Hereditary Diffuse Gastric Cancer via CDH1 Gene Sequencing with CNV Detection
- Summary and Pricing
- Clinical Features and Genetics
Sequencing and CNV
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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.
The clinical sensitivity of CDH1 germline mutations is 30% for HDGC families (Carneiro et al. J Clin Pathol 61:25–30, 2008). The majority of mutations reported to date are single nucleotide substitutions, and small insertions and deletions. Gross deletions and duplications which can be found in up to 4% of HDGC cases (Kaurah and Huntsman. GeneReviews. 2011).
Hereditary diffuse gastric cancer (HDGC) is a highly penetrant diffuse-type of gastric cancer. Patients with HDGC typically present at 40 years of age and have a cumulative cancer risk of 67% by 80 years of age for men and 83% for women (Pharoah et al. Gastroenterology 121:1348–53, 2001). HDGC is caused by CDH1 mutations, which are found in all ethnic groups, but are rare in countries with high rates of sporadic gastric cancer, including Japan and Korea (Guilford et al. Gastric Cancer 13: 1–10, 2010). Approximately 10% of gastric cancers show familial clustering and about 1–3% of cases are known to be hereditary (e.g. CDH1 mutations). Mutations in CDH1 can also cause lobular breast cancer in women (Carneiro et al. J Clin Pathol 61:25–30, 2008).
Hereditary diffuse gastric cancer is an autosomal dominant disease that is caused by mutations in the CDH1 gene. CHD1 encodes epithelial cadherin (e-cadherin), which is a transmembrane membrane protein that is responsible for cell-to-cell adhesion and cellular invasion suppression. It also plays important roles in signal transduction, differentiation, gene expression, cell motility, and inflammation (Kaurah and Huntsman. GeneReviews. 2011). The activity of E-cadherin in coordination with the actin cytoskeleton through catenins (e.g. α-, β-, and γ-) is responsible for cellular adhesion. Many human cancers show low levels of e-cadherin compared to normal tissue, which causes defects in cellular adhesion and ultimately leads to metastasis. HDGC is inherited from an affected family member, and de novo mutations haven't been reported. The majority of germline mutations are truncating mutations in e-cadherin, the second hit of the normal allele in normal gastric tissue is usually due to CDH1 promoter hypermethylation and secondly due to CDH1 deletions (Schrader and Huntsman. Cancer Treatment and Research 155: 33-62, 2010).
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 CDH1 gene, plus ~10 bases of flanking noncoding DNA. We define full coverage as >20X NGS reads or Sanger sequencing.
Indications for Test
The International Gastric Cancer Linkage Consortium (IGCLC) recently made recommendations for testing for HDGC (Fitzgerald, et al. J Med Genet 47(7): 436–444, 2010).
Two gastric cancer cases in family, one individual under age 50 years with confirmed diffuse gastric cancer (DGC) Three confirmed DGC cases in first- or second-degree relatives independent of age Simplex case (i.e., a single occurrence in a family) of DGC occurring before age 40 years Personal or family history of DGC and lobular breast cancer, one diagnosed before age 50 years.
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|
|Breast Cancer - High / Moderate Risk Sequencing Panel with CNV Detection|
|Breast Cancer - High Risk Sequencing Panel with CNV Detection|
|Hereditary Breast and Ovarian Cancer - High Risk and Lynch Syndrome Sequencing Panel with CNV Detection|
- Genetic Counselor Team - email@example.com
- Jerry Machado, PhD, DABMG, FCCMG - firstname.lastname@example.org
- Carneiro F, Oliveira C, Suriano G, Seruca R. 2007. Molecular pathology of familial gastric cancer, with an emphasis on hereditary diffuse gastric cancer. Journal of Clinical Pathology 61: 25–30. PubMed ID: 17513507
- Fitzgerald, et al. (2010). PubMed ID: 20591882
- Guilford P, Humar B, Blair V. 2010. Hereditary diffuse gastric cancer: translation of CDH1 germline mutations into clinical practice. Gastric Cancer 13: 1–10. PubMed ID: 20373070
- Kaurah and Huntsman. GeneReviews. 2011
- Kaurah P, Huntsman DG. 2011. Hereditary Diffuse Gastric Cancer. In: Pagon RA, Adam MP, Bird TD, Dolan CR, Fong C-T, and Stephens K, editors. GeneReviews™, Seattle (WA): University of Washington, Seattle. PubMed ID: 20301318
- Pharoah PDP, Guilford P, Caldas C. 2001. Incidence of gastric cancer and breast cancer in CDH1 (E-cadherin) mutation carriers from hereditary diffuse gastric cancer families. Gastroenterology 121: 1348–1353. PubMed ID: 11729114
- Schrader K, Huntsman D. 2010. Hereditary Diffuse Gastric Cancer. In: Pasche B, editors. Cancer Genetics, Boston, MA: Springer US, p 33–63. PubMed ID: 20517687
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.