Li-Fraumeni Syndrome via TP53 Gene Sequencing with CNV Detection
- Summary and Pricing
- Clinical Features and Genetics
Sequencing and CNV
|Test Code||Test Copy Genes||Price||CPT Code Copy CPT Codes|
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.
For ordering sequencing of targeted known variants, please proceed to our Targeted Variants landing page.
The great majority of tests are completed within 20 days.
This test is predicted to detect pathogenic mutations in ~90% of children with an adrenal cortical tumor (Varley et al. Am J Hum Genet 65:995-1006, 1999), ~33% of patients diagnosed with a bone or soft-tissue sarcoma (Toguchida et al. N Engl J Med 326:1301-1308, 1992), 7-20% of patients with multiple primary tumors (Malkin et al. N Engl J Med 326:1309-1315, 1992) and ~7% of women with pre-menopausal breast cancer and a relative diagnosed with a typical LFS cancer (Chompret et al. J Med Genet 38:43-47, 2001). Deletions in the TP53 gene have been detected in 1% of Li-Fraumeni cases (Schneider and Garber. GeneReviews. 2010).
Li-Fraumeni Syndrome (LFS; OMIM 151623) is a hereditary cancer syndrome that predisposes individuals to multiple neoplasms at an early age. The most common neoplasms associated with LFS are pre-menopausal breast carcinomas, bone and soft-tissue sarcomas, adrenocortical carcinomas and brain tumors. Although much less common, melanomas, germ cell tumors, gastric carcinomas and Wilms tumors have also been described in LFS patients (Varley et al. Cancer Res 57:3245-3252, 1997). The average age of malignancy for individuals with LFS is typically between 20 and 45, which is at least 2-3 decades sooner than reported for the general population (Nichols et al. Cancer Epidemiol Biomarkers Prev 10:83-87, 2001).
Li-Fraumeni Syndrome is inherited in an autosomal dominant manner and caused by heterozygous germline mutations in the TP53 gene (Malkin et al. Science 250:1233-1238, 1990; Srivastava et al. Nature 348:747-749, 1990). TP53 encodes the well described cellular tumor p53 antigen (Soussi EMBO Rep 11:822-826, 2010). p53 is a ubiquitously expressed DNA-binding protein that plays a major role in the regulation of cell division, DNA repair, programmed cell death, and metabolism. More than 200 pathogenic variations have been reported throughout the TP53 gene, and nearly all are detectable by DNA sequencing (Human Gene Mutation Database, www.hgmd.cf.ac.uk). Three gross deletions encompassing one or more exons of the TP53 gene have been described but these account for less than 1% of all LFS patients. The risk of developing cancer for carriers of TP53 mutations has been estimated to be ~73% for men and nearly 100% for women (Chompret et al. Br J Cancer 82:1932-1937, 2000).
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 TP53 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 a region in intron 6 for the detection of a previously documented pathogenic mutation (Barel et al. Cancer Genet Cytogenet 103:1-6, 1998).
Indications for Test
This test is recommended for individuals with any childhood cancer, sarcoma or brain tumor, or an adrenal cortical tumor diagnosed under the age of 45, plus one first or second degree relative with a typical LFS cancer diagnosed at any age and another first or second degree relative diagnosed with any cancer under the age of 60 (Chompret et al. J Med Genet 38:43-47, 2001). Women with pre-menopausal breast cancer and a relative diagnosed with a typical LFS cancer are also candidates for this test. This test is specifically designed for heritable germline mutations and is not appropriate for the detection of somatic mutations in tumor tissue.
|Official Gene Symbol||OMIM ID|
- Genetic Counselor Team - email@example.com
- Jerry Machado, PhD, DABMG, FCCMG - firstname.lastname@example.org
- Barel, D., et.al. (1998). "A novel germ-line mutation in the noncoding region of the p53 gene in a Li-Fraumeni family." Cancer Genet Cytogenet 103(1): 1-6. PubMed ID: 9595036
- Chompret A, Brugières L, Ronsin M, Gardes M, Dessarps-Freichey F, Abel A, Hua D, Ligot L, Dondon M-G, Bressac-de Paillerets B. 2000. P53 germline mutations in childhood cancers and cancer risk for carrier individuals. British journal of cancer 82: 1932. PubMed ID: 10864200
- Chompret, A., et.al. (2001). "Sensitivity and predictive value of criteria for p53 germline mutation screening." J Med Genet 38(1): 43-7. PubMed ID: 11332399
- Human Gene Mutation Database.
- Malkin, D., et.al. (1992). "Germline mutations of the p53 tumor-suppressor gene in children and young adults with second malignant neoplasms." N Engl J Med 326(20): 1309-15. PubMed ID: 1565144
- Malkin, D., et.al. 1990. Germ line p53 mutations in a familial syndrome of breast cancer, sarcomas, and other neoplasms. Science 250(4985): 1233-8. PubMed ID: 1978757
- Nichols KE, Malkin D, Garber JE, Fraumeni JF, Li FP. 2001. Germ-line p53 mutations predispose to a wide spectrum of early-onset cancers. Cancer Epidemiology Biomarkers & Prevention 10: 83–87. PubMed ID: 11219776
- Schneider and Garber. GeneReviews. 2010
- Soussi T. 2010. The history of p53. EMBO reports 11: 822–826. PubMed ID: 20930848
- Srivastava S, Zou ZQ, Pirollo K, Blattner W, Chang EH. 1990. Germ-line transmission of a mutated p53 gene in a cancer-prone family with Li-Fraumeni syndrome. Nature 348: 747–749. PubMed ID: 2259385
- Toguchida, J., et.al. (1992). "Prevalence and spectrum of germline mutations of the p53 gene among patients with sarcoma." N Engl J Med 326(20): 1301-8. PubMed ID: 1565143
- Varley JM, McGown G, Thorncroft M, Santibanez-Koref MF, Kelsey AM, Tricker KJ, Evans DGR, Birch JM. 1997. Germ-line mutations of TP53 in Li-Fraumeni families: an extended study of 39 families. Cancer research 57: 3245–3252. PubMed ID: 9242456
- Varley, J. M., et.al. (1999). "Are there low-penetrance TP53 Alleles? evidence from childhood adrenocortical tumors." Am J Hum Genet 65(4): 995-1006. PubMed ID: 10486318
Sequencing and CNV Detection via NextGen Sequencing using PG-Select Capture Probes
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
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.
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.
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.
- 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.
(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.
(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.
(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.