Hereditary Ovarian Cancer Sequencing Panel with CNV Detection
- Summary and Pricing
- Clinical Features and Genetics
Sequencing and CNV
|Test Code||Test Copy Genes||CPT Code Copy CPT Codes|
|BRCA1 and BRCA2||81162|
|Full Panel Price*||$540|
|Test Code||Test Copy Genes||Total Price||CPT Codes Copy CPT Codes|
|5469||Genes x (25)||$540||81162, 81292, 81294, 81295, 81297, 81298, 81300, 81317, 81319, 81321, 81323, 81403, 81404, 81405(x2), 81406(x3), 81408(x2), 81479(x27)||Add|
CPT codes 81432 and 81433 can be used in place of the individual gene codes.
We are happy to accommodate requests for single genes or a subset of these genes. The price will remain the list price. If desired, free reflex testing to remaining genes on panel is available.
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.
For ordering sequencing of targeted known variants, please proceed to our Targeted Variants landing page.
The great majority of tests are completed within 20 days.
Current estimates are that less than 1% of the general population has a pathogenic variant in the BRCA1 or BRCA2 genes, and 10-15% of women diagnosed with breast cancer have a pathogenic variant in one of these genes with an increased incidence of monoallelic carriers having ovarian cancer (Turnbull et al. 2008. PubMed ID: 18544032; Tan et al. 2008. PubMed ID: 18682420). In the German population, CHEK2 pathogenic variants are found 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). Approximately 6% of patients with hereditary ovarian cancers who do not have pathogenic variants in BRCA1 or BRCA2 have pathogenic variants in BARD1, BRIP1, CHEK2, MRE11A, NBN, PALB2, RAD50, RAD51C, RAD51D, and TP53 (Walsh et al. 2011. PubMed ID: 22006311; Loveday et al. 2011. PubMed ID: 21822267). Highly penetrant variants in other genes such as STK11, CDH1, and PTEN account for less than 1% of ovarian cancer cases (Minion et al. 2015. PubMed ID: 25622547). Pathogenic sequence variants in the MLH1, MSH2, MSH6, and PMS2 genes account for approximately 50%, 40%, 7-10%, and <5% of Lynch syndrome cases, respectively (Kohlmann and Gruber. 2018. PubMed ID: 20301390).
Ovarian cancer accounts for approximately 3.7% of all cancer in women and accounts for 4.2% of all deaths in women annually (Toss et al. 2015. PubMed ID: 26075229). Twenty-three percent of cases have a hereditary component, and 65%-85% of these cases are attributed to variants in BRCA1 and BRCA2 (Toss et al. 2015. PubMed ID: 26075229; Nakonechny and Gilks. 2016. PubMed ID: 27241103). The remaining hereditary cases are attributed to variants within genes that encode additional components of the mismatch repair machinery, double-strand break repair, and tumor suppressor genes important for regulation and activation of cell proliferation, apoptosis, and genomic stability (Toss et al. 2015. PubMed ID: 26075229; Nakonechny and Gilks. 2016. PubMed ID: 27241103).
More than 90% of ovarian cancers are derived from epithelial cells, while 10% develop from germ cells or granulosa-theca cells (Toss et al. 2015. PubMed ID: 26075229). Ovarian cancers can be classified into 5 histotypes: high-grade serous carcinoma (HGSC), low-grade serous carcinoma (LGSC), clear cell carcinoma (CCC), endometrioid carcinoma (EC), and mucinous carcinoma (MC) (Nakonechny and Gilks. 2016. PubMed ID: 27241103). HGSC is the most common ovarian cancer histotype (68% of cases) and is predominantly derived from cells of the ovarian epithelium or fallopian tubes, specifically the fimbriated or distal portion (Singh et al. 2015. PubMed ID: 26126051). Clear cell carcinoma (CCC) is the second most common histotype of ovarian carcinoma in western populations (~12%) (Köbel et al. 2010. PubMed ID: 20407318) with higher incidence in individuals of Japanese descent (Nakonechny and Gilks. 2016. PubMed ID: 27241103). Endometrioid carcinoma (EC) is the third most common type, and approximately 20% of individuals present with endometriosis in either the ovary or elsewhere in the pelvic region. Low-grade serous carcinoma (LGSC) and mucinous carcinoma (MC) collectively account for 6-7% of ovarian carcinomas, with the mean age of MC presentation around 45 years (Nakonechny and Gilks. 2016. PubMed ID: 27241103).
