Hereditary Breast and Ovarian Cancer - High Risk and Lynch Syndrome Sequencing Panel with CNV Detection
- Summary and Pricing
- Clinical Features and Genetics
Sequencing and Del/Dup via NGS
|Test Code||Test Copy Genes||CPT Code Copy CPT Codes|
|BRCA1 and BRCA2||81162|
|Full Panel Price*||$540.00|
|Test Code||Test Copy Genes||Total Price||CPT Codes Copy CPT Codes|
|5453||Genes x (17)||$540.00||81162, 81292, 81294, 81295, 81297, 81298, 81300, 81317, 81319, 81321, 81323, 81403, 81404, 81405(x2), 81406(x2), 81408, 81479(x13)||Add|
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The great majority of tests are completed within 28 days.
Current estimates are that less than 1% of the general population have a pathogenic variant in the BRCA1 or BRCA2 genes, and about 10-15% of women diagnosed with breast cancer have a pathogenic variant in one of these genes (Tan et al. 2008. PubMed ID: 18682420). In the German population, CHEK2 is mutated in around 4% of all cases of hereditary breast cancer. The prevalence of PALB2 pathogenic variants in the populations of both Germany and England is approximately 1% (Meindl et al. 2011. PubMed ID: 21637635). All genes tested in this panel have been implicated in hereditary breast and ovarian cancer, and although individually these genes may be involved in a minority of inherited breast cancer cases, the combination of these high-risk genes may be responsible for a significant portion of these hereditary cancers (Turnbull et al. 2008. PubMed ID: 18544032). Approximately 6% of patients with hereditary ovarian cancers who do not have pathogenic variants in BRCA1 or BRCA2 have causative variants in genes such BRIP1, CHEK2, NBN, PALB2, RAD51C, RAD51D and TP53 (Walsh et al. 2011. PubMed ID: 17292821; Loveday et al. 2011. PubMed ID: 21822267). Highly penetrant pathogenic variants in other genes such as STK11, CDH1 and PTEN account for approximately 1% of all breast cancer cases that aggregate in families. Another 5% of familial breast cancer cases might be explained by causative variants in susceptibility genes of moderate effect such as ATM, CHEK2, NBN, RAD50, RAD51B and RAD51D (Klonowska et al. 2015. PubMed ID: 25994375).
Hereditary breast and ovarian cancer (HBOC) is an inherited disorder that is highly associated with tumors of the breasts and ovaries. HBOC cases tend to arise prior to age 50, tumors often occur bilaterally, consist of multiple affected family members (including males with breast cancer), and occur with a high predisposition in specific ethnic groups. Individuals with Ashkenazi Jewish ancestry have an increased risk for HBOC. There are three specific founder genetic variants that are known to be common in this population: c.68_69delAG (BRCA1), c.5266dupC (BRCA1), and c.5946delT (BRCA2) (Petrucelli et al. 2013. PubMed ID: 20301425). It is estimated that approximately 1:40 individuals with Ashkenazi Jewish ancestry has at least one of these variants; approximately 1:10 women with breast cancer and 1:3 women with ovarian cancer (cancer.net).
Lynch Syndrome, the most prevalent colorectal cancer syndrome, is caused by variations in mismatch repair (MMR) genes, mainly the MSH2 gene. Mismatch repair proteins are involved in numerous cellular functions including repairing DNA synthesis errors, repairing double stranded breaks, and apoptosis. Lynch syndrome is generally early onset, and is characterized by predominantly right-sided colon cancer (Jang et al. 2010. PubMed ID: 20559516). Lynch syndrome is estimated to account for approximately 3-5% of colorectal cancer (Jang et al. 2010. PubMed ID: 20559516; Bonadona et al. 2015. PubMed ID: 21642682). Based on the Amsterdam criteria, families with Lynch syndrome have at least 3 relatives with colorectal cancer, one being a first degree relative, at least 2 successive generations involved, and at least 1 relative with onset of cancer before age 50 (Kohlman et al. 2012. PubMed ID: 20301390; cancer.org).
