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Hereditary Breast and Ovarian Cancer BRCA1/2 Sequencing Panel with CNV Detection

  • Summary and Pricing
  • Clinical Features and Genetics
  • Citations
  • Methods
  • Ordering/Specimens
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TEST METHODS

Sequencing and CNV

Test Code Test Copy GenesPriceCPT Code Copy CPT Codes
5451 BRCA1 and BRCA2$540 81162 Add to Order

New York State Approved Test

Pricing Comments

This test is also offered via our exome backbone with CNV detection (click here). The exome-based test may be higher priced, but permits reflex to the entire exome or to any other set of clinically relevant genes.

Targeted Testing

For ordering sequencing of targeted known variants, please proceed to our Targeted Variants landing page.

Turnaround Time

The great majority of tests are completed within 20 days.

Clinical Sensitivity

The overall prevalence of germline BRCA1 or BRCA2 pathogenic variants in the general population is 1:400 to 1:800, with higher rates depending on the specific ethnicity, such as 1:40 in the Ashkenazi Jewish population. Nucleotide substitutions and small insertions/deletions are found in about 90% of individuals with an identifiable pathogenic variant. For individuals with pathogenic variants in these genes, BRCA1 variants were observed in 63% and BRCA2 variants in 37% (Petrucelli et al. 2016. PubMed ID: 20301425).

Copy Number Variants (CNVs) are found in approximately 10% of individuals with an identifiable germline pathogenic variant, with 90% of these in BRCA1 and 10% in BRCA2 (Petrucelli et al. 2013. PubMed ID: 20301425). High-risk patients, defined as individuals with early onset (<50 years of age) invasive or in situ breast cancer or ovarian cancer or male breast cancer, and with 2 or more relatives with HBOC have higher rates of CNVs. Specific ethnic populations (Latin American/Caribbean) have also been shown to have higher rates of CNVs (Judkins et al. 2012. PubMed ID: 22544547).

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Clinical Features

Hereditary breast and ovarian cancer (HBOC) syndrome is an inherited disorder that is highly associated with tumors of the breasts and ovaries. Other malignancies in HBOC families can occur, including melanoma, pancreatic and prostate cancer. In comparison to sporadic breast and ovarian cancers, HBOC syndrome tumors tend to occur at an earlier age (< 50 years), to occur bilaterally, to occur in multiple family members, to include males with breast cancer, and to occur with a higher predisposition in specific ethnicities, such as the Ashkenazi Jewish population (Petrucelli et al. 2016. PubMed ID: 20301425; Pruthi et al. 2010. PubMed ID: 21123638). Identifying individuals with a high-risk for developing HBOC may allow for early detection of tumor growth and allow for prophylactic mastectomy and/or oophorectomy or other treatments (Smith 2012. PubMed ID: 23050669). The majority of breast and ovarian cancers occur sporadically. However, approximately 5-10% of breast, and 10-15% of ovarian cancer cases are due to pathogenic variants in specific genes, particularly BRCA1 and BRCA2, that significantly increase an individual's risk of developing these cancers (Marchina et al. 2010. PubMed ID: 21042765). In addition, HBOC syndrome may also be the result of lower penetrance variants in other genes, which confer a moderate risk (Berliner et al. 2013. PubMed ID: 23188549).

Genetics

Hereditary breast and ovarian cancer is inherited in an autosomal dominant manner and presents with high, although incomplete, penetrance. Pathogenic variants in a number of genes have been reported to significantly increase an individual’s likelihood for developing breast cancer (Tan et al. 2008. PubMed ID: 18682420). Among those, germline pathogenic variants in the most common highly penetrant mutated breast cancer genes, BRCA1 and BRCA2 (Miki et al. 1994. PubMed ID: 7545954; Wooster et al. 1995. PubMed ID: 8524414), appear to provide the highest relative risk with lifetime prevalence up to 40%-85% for breast cancer (Rebbeck et al. 2009. PubMed ID: 19141781; Pruthi et al. 2010. PubMed ID: 21123638), and 10-46% for ovarian cancer depending on the gene mutated (Smith 2012. PubMed ID: 23050669; Pruthi et al. 2010. PubMed ID: 21123638). Hereditary BRCA1 and BRCA2 variants account for approximately 25-60% of inherited breast cancer (Pruthi et al. 2010. PubMed ID: 21123638; Meindl et al. 2011. PubMed ID: 21637635) and 11-39% for ovarian cancer (Berliner et al. 2013. PubMed ID: 23188549).

