Pancreatic Cancer Sequencing Panel
- Summary and Pricing
- Clinical Features and Genetics
- NextGen Sequencing using PG-Select Capture Probes
- Deletion/Duplication Testing Via Array Comparative Genomic Hybridization
|Test Code||Test Copy Genes||CPT Code Copy CPT Codes|
|Full Panel Price*||$1490.00|
|Test Code||Test Copy Genes||Total Price||CPT Codes Copy CPT Codes|
|1343||Genes x (12)||$1490.00||81201, 81214, 81216, 81292, 81295, 81298, 81317, 81404, 81405(x2), 81406, 81408||Add|
If you would like to order a subset of these genes contact us to discuss pricing.
For ordering targeted known variants, please proceed to our Targeted Variants landing page.
The great majority of tests are completed within 28 days.
Clinical sensitivity of the tested genes is given for each syndrome.
Sequencing the TP53 gene yields positive results in approximately 95% of patients with Li-Fraumeni syndrome (Schneider et al. 2013). Depending on the clinical criteria used to make a diagnosis (i.e. Amsterdam or “Revised Bethesda”), 30-50% and 15-20% of Lynch patients have a detectable MLH1 and MSH2 mutation, respectively (Syngal et al. 2000). A causative mutation in MSH6 is detected in < 2% of patients that meet the stringent Amsterdam I criteria, but is detected in ~12% of atypical Lynch/HNPCC families (Peltomäki and Vasen 2004). A causative mutation in PMS2 is detected in 1-2% of Lynch patients (Peltomäki and Vasen 2004) and ~50% of constitutional mismatch repair-deficiency patients (Wimmer and Etzler 2008). Approximately 55% of patients with a positive family history or 70% of patients with no family history of Peutz-Jeghers syndrome will be detected by STK11 sequencing (McGarrity et al. 1993). Approximately 90% of individuals with Ataxia-telangiectasia have detectable mutations via sequencing of the ATM gene (Gatti 1993). This test is predicted to detect >90% of causative APC mutations (Laken et al. 1999). PALB2 mutations account for <1% of Fanconi Anemia (Alter and Kupfer 2013). It has been reported that 25% to 50% of familial melanoma kindreds are affected by a CDKN2A causative mutation (Goldstein et al. 2006). Individuals with multiple primary melanomas have a 1-3% chance of having a CDKN2A causative mutation (Berwick 2006). The overall prevalence of germline BRCA1 or BRCA2 causative mutations in the general population is 1:400 to 1:800, and higher rates depending on the specific ethnicity, such as 1:40 in the Ashkenazi Jewish population. Pathogenic variants will be detected by sequencing in 90% of individuals with an identifiable mutation. Previously, BRCA1 variants were observed in 63% of these cases and BRCA2 variants in 37% of these cases (Petrucelli 2013). BRCA1 mutation carriers tend to have breast tumors that are estrogen receptor (ER) negative, progesterone receptor (PR) negative and basal type tumors, whereas BRCA2 mutation carriers have breast tumors that are ER positive, PR positive, and have a luminal phenotype (Pruthi et al. 2010). Individuals with HBOC with a more severe personal or family history tend to have mutations in BRCA1 vs. BRCA2 due to higher penetrance of mutations in the BRCA1 gene (Antoniou et al. 2000).
Deletion/Duplication Testing via aCGH
|Test Code||Test Copy Genes||Individual Gene Price||CPT Code Copy CPT Codes|
|Full Panel Price*||$1290.00|
|Test Code||Test Copy Genes||Total Price||CPT Codes Copy CPT Codes|
|600||Genes x (12)||$1290.00||81203, 81294, 81297, 81300, 81319, 81404, 81479(x6)||Add|
STK11 is analyzed by Multiplex Ligation-dependent Probe Amplification.
The great majority of tests are completed within 28 days.
Clinical sensitivity of the tested genes is given for each syndrome.
