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Familial Adenomatous Polyposis via APC Gene Sequencing 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 GenesPriceCPT Code Copy CPT Codes
3115 APC$540 81201,81203 Add to Order

New York State Approved Test

Pricing Comments

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

This test is predicted to detect >90% of causative FAP mutations (Laken et al. PNAS 96:2322-2326, 1999).

Gross deletions/duplications have been reported in up to 12% of APC patient samples (Jasperson and Burt. GeneReviews. 2011).

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Clinical Features

Familial Adenomatous Polyposis (FAP) (OMIM 175100) is an inherited cancer syndrome characterized clinically by the development of hundreds to thousands of adenomatous polyps in the colon and rectum. If not treated, nearly all FAP patients will develop colorectal cancer (CRC) by age 40 (Fearnhead et al. Hum Mol Genet 10:721-733, 2001). In addition to CRC, FAP patients are also predisposed to desmoid tumors, small bowel cancer, thyroid cancer, hepatoblastoma and medulloblastoma (Galiatsatos et al. Am J Gastroenterol 101:385-398, 2006). About 60% of families with FAP also display congenital hypertrophy of the retinal pigment epithelium (CHRPE), a condition that does not affect sight or have malignant potential but can be easily detected by ophthalmoscopy at any age. CHRPE is highly diagnostic of FAP and can be useful for identifying FAP patients and at-risk family members, well before the appearance of polyps (Diaz-Llopis and Menezo Arch Ophthalmol 106:412-413, 1988).

Genetics

Familial Adenomatous Polyposis (FAP) is an autosomal dominant disorder caused by germline mutations in the Adenomatous Polyposis Coli (APC) gene. More than 1200 mutations have been reported in APC (Human Gene Mutation Database, www.hgmd.cf.ac.uk) and >90% are nonsense or frameshift mutations that result in a dysfunctional truncated protein product (Nagase and Nakamura Hum Mut 2:425-434, 1993). Germline mutations are spread throughout the coding region (Beroud and Soussi Nucleic Acids Res 24:121-124, 1996). Severe FAP (i.e. more than 1000 polyps) typically occurs in patients with mutations between codons 1250 and 1464 (Caspari et al. Lancet 343:629-632, 1994). In contrast, patients with attenuated FAP (i.e. fewer than 100 colorectal polyps) usually have mutations at the very 5’ and 3’ ends of the gene, or in an alternatively spliced region of exon 9 (Young et al. Hum Mut 11:450-455, 1998; Soravia et al. Am J Hum Genet 62:1290-1301, 1998). Congenital hypertrophy of retinal pigment epithelium (CHRPE) is limited to patients with mutations between codons 457 and 1444 (Caspari et al. Hum Mol Genet 4:337-340, 1995). 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. PNAS 95:10722-10727, 1998). 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).

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.

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 APC gene, plus ~10 bases of flanking noncoding DNA. We define full coverage as >20X NGS reads or Sanger sequencing.

Indications for Test

Candidates for this test are FAP patients and relatives of patients with a known APC mutation. This test is designed for heritable germline mutations and is not appropriate for the detection of somatic mutations in tumor tissue.

Gene

Official Gene Symbol OMIM ID
APC 611731
Inheritance Abbreviation
Autosomal Dominant AD
Autosomal Recessive AR
X-Linked XL
Mitochondrial MT

Disease

Name Inheritance OMIM ID
Adenomatous Polyposis Coli AD 175100

CONTACTS

Genetic Counselors
Geneticist
Citations
  • Beroud C, Soussi T. 1996. APC gene: database of germline and somatic mutations in human tumors and cell lines. Nucleic acids research 24: 121-124. PubMed ID: 8594558
  • Caspari R, Friedl W, Mandl M, Propping P, Möslein G, Kadmon M, Timmermanns G, Knapp M, Jacobasch KH, Ecker KW. 1994. Familial adenomatous polyposis: mutation at codon 1309 and early onset of colon cancer. The Lancet 343: 629–632. PubMed ID: 7906810
  • Caspari R, Olschwang S, Friedl W, Mandl M, Boisson C, Böker T, Augustin A, Kadmon M, Möslein G, Thomas G. 1995. Familial adenomatous polyposis: desmoid tumours and lack of ophthalmic lesions (CHRPE) associated with APC mutations beyond codon 1444. Human molecular genetics 4: 337–340. PubMed ID: 7795585
  • Díaz-Llopis M, Menezo JL. 1988. Congenital hypertrophy of the retinal pigment epithelium in familial adenomatous polyposis. Arch. Ophthalmol. 106: 412–413. PubMed ID: 2830869
  • Fearnhead NS, Britton MP, Bodmer WF. 2001. The ABC of APC. Hum. Mol. Genet. 10: 721–733. PubMed ID: 11257105
  • Frayling IM, Beck NE, Ilyas M, Dove-Edwin I, Goodman P, Pack K, Bell JA, Williams CB, Hodgson SV, Thomas HJ. 1998. The APC variants I1307K and E1317Q are associated with colorectal tumors, but not always with a family history. Proceedings of the National Academy of Sciences 95: 10722–10727. PubMed ID: 9724771
  • Galiatsatos P, Foulkes WD. 2006. Familial Adenomatous Polyposis. The American Journal of Gastroenterology 101: 385–398. PubMed ID: 16454848
  • Human Gene Mutation Database.
  • Laken SJ, Papadopoulos N, Petersen GM, Gruber SB, Hamilton SR, Giardiello FM, Brensinger JD, Vogelstein B, Kinzler KW. 1999. Analysis of masked mutations in familial adenomatous polyposis. Proceedings of the National Academy of Sciences 96: 2322–2326. PubMed ID: 10051640
  • Li J. et al. 2016. American Journal of Human Genetics. 98: 830-42. PubMed ID: 27087319
  • Nagase H, Nakamura Y. 1993. Mutations of the APC adenomatous polyposis coli gene. Human Mutation 2: 425–434. PubMed ID: 8111410
  • Soravia C, Berk T, Madlensky L, Mitri A, Cheng H, Gallinger S, Cohen Z, Bapat B. 1998. Genotype-phenotype correlations in attenuated adenomatous polyposis coli. The American Journal of Human Genetics 62: 1290–1301. PubMed ID: 9585611
  • Young J, Simms LA, Tarish J, Buttenshaw R, Knight N, Anderson GJ, Bell A, Leggett B. 1998. A family with attenuated familial adenomatous polyposis due to a mutation in the alternatively spliced region of APC exon 9. Hum. Mutat. 11: 450–455. PubMed ID: 9603437
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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.
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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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