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Hereditary Polyposis 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
5465 APC 81201,81203 Add to Order
MSH3 81479,81479
MUTYH 81406,81479
NTHL1 81479,81479
POLD1 81479,81479
POLE 81479,81479
Full Panel Price* $540
Test Code Test Copy Genes Total Price CPT Codes Copy CPT Codes
5465 Genes x (6) $540 81201, 81203, 81406, 81479(x9) Add to Order

New York State Approved Test

Pricing Comments

We are happy to accommodate requests for single genes 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

It is estimated that 20-30% of all colorectal cancers (CRCs) are familial. Inherited, highly penetrant single-gene variants may account for up to 5% of all colon cancer cases. Eighty percent of individuals with familial adenomatous polyposis (FAP) carry a pathogenic variant in APC, and sequencing detects 87% to 90% of these variants (Hegde et al. 2014. PubMed ID: 24310308; Laken et al. 1999. PubMed ID: 10051640). MUTYH-associated polyposis accounts for 0.7% of all CRC cases, and 2% of familial or early-onset CRC with a low number of adenomas (<15-20) (Hegde et al. 2014. PubMed ID: 24310308). About 25% of patients initially diagnosed with FAP have biallelic variants in MUTYH (Sampson et al. 2003. PubMed ID: 12853198). Pathogenic variants in POLE and POLD1 have been observed in 0.3%-0.6% and 0.2% of individuals with CRC, respectively (Chubb et al. 2015. PubMed ID: 25559809). The clinical sensitivity of MSH3 and NTHL1 sequence variants is unknown.

Gross deletions/duplications may have been underreported in the past and may occur in up to 12% of patients with pathogenic APC variants (Hegde et al. 2014. PubMed ID: 24310308; Jasperson and Burt 2011). The clinical sensitivity for MSH3, MUTYH, NTHL1, POLE, and POLD1 deletions/duplications is currently unknown.

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Clinical Features

Colorectal cancer (CRC) is defined by development of tumors in the colon and rectum. CRC is generally classified by the presence or absence of polyposis (numerous internal polyps) and has been categorized into specific inherited diseases based on the degree of polyposis and other physiologically-defining features. CRC includes Lynch syndrome (also known as hereditary non-polyposis CRC), which is defined by age of onset of cancer before age 50 and delineated by specific criteria (Amsterdam Criteria) for diagnosis (Vasen et al. 1999. PubMed ID: 10348829). Peutz-Jeghers syndrome, Cowden syndrome, and juvenile polyposis are each associated with increased risk of hereditary cancers (such as CRC) but also present with additional physiological anomalies (Eng et al. 2014. PubMed ID: 20301661; Eng et al. 2001. PubMed ID: 11160785; Abdalla et al. 2003. PubMed ID: 12843319; Hearle et al. 2006. PubMed ID: 16707622).

Familial adenomatous polyposis (FAP) and MUTYH–associated polyposis (MAP) are specifically associated with an abundance of internal polyps that have the potential to progress to CRC. Thirty percent of CRCs are considered familial, while 5% are caused by a Mendelian disorder (Esteban-Jurado 2014. PubMed ID: 24587672). Early surveillance and treatment has been shown to decrease morbidity and mortality, and identification of pathogenic variants is important for determining the degree of cancer surveillance (colonoscopy) necessary for affected individuals and family members (Kohlmann and Gruber. 2018. PubMed ID: 20301390).

Familial adenomatous polyposis (FAP) is characterized by polyposis of the colon (>100 polyps) which begins to form around the age of 16. FAP accounts for 1% of all CRCs (Hegde et al. 2014. PubMed ID: 24310308). If untreated, nearly all FAP patients will develop CRC by age 40 (Fearnhead et al. 2001. PubMed ID: 11257105). Individuals with FAP can also present with desmoid tumors, congenital hypertrophy of retinal pigment epithelium (CHRPE, 60% of individuals), osteomas, supernumerary teeth, odontomas, epidermoid cysts, and small bowel adenomas (Hegde et al. 2014. PubMed ID: 24310308). CHRPE does not affect sight or have malignant potential, but can be detected by ophthalmoscopy and is highly diagnostic of FAP before the appearance of polyps (Díaz-Llopis, Menezo. 1988. PubMed ID: 2830869).

MAP is a specific form of FAP in which individuals present by age 55 with colorectal adenomas (10-1000) (Poulsen and Bisgaard. 2008. PubMed ID: 19506731). Polyp burden in these cases is variable, as biallelic variants in MUTYH have been found in 30% of individuals with 15-100 polyps, and 7% of individuals with >100 polyps. Due to the function of MUTYH in the base excision repair system, colorectal adenomas and carcinomas from these patients include numerous G:C to T:A transversions in various genes such as APC and KRAS (K-ras tumor suppressor) (Lipton et al. 2003. PubMed ID: 14633673; Jones et al. 2004. PubMed ID: 15083190; Hegde et al. 2014. PubMed ID: 24310308).