Deletions in the TP53 gene have been detected in 1% of Li-Fraumeni cases (Schneider et al. 2013). Large deletions and genetic rearrangements account for 20%, 5%, 20%, and 7% of identifiable pathogenic variants in the MSH2, MLH1, PMS2, and MSH6 genes (Kohlmann and Gruber. 2012). Approximately 45% of patients with a positive family history or 21% of patients with no family history of Peutz-Jeghers syndrome will have a pathogenic variant in STK11 by deletion analysis (McGarrity et al. 2013). Gross deletions/duplications have been reported in up to 12% of APC patient samples (Jasperson and Burt 2011). Approximately 1-2% of Ataxia Telangiectasia patients have large genomic deletions involving the ATM gene that can be detected using aCGH (Gatti 2010). The clinical sensitivity of large deletions and duplications for CDKN2A and PALB2 is not known but large deletions have been reported. Pathogenic variants will be detected by copy number analysis in 10% of HBOC individuals with an identifiable germline mutation. Previously, BRCA1 variants were observed in 90% of these cases and BRCA2 variants in 10% of these cases (Petrucelli 2013). Large rearrangements (e.g. deletions, duplications, tripications), including the five most commonly reported BRCA1 alterations (Hendrickson et al. 2005), can be detected using this test. High-risk patients, defined as individuals with early onset (
Pancreatic cancer has been estimated to have a familial inheritance in 5-10% of pancreatic cancer cases; and individuals with a family history have a greater risk of developing pancreatic cancer with each affected family member (Axilbund and Wiley 2012). Familial pancreatic cancer applies to individuals with two or more first degree relatives with pancreatic cancer or three or more relatives with pancreatic cancer of any degree (Bartsch et al. 2012). Individuals with hereditary pancreatic cancers often show earlier ages of diagnosis compared to sporadic pancreatic cancer or are of a certain ethnicity (i.e. Ashkenazi Jewish ancestry) (Matsubayashi 2011). Although, no specific gene(s) explain most cases of familial pancreatic cancer, individuals with familial pancreatic cancer may have gene mutations that are causative for specific syndromes. The syndromes that have pancreatic cancer as a clinical feature include Familial Adenomatous Polyposis, Hereditary Breast and Ovarian Cancer Syndrome, Li-Fraumeni Syndrome, Lynch Syndrome, Melanoma Predisposition, and Peutz-Jeghers Syndrome. Also, heterozygous carriers of pathogenic variants in genes causative for Ataxia Telangiectasia and Fanconi Anemia have also been found to have increased risk of developing pancreatic cancer (Solomon et al. 2012).
The pancreatic cancer next generation sequencing panel assesses genes that have been shown to be causative when mutated for disorders that have pancreatic cancer as a clinical feature. The mode of inheritance appears to be autosomal dominant and may show anticipation (Bartsch et al. 2012).
Familial adenomatous polyposis is an autosomal dominant disorder caused by germline mutations in the adenomatous polyposis coli (APC) gene. More than 1,200 mutations have been reported in APC (Human Gene Mutation Database), and >90% are nonsense or frameshift mutations that result in a dysfunctional truncated protein product (Nagase and Nakamura 1993). Germline mutations are spread throughout the coding region (Béroud and Soussi 1996). Several pathogenic mutations have also been documented in the promoter, 3’ untranslated region (UTR) and deep within intron 14 (Heinimann et al. 2001; Rosa De et al. 2009). Severe FAP (i.e. more than 1,000 polyps) typically occurs in patients with mutations between codons 1250 and 1464 (Caspari et al. 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. 1998; Soravia et al. 1998). Congenital hypertrophy of retinal pigment epithelium (CHRPE) is limited to patients with mutations between codons 457 and 1444 (Caspari et al. 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. 1998). The risk of developing pancreatic cancer in individuals with an APC mutation is <5% (Bartsch et al. 2012). 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).
