomal dominant

Penetrance

Complete penetrance, except for fewer than 10% of families that show a "low-penetrance" phenotype with reduced expressivity. See GeneReviews for additional details.

Test Interpretation

Contraindications for Ordering

Should not be ordered to detect somatic variants associated with tumors/malignancy because sensitivity for mosaic variants is low with methodology used for germline assays
Individuals with hematologic malignancy and/or a previous allogeneic bone marrow transplantation should not undergo molecular genetic testing on a peripheral blood specimen.
- Testing of cultured fibroblasts is required for accurate interpretation of test results.

Methodology

This test is performed using the following sequence of steps:

Selected genomic regions, primarily coding exons and exon-intron boundaries, from the targeted genes are isolated from extracted genomic DNA using a probe-based hybrid capture enrichment workflow.
Enriched DNA is sequenced by massively parallel sequencing (MPS; also known as next generation sequencing [NGS]) followed by paired-end read alignment and variant calling using a custom bioinformatics pipeline. The pipeline includes an algorithm for detection of large (single exon-level or larger) deletions and duplications.
Sanger sequencing is performed as necessary to fill in regions of low coverage and in certain situations, to confirm variant calls.
Large deletion/duplication calls made using MPS are confirmed by an orthogonal exon-level microarray when sample quality and technical conditions allow.

Sensitivity/Specificity

Clinical Sensitivity

The clinical sensitivity is variable and dependent on phenotype. The table below details the probability of a germline pathogenic variant being present in a proband with retinoblastoma based on family history and tumor presentation.

Retinoblastoma Presentation	Family History	Probability That an RB1 Germline PV Is Present (%)
Unilateral (unifocal)	Positive^a	100
Unilateral (unifocal)	Negative^b	Approximately 14
Unilateral (multifocal)	Positive^a	100
Unilateral (multifocal)	Negative^b	14-95
Bilateral	Positive^a	100
Bilateral	Negative^b	Close to 100^c
^aPositive family history is defined as more than one affected family member. ^bNegative family history is defined as only one affected individual in the family. ^cRB1 pathogenic variants are identified by conventional molecular testing in 90-97% of simplex cases with bilateral involvement; the remaining 5% may have translocations, deep intronic splice variants, or low-level mosaic pathogenic variants that may or may not be in the germline. Source: Lohmann, 2018

Analytic Sensitivity/Specificity

Variant Class	Analytic Sensitivity (PPA) Estimate^a (%) and 95% Credibility Region	Analytic Specificity (NPA) Estimate (%)
SNVs	>99 (96.9-99.4)	>99.9
Deletions 1-10 bp^b	93.8 (84.3-98.2)	>99.9
Insertions 1-10 bp^b	94.8 (86.8-98.5)	>99.9
Exon-level^c deletions	97.8 (90.3-99.8) [2 exons or larger] 62.5 (38.3-82.6) [single exon]	>99.9
Exon-level^c duplications	83.3 (56.4-96.4) [3 exons or larger]	>99.9
^aPPA values are derived from larger methods-based MPS and/or Sanger validations. These values do not apply to testing performed by multiplex ligation-dependent probe amplification (MLPA) unless otherwise indicated. ^bVariants greater than 10 bp may be detected, but the analytic sensitivity may be reduced. ^cIn most cases, a single exon deletion or duplication is less than 450 bp and 3 exons span a genomic region larger than 700 bp. bp, base pairs; NPA, negative percent agreement; PPA, positive percent agreement; SNVs, single nucleotide variants

Results

Result	Variant(s) Detected	Clinical Significance
Positive	One RB1 pathogenic variant detected	Consistent with a diagnosis of hereditary retinoblastoma
Negative	No RB1 pathogenic variants detected	Risk of hereditary retinoblastoma is reduced but not eliminated (less than 1% of individuals with retinoblastoma or retinoma and a negative germline result on blood have mosaicism)
Uncertain	RB1 variant(s) of unknown clinical significance detected	Uncertain; it is unknown whether variant is benign or pathogenic

Limitations

A negative result does not exclude a heritable form of retinoblastoma.
Diagnostic errors can occur due to rare sequence variations.
Interpretation of this test result may be impacted if this patient has had an allogeneic stem cell transplantation.
The following will not be evaluated:
- Variants outside the coding regions and intron-exon boundaries of targeted gene(s)
- Regulatory region and deep intronic variants
- Breakpoints of large deletions/duplications
The following may not be detected:
- Deletions/duplications/insertions of any size by MPS
- Large duplications less than 3 exons in size
- Deletions/duplications in the following exon RB1 (NM_000321) 22
- Noncoding transcripts
- Some variants due to technical limitations in the presence of pseudogenes, repetitive, or homologous regions
- Low-level somatic variants
Exon RB1 (NM_000321) 22 may have reduced sequencing sensitivity due to technical limitations of the assay.

References

GeneReviews - Retinoblastoma

Lohmann DR, Gallie BL. Retinoblastoma. In: Adam MP, Everman DB, Mirzaa GM, et al, eds. GeneReviews. University of Washington, Seattle. Updated Jul 2000; accessed Jun 2022.

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