Genetics

The APC gene is best characterized for its association with autosomal dominant FAP. APC is a tumor suppressor gene responsible for regulating the Wnt pathway. In FAP tumors, both alleles of APC are inactivated (with one inactive allele being inherited and the other occurring somatically; the two hit hypothesis). The APC protein is responsible for regulation of c-myc, cyclin-D, and cell adhesion and microtubule assembly proteins; absence results in aberrant transcription of these targets (Hegde et al. 2014. PubMed ID: 24310308).

More than 1,900 pathogenic variants have been reported in APC (Human Gene Mutation Database) of which ~90% are nonsense, splicing, or frameshift variants with penetrance reported to be 100% (Hegde et al. 2014. PubMed ID: 24310308). While variants have been identified throughout the APC coding region, severe FAP is associated with variants between codons 1250 and 1464. Recurrent variants at codon 1061 and 1309 are estimated to account for 30% of germline APC-specific cases (Hegde et al. 2014. PubMed ID: 24310308). Conversely, attenuated FAP (less than 100 polyps) is associated with variants in APC located at the distal 5’ and 3’ ends of the gene, or in an alternatively spliced region of exon 9 (Young et al. 1998. PubMed ID: 9603437; Soravia et al. 1998. PubMed ID: 9585611). Congenital hypertrophy of retinal pigment epithelium (CHRPE) is limited to patients with variants between codons 457 and 1444 (Caspari et al. 1994. PubMed ID: 7906810). Two missense variants, p.Ile1307Lys and p.Glu1317Lys (commonly found in Ashkenazi Jewish populations), predispose carriers to multiple colorectal adenomas (generally less than 100) and carcinoma, but with low and variable penetrance (Frayling et al. 1998. PubMed ID: 9724771). Pathogenic variants in the exon 1B promoter of APC have also been associated with gastric adenocarcinoma and proximal polyposis of the stomach (Li et al. 2016. PubMed ID: 27087319).

MUTYH-associated polyposis (MAP) is an autosomal recessive form of FAP resulting from biallelic variants in MUTYH, which encodes an adenine-specific DNA glycosylase that is a component of the base excision repair system. MUTYH removes adenine residues mispaired with 8-oxo-dG or guanine (Hegde et al. 2014. PubMed ID: 24310308). As a result, tumors of patients with biallelic variants in MUTYH have an abundance of G>T transversions, particularly in APC (Hegde et al. 2014. PubMed ID: 24310308).

To date, about 160 pathogenic variants have been reported in the MUTYH gene, nearly all of which are single nucleotide variations or small frameshift insertions or deletions (Human Gene Mutation Database). While MUTYH-associated polyposis (MAP) occurs in patients from various ethnic groups, specific MUTYH pathogenic variants are found in different populations. In European and North American individuals with MAP, two missense variants (p.Tyr179Cys and p.Gly396Asp) are the most common, accounting for 70-80% of disrupted alleles in this population (Hegde et al. 2014. PubMed ID: 24310308). Both homozygous and compound heterozygous variants contribute to the disease (Jones et al. 2004. PubMed ID: 15083190). In Asian individuals with MAP, commonly reported variants include p.Arg245Cys, c.934-2A>G (splicing), and p.Glu480*; in these cases only homozygous variants have been reported to contribute to disease (Tao et al. 2004. PubMed ID: 15180946; Miyaki et al. 2005. PubMed ID: 15890374). The penetrance of colorectal cancer (CRC) for biallelic carriers of MUTYH variants is reported to be nearly 100% by the age of 60 (Farrington et al. 2005. PubMed ID: 15931596); however, biallelic pathogenic variants in MUTYH have also been reported in unaffected individuals (Hegde et al. 2014. PubMed ID: 24310308).

POLD1 and POLE encode the DNA polymerases POL-δ and POL-ε, respectively. Variants within the proofreading domain of each are associated with autosomal dominant polyposis and CRC (Bellido et al. 2016. PubMed ID: 26133394). POL-ε catalyzes the synthesis of the leading DNA strand, while POL-δ catalyzes synthesis of Okazaki fragments of the lagging DNA strand. Variants impacting the proofreading domain of these proteins result in the inability to correct mismatched bases during DNA replication and consequently, an accumulation of base substitutions (Palles et al. 2012. PubMed ID: 23263490; Church et al. 2013. PubMed ID: 23528559). Reported pathogenic variants in POLE include missense and small frameshift deletions while POLD1 reported pathogenic variants include mostly missense variants (Human Gene Mutation Database).