Ataxia-telangiectasia is an autosomal recessive disorder that is caused by mutations in the ATM gene. ATM encodes a serine protein kinase (ATM) that is involved in DNA repair via phosphorylation of downstream proteins. It senses double-stranded DNA breaks, coordinates cell-cycle checkpoints prior to repair, and recruits repair proteins to damaged DNA sites (Taylor et al. 2004). Mutations in ATM result in defective checkpoint cycling. Over 500 private causative mutations are described with no common hot spots for mutations. In North America, most affected individuals are compound heterozygotes for two ATM mutations. However, founder ATM mutations have been observed in several populations (Gatti 2010). Pathogenic heterozygous variants of ATM in familial pancreatic cancer can be observed in up to 5% individuals with pancreatic cancer (Bartsch et al. 2012).
Hereditary Breast and Ovarian Cancer is inherited in an autosomal dominant manner. BRCA1 is a tumor suppressor that is involved in cellular processes including DNA damage repair, cell cycle progression, gene transcription and ubiquitination. BRCA2 is a tumor suppressor that along with RAD51 has a large role in DNA repair processes and genome stability. Most pathogenic variants of the BRCA1 and BRCA2 genes are private mutations which are observed in a single family or in a small number of families. Three pathogenic variants in the BRCA genes are commonly found in Ashkenazi Jewish individuals: c.68_69delAG (BRCA1), c.5266dupC (BRCA1), and c.5946delT (BRCA2), and the coexistence of more than one founder mutation has been reported in some Ashkenazi Jewish families. Most BRCA mutations are inherited from a parent who may or may not have been affected with HBOC due to incomplete penetrance of the mutation, gender, and other factors (Petrucelli 2013). Most mutations result in predicted truncated BRCA1 or BRCA2 proteins.
Melanoma predisposition is inherited in an autosomal dominant manner. The strongest genetic risk for the development of melanoma results from heritable alterations in the cyclin-dependent kinase inhibitor 2A (CDKN2A) gene, which encodes two separate but related proteins, p16/INK4a and p14/ARF, by using two different promoters. The CDKN2A gene is a tumor suppressor, and its protein products help regulate cell division and apoptosis (Nelson et al. 2009). The p16/INK4a protein is produced from a transcript generated from exons 1α, 2 and 3, whereas p14ARF is produced, using an alternative reading frame, from a transcript comprising exons 1β, 2 and 3 (Lin and Fisher 2007). The function of the p16/INK4a protein is to inhibit CDK4/6 protein-mediated phosphorylation of the Rb (Retinoblastoma) tumor suppressor protein; dephosphorylated Rb is the active state. Mutated p16/INK4a leads to phosphorylation of Rb, which in turn results in release of the bound transcription factor E2F, which then allows the cell to undergo unregulated cell division leading to the development of melanoma.
p14/ARF exerts its regulation of cell division through its indirect interaction with the p53 protein. p14/ARF binds to and inhibits the HDM2 protein. HDM2 functions to ubiquitinate proteins and target them for degradation. Mutations in p14/ARF abrogate binding to HDM2. As a result, HDM2 is released and increases ubiquitination of p53 leading to increased destruction of this tumor suppressor. The main functions of p53 are to sense genetic damage to allow pause for DNA repair and to activate cellular apoptosis. Thus, the decreased levels of p53 associated with mutations in p14/ARF lead to genetic instability and to a higher risk of melanoma in individuals with CDKN2A mutations (Lin and Fisher 2007). Families with mutated p14/ARF proteins also have an increase in neural system tumors in addition to melanoma (Nelson et al. 2009).
CDKN2A causative mutations reported to date include mostly missense mutations, however nonsense, splicing, small insertions and deletions, regulatory, and gross insertions and deletions have also been reported (Human Gene Mutation Database).
The co-occurrence of melanoma and pancreatic cancer is an important indicator of a CDKN2A mutation. The risk of developing pancreatic cancer in melanoma predisposition syndrome is 17% (Bartsch et al. 2012).
MLH1, MSH2, MSH6, and PMS2
Mutations in the MLH1, MSH2, MSH6, and PMS2 genes cause Lynch Syndrome and are associated with pancreatic cancer. These genes are involved in mismatch repair, and mutations result in defective DNA repair leading to cancer.
