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Lynch Syndrome Sequencing and Deletion/Duplication Panel

  • Summary and Pricing
  • Clinical Features and Genetics
  • Citations
  • Methods
  • Ordering/Specimens
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TEST METHODS

Sequencing and Deletion/Duplication Testing via aCGH

Test Code TestCPT Code Copy CPT Codes
1325 EPCAM 81479, 81403 Add to Order
MLH1 81292, 81294
MSH2 81295, 81297
MSH6 81298, 81300
PMS2 81317, 81319
Full Panel Price* $990.00
Test Code Test Total Price CPT Codes Copy CPT Codes
1325 Genes x (5) $990.00 81292, 81294, 81295, 81297, 81298, 81300, 81317, 81319, 81403, 81479 Add to Order

New York State Approved Test (Sequencing Only)

Pricing Comment

Targeted testing for gross deletion/duplications discovered by aCGH will be available for family members of the proband only if we are able to test the del/dup by PCR or qPCR. If you would like to order a subset of these genes contact us to discuss pricing.

Targeted Testing

For ordering targeted known variants, please proceed to our Targeted Variants landing page.

Turnaround Time

The great majority of tests are completed within 28 days.

Clinical Sensitivity

Lynch syndrome is attributed to pathogenic variants in the MLH1, MSH2, MSH6, and PMS2 genes in approximately 50%, 40%, 7-10% and < 5% of cases, respectively (Kohlmann and Gruber. 2012). The majority of these variants are single nucleotide substitutions or small insertions and deletions. Missense, nonsense and splicing EPCAM mutations are involved in congenital tufting enteropathy (Human Gene Mutation Database), whereas EPCAM deletions account for 1-3% of Lynch syndrome cases (Kohlmann and Gruber. 2012). 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. 2012).

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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 mutations in DNA mismatch repair (MMR) genes. MMR genes are responsible for repairing small sequence errors, or mismatches, during DNA replication. Mutations 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). This phenomenon of microsatellite instability (MSI) leads to somatic mutations in oncogenes and/or tumor suppressor genes, including TGFBR2NF1 and others (Wang et al. 2003). 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). 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).

Genetics

Lynch syndrome is an autosomal dominant disease mainly caused by germline mutations in one of four described MMR genes: MLH1, MSH2, MSH6, and PMS2 (Peltomäki and Vasen 2004; Kohlmann and Gruber. 2012). Mutations 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 I criteria. Mutations 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 MSI. Mutations 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 mutations in the EPCAM gene cause inactivation of the nearby MSH2 gene via hypermethylation in approximately 1-3% of individuals with Lynch syndrome (Kohlmann and Gruber. 2012). The only reported mutations in the EPCAM gene that are causative for Lynch Syndrome include 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). See individual gene test descriptions for information on molecular biology of gene products.

Testing Strategy

The Lynch Syndrome NextGen Sequencing Panel analyzes 5 genes that have been associated with Lynch syndrome. For this NGS panel, the full coding regions, plus ~20bp of non-coding DNA flanking each exon, are sequenced for each of the genes listed below. Sequencing is accomplished by capturing specific regions with an optimized solution-based hybridization method, followed by massively parallel sequencing of the captured DNA fragments. Additional Sanger sequencing is performed for any regions not captured or with insufficient number of sequence reads. All pathogenic, undocumented and questionable variant calls are confirmed by Sanger sequencing.

Due to known PMS2 pseudogenes, the PMS2 gene is also analyzed via Sanger sequencing using a long-range PCR strategy.

Each gene can also be tested individually using our Sanger sequencing assays. Please see our test menu.

Deletion and duplication testing is analyzed using array comparative genomic hybridization.

***If deletion and duplication testing is not required for any of the genes please note on test requisition.***

Indications for Test

This test is suitable for individuals with multifocal, recurrent, and early onset (e.g. < 50 years) colorectal tumors or a family history of colorectal tumors. Germline mutations in the Lynch syndrome genes have also been shown to be associated with ovarian cancer (Watson et al. 2008). 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 and Deletion/Duplication Panel
Colorectal Cancer Sequencing And Deletion/Duplication Panel
Hereditary Breast and Ovarian Cancer Syndrome - HBOC EXPANDED Sequencing and Deletion/Duplication Panel
Lynch Syndrome via the EPCAM Gene
Lynch Syndrome via the MLH1 Gene
Lynch Syndrome via the MSH2 Gene
Lynch Syndrome via the PMS2 Gene
Lynch Syndrome via the MSH6 Gene
Pancreatic Cancer Sequencing Panel
Renal Cancer Sequencing Panel