Hereditary breast and ovarian cancer (HBOC) syndrome is a disorder that follows an autosomal dominant pattern of inheritance. It is associated with tumors mainly in the breast and ovaries, and is primarily a result of alterations in high-penetrance genes BRCA1, BRCA2, and TP53 (Seal et al. 2006. PubMed ID: 17033622).
The BRCA1 tumor suppressor gene is associated with hereditary breast and ovarian cancer. Its protein product is essential for processes such as DNA repair, cell cycle checkpoint control, and maintenance of genomic stability (Wang et al. 2009. PubMed ID: 19261749). Women carrying a pathogenic variant in BRCA1 have a 46-65% risk of developing breast cancer, and approximately a 39% risk of developing ovarian cancer by age 70 (Berlinear et al. 2013. PubMed ID: 23188549). Men who have pathogenic variants in the BRCA1 gene have a life time risk of 7-8% of developing breast cancer compared to the average man whose risk is approximately 0.1% (Berlinear et al. 2013. PubMed ID: 23188549).
BRCA2 is a tumor suppressor gene, and along with RAD51 has a major role in DNA damage repair and genome stability (Tan et al. 2008. PubMed ID: 18682420). Women with a pathogenic variant in BRCA2 gene are at a 43-45% risk of developing breast cancer by age 70, and 11% risk of developing ovarian cancer (Berlinear et al. 2013. PubMed ID: 23188549).
Hereditary breast and/or ovarian cancers can sometimes be associated with other hereditary cancer syndromes including Li-Fraumeni, Cowden, Peutz-Jeghers, Hereditary Diffuse Gastric Cancer, and Lynch syndrome (Berlinear et al. 2013. PubMed ID: 23188549). Variants in TP53 have been implicated as the cause of Li-Fraumeni syndrome. Breast cancer appears as a feature of this syndrome, and carriers of TP53 pathogenic variants are at high risk of developing early onset breast cancer (Antoniou et al. 2006. PubMed ID: 16998504).
Individuals with Cowden syndrome caused by pathogenic variants in PTEN have a lifetime risk of 50% for breast cancer and 5-10% for endometrial cancer (Hearle et al. 2006. PubMed ID: 16707622).
Peutz-Jeghers syndrome, caused by pathogenic variants in STK11, can reach a breast cancer incidence of 32% by 60 years of age (Lim et al. 2004. PubMed ID: 15188174).
Other genes that are thought to confer low to moderate risk of breast and ovarian cancer have been identified. Pathogenic variants in CDH1 have been shown to be associated with the development of invasive lobular carcinoma. It is also thought to be a gene that causes intermediate risk of hereditary breast cancer (Masciari et al. 2007. PubMed ID: 17660459). CHK2 truncating variants have been shown to confer moderate risk of breast cancer development (Meijers-Heijboer et al. 2002. PubMed ID: 11967536; Tan et al. 2008. PubMed ID: 18682420). Variants in ATM that cause ataxia telangiectasia in biallelic carriers confer a two-fold increased risk of breast cancer in monoallelic carriers (Tan et al. 2008. PubMed ID: 18682420). It was estimated that BRIP1 variants confer a relative risk of breast cancer of 2.0, and that inactivating truncating BRIP1 variants cause Fanconi anemia in biallelic carriers and confers susceptibility to breast cancer in monoallelic carriers (Seal et al. 2006. PubMed ID: 17033622). Ovarian tumors from carriers of BRIP1 pathogenic variants show loss of the wild type allele, suggesting its tumor suppressor capabilities. Frameshift pathogenic variants in BRIP1 also lead to an increased risk of invasive ovarian cancer (Rafnar et al. 2011. PubMed ID: 21964575).