The risk of developing pancreatic cancer in Lynch syndrome is <5% (Bartsch et al. 2012). The pancreatic tumors tend to have a medullary appearance and present with microsatellite instability (Grover and Syngal 2010). These patients tend to have a better prognosis in comparison to individuals with conventional pancreatic ductal adenocarcinomas (Shi et al. 2009).
Homozygous pathogenic variants of the PALB2 gene have been shown to be causative for Fanconi Anemia. The PALB2 gene acts as a tumor suppressor and colocalizes with the BRCA2 protein (Solomon et al. 2012). Pathogenic variants in the PALB2 gene may be accountable for familial pancreatic cancer in up to 3-5% of cases (Axilbund and Wiley 2012; Bartsch et al. 2012).
Peutz-Jeghers syndrome is caused by heterozygous germline mutations in the tumor suppressor gene STK11. STK11, also called LKB1, encodes a serine/threonine kinase that inhibits cellular proliferation by promoting cell-cycle arrest (Tiainen et al. 1999). Second hit mutations in STK11 ultimately lead to unfettered growth and tumorigenesis. To date, ~100 unique mutations have been described throughout the STK11 gene (Human Gene Mutation Database). Most (80%) are truncating mutations (i.e. frameshift, nonsense, splice-site, or exonic deletions) that result in early protein termination (Hearle 2006). The remaining mutations are missense or in-frame deletions. Large genomic deletions in STK11 have also been described.
Lifetime risk for pancreatic cancer can be 36% (Grover and Syngal 2010), and individuals with STK11 mutations are predisposed to intraductal papillary mucincous neoplasms (Shi et al. 2009).
Li-Fraumeni Syndrome is inherited in an autosomal dominant manner and caused by heterozygous germline mutations in the TP53 gene (Malkin et al. 1990; Srivastava et al. 1990). TP53 encodes the well described cellular tumor p53 antigen (Soussi 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). 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. 2000). Li-Fraumeni syndrome is responsible for multiple cancers, including pancreatic cancer.
The Pancreatic Cancer NextGen Sequencing Panel analyzes 12 genes that have been associated with familial pancreatic cancers. For this NGS panel, the full coding regions, plus ~20bp of non-coding DNA flanking each exon, are sequenced for each of the genes listed below. Sequencing is accomplished by capturing specific regions with an optimized solution-based hybridization method, followed by massively parallel sequencing of the captured DNA fragments. Additional Sanger sequencing is performed for any regions not captured or with insufficient number of sequence reads, including the exon 1B promoter region of APC. All pathogenic, undocumented and questionable variant calls are confirmed by Sanger sequencing.
Due to known PMS2 pseudogenes, the PMS2 gene is also analyzed via Sanger sequencing using a long-range PCR strategy.
For deletion/duplication testing, STK11 is analyzed by Multiplex Ligation-dependent Probe Amplification.
Each gene/group of genes can also be tested using our Sanger sequencing and Deletion/duplication assays. Please see our test menu.
Indications for Test
This test is suitable for individuals with a clinical history of familial pancreatic cancers or presenting an autosomal dominant disorder that includes pancreatic cancer. This test especially aids in a differential diagnosis of similar phenotypes, rules out particular syndromes, and provides the analysis of multiple genes simultaneously. Individuals with or without a family history of pancreatic tumors that are synchronous and/or metachronous, early onset (e.g. < 50 years) should be assessed with this panel.