BRCA1 mutation carriers tend to have breast tumors that are estrogen receptor (ER) negative, progesterone receptor (PR) negative and basal type tumors, whereas BRCA2 mutation carriers have breast tumors that are ER positive, PR positive, and have a luminal phenotype (Pruthi et al. 2010. PubMed ID: 21123638). Individuals with HBOC with a more severe personal or family history tend to have pathogenic variants in BRCA1 vs. BRCA2 due to higher penetrance of pathogenic variants in the BRCA1 gene (Antoniou et al. 2000. PubMed ID: 10642429).

BRCA1 is a tumor suppressor gene that is involved in cellular processes including DNA damage repair, cell cycle progression, gene transcription and ubiquitination. BRCA2 is a tumor suppressor gene that along with RAD51 has a large role in DNA repair processes and genome stability. Most pathogenic variants of the BRCA1 and BRCA2 genes are private variants which are observed in a single family or in a small number of families. Three pathogenic variants in the BRCA genes are commonly found in Ashkenazi Jewish individuals: c.68_69delAG (BRCA1), c.5266dupC (BRCA1), and c.5946delT (BRCA2), and the coexistence of more than one founder mutation has been reported in some Ashkenazi Jewish families. Most BRCA pathogenic variants are inherited from a parent who may or may not have been affected with HBOC due to incomplete penetrance of the mutation, gender, and other factors (Petrucelli et al. 2016. PubMed ID: 20301425). Most pathogenic variants in these genes are chain termination (nonsense, frameshift or splicing).

Large rearrangements (deletions, duplications, triplications), including the five most commonly reported BRCA1 CNVs (Hendrickson et al. 2005. PubMed ID: 15846789), can be detected using this test.

Testing Strategy

For this Next Generation Sequencing (NGS) test, sequencing is accomplished by capturing specific regions with an optimized solution-based hybridization kit, followed by massively parallel sequencing of the captured DNA fragments. Additional Sanger sequencing is performed for regions not captured or with insufficient number of sequence reads.

For Sanger sequencing, polymerase chain reaction (PCR) is used to amplify targeted regions. After purification of the PCR products, cycle sequencing is carried out using the ABI Big Dye Terminator v.3.0 kit. PCR products are resolved by electrophoresis on an ABI 3730xl capillary sequencer. In nearly all cases, cycle sequencing is performed separately in both the forward and reverse directions.

Copy number variants (CNVs) are also detected from NGS data. We utilize a CNV calling algorithm that compares mean read depth and distribution for each target in the test sample against multiple matched controls. Neighboring target read depth and distribution and zygosity of any variants within each target region are used to reinforce CNV calls. All CNVs are confirmed using another technology such as aCGH, MLPA, or PCR before they are reported.

This panel provides 100% coverage of all coding exons of the genes listed, plus ~10 bases of flanking noncoding DNA. We define coverage as ≥20X NGS reads or Sanger sequencing.

Indications for Test

Individuals with a clinical presentation of Hereditary Breast and Ovarian Cancer are candidates. Clinical presentation includes family history, early-onset of breast cancer (< 50 years), bilateral breast tumors, multiple affected family members, including males with breast cancer, and a member of a high-risk ethnicity, such as the Ashkenazi Jewish population (Petrucelli et al. 2016. PubMed ID: 20301425; Pruthi et al. 2010. PubMed ID: 21123638). This is a predictive test which only provides information regarding the likelihood of breast and/or ovarian cancer. A positive test does not mean that a person will develop breast and/or ovarian cancer, and a negative test does not mean that a person will not. This test is specifically designed for heritable germline mutations and is not appropriate for the detection of somatic mutations in tumor tissue.

Genes

Official Gene Symbol OMIM ID
BRCA1 113705
BRCA2 600185
Inheritance Abbreviation
Autosomal Dominant AD
Autosomal Recessive AR
X-Linked XL
Mitochondrial MT

Related Test

Name
PGxome®

CONTACTS

Genetic Counselors
Geneticist
Citations
  • Antoniou et al. 2000. PubMed ID: 10642429
  • Berliner et al. 2013. PubMed ID: 23188549
  • Hendrickson et al. 2005. PubMed ID: 15846789
  • Judkins et al. 2012. PubMed ID: 22544547
  • Marchina et al. 2010. PubMed ID: 21042765
  • Meindl et al. 2011. PubMed ID: 21637635
  • Miki et al. 1994. PubMed ID: 7545954
  • Petrucelli et al. 2016. PubMed ID: 20301425
  • Pruthi et al. 2010. PubMed ID: 21123638
  • Rebbeck et al. 2009. PubMed ID: 19141781
  • Smith. 2012. PubMed ID: 23050669
  • Tan et al. 2008. PubMed ID: 18682420
  • Wooster et al. 1995. PubMed ID: 8524414
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TEST METHODS

Sequencing and CNV Detection via NextGen Sequencing using PG-Select Capture Probes

Test Procedure

NextGen Sequencing

We use a combination of Next Generation Sequencing (NGS) and Sanger sequencing technologies to cover the full coding regions of the listed genes plus ~10 bases of non-coding DNA flanking each exon.  As required, genomic DNA is extracted from the patient specimen.  For NGS, patient DNA corresponding to these regions is captured using an optimized set of DNA hybridization probes.  Captured DNA is sequenced using Illumina’s Reversible Dye Terminator (RDT) platform (Illumina, San Diego, CA, USA).  Regions with insufficient coverage by NGS are covered by Sanger sequencing.

