Hereditary Retinoblastoma Panel

New
Summary
  • Is a 1 gene panel that includes assessment of non-coding variants
  • The Blueprint Genetics Hereditary Retinoblastoma Panel analyzes mutations in RB1 gene associated with inherited susceptibility to retinoblastoma.

Analysis methods
  • PLUS
Availability

4 weeks

Number of genes

1

Test code

ON1701

Panel size

Small

CPT codes
81479

Summary

The Blueprint Genetics Hereditary Retinoblastoma Panel (test code ON1701):

ICD codes

Commonly used ICD-10 code(s) when ordering the Hereditary Retinoblastoma Panel

ICD-10 Disease
C69.20 Hereditary retinoblastoma

Sample Requirements

  • Blood (min. 1ml) in an EDTA tube
  • Extracted DNA, min. 2 μg in TE buffer or equivalent
  • Saliva (Oragene DNA OG-500 kit/OGD-500 or OG-575 & OGD-575)

Label the sample tube with your patient's name, date of birth and the date of sample collection.

Note that we do not accept DNA samples isolated from formalin-fixed paraffin-embedded (FFPE) tissue. Read more about our sample requirements here.

Although majority of retinoblastomas are sporadic, approximately 40% of the cases are inherited. Hereditary retinoblastoma is caused by mutations in the RB1 gene. These mutations have high penetrance and are detected in 95% of patients with hereditary retinoblastoma. Hereditary retinoblastoma affects predominantly young children and almost all children with retinoblastoma are diagnosed before the age of five. Inherited forms of retinoblastoma are more likely to be bilateral and patients with hereditary retinoblastoma have an increased risk of developing secondary tumors. Prevalence of hereditary retinoblastoma is 1-2:100 000, with higher prevalence in developing countries. Hereditary retinoblastoma is the most common malignant cancer of the eye in children.

Genes in the Hereditary Retinoblastoma Panel and their clinical significance

Gene Associated phenotypes Inheritance ClinVar HGMD
RB1 Retinoblastoma AD 266 1102

Gene refers to the HGNC approved gene symbol; Inheritance refers to inheritance patterns such as autosomal dominant (AD), autosomal recessive (AR), X-linked (XL), X-linked dominant (XLD) and X-linked recessive (XLR); ClinVar refers to the number of variants in the gene classified as pathogenic or likely pathogenic in this database (ClinVar); HGMD refers to the number of variants with possible disease association in the gene listed in Human Gene Mutation Database (HGMD). The list of associated, gene specific phenotypes are generated from CGD or Orphanet databases.

Non-coding variants covered by Hereditary Retinoblastoma Panel

Gene Genomic location HG19 HGVS RefSeq RS-number
RB1 Chr13:48877814 NM_000321.2 rs576931877
RB1 Chr13:48877836 NM_000321.2
RB1 Chr13:48877837 c.-212G>A NM_000321.2
RB1 Chr13:48877851 c.-198G>A NM_000321.2 rs387906521
RB1 Chr13:48877851 c.-198G>T NM_000321.2
RB1 Chr13:48877852 c.-197G>A NM_000321.2
RB1 Chr13:48877853 NM_000321.2
RB1 Chr13:48877856 c.-193T>A/G NM_000321.2
RB1 Chr13:48877856 NM_000321.2
RB1 Chr13:48877856 NM_000321.2
RB1 Chr13:48877857 c.-192G>A NM_000321.2
RB1 Chr13:48877860 c.-189G>T NM_000321.2 rs387906520
RB1 Chr13:48877899 c.-150G>C NM_000321.2
RB1 Chr13:48877900 c.-149G>T NM_000321.2
RB1 Chr13:48921946 c.501-15T>G NM_000321.2
RB1 Chr13:48930735 c.608-3418A>G NM_000321.2
RB1 Chr13:48937921 c.861+828T>G NM_000321.2
RB1 Chr13:48947691 c.1215+63T>G NM_000321.2
RB1 Chr13:48954175 c.1390-14A>G NM_000321.2 rs9535023
RB1 Chr13:48954239 c.1421+20_1421+33delTAAAAAATTTTTTT NM_000321.2
RB1 Chr13:49027115 c.1696-14C>T NM_000321.2 rs776912915
RB1 Chr13:49027117 c.1696-12T>G NM_000321.2
RB1 Chr13:49027258 c.1814+11T>C NM_000321.2 rs771369373
RB1 Chr13:49030329 c.1815-11A>G NM_000321.2
RB1 Chr13:49039121 c.2212-13T>A NM_000321.2
RB1 Chr13:49039327 c.2326-14T>C NM_000321.2
RB1 Chr13:49046098 c.2490-1398A>G NM_000321.2
RB1 Chr13:49047468 c.2490-28T>C NM_000321.2
RB1 Chr13:49047470 c.2490-26A>C/G/T NM_000321.2
RB1 Chr13:49047470 c.2490-26A>C NM_000321.2
RB1 Chr13:49047470 c.2490-26A>T NM_000321.2
RB1 Chr13:49047470 c.2490-26A>G NM_000321.2