Most cases of hereditary ovarian cancer are associated with a concurrent diagnosis of hereditary breast and ovarian cancer (known as HBOC) or Lynch syndrome (LS). 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, with tumors often occurring bilaterally. Multiple family members are often affected (including males with breast cancer). Patients with HBOC have a 45%-50% increased lifetime risk of developing the HGSC ovarian carcinoma histotype, while individuals with Lynch syndrome have an ~10% increased lifetime risk of developing EC or CCC ovarian carcinoma histotypes (Risch et al. 2001. PubMed ID: 11179017; McAlpine et al. 2012. PubMed ID: 22282309). Conversely, 15%-25% of women with a diagnosis of HGSC will also have HBOC; therefore, HBOC testing is strongly suggested for these individuals (Nakonechny and Gilks. 2016. PubMed ID: 27241103).
Hereditary breast and ovarian cancer (HBOC) and Lynch syndrome (LS) are the two most common autosomal dominant cancer susceptibility syndromes that include individuals who present with ovarian cancers. Double-strand break repair via homologous recombination (HR) or non-homologous end joining (NHEJ) are associated with HBOC. HR components are encoded by numerous genes, including BRCA1, BRCA2, ATM, CHEK2, RAD51, BRIP1, and PALB2. Collectively, the products of these genes respond to double-strand breaks through kinase activity, which leads to cell cycle arrest in either G1-S, S, or G2-M phase via BRCA1-dependent mechanisms. Double-strand DNA breaks are then repaired through a mechanism that utilizes the sister chromatid as a template (Seal et al. 2006. PubMed ID: 17033622; Rahman et al. 2007. PubMed ID: 17200668; Toss et al. 2015. PubMed ID: 26075229). In the absence of a functioning HR pathway, double-strand breaks must be repaired via non-homologous end joining (NHEJ), which does not utilize a sister chromatid as a template and is error prone, subsequently increasing the risk of novel defects and cancer (Lieber et al. 2010. PubMed ID: 20012587). Variants in BRCA1/BRCA2 account for 65%-85% of hereditary ovarian cancers, with the majority of variants being missense, nonsense, or small frameshift deletions (Human Gene Mutation Database; Toss et al. 2015. PubMed ID: 26075229; Nakonechny and Gilks. 2016. PubMed ID: 27241103).
Lynch syndrome (LS) is an autosomal dominant cancer susceptibility disease that results from variants within genes that are associated with the mismatch repair (MMR) system. Single-strand breaks are repaired via base excision repair or nucleotide excision repair systems which include components encoded by MLH1, MSH2, MSH6, and PMS2. Approximately 90% of germline 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 diagnosed with Lynch syndrome (Jang et al. 2010. PubMed ID: 20559516; Bonadona et al. 2015. PubMed ID: 21642682; Kohlman and Gruber. 2018. PubMed ID: 20301390). LS-associated ovarian cancers tend to occur at an early age, are low-grade, and are usually detected early in tumor progression (Niskakoski et al. 2013. PubMed ID: 23716351).
Hereditary breast and/or ovarian cancers can sometimes be associated with other hereditary cancer syndromes including Cowden, Li-Fraumeni, 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 variants are at high risk of developing early onset breast and ovarian cancer (Antoniou et al. 2006. PubMed ID: 16998504; Kraus et al. 2017. PubMed ID: 27616075). Individuals with Cowden syndrome caused by mutations in PTEN have a lifetime risk of 50% for breast cancer and 5-10% for endometrial cancer (Apostolou et al. 2013. PubMed ID: 23586058). 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; Hearle et al. 2006. PubMed ID: 16707622).
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 and ovarian cancer (Masciari et al. 2007. PubMed ID: 17660459; Kraus et al. 2017. PubMed ID: 27616075).
CHEK2 truncating variants have been shown to confer moderate risk of breast and ovarian cancer development (Meijers-Heijboer et al. 2002. PubMed ID: 11967536; Tan et al. 2008. PubMed ID: 18682420; Walsh et al. 2011. PubMed ID: 22006311).
ATM is a known ovarian cancer predisposition gene. Additionally, variants in ATM that cause ataxia telangiectasia in biallelic carriers confer a twofold increased risk of breast cancer development in monoallelic carriers (Tan et al. 2008. PubMed ID: 18682420).
Pathogenic variants in BRIP1 result in ~0.4% increased risk of ovarian cancer in Icelandic populations (Ramus et al. 2015. PubMed ID: 26315354). Ovarian tumors from carriers of BRIP1 variants show loss of the wild type allele, suggesting its tumor suppressor capabilities. Frameshift variants in BRIP1 also show 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 causes a two- to fourfold increased risk of breast cancer (Caminsky et al. 2016. PubMed ID: 26898890). It is considered an ovarian cancer susceptibility gene (Ramus et al. 2015. PubMed ID: 26315354).
