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Lynch Syndrome 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 GenesCPT Code Copy CPT Codes
5463 EPCAM 81479,81403 Add to Order
MLH1 81292,81294
MSH2 81295,81297
MSH6 81298,81300
PMS2 81317,81319
Full Panel Price* $540
Test Code Test Copy Genes Total Price CPT Codes Copy CPT Codes
5463 Genes x (5) $540 81292, 81294, 81295, 81297, 81298, 81300, 81317, 81319, 81403, 81479 Add to Order

New York State Approved Test

Pricing Comments

We are happy to accommodate requests for testing single genes in this panel 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.

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

Lynch syndrome is attributed to pathogenic sequence variants in the MLH1, MSH2, MSH6, and PMS2 genes in approximately 50%, 40%, 7-10% and <5% of cases, respectively (Kohlmann and Gruber. 2014. PubMed ID: 20301390). The majority of these variants are single nucleotide substitutions or small insertions and deletions. Missense, nonsense and splicing EPCAM pathogenic variants are involved in congenital tufting enteropathy (Human Gene Mutation Database), while EPCAM deletions account for 1-3% of Lynch syndrome cases (Kohlmann and Gruber. 2014. PubMed ID: 20301390). Large deletions and genetic rearrangements account for 20%, 5%, 20%, 7%, and 100% of identifiable pathogenic variants in the MSH2, MLH1, PMS2, MSH6, and EPCAM genes (Kohlmann and Gruber. 2014. PubMed ID: 20301390).

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Clinical Features

Lynch syndrome, also known as Hereditary Nonpolyposis Colorectal Cancer (HNPCC), is an inherited cancer syndrome mainly caused by germline pathogenic variants in DNA mismatch repair (MMR) genes. MMR genes encode proteins that repair small sequence errors, or mismatches, during DNA replication. Pathogenic variants in mismatch repair genes can cause widespread genomic instability characterized by the expansion and contraction of short tandem repeat sequences (microsatellites) (Grady and Carethers. 2008. PubMed ID: 18773902). As a result, Lynch syndrome is marked by early onset and a high lifetime risk of cancer, particularly in the right colon but also in the endometrium, ovary, stomach, bile duct, kidney, bladder, ureter, and brain (Jang and Chung. 2010. PubMed ID: 20559516). Clinical hallmarks of Lynch Syndrome, as delineated by the Amsterdam criteria, include heritable colorectal (Type I) or extracolonic (Type II) cancer, present in at least three relatives over at least two consecutive generations, with an onset of cancer before the age of 50 in at least one case, and exclusion of familial adenomatous polyposis (FAP) (Vasen et al. 1999. PubMed ID: 10348829).

Genetics

Lynch syndrome is an autosomal dominant disease mainly caused by germline pathogenic variants in one of four MMR genes: MLH1, MSH2, MSH6, and PMS2 (Peltomäki and Vasen. 2004. PubMed ID: 15528792; Kohlmann and Gruber. 2014. PubMed ID: 20301390). Pathogenic variants in the MLH1 and MSH2 genes account for approximately 80-90% of all Lynch syndrome patients, and most frequently occur in families meeting the stringent Amsterdam criteria. Pathogenic variants in the MSH6 and PMS2 genes account for most of the remaining Lynch patients, and are often found in families with atypical HNPCC symptoms, such extracolonic carcinomas; and have also been found to have a low rate of microsatellite instability.

Pathogenic variants in another gene, EPCAM, which encodes a calcium-independent cell adhesion molecule and not a mismatch repair protein, are also involved in Lynch syndrome. Germline pathogenic variants in the EPCAM gene can cause inactivation of the nearby MSH2 gene via hypermethylation in 1-3% of individuals with Lynch syndrome (Kohlmann and Gruber. 2014. PubMed ID: 20301390). The only reported pathogenic variants in the EPCAM gene that are causative for Lynch Syndrome are large deletions (Human Gene Mutation Database; www.insight-group.org). The cumulative incidence of colon cancer risk from EPCAM deletions has been estimated to be 75% by 70 years of age, and for endometrial cancer in women to be 12% (Kempers et al. 2011. PubMed ID: 21145788).

A germline inversion of exons 1-7 in MSH2 has been reported in fourteen individuals from eleven un-related families clinically presenting with Lynch syndrome associated phenotypes including colorectal, endometrial, gastric, and ovarian cancer (Wagner et al. 2002. PubMed ID: 12203789; Rhees et al. 2013. PubMed ID: 24114314; Mork et al. 2016. PubMed ID: 28004223).

See individual gene test descriptions for information on molecular biology of gene products and mutation spectra.

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.

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.

This test also includes analysis of the inversion of exons 1-7 in MSH2.

Indications for Test

This test is suitable for individuals with multifocal, recurrent, and early onset (< 50 years) colorectal tumors or a family history of colorectal tumors. Germline pathogenic variants in the Lynch syndrome genes have also been shown to be associated with ovarian cancer (Watson et al. 2008. PubMed ID: 18398828). 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
EPCAM 185535
MLH1 120436
MSH2 609309
MSH6 600678
PMS2 600259
Inheritance Abbreviation
Autosomal Dominant AD
Autosomal Recessive AR
X-Linked XL
Mitochondrial MT

Related Tests

Name
Cancer Sequencing Panel with CNV Detection
Colorectal Cancer Sequencing Panel with CNV Detection
Hereditary Breast and Ovarian Cancer - Expanded and Lynch Syndrome Sequencing Panel with CNV Detection
Hereditary Breast and Ovarian Cancer - High Risk and Lynch Syndrome Sequencing Panel with CNV Detection
Hereditary Endometrial Cancer Sequencing Panel with CNV Detection
Hereditary Ovarian Cancer Sequencing Panel with CNV Detection
Lynch Syndrome via EPCAM Gene Sequencing with CNV Detection
Lynch Syndrome via MLH1 Gene Sequencing with CNV Detection
Lynch Syndrome via MSH2 Gene Sequencing with CNV Detection
Lynch Syndrome via PMS2 Gene Sequencing with CNV Detection
Lynch Syndrome via the MSH2 Exons 1-7 Inversion
Lynch Syndrome via MSH6 Gene Sequencing with CNV Detection
Pancreatic Cancer Sequencing Panel with CNV Detection
Prostate Cancer Sequencing Panel with CNV Detection
Renal Cancer Sequencing Panel with CNV Detection

CONTACTS

Genetic Counselors
Geneticist
Citations
  • Grady and Carethers. 2008. PubMed ID: 18773902
  • Human Gene Mutation Database (Bio-base).
  • Jang and Chung. 2010. PubMed ID: 20559516
  • Kempers et al. 2011. PubMed ID: 21145788
  • Kohlmann and Gruber. 2014. PubMed ID: 20301390
  • Mork et al. 2016. PubMed ID: 28004223
  • Peltomäki and Vasen. 2004. PubMed ID: 15528792
  • Rhees et al. 2013. PubMed ID: 24114314
  • Vasen et al. 1999. PubMed ID: 10348829
  • Wagner et al. 2002. PubMed ID: 12203789
  • Watson et al. 2008. PubMed ID: 18398828
  • www.insight-group.org
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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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