For Sanger sequencing, Polymerase Chain Reaction (PCR) is used to amplify targeted regions.  After purification of the PCR products, cycle sequencing is carried out using the ABI Big Dye Terminator v.3.0 kit.  PCR products are resolved by electrophoresis on an ABI 3730xl capillary sequencer.  In nearly all cases, cycle sequencing is performed separately in both the forward and reverse directions.

Patient DNA sequence is aligned to the genomic reference sequence for the indicated gene region(s). All differences from the reference sequences (sequence variants) are assigned to one of five interpretation categories, listed below, per ACMG Guidelines (Richards et al. 2015).

(1) Pathogenic Variants
(2) Likely Pathogenic Variants
(3) Variants of Uncertain Significance
(4) Likely Benign Variants
(5) Benign Variants

Human Genome Variation Society (HGVS) recommendations are used to describe sequence variants (http://www.hgvs.org).  Rare variants and undocumented variants are nearly always classified as likely benign if there is no indication that they alter protein sequence or disrupt splicing.

Deletion and Duplication Testing via NGS

Copy number variants (CNVs) such as deletions and duplications are detected from next generation sequencing data. We utilize a CNV calling algorithm that compares mean read depth and distribution for each target in the test sample against multiple matched controls. Neighboring target read depth and distribution, and zygosity of any variants within each target region are used to reinforce CNV calls. All CNVs are confirmed using another technology such as PCR, aCGH or MLPA before they are reported.
Analytical Validity

NextGen Sequencing

As of March 2016, 6.36 Mb of sequence (83 genes, 1557 exons) generated in our lab was compared between Sanger and NextGen methodologies. We detected no differences between the two methods. The comparison involved 6400 total sequence variants (differences from the reference sequences). Of these, 6144 were nucleotide substitutions and 256 were insertions or deletions. About 65% of the variants were heterozygous and 35% homozygous. The insertions and deletions ranged in length from 1 to over 100 nucleotides.

In silico validation of insertions and deletions in 20 replicates of 5 genes was also performed. The validation included insertions and deletions of lengths between 1 and 100 nucleotides. Insertions tested in silico: 2200 between 1 and 5 nucleotides, 625 between 6 and 10 nucleotides, 29 between 11 and 20 nucleotides, 25 between 21 and 49 nucleotides, and 23 at or greater than 50 nucleotides, with the largest at 98 nucleotides. All insertions were detected. Deletions tested in silico: 1813 between 1 and 5 nucleotides, 97 between 6 and 10 nucleotides, 32 between 11 and 20 nucleotides, 20 between 21 and 49 nucleotides, and 39 at or greater than 50 nucleotides, with the largest at 96 nucleotides. All deletions less than 50 nucleotides in length were detected, 13 greater than 50 nucleotides in length were missed. Our standard NextGen sequence variant calling algorithms are generally not capable of detecting insertions (duplications) or heterozygous deletions greater than 100 nucleotides. Large homozygous deletions appear to be detectable.

Deletion and Duplication Testing via NGS
 
In general, sensitivity for single, double, or triple exon CNVs is ~80% and for CNVs of four exon size or larger is close to 100%, but may vary from gene-to-gene based on exon size, depth of coverage, and characteristics of the region.
Analytical Limitations

NextGen Sequencing

Interpretation of the test results is limited by the information that is currently available.  Better interpretation should be possible in the future as more data and knowledge about human genetics and this specific disorder are accumulated.

When Sanger sequencing does not reveal any difference from the reference sequence, or when a sequence variant is homozygous, we cannot be certain that we were able to detect both patient alleles.  Occasionally, a patient may carry an allele which does not amplify, due to a large deletion or insertion.   In these cases, the report will contain no information about the second allele.  Our Sanger and NGS Sequencing tests are generally not capable of detecting Copy Number Variants (CNVs).

We sequence all coding exons for each given transcript, plus ~10 bp of flanking non-coding DNA for each exon.  Test reports contain no information about other portions of the gene, such as regulatory domains, deep intronic regions or any currently uncharacterized alternative exons.