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 - firstname.lastname@example.org
- Jerry Machado, PhD, DABMG, FCCMG - email@example.com
- Alter BP, Kupfer G. 2013. Fanconi Anemia. 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: 20301575
- Antoniou AC, Gayther SA, Stratton JF, Ponder BA, Easton DF. 2000. Risk models for familial ovarian and breast cancer. Genet. Epidemiol. 18: 173–190. PubMed ID: 10642429
- Axilbund JE, Wiley EA. 2012. Genetic Testing by Cancer Site: Pancreas. The Cancer Journal 18: 350–354. PubMed ID: 22846737
- Bartsch DK, Gress TM, Langer P. 2012. Familial pancreatic cancer—current knowledge. Nature Reviews Gastroenterology & Hepatology 9: 445–453. PubMed ID: 22664588
- 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
- Berwick M. 2006. The Prevalence of CDKN2A Germ-Line Mutations and Relative Risk for Cutaneous Malignant Melanoma: An International Population-Based Study. Cancer Epidemiology Biomarkers & Prevention 15: 1520–1525. PubMed ID: 16896043
- 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
- 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
- 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
- Gatti R. 2010. Ataxia-Telangiectasia. 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: 20301790
- Goldstein AM, Chan M, Harland M, Hayward NK, Demenais F, Timothy Bishop D, Azizi E, Bergman W, Bianchi-Scarra G, Bruno W, Calista D, Cannon Albright LA, et al. 2006. Features associated with germline CDKN2A mutations: a GenoMEL study of melanoma-prone families from three continents. Journal of Medical Genetics 44: 99–106. PubMed ID: 16905682
- Grover S, Syngal S. 2010. Hereditary Pancreatic Cancer. Gastroenterology 139: 1076–1080.e2. PubMed ID: 20727885
- Hearle N. 2006. Frequency and Spectrum of Cancers in the Peutz-Jeghers Syndrome. Clinical Cancer Research 12: 3209–3215. PubMed ID: 16707622
- Heinimann K, Thompson A, Locher A, Furlanetto T, Bader E, Wolf A, Meier R, Walter K, Bauerfeind P, Marra G. 2001. Nontruncating APC germ-line mutations and mismatch repair deficiency play a minor role in APC mutation-negative polyposis. Cancer research 61: 7616–7622. PubMed ID: 11606402
- Hendrickson BC, Judkins T, Ward BD, Eliason K, Deffenbaugh AE, Burbidge LA, Pyne K, Leclair B, Ward BE, Scholl T. 2005. Prevalence of five previously reported and recurrentBRCA1 genetic rearrangement mutations in 20,000 patients from hereditary breast/ovarian cancer families. Genes, Chromosomes and Cancer 43: 309–313. PubMed ID: 15846789
- Human Gene Mutation Database (Bio-base).
- Jasperson KW, Burt RW. 2011. APC-Associated Polyposis Conditions. 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: 20301519
- Judkins T, Rosenthal E, Arnell C, Burbidge LA, Geary W, Barrus T, Schoenberger J, Trost J, Wenstrup RJ, Roa BB. 2012. Clinical significance of large rearrangements in BRCA1 and BRCA2. Cancer 118: 5210–5216. PubMed ID: 22544547
- Kohlmann W, Gruber SB. 2012. 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
- 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
- Lin JY, Fisher DE. 2007. Melanocyte biology and skin pigmentation. Nature 445: 843–850. PubMed ID: 17314970
- 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
- Matsubayashi H. 2011. Familial pancreatic cancer and hereditary syndromes: screening strategy for high-risk individuals. Journal of Gastroenterology 46: 1249–1259. PubMed ID: 21847571
- McGarrity TJ, Amos CI, Frazier ML, Wei C. 2013. Peutz-Jeghers 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: 20301443
- Nagase H, Nakamura Y. 1993. Mutations of the APC adenomatous polyposis coli gene. Human Mutation 2: 425–434. PubMed ID: 8111410
- Nelson AA, Tsao H. 2009. Melanoma and genetics. Clinics in Dermatology 27: 46–52. PubMed ID: 19095153
- Peltomäki P, Vasen H. 2004. Mutations associated with HNPCC predisposition–Update of ICG-HNPCC/INSiGHT mutation database. Disease markers 20: 269–276. PubMed ID: 15528792
- Petrucelli N, Daly MB, Feldman GL. 2013. BRCA1 and BRCA2 Hereditary Breast and Ovarian Cancer. 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: 20301425