Added and removed genes from the panel

Genes added Genes removed


Test Strengths

The strengths of this test include:
  • CAP and ISO-15189 accredited laboratory
  • CLIA-certified personnel performing clinical testing in a CLIA-certified laboratory
  • Powerful sequencing technologies, advanced target enrichment methods and precision bioinformatics pipelines ensure superior analytical performance
  • Careful construction of clinically effective and scientifically justified gene panels
  • Our Nucleus online portal providing transparent and easy access to quality and performance data at the patient level
  • Our publicly available analytic validation demonstrating complete details of test performance
  • ~2,000 non-coding disease causing variants in our clinical grade NGS assay for panels (please see below ‘Non-coding disease causing variants covered by this panel’)
  • Our rigorous variant classification scheme
  • Our systematic clinical interpretation workflow using proprietary software enabling accurate and traceable processing of NGS data
  • Our comprehensive clinical statements

Test Limitations

This test does not detect the following:
  • Complex inversions
  • Gene conversions
  • Balanced translocations
  • Mitochondrial DNA variants
  • Repeat expansion disorders unless specifically mentioned
  • Non-coding variants deeper than ±20 base pairs from exon-intron boundary unless otherwise indicated (please see above Panel Content / non-coding variants covered by the panel).
This test may not reliably detect the following:
  • Low level mosaicism (variant with a minor allele fraction of 14.6% is detected with 90% probability)
  • Stretches of mononucleotide repeats
  • Indels larger than 50bp
  • Single exon deletions or duplications
  • Variants within pseudogene regions/duplicated segments

The sensitivity of this test may be reduced if DNA is extracted by a laboratory other than Blueprint Genetics.

For additional information, please refer to the Test performance section and see our Analytic Validation.

Blueprint Genetics offers a comprehensive Hereditary Retinoblastoma Panel that covers classical genes associated with hereditary retinoblastoma. The genes are carefully selected based on the existing scientific evidence, our experience and most current mutation databases. Candidate genes are excluded from this first-line diagnostic test. The test does not recognise balanced translocations or complex inversions, and it may not detect low-level mosaicism. The test should not be used for analysis of sequence repeats or for diagnosis of disorders caused by mutations in the mitochondrial DNA.

Analytical validation is a continuous process at Blueprint Genetics. Our mission is to improve the quality of the sequencing process and each modification is followed by our standardized validation process. Average sensitivity and specificity in Blueprint NGS Panels is 99.3% and 99.9% for detecting SNPs. Sensitivity to for indels vary depending on the size of the alteration: 1-10bps (96.0%), 11-20 bps (88.4%) and 21-30 bps (66.7%). The longest detected indel was 46 bps by sequence analysis. Detection limit for Del/Dup (CNV) analysis varies through the genome depending on exon size, sequencing coverage and sequence content. The sensitivity is 71.5% for single exon deletions and duplications and 99% for three exons’ deletions and duplications. We have validated the assays for different starting materials including EDTA-blood, isolated DNA (no FFPE) and saliva that all provide high-quality results. The diagnostic yield varies substantially depending on the used assay, referring healthcare professional, hospital and country. Blueprint Genetics’ Plus Analysis (Seq+Del/Dup) maximizes the chance to find molecular genetic diagnosis for your patient although Sequence Analysis or Del/Dup Analysis may be cost-effective first line test if your patient’s phenotype is suggestive for a specific mutation profile.

Performance of Blueprint Genetics high-quality, clinical grade NGS sequencing assay for panels.

