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Colorectal Cancer Predisposition via POLE Gene Sequencing with CNV Detection

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

Sequencing and Del/Dup via NGS

Test Code Test Copy GenesIndividual Gene PriceCPT Code Copy CPT Codes
7511 POLE$540.00 81479,81479 Add to Order

New York State Approved Test

Pricing Comment

Our most cost-effective testing approach is NextGen sequencing with Sanger sequencing supplemented as needed to ensure sufficient coverage and to confirm NextGen calls that are pathogenic, likely pathogenic or of uncertain significance. If, however, full gene Sanger sequencing only is desired (for purposes of insurance billing or STAT turnaround time for example), please see link below for Test Code, pricing, and turnaround time information.

For Sanger Sequencing click here.
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 20 days.

Clinical Sensitivity

Pathogenic variants in the POLE gene have been observed in 0.3-0.6% of individuals with colorectal cancer (Chubb et al. 2015).

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Clinical Features

Colorectal cancer (CRC) is the development of tumors in the colon and rectum that occur in approximately 8% of individuals with cancer (Siegel et al. 2015). Similar to other cancers, CRC is caused by genetic and environmental factors (e.g. smoking, diet). Approximately 30% of CRCs are considered familial and 5% of CRCs are caused by a Mendelian disorder (Esteban-Jurado 2014). Colorectal cancer is generally broken down into the presence or absence of polyposis (numerous internal polyps). CRC inherited diseases include Lynch syndrome, Familial Adenomatous polyposis, Peutz-Jeghers, Juvenile Polyposis and Cowden syndrome. A recent syndrome has been coined CRC-polymerase proofreading-associated polyposis (PPAP). It is caused by pathogenic variants in proofreading DNA repair genes (Palles et al. 2012). Mutations in these genes predispose individuals to adenomatous polyposis or early-onset cancer (Spier et al. 2015). Identification of pathogenic variants in the germline of CRC patients is important for cancer surveillance (i.e. colonoscopy) for the affected individual and family members, since early surveillance and treatment has been shown to decrease morbidity and mortality (Kohlmann and Gruber 2014).

Genetics

Colorectal predisposition syndrome is inherited in an autosomal dominant manner. Polymerase proof-reading-associated polyposis (PPAP) is caused by pathogenic variants in two proofreading DNA repair genes POLD1 and POLE (Chubb et al. 2015). The protein products of these genes are involved in the replication of DNA and are also involved in DNA repair. POLE catalyzes the synthesis of the leading strand, while POLD catalyzes synthesis of Okazaki fragments of the lagging strand. (Church et al. 2013). Mutations in the proof-reading exonuclease domains of these proteins result in the inability to correct mismatched bases during DNA replication. Pathogenic variants in the POLE gene result in predisposition to colorectal cancer (Church et al. 2013). Tumors from individuals with these pathogenic variants are microsatellite stable, but acquire base substitution mutations (Palles et al. 2012). Reported pathogenic variants in POLE include missense variants and small deletions (Human Gene Mutation Database).

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. Additional Sanger sequencing is performed for regions not captured or with insufficient number of sequence reads. All reported pathogenic, likely pathogenic, and 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.

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 POLE 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 suitable for individuals with multifocal, recurrent, and early onset (e.g. < 50 years) colorectal and endometrial tumors or a family history of these tumors. This test is specifically designed for heritable germline mutations and is not appropriate for the detection of somatic mutations in tumor tissue.

Gene

Official Gene Symbol OMIM ID
POLE 174762
Inheritance Abbreviation
Autosomal Dominant AD
Autosomal Recessive AR
X-Linked XL
Mitochondrial MT

Disease

Name Inheritance OMIM ID
Colorectal cancer, susceptibility to, 12 615083

Related Test

Name
Hereditary Polyposis Sequencing Panel with CNV Detection

CONTACTS

Genetic Counselors
Geneticist
Citations
  • Chubb D, Broderick P, Frampton M, Kinnersley B, Sherborne A, Penegar S, Lloyd A, Ma YP, Dobbins SE, Houlston RS. 2015. Genetic Diagnosis of High-Penetrance Susceptibility for Colorectal Cancer (CRC) Is Achievable for a High Proportion of Familial CRC by Exome Sequencing. Journal of Clinical Oncology 33: 426–432. PubMed ID: 25559809
  • Church DN, Briggs SEW, Palles C, Domingo E, Kearsey SJ, Grimes JM, Gorman M, Martin L, Howarth KM, Hodgson SV, The NSECG Collaborators, Kaur K, Taylor J, Tomlinson IP. 2013. DNA polymerase and exonuclease domain mutations in endometrial cancer. Human Molecular Genetics 22: 2820–2828. PubMed ID: 23528559
  • Esteban-Jurado C. 2014. New genes emerging for colorectal cancer predisposition. World Journal of Gastroenterology 20: 1961. PubMed ID: 24587672
  • Human Gene Mutation Database (Bio-base).
  • Kohlmann W, Gruber SB. 2014. 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
  • Palles C, Cazier J-B, Howarth KM, Domingo E, Jones AM, Broderick P, Kemp Z, Spain SL, Almeida EG, Salguero I, Sherborne A, Chubb D, Carvajal-Carmona LG, Ma Y, Kaur K, Dobbins S, Barclay E, Gorman M, Martin L, Kovac MB, Humphray S; CORGI Consortium; WGS500 Consortium, Lucassen A, Holmes CC, Bentley D, Donnelly P, Taylor J, Petridis C, Roylance R, Sawyer EJ, Kerr DJ, Clark S, Grimes J, Kearsey SE, Thomas HJ, McVean G, Houlston RS, Tomlinson I. 2012. Germline mutations affecting the proofreading domains of POLE and POLD1 predispose to colorectal adenomas and carcinomas. Nature Genetics 45: 136–144. PubMed ID: 23263490
  • Siegel RL, Miller KD, Jemal A. 2015. Cancer statistics, 2015: Cancer Statistics, 2015. CA: A Cancer Journal for Clinicians 65: 5–29. PubMed ID: 25559415
  • Spier I, Holzapfel S, Altmüller J, Zhao B, Horpaopan S, Vogt S, Chen S, Morak M, Raeder S, Kayser K, Stienen D, Adam R, Nürnberg P, Plotz G, Holinski-Feder E, Lifton RP, Thiele H, Hoffmann P, Steinke V, Aretz S. 2015. Frequency and phenotypic spectrum of germline mutations in POLE and seven other polymerase genes in 266 patients with colorectal adenomas and carcinomas: POLE Mutations in Polyposis. International Journal of Cancer n/a–n/a. PubMed ID: 25529843
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TEST METHODS

Sequencing and Deletion/Duplication Testing 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 ~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 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 aCGH, MLPA, PCR or qPCR 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 ~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 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 minor CNVs, due for example to somatic mosaicism is limited.
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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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