CONTACTS

Genetic Counselors
Geneticist
Citations
  • Grady WM, Carethers JM. 2008. Genomic and Epigenetic Instability in Colorectal Cancer Pathogenesis. Gastroenterology 135: 1079–1099. PubMed ID: 18773902
  • Human Gene Mutation Database (Bio-base).
  • Jang E, Chung DC. 2010. Hereditary Colon Cancer: Lynch Syndrome. Gut and Liver 4: 151. PubMed ID: 20559516
  • Kempers MJ, Kuiper RP, Ockeloen CW, Chappuis PO, Hutter P, Rahner N, Schackert HK, Steinke V, Holinski-Feder E, Morak M, Kloor M, Büttner R, et al. 2011. Risk of colorectal and endometrial cancers in EPCAM deletion-positive Lynch syndrome: a cohort study. The Lancet Oncology 12: 49–55. PubMed ID: 21145788
  • Kohlmann W, Gruber SB. 2012. Lynch Syndrome. 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: 20301390
  • Peltomäki P, Vasen H. 2004. Mutations associated with HNPCC predisposition–Update of ICG-HNPCC/INSiGHT mutation database. Disease markers 20: 269–276. PubMed ID: 15528792
  • Vasen HF, Watson P, Mecklin J-P, Lynch HT. 1999. New clinical criteria for hereditary nonpolyposis colorectal cancer (HNPCC, Lynch syndrome) proposed by the International Collaborative group on HNPCC. Gastroenterology 116: 1453–1456. PubMed ID: 10348829
  • Wang Q, Montmain G, Ruano E, Upadhyaya M, Dudley S, Liskay MR, Thibodeau SN, Puisieux A. 2003. Neurofibromatosis type 1 gene as a mutational target in a mismatch repair-deficient cell type. Human genetics 112: 117–123. PubMed ID: 12522551
  • Watson P, Vasen HFA, Mecklin J-P, Bernstein I, Aarnio M, Järvinen HJ, Myrhøj T, Sunde L, Wijnen JT, Lynch HT. 2008. The risk of extra-colonic, extra-endometrial cancer in the Lynch syndrome. International Journal of Cancer 123: 444–449. PubMed ID: 18398828
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TEST METHODS

NextGen Sequencing and Deletion/Duplication Testing Via Array Comparative Genomic Hybridization

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 ~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/Duplication Testing via aCGH

As required, DNA is extracted from the patient specimen. Equal amounts of genomic DNA from the patient and a gender matched reference sample are amplified and labeled with Cy3 and Cy5 dyes, respectively. To prevent any sample cross contamination, a unique sample tracking control is added into each patient sample. Each labeled patient product is then purified, quantified, and combined with the same amount of reference product.  The combined sample is loaded onto the designed array and hybridized for at least 22 hours at 65ºC.  Arrays are then washed and scanned immediately with 2.5 µM resolution.  Only data for the gene(s) of interest for each patient are extracted and analyzed.

PreventionGenetics’ high density gene-centric (HDGC) aCGH is designed to have comprehensive coverage for both coding and non-coding regions for each targeted gene with very high density probe coverage.  The average probe spacing within each exon is 47 bp or a minimum of three probes per exon covering all targeted exons and UTRs.  The average probe spacing is 289 bp covering all intronic, 2kb upstream and downstream regions of each targeted gene.  In addition, the flanking 300-bp intronic sequence on either side of targeted exons has enriched probe coverage.  Therefore, PreventionGenetics’ aCGH enables the detection of relatively small deletion and amplification mutations within a single exon of a given gene or deletion and amplification mutations encompassing the entire gene.

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/Duplication Testing via aCGH

PreventionGenetics’ high density gene-centric custom designed aCGH enables the detection of relatively small deletion and amplification mutations (down to ~300 bp) within a single exon of a given gene or deletion and amplification mutations encompassing the entire gene. PreventionGenetics has established and verified this test’s accuracy and precision.

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 ~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.

Deletion/Duplication Testing via aCGH

Any copy number changes smaller than 300bps (within the targeted region) may not be detected by our array.

This array may not detect deletion and amplification mutations present at low levels of mosaicism or those present in genes that have pseudogene copies or repeats elsewhere in the genome.

aCGH will not detect balanced translocations, inversions, or point mutations that may be responsible for the clinical phenotype

Breakpoints, if happened outside the targeted gene, may be hard to define.

The sensitivity of this assay may be reduced when DNA is extracted by an outside laboratory

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
  • The first four pages of the requisition form must accompany all specimens.
  • Billing information is on the third and fourth pages.
  • Specimen and shipping instructions are listed on the fifth and sixth pages.
  • All testing must be ordered by a qualified healthcare provider.

SPECIMEN TYPES
WHOLE BLOOD

(Delivery accepted Monday - Saturday)

  • Collect 3-5 ml (5 ml preferred) of whole blood in EDTA (purple top tube) or ACD (yellow top tube). For Test #500-DNA Banking only, collect 10-20 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 good for up to 48 hours.
  • If refrigerated, blood specimen is good for up to one week.
  • Label the tube with the patient name, date of birth and/or ID number.

DNA

(Delivery accepted Monday - Saturday)

  • NextGen Sequencing Tests: Send in screw cap tube at least 10 µg of purified DNA at a concentration of at least 50 µg/ml
  • Sanger Sequencing Tests: Send in a screw cap tube at least 15 µg of purified DNA at a concentration of at least 20 µg/ml. For tests involving the sequencing of more than three genes, send an additional 5 µg DNA per gene. DNA may be shipped at room temperature.
  • Deletion/Duplication via aCGH: Send in screw cap tube at least 1 µg of purified DNA at a concentration of at least 100 µg/ml.
  • Whole-Genome Chromosomal Microarray: Collect at least 5 µg of DNA in TE (10 mM Tris-cl pH 8.0, 1mM EDTA), dissolved in 200 µl at a concentration of at least 100 ng/ul (indicate concentration on tube label). DNA extracted using a column-based method (Qiagen) or bead-based technology is preferred.

CELL CULTURE

(Delivery accepted Monday - Thursday)

  • PreventionGenetics should be notified in advance of arrival of a cell culture.
  • Ship at least two T25 flasks of confluent cells.
  • Label the flasks with the patient name, date of birth, and/or ID number.
  • We do not culture cells.