Hereditary Retinoblastoma (RB1) Sequencing and Deletion/Duplication

Last Literature Review: August 2022 Last Update:

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. 



RB1 (NM_000321)


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


Autosomal dominant


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.


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.


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)




Approximately 14

Unilateral (multifocal)









Close to 100c

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) Estimatea (%) and 95% Credibility Region Analytic Specificity (NPA) Estimate (%)


>99 (96.9-99.4)


Deletions 1-10 bpb

93.8 (84.3-98.2)


Insertions 1-10 bpb

94.8 (86.8-98.5)


Exon-levelc deletions

97.8 (90.3-99.8) [2 exons or larger]

62.5 (38.3-82.6) [single exon]


Exon-levelc duplications

83.3 (56.4-96.4) [3 exons or larger]


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


Result Variant(s) Detected Clinical Significance


One RB1 pathogenic variant detected

Consistent with a diagnosis of hereditary retinoblastoma


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) 


RB1 variant(s) of unknown clinical significance detected

Uncertain; it is unknown whether variant is benign or pathogenic


  • 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.