MSH3 encodes a mismatch repair protein which forms a heterodimer with MSH2 for DNA repair. Pathogenic variants have been associated with colorectal cancer and polyposis (Duraturo et al. 2011. PubMed ID: 21128252; Adam et al. 2016. PubMed ID: 27476653; Raskin et al. 2017. PubMed ID: 29212164). MSH3 pathogenic variants appear to be inherited in autosomal dominant and recessive manners for colorectal cancer and polyposis, respectively (Adam et al. 2016. PubMed ID: 27476653; DeRycke et al. 2017. PubMed ID: 28944238).

NTHL1 is a base excision repair gene, which has been associated with autosomal recessive adenomatous polyposis (Weren et al. 2015. PubMed ID: 25938944). One individual with polyposis, colorectal cancer, and multiple primary tumors was reported to be compound heterozygous for NTHL1 pathogenic variants (Rivera et al. 2015. PubMed ID: 26559593).

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.

Indications for Test

This test is suitable for individuals with multifocal, recurrent, and early onset (<50 years) colorectal cancer and polyposis or a family history of these lesions. This test is specifically designed for heritable germline variants and is not appropriate for the detection of somatic variants in tumor tissue.

Genes

Official Gene Symbol OMIM ID
APC 611731
MSH3 600887
MUTYH 604933
NTHL1 602656
POLD1 174761
POLE 174762
Inheritance Abbreviation
Autosomal Dominant AD
Autosomal Recessive AR
X-Linked XL
Mitochondrial MT

Related Tests

Name
Breast Cancer - Comprehensive Risk Sequencing Panel with CNV Detection
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Colorectal Cancer Predisposition via POLD1 Gene Sequencing with CNV Detection
Colorectal Cancer Predisposition via POLE Gene Sequencing with CNV Detection
Colorectal Cancer Sequencing Panel with CNV Detection
Familial Adenomatous Polyposis via APC Gene Sequencing with CNV Detection
Hereditary Breast and Ovarian Cancer - Expanded and Lynch Syndrome Sequencing Panel with CNV Detection
Hereditary Endometrial Cancer Sequencing Panel with CNV Detection
Hereditary Ovarian Cancer Sequencing Panel with CNV Detection
MUTYH Associated Polyposis (MAP) Syndrome via MUTYH Gene Sequencing with CNV Detection
Neonatal Crisis Sequencing Panel with CNV Detection
Pancreatic Cancer Sequencing Panel with CNV Detection

CONTACTS

Genetic Counselors
Geneticist
Citations
  • Abdalla et al. 2003. PubMed ID: 12843319
  • Adam et al. 2016. PubMed ID: 27476653
  • Bellido et al. 2016. PubMed ID: 26133394
  • Caspari et al. 1994. PubMed ID: 7906810
  • Chubb et al. 2015. PubMed ID: 25559809
  • Church et al. 2013. PubMed ID: 23528559
  • DeRycke et al. 2017. PubMed ID: 28944238
  • Díaz-Llopis, Menezo, 1988. PubMed ID: 2830869
  • Duraturo et al. 2011. PubMed ID: 21128252
  • Eng et al. 2001. PubMed ID: 11160785
  • Eng et al. 2014. PubMed ID: 20301661
  • Esteban-Jurado 2014. PubMed ID: 24587672
  • Farrington et al. 2005. PubMed ID: 15931596
  • Fearnhead et al. 2001. PubMed ID: 11257105
  • Frayling et al. 1998. PubMed ID: 9724771
  • Hearle et al. 2006. PubMed ID: 16707622
  • Hegde et al. 2014. PubMed ID: 24310308
  • Human Gene Mutation Database (Bio-base).
  • Jasperson et al. 2017. PubMed ID: 20301519
  • Jones et al. 2004. PubMed ID: 15083190
  • Kohlmann and Gruber. 2018. PubMed ID: 20301390
  • Laken et al. 1999. PubMed ID: 10051640
  • Li et al. 2016. PubMed ID: 27087319
  • Lipton et al. 2003. PubMed ID: 14633673
  • Miyaki et al. 2005. PubMed ID: 15890374
  • Palles et al. 2012. PubMed ID: 23263490
  • Poulsen and Bisgaard. 2008. PubMed ID: 19506731
  • Raskin et al. 2017. PubMed ID: 29212164
  • Rivera et al. 2015. PubMed ID: 26559593
  • Sampson et al. 2003. PubMed ID: 12853198
  • Soravia et al. 1998. PubMed ID: 9585611
  • Tao et al. 2004. PubMed ID: 15180946
  • Vasen et al. 1999. PubMed ID: 10348829
  • Weren et al. 2015. PubMed ID: 25938944
  • Young et al. 1998. PubMed ID: 9603437
Order Kits
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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