Sensitivity % (TP/(TP+FN) Specificity %
Single nucleotide variants 99.89% (99,153/99,266) >99.9999
Insertions, deletions and indels by sequence analysis
1-10 bps 96.9% (7,563/7,806) >99.9999
11-50 bps 99.13% (2,524/2,546) >99.9999
Copy number variants (exon level dels/dups)
1 exon level deletion (heterozygous) 100% (20/20) NA
1 exon level deletion (homozygous) 100% (5/5) NA
1 exon level deletion (het or homo) 100% (25/25) NA
2-7 exon level deletion (het or homo) 100% (44/44) NA
1-9 exon level duplication (het or homo) 75% (6/8) NA
Simulated CNV detection
5 exons level deletion/duplication 98.7% 100.00%
Microdeletion/-duplication sdrs (large CNVs, n=37))
Size range (0.1-47 Mb) 100% (37/37)
     
The performance presented above reached by WES with the following coverage metrics
     
Mean sequencing depth at exome level 143X
Nucleotides with >20x sequencing coverage (%) 99.86%

Bioinformatics

The target region for each gene includes coding exons and ±20 base pairs from the exon-intron boundary. In addition, the panel includes non-coding and regulatory variants if listed above (Non-coding variants covered by the panel). Some regions of the gene(s) may be removed from the panel if specifically mentioned in the ‘Test limitations” section above. The sequencing data generated in our laboratory is analyzed with our proprietary data analysis and annotation pipeline, integrating state-of-the art algorithms and industry-standard software solutions. Incorporation of rigorous quality control steps throughout the workflow of the pipeline ensures the consistency, validity and accuracy of results. Our pipeline is streamlined to maximize sensitivity without sacrificing specificity. We have incorporated a number of reference population databases and mutation databases including, but not limited, to 1000 Genomes Project, gnomAD, ClinVar and HGMD into our clinical interpretation software to make the process effective and efficient. For missense variants, in silico variant prediction tools such as  SIFT, PolyPhen, MutationTaster are used to assist with variant classification. Through our online ordering and statement reporting system, Nucleus, ordering providers have access to the details of the analysis, including patient specific sequencing metrics, a gene level coverage plot and a list of regions with <20X sequencing depth if applicable. This reflects our mission to build fully transparent diagnostics where ordering providers can easily visualize the crucial details of the analysis process.

Clinical interpretation

We provide customers with the most comprehensive clinical report available on the market. Clinical interpretation requires a fundamental understanding of clinical genetics and genetic principles. At Blueprint Genetics, our PhD molecular geneticists, medical geneticists and clinical consultants prepare the clinical statement together by evaluating the identified variants in the context of the phenotypic information provided in the requisition form. Our goal is to provide clinically meaningful statements that are understandable for all medical professionals regardless of whether they have formal training in genetics.

Variant classification is the corner stone of clinical interpretation and resulting patient management decisions. Our classifications follow the Blueprint Genetics Variant Classification Schemes based on the ACMG guideline 2015. Minor modifications were made to increase reproducibility of the variant classification and improve the clinical validity of the report. Our experience with tens of thousands of clinical cases analyzed at our laboratory allowed us to further develop the industry standard.

The final step in the analysis is orthogonal confirmation. Sequence variants classified as pathogenic, likely pathogenic and variants of uncertain significance (VUS) are confirmed using bi-directional Sanger sequencing when they do not meet our stringent NGS quality metrics for a true positive call.
Reported heterozygous and homo/hemizygous copy number variations with a size <10 and <3 target exons are confirmed by orthogonal methods such as qPCR if the specific CNV has been seen and confirmed less than three times at Blueprint Genetics.

Our clinical statement includes tables for sequencing and copy number variants that include basic variant information (genomic coordinates, HGVS nomenclature, zygosity, allele frequencies, in silico predictions, OMIM phenotypes and classification of the variant). In addition, the statement includes detailed descriptions of the variant, gene and phenotype(s) including the role of the specific gene in human disease, the mutation profile, information about the gene’s variation in population cohorts and detailed information about related phenotypes. We also provide links to the references, abstracts and variant databases used to help ordering providers further evaluate the reported findings if desired. The conclusion summarizes all of the existing information and provides our rationale for the classification of the variant.

Identification of pathogenic or likely pathogenic variants in dominant disorders or their combinations in different alleles in recessive disorders are considered molecular confirmation of the clinical diagnosis. In these cases, family member testing can be used for risk stratification. We do not recommend using variants of uncertain significance (VUS) for family member risk stratification or patient management. Genetic counseling is recommended.

Our interpretation team analyzes millions of variants from thousands of individuals with rare diseases. Our internal database and our understanding of variants and related phenotypes increases with every case analyzed. Our laboratory is therefore well-positioned to re-classify previously reported variants as new information becomes available. If a variant previously reported by Blueprint Genetics is re-classified, our laboratory will issue a follow-up statement to the original ordering health care provider at no additional cost.

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