PALB2 is also considered a gene with moderate risk alleles and can cause a 2 and 4 fold increased risk of breast cancer (Caminsky et al. 2016. PubMed ID: 26898890). RAD51C, essential for homologous recombination repair, has been reported to be a hereditary breast and ovarian cancer susceptibility gene in that several pathogenic variants have been identified in BRCA1/2-negative HBOC families (Clague et al. 2011. PubMed ID: 21980511). RAD51C variants are found in 1-5% of individuals with a family history of breast and ovarian cancer (Meindl et al. 2011. PubMed ID: 21637635). The relative risk of ovarian and breast cancer for RAD51D pathogenic variant carriers was estimated to be 6.3 and 1.3, respectively (Loveday et al. 2011. PubMed ID: 21822267).
Lynch syndrome is an autosomal dominant cancer susceptibility disease that is mainly caused by pathogenic variants in MMR genes: MLH1, MSH2, MSH6, and PMS2. Approximately 90% of causative variants are located in MLH1, and MLH2, and approximately 10% in MSH6 and PMS2. Germline deletions in EPCAM, which is not a mismatch repair gene, inactivates MSH2 in about 1% of individuals with Lynch syndrome (Jang et al. 2010. PubMed ID: 20559516; Bonadona et al. 2015. PubMed ID: 21642682; Kohlman et al. 2012. PubMed ID: 20301390). Germline mutations in the Lynch syndrome genes have also been shown to be associated with ovarian cancer (Watson et al. 2008. PubMed ID: 18398828).
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. All reported pathogenic, likely pathogenic, and variants of uncertain significance are confirmed 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.
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.
Deletion and duplication testing for STK11 and PMS2 is performed using NGS, but CNVs detected in these genes are confirmed via Multiplex Ligation-dependent Probe Amplification (MLPA).
Indications for Test
Individuals with a clinical presentation of Hereditary Breast and Ovarian Cancer syndrome. This test is also suitable for individuals with multifocal, recurrent, and early onset (< 50 years) colorectal tumors or a family history of colorectal tumors. This test is specifically designed for heritable germline mutations and is not appropriate for the detection of somatic mutations in tumor tissue. This is a predictive test and it only provides information regarding the likelihood of breast and/or ovarian cancer. A positive test does not mean that a person will develop any of these diseases and a negative test does not mean that a person will not.
|Official Gene Symbol||OMIM ID|
- Genetic Counselor Team - firstname.lastname@example.org
- Elaine Welch, PhD - email@example.com
- Antoniou et al. 2006. PubMed ID: 16998504
- Berlinear et al. 2013. PubMed ID: 23188549
- Bonadona et al. 2015. PubMed ID: 21642682
- Caminsky et al. 2016. PubMed ID: 26898890
- Clague et al. 2011. PubMed ID: 21980511
- Hearle et al. 2006. PubMed ID: 16707622
- Jang et al. 2010. PubMed ID: 20559516
- Klonowska et al. 2015. PubMed ID: 25994375
- Kohlman et al. 2012. PubMed ID: 20301390
- Lim et al. 2004. PubMed ID: 15188174
- Loveday et al. 2011. PubMed ID: 21822267
- Masciari et al. 2007. PubMed ID: 17660459
- Meijers-Heijboer et al. 2002. PubMed ID: 11967536
- Meindl et al. 2011. PubMed ID: 21637635
- Petrucelli et al. 2013. PubMed ID: 20301425
- Rafnar et al. 2011. PubMed ID: 21964575
- Seal et al. 2006. PubMed ID: 17033622
- Tan et al. 2008. PubMed ID: 18682420
- Turnbull et al. 2008. PubMed ID: 18544032
- Walsh et al. 2011. PubMed ID: 17292821
- Wang et al. 2009. PubMed ID: 19261749
- Watson et al. 2008. PubMed ID: 18398828
Sequencing and Deletion/Duplication Testing via NextGen Sequencing using PG-Select Capture Probes
We use a combination of Next Generation Sequencing (NGS) and Sanger sequencing technologies to cover the full coding regions of the listed genes plus ~20 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. All pathogenic, likely pathogenic, or variants of uncertain significance are confirmed 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, Common 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
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.
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 ~20 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.
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.
- 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.
(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.
(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.
(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.