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PTEN Hamartoma Tumor Syndrome via PTEN 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
7513 PTEN$540 81321,81323 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 causative mutations in ~80% of patients with CS, ~65% of patients with BRRS and ~20% of patients with PS (Eng Hum Mutat 22:183-198, 2003).

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Clinical Features

PTEN Hamartoma Tumor Syndrome (PHTS) is a cluster of related clinical conditions, all caused by germline mutations in the PTEN tumor suppressor gene (OMIM 601728). Included in PHTS are Cowden Syndrome (CS; OMIM 158350), Bannayan-Riley-Ruvalcaba Syndrome (BRRS; OMIM 153480), Proteus (PS; OMIM 176920) and Proteus-like Syndromes, and VACTERL Association with Hydrocephalus (OMIM 276950). While each PHTS condition has its own unique pathognomonic features (see for example Blumenthal & Dennis, Eur J Hum Genet 16:1289-1300, 2008), hamartomatous overgrowth, macrocephaly and vascular malformations appear to be common to all conditions (Zhou et al. Lancet 358:210-211, 2001). A presumptive diagnosis of PHTS is typically made based on clinical symptoms, but a definitive diagnosis requires the identification of a heterozygous PTEN mutation. Patients with a germline mutation in PTEN have a 5-10 fold higher chance of developing cancer at a much earlier age (<30 y/o) than the general population (Eng, Hum Mut 22:183-198, 2003). In addition to confirming the diagnosis of PHTS, testing patients for a germline PTEN mutation is essential to accurately assess their risk for cancer and to make appropriate recommendations regarding prevention and treatment of malignancy.

Genetics

PTEN Hamartoma Tumor Syndrome inherited in an autosomal dominant manner, and PTEN is the only known gene to be associated with the disease. In addition to PHTS, germline mutations in PTEN have been identified in 16% of patients with Autism Spectrum Disorders (ASD) and macrocephaly, 12.5% of patients with adenomatous and hyperplastic polyps, and 5% of women with at least two different types of cancer (Zbuk & Eng Nat Rev Cancer 7:35-45, 2007; Lintas & Persico J Med Genet 46:1-8, 2009). To date, ~230 mutations have been reported for the PTEN gene, and most (~95%) are of the type that can be detected by DNA sequencing (Human Gene Mutation Database, www.hgmd.cf.ac.uk). The PTEN gene consists of 9 exons and encodes a dual lipid and protein phosphatase. Mutations have been reported throughout the coding region, and sequencing of all 9 exons is recommended (Eng Hum Mut 22:183-198, 2003). Five mutations have also been reported within the minimal promoter about 800 bp upstream of the start codon and sequencing of this region is also recommended (Teresi et al. Am J Hum Genet 81:756-767, 2007).

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

In addition to the regions described above, this testing includes coverage of the PTEN minimal promoter region (positions -1239 to -765 relative to the start codon).

Indications for Test

Candidates for this test are patients with PHTS or Autism with macrocephaly, women presenting with multiple primary cancers, and relatives of patients with a known germline PTEN mutation. 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
PTEN 601728
Inheritance Abbreviation
Autosomal Dominant AD
Autosomal Recessive AR
X-Linked XL
Mitochondrial MT

Related Tests

Name
Autism Spectrum Disorders Sequencing Panel with CNV Detection
Breast Cancer - High / Moderate Risk Sequencing Panel with CNV Detection
Breast Cancer - High Risk Sequencing Panel with CNV Detection
Cowden and Cowden-like Syndromes via PIK3CA Gene Sequencing 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

CONTACTS

Genetic Counselors
Geneticist
Citations
  • Blumenthal GM, Dennis PA. 2008. PTEN hamartoma tumor syndromes. European Journal of Human Genetics 16: 1289–1300. PubMed ID: 18781191
  • Eng C. 2003. PTEN: One Gene, Many Syndromes. Human Mutation 22: 183–198. PubMed ID: 12938083
  • Human Gene Mutation Database.
  • Lintas C, Persico AM. 2009. Autistic phenotypes and genetic testing: state-of-the-art for the clinical geneticist. Journal of medical genetics 46: 1–8. PubMed ID: 18728070
  • Teresi RE, Zbuk KM, Pezzolesi MG, Waite KA, Eng C. 2007. Cowden Syndrome–Affected Patients with PTEN Promoter Mutations Demonstrate Abnormal Protein Translation. The American Journal of Human Genetics 81: 756–767. PubMed ID: 17847000
  • Zbuk KM, Eng C. 2006. Cancer phenomics: RET and PTEN as illustrative models. Nature Reviews Cancer 7: 35–45. PubMed ID: 17167516
  • Zhou X-P, Hampel H, Thiele H, Gorlin RJ, Hennekam R, Parisi M, Winter RM, Eng C. 2001. Association of germline mutation in the PTEN tumour suppressor gene and Proteus and Proteus-like syndromes. The Lancet 358: 210–211. PubMed ID: 11476841
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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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