Hereditary Breast Cancer via CHEK2 Gene Sequencing with CNV Detection
- Summary and Pricing
- Clinical Features and Genetics
Sequencing and CNV
|Test Code||Test Copy Genes||Price||CPT Code Copy CPT Codes|
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.
This test is predicted to detect pathogenic mutations in ~6% of women with non-BRCA1/2 Hereditary Breast Cancer (Nevanlinna & Bartek Oncogene 25:5912-5919, 2006).
More than 1 million new cases of breast cancer occur each year worldwide, making it the most common malignancy among women. It is estimated that ~10% of these cases have a strong hereditary component. Hereditary Breast Cancer (HBC; OMIM 114480) refers to the familial occurrence of early-onset (prior to the age of 40), bilateral mammary carcinomas. Importantly, tumors from individuals with HBC tend to be of a much higher histological grade, when first detected, than tumors from sporadic age-matched breast cancer controls (Honrado et al. Modern Pathology 18:1305-1320, 2005). As a result, survival rate after treatment is two-fold lower for patients with HBC, compared to those with sporadic breast cancer (Lonning et al. Ann Oncol 18:1293-1306, 2007). Thus, identifying individuals with a high-risk for developing HBC allows for early detection of tumor formation in these individuals, and is predicted to increase the rate of patient survival.
Mutations in a number of genes have been reported to significantly increase an individual’s likelihood for developing breast cancer (reviewed by Tan et al. J Clin Pathol 61:1073-1082, 2008). Among those, germline mutations in the Breast Cancer genes, BRCA1 and BRCA2, appear to provide the highest relative risk, ~10- to 20-fold. Early-onset breast cancer is also a major component of the Li-Fraumeni Syndrome (LFS; OMIM 151623), and mutations in the LFS-associated gene TP53 also provide a 10- to 20-fold increased risk for developing bilateral mammary carcinomas, in addition to other cancers. Mutations in the CHEK2 gene (OMIM 604373) were also reported to cause a Li-Fraumeni-like syndrome (Bell et al. Science 286:2528-2531, 1999), although subsequent studies have indicated that CHEK2 mutations are only very rarely found in patients with classic symptoms of LFS (Lee et al. Cancer Res 61:8062-8067, 2001). However, mutations in CHEK2 have been frequently found in patients who have hereditary breast cancer (HBC) but do not have detectable BRCA1 or BRACA2 mutations (Vahteristo et al. Am J Hum Genet 71:432-438, 2002; Meijers-Heijboer et al. Am J Hum Genet 72:1308-1314, 2003), indicating CHEK2 mutations likely contribute to a significant fraction of non-BRCA1/2 hereditary breast carcinomas. CHEK2 encodes a protein kinase that protects the genome from ionizing radiation and genotoxic insults. To date, approximately 40 mutations have been reported throughout the CHEK2 gene, and >95% are detectable by this DNA sequencing test (Human Gene Mutation Database, www.hgmd.cf.ac.uk).
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 test provides full coverage of all coding exons of the CHEK2 gene, plus ~10 bases of flanking noncoding DNA. We define full coverage as >20X NGS reads or Sanger sequencing.
Indications for Test
This test is recommended for individuals diagnosed with early-onset bilateral mammary carcinomas and a family history of breast cancer and/or sarcomas, particularly those who do not have a detectable mutation in BRCA1, BRCA2 or TP53 genes. This test is specifically designed for heritable germline mutations and is not appropriate for the detection of somatic mutations in tumor tissue.
|Official Gene Symbol||OMIM ID|
- Genetic Counselor Team - firstname.lastname@example.org
- Jerry Machado, PhD, DABMG, FCCMG - email@example.com
- Bell, D. W., et.al. (1999). "Heterozygous germ line hCHK2 mutations in Li-Fraumeni syndrome." Science 286(5449): 2528-31. PubMed ID: 10617473
- Honrado, E., et.al. (2005). "The molecular pathology of hereditary breast cancer: genetic testing and therapeutic implications." Mod Pathol 18(10): 1305-20. PubMed ID: 15933754
- Human Gene Mutation Database.
- Lee SB, Kim SH, Bell DW, Wahrer DC, Schiripo TA, Jorczak MM, Sgroi DC, Garber JE, Li FP, Nichols KE. 2001. Destabilization of CHK2 by a missense mutation associated with Li-Fraumeni Syndrome. Cancer research 61: 8062–8067. PubMed ID: 11719428
- Lonning, P. E., et.al. (2007). "Breast cancer prognostication and prediction in the postgenomic era." Ann Oncol 18(8): 1293-306. PubMed ID: 17317675
- Meijers-Heijboer H, Wijnen J, Vasen H, Wasielewski M, Wagner A, Hollestelle A, Elstrodt F, Bos R van den, Snoo A de, Fat GTA, Brekelmans C, Jagmohan S, et al. 2003. The CHEK2 1100delC mutation identifies families with a hereditary breast and colorectal cancer phenotype. Am. J. Hum. Genet. 72: 1308–1314. PubMed ID: 12690581
- Nevanlinna, H., Bartek, J. (2006). "The CHEK2 gene and inherited breast cancer susceptibility." Oncogene 25(43): 5912-9. PubMed ID: 16998506
- Tan DSP, Marchio C, Reis-Filho JS. 2008. Hereditary breast cancer: from molecular pathology to tailored therapies. Journal of Clinical Pathology 61: 1073–1082. PubMed ID: 18682420
- Vahteristo P, Bartkova J, Eerola H, Syrjäkoski K, Ojala S, Kilpivaara O, Tamminen A, Kononen J, Aittomäki K, Heikkilä P, Holli K, Blomqvist C, et al. 2002. A CHEK2 Genetic Variant Contributing to a Substantial Fraction of Familial Breast Cancer. American Journal of Human Genetics 71: 432. PubMed ID: 12094328
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.