In most cases, we are unable to determine the phase of sequence variants.  In particular, when we find two likely causative mutations for recessive disorders, we cannot be certain that the mutations are on different alleles.

Our ability to detect minor sequence variants due to somatic mosaicism is limited.  Sequence variants that are present in less than 50% of the patient’s nucleated cells may not be detected.

Runs of mononucleotide repeats (eg (A)n or (T)n) with n >8 in the reference sequence are generally not analyzed because of strand slippage during PCR.

Unless otherwise indicated, DNA sequence data is obtained from a specific cell-type (usually leukocytes from whole blood).   Test reports contain no information about the DNA sequence in other cell-types.

We cannot be certain that the reference sequences are correct.

Rare, low probability interpretations of sequencing results, such as for example the occurrence of de novo mutations in recessive disorders, are generally not included in the reports.

We have confidence in our ability to track a specimen once it has been received by PreventionGenetics.  However, we take no responsibility for any specimen labeling errors that occur before the sample arrives at PreventionGenetics.

Deletion and Duplication Testing via NGS
 
This CNV calling algorithm used in this test detects most deletions and duplications; however aberrations in a small percentage of regions may not be accurately detected due to sequence paralogy (e.g. pseudogenes, segmental duplications), sequence properties, deletion/duplication size (e.g. single vs. two or more exons), and inadequate coverage. 
 
Balanced translocations or inversions within a targeted gene, or large unbalanced translocations or inversions that extend beyond the genomic location of a targeted gene are not detected.
 
In nearly all cases, our ability to determine the exact copy number change within a targeted gene is limited. In particular, when we find copy excess within a targeted gene, we cannot be certain that the region is duplicated, triplicated etc. In many duplication cases, we are unable to determine the genomic location or the orientation of the duplicated segment with respect to the gene. In particular, we often cannot determine if the duplicated segment is inserted in tandem within the gene or inserted elsewhere in the genome. Similarly, we may not be able to determine the orientation of the duplicated segment (direct or inverted), and whether it will disrupt the open reading frame of the given gene.
 
Our ability to detect CNVs due to somatic mosaicism is limited.
Order Kits

Ordering Options


myPrevent - Online Ordering
  • The test can be added to your online orders in the Summary and Pricing section.
  • Once the test has been added log in to myPrevent to fill out an online requisition form.
REQUISITION FORM
  • A completed requisition form must accompany all specimens.
  • Billing information along with specimen and shipping instructions are within the requisition form.
  • All testing must be ordered by a qualified healthcare provider.

SPECIMEN TYPES
WHOLE BLOOD

(Delivery accepted Monday - Saturday)

  • Collect 3 ml -5 ml (5 ml preferred) of whole blood in EDTA (purple top tube) or ACD (yellow top tube). For Test #500-DNA Banking only, collect 10 ml -20 ml of whole blood.
  • For small babies, we require a minimum of 1 ml of blood.
  • Only one blood tube is required for multiple tests.
  • Ship blood tubes at room temperature in an insulated container. Do not freeze blood.
  • During hot weather, include a frozen ice pack in the shipping container. Place a paper towel or other thin material between the ice pack and the blood tube.
  • In cold weather, include an unfrozen ice pack in the shipping container as insulation.
  • At room temperature, blood specimen is stable for up to 48 hours.
  • If refrigerated, blood specimen is stable for up to one week.
  • Label the tube with the patient name, date of birth and/or ID number.

DNA

(Delivery accepted Monday - Saturday)

  • Send in screw cap tube at least 5 µg -10 µg of purified DNA at a concentration of at least 20 µg/ml for NGS and Sanger tests and at least 5 µg of purified DNA at a concentration of at least 100 µg/ml for gene-centric aCGH, MLPA, and CMA tests, minimum 2 µg for limited specimens.
  • For requests requiring more than one test, send an additional 5 µg DNA per test ordered when possible.
  • DNA may be shipped at room temperature.
  • Label the tube with the composition of the solute, DNA concentration as well as the patient’s name, date of birth, and/or ID number.
  • We only accept genomic DNA for testing. We do NOT accept products of whole genome amplification reactions or other amplification reactions.

CELL CULTURE

(Delivery preferred Monday - Thursday)

  • PreventionGenetics should be notified in advance of arrival of a cell culture.
  • Culture and send at least two T25 flasks of confluent cells.
  • Some panels may require additional flasks (dependent on size of genes, amount of Sanger sequencing required, etc.). Multiple test requests may also require additional flasks. Please contact us for details.
  • Send specimens in insulated, shatterproof container overnight.
  • Cell cultures may be shipped at room temperature or refrigerated.
  • Label the flasks with the patient name, date of birth, and/or ID number.
  • We strongly recommend maintaining a local back-up culture. We do not culture cells.
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