- Pruthi S, Gostout BS, Lindor NM. 2010. Identification and Management of Women With BRCA Mutations or Hereditary Predisposition for Breast and Ovarian Cancer. Mayo Clinic Proceedings 85: 1111–1120. PubMed ID: 21123638
- Rosa M De, Galatola M, Borriello S, Duraturo F, Masone S, Izzo P. 2009. Implication of adenomatous polyposis coli and MUTYH mutations in familial colorectal polyposis. Dis. Colon Rectum 52: 268–274. PubMed ID: 19279422
- Schneider K, Zelley K, Nichols KE, Garber J. 2013. Li-Fraumeni Syndrome. In: Pagon RA, Adam MP, Bird TD, Dolan CR, Fong C-T, Smith RJ, and Stephens K, editors. GeneReviews™, Seattle (WA): University of Washington, Seattle. PubMed ID: 20301488
- Shi C, Hruban RH, Klein AP. 2009. Familial pancreatic cancer. Archives of pathology & laboratory medicine 133: 365–374. PubMed ID: 19260742
- Solomon S, Das S, Brand R, Whitcomb DC. 2012. Inherited Pancreatic Cancer Syndromes: The Cancer Journal 18: 485–491. PubMed ID: 23187834
- 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
- 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
- Syngal S, Fox EA, Eng C, Kolodner RD, Garber JE. 2000. Sensitivity and specificity of clinical criteria for hereditary non-polyposis colorectal cancer associated mutations inMSH2 and MLH1. Journal of medical genetics 37: 641–645. PubMed ID: 10978352
- Taylor AMR, Groom A, Byrd PJ. 2004. Ataxia-telangiectasia-like disorder (ATLD)—its clinical presentation and molecular basis. DNA Repair 3: 1219–1225. PubMed ID: 15279810
- Tiainen M, Ylikorkala A, Mäkelä TP. 1999. Growth suppression by Lkb1 is mediated by a G1 cell cycle arrest. Proceedings of the National Academy of Sciences 96: 9248–9251. PubMed ID: 10430928
- Wimmer K, Etzler J. 2008. Constitutional mismatch repair-deficiency syndrome: have we so far seen only the tip of an iceberg? Human Genetics 124: 105–122. PubMed ID: 18709565
- 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
- NextGen Sequencing using PG-Select Capture Probes
- Deletion/Duplication Testing Via Array Comparative Genomic Hybridization
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 ~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.
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 ~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/Duplication Testing Via Array Comparative Genomic Hybridization
Equal amounts of genomic DNA from the patient and a gender matched reference sample are amplified and labeled with Cy3 and Cy5 dyes, respectively. To prevent any sample cross contamination, a unique sample tracking control is added into each patient sample. Each labeled patient product is then purified, quantified, and combined with the same amount of reference product. The combined sample is loaded onto the designed array and hybridized for at least 22-42 hours at 65°C. Arrays are then washed and scanned immediately with 2.5 µM resolution. Only data for the gene(s) of interest for each patient are extracted and analyzed.
PreventionGenetics' high density gene-centric custom designed aCGH enables the detection of relatively small deletions and duplications within a single exon of a given gene or deletions and duplications encompassing the entire gene. PreventionGenetics has established and verified this test's accuracy and precision.
Our dense probe coverage may allow detection of deletions/duplications down to 100 bp; however due to limitations and probe spacing this cannot be guaranteed across all exons of all genes. Therefore, some copy number changes smaller than 100-300 bp within a targeted large exon may not be detected by our array.
This array may not detect deletions and duplications present at low levels of mosaicism or those present in genes that have pseudogene copies or repeats elsewhere in the genome.
aCGH will not detect balanced translocations, inversions, or point mutations that may be responsible for the clinical phenotype.
Breakpoints, if occurring outside the targeted gene, may be hard to define.
The sensitivity of this assay may be reduced when DNA is extracted by an outside laboratory.
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.