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Methodology

Methodology

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Molecular Genetics
DDeletion/duplication analysis
Multiplex Ligation-dependent Probe Amplification (MLPA)Next-Generation (NGS)/Massively parallel sequencing (MPS)
  • Illumina MiSeq/NextSeq
CSequence analysis of the entire coding region
Next-Generation (NGS)/Massively parallel sequencing (MPS)
  • Illumina MiSeq/NextSeq
TTargeted variant analysis
Bi-directional Sanger Sequence Analysis
  • Applied Biosystems 3730 capillary sequencing instrument

Test comments

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Test development

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Test developed by laboratory (no manufacturer test name)

Test procedure

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Methodology: All coding exons and 20 bp of flanking non-coding sequence are enriched using an LHSC custom targeted hybridization protocol (Roche Nimblegen), followed by high throughput sequencing (Illumina). Sequence variants and copy number changes are assessed and interpreted using clinically validated algorithms and commercial software (SoftGenetics: Nextgene, Geneticist Assistant, Mutation Surveyor; and Alamut Visual). All exons have >300x mean read depth coverage, with a minimum 100x coverage at a single nucleotide resolution. This assay meets the sensitivity and specificity of combined Sanger sequencing and MLPA copy number analysis. Variants interpreted as either ACMG category 1, 2, or 3 (pathogenic, likely pathogenic, VUS; PMID: 25741868), if necessary, are confirmed using Sanger sequencing, MLPA, or other assays. ACMG category 4 and 5 variants (likely benign, benign) are not reported, but are available upon request. Variants detected in the 5’ UTR and 3’ UTR are not reported unless there is evidence suggesting pathogenicity. This assay has been validated at a level of sensitivity equivalent to the Sanger sequencing and standard copy number analysis (>99%; PMID: 27376475,28818680).

Citations
  • Clinical Next-Generation Sequencing Pipeline Outperforms a Combined Approach Using Sanger Sequencing and Multiplex Ligation-Dependent Probe Amplification in Targeted Gene Panel Analysis. - PubMed ID: 27376475

Confirmation of results

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Mutations identified are repeated if necessary in an independent assay using either Sanger sequence, MLPA, Q-PCR or other.

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Suggested reading

  • USPSTF, 2019
    Risk Assessment, Genetic Counseling, and Genetic Testing for BRCA1/2-Related Cancer in Women: A Systematic Review for the U.S. Preventive Services Task Force
  • USPSTF, 2013
    Risk Assessment, Genetic Counseling, and Genetic Testing for BRCA-Related Cancer: Systematic Review to Update the U.S. Preventive Services Task Force Recommendation (See 2019 Update)
  • Phillips et al., 2013
    Tamoxifen and risk of contralateral breast cancer for BRCA1 and BRCA2 mutation carriers.
  • Domchek et al., 2010
    Association of Risk-Reducing Surgery in BRCA1 or BRCA2 Mutation Carriers with Cancer Risk and Mortality
  • NCI PDQ, Cancer Genetics Counseling
    Cancer Genetics Risk Assessment and Counseling (PDQ®): Health Professional Version

Practice guidelines

  • NICE, 2023
    UK NICE Clinical Guideline CG164, Familial breast cancer: classification, care and managing breast cancer and related risks in people with a family history of breast cancer, 2023
  • NICE, 2023
    UK NICE Clinical guideline (CG122), Ovarian cancer: recognition and initial management, 2023
  • NCCN, 2022
    NCCN Clinical Practice Guidelines in Oncology, Genetic/Familial High-Risk Assessment: Breast, Ovarian, and Pancreatic version 2.2022
  • ACMG ACT, 2019
    American College of Medical Genetics and Genomics, Genomic Testing (Secondary Findings) ACT Sheet, BRCA1 and BRCA2 Pathogenic Variants (Hereditary Breast and Ovarian Cancer), 2019

IMPORTANT NOTE: NIH does not independently verify information submitted to the GTR; it relies on submitters to provide information that is accurate and not misleading. NIH makes no endorsements of tests or laboratories listed in the GTR. GTR is not a substitute for medical advice. Patients and consumers with specific questions about a genetic test should contact a health care provider or a genetics professional.