RAD51C, essential for homologous recombination repair, has been reported to be a hereditary breast and ovarian cancer susceptibility gene; several pathogenic variants have been identified in BRCA1/2-negative HBOC families (Clague et al. 2011. PubMed ID: 21980511). RAD51C is mutated in approximately 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 variant carriers was estimated to be 6.3 and 1.3, respectively (Loveday et al. 2011. PubMed ID: 21822267).
It has been suggested that large multi-exon deletions and insertions in BARD1 may substantially contribute to familial breast and ovarian cancer risk (Klonowska et al. 2015. PubMed ID: 25994375).
Pathogenic NBN variants have been identified in individuals with ovarian cancer and are associated with a twofold increased risk of breast cancer (Walsh et al. 2011. PubMed ID: 22006311).
Pathogenic variants in the MUTYH gene have been associated with hereditary breast and ovarian cancer (Maxwell et al. 2014. PubMed ID: 25503501; Schrader et al. 2016. PubMed ID: 26556299).
Other genes that are thought to confer an increased risk of HBOC and Lynch syndrome include DICER1 (Jalkh et al. 2017. PubMed ID: 28202063), MRE11, RAD50 (van der Merwe et al. 2017. PubMed ID: 28241424; Sung et al. 2017. PubMed ID: 28961279) and SMARCA4 (Witkowski et al. 2014. PubMed ID: 24658002; Hayano et al. 2016. PubMed ID: 27701467).
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.
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 typically provides ≥98% 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.
Deletion and duplication testing for STK11, NF1, and PMS2 is performed using NGS, but CNVs detected in these genes are usually confirmed via multiplex ligation-dependent probe amplification (MLPA).
Indications for Test
Individuals with a clinical presentation of ovarian cancer, hereditary breast and ovarian cancer, Lynch syndrome, or a family history which includes ovarian cancer are candidates for this test. A positive test does not mean that a currently unaffected individual will develop ovarian cancer, and a negative test does not mean that an individual will not develop ovarian cancer. Furthermore, this test is specifically designed for heritable germline variants and is not appropriate for the detection of somatic variants in tumor tissue.
|Official Gene Symbol||OMIM ID|
- Genetic Counselor Team - email@example.com
- Elaine Welch, PhD - firstname.lastname@example.org
- Antoniou et al. 2006. PubMed ID: 16998504
- Apostolou et al. 2013. PubMed ID: 23586058
- 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
- Hayano et al. 2016. PubMed ID: 27701467
- Hearle et al. 2006. PubMed ID: 16707622
- Human Gene Mutation Database (Bio-base).
- Jalkh et al. 2017. PubMed ID: 28202063
- Jang et al. 2010. PubMed ID: 20559516
- Klonowska et al. 2015. PubMed ID: 25994375
- Köbel et al. 2010. PubMed ID: 20407318
- Kohlmann and Gruber. 2018. PubMed ID: 20301390
- Kraus et al. 2017. PubMed ID: 27616075
- Lieber et al. 2010. PubMed ID: 20012587
- Lim et al. 2004. PubMed ID: 15188174
- Loveday et al. 2011. PubMed ID: 21822267
- Masciari et al. 2007. PubMed ID: 17660459
- Maxwell et al. 2014. PubMed ID: 25503501
- McAlpine et al. 2012. PubMed ID: 22282309
- Meijers-Heijboer et al. 2002. PubMed ID: 11967536
- Meindl et al. 2011. PubMed ID: 21637635
- Minion et al. 2015. PubMed ID: 25622547
- Nakonechny and Gilks. 2016. PubMed ID: 27241103
- Niskakoski et al. 2013. PubMed ID: 23716351
- Rafnar et al. 2011. PubMed ID: 21964575
- Rahman et al. 2007. PubMed ID: 17200668
- Ramus et al. 2015. PubMed ID: 26315354
- Risch et al. 2001. PubMed ID: 11179017
- Schrader et al. 2016. PubMed ID: 26556299
- Seal et al. 2006. PubMed ID: 17033622
- Singh et al. 2015. PubMed ID: 26126051
- Sung et al. 2017. PubMed ID: 28961279
- Tan et al. 2008. PubMed ID: 18682420
- Toss et al. 2015. PubMed ID: 26075229
- Turnbull et al. 2008. PubMed ID: 18544032
- van der Merwe et al. 2017. PubMed ID: 28241424
- Walsh et al. 2011. PubMed ID: 22006311
- Witkowski et al. 2014. PubMed ID: 24658002
Sequencing and CNV Detection 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 ~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
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 ~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.
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.