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Pancreatic Cancer 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
5461 APC 81201,81203 Add to Order
ATM 81408,81479
BRCA1 and BRCA2 81162
CDK4 81479,81479
CDKN2A 81404,81479
EPCAM 81479,81403
MLH1 81292,81294
MSH2 81295,81297
MSH6 81298,81300
PALB2 81406,81479
PALLD 81479,81479
PMS2 81317,81319
STK11 81405,81404
TP53 81405,81479
VHL 81404,81403
Full Panel Price* $540
Test Code Test Copy Genes Total Price CPT Codes Copy CPT Codes
5461 Genes x (16) $540 81162, 81201, 81203, 81292, 81294, 81295, 81297, 81298, 81300, 81317, 81319, 81403(x2), 81404(x3), 81405(x2), 81406, 81408, 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

ATM: Pathogenic ATM heterozygous variants in familial pancreatic cancer can be observed in up to 5% of affected individuals (Bartsch et al. 2012. PubMed ID: 22664588; Solomon et al. 2012. PubMed ID: 23187834).

BRCA1 and BRCA2: Pathogenic variants in BRCA1 and BRCA2 have recently been reported in 1.2% and 3.7% of affected individuals, respectively, in a study of familial pancreatic cancer (Zhen et al. 2015. PubMed ID: 25356972).

CDKN2A: Pathogenic variants in CDKN2A were found in 2.5% of affected individuals with familial pancreatic cancer (Zhen et al. 2015. PubMed ID: 25356972).

MLH1, MSH2, MSH6, PMS2 and EPCAM: One large study showed that 21% of families with Lynch syndrome had at least one case of pancreatic cancer (Kastrinos et al. 2009. PubMed ID: 19861671).

PALB2: Pathogenic variants in the PALB2 gene may account for familial pancreatic cancer in up to 0.6-5% of cases (Axilbund and Wiley. 2012. PubMed ID: 22846737; Bartsch et al. 2012. PubMed ID: 22664588; Zhen et al. 2015. PubMed ID: 25356972).

The clinical sensitivity for pancreatic cancer caused by pathogenic variants in APC, CDK4, PALLD, STK11, TP53 and VHL is not known, but pancreatic cancer has been reported in syndromes resulting from pathogenic variants in these genes (Solomon et al. 2012. PubMed ID: 23187834; Pogue-Geile et al. 2006. PubMed ID: 17194196; Das and Early. 2017. PubMed ID: 28879469; Matsubayashi et al. 2017. PubMed ID: 28246467).

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Clinical Features

Familial inheritance has been estimated to occur 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. PubMed ID: 22846737). Individuals with either two or more first degree relatives with pancreatic cancer or three or more relatives with pancreatic cancer of any degree are considered to have familial pancreatic cancer (Bartsch et al. 2012. PubMed ID: 22664588). Hereditary pancreatic cancer patients often show earlier ages of diagnosis compared to those with sporadic pancreatic cancer, and individuals of Ashkenazi Jewish ancestry have a higher risk to develop familal pancreatic cancer (Matsubayashi. 2011. PubMed ID: 21847571).

Even though no specific gene explains most cases of familial pancreatic cancer, affected individuals may have pathogenic variants in genes that are causative for other cancer syndromes. The syndromes that have pancreatic cancer as a clinical feature include familial adenomatous polyposis, hereditary breast and ovarian cancer (HBOC), Li-Fraumeni syndrome, Lynch syndrome, melanoma predisposition, and Peutz-Jeghers syndrome. 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. PubMed ID: 23187834).

Genetics

The pancreatic cancer next generation sequencing panel with CNV detection 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 (Bartsch et al. 2012. PubMed ID: 22664588).

APC: Familial adenomatous polyposis (FAP) is an autosomal dominant disorder caused by germline pathogenic variants in the adenomatous polyposis coli (APC) gene. More than 1,200 pathogenic variants have been reported in APC (Human Gene Mutation Database), and >90% are nonsense or frameshift pathogenic variants that are predicted to result in a dysfunctional truncated protein product (Nagase and Nakamura. 1993. PubMed ID: 8111410). Germline pathogenic variants are spread throughout the coding region (Beroud and Soussi. 1996. PubMed ID: 8594558). Several pathogenic variants have also been documented in the promoter, 3’ untranslated region (UTR), and deep intronic regions (Heinimann et al. 2001. PubMed ID: 11606402; Rosa et al. 2009. PubMed ID: 19279422). Severe FAP (more than 1,000 polyps) typically occurs in patients with pathogenic variants between codons 1250 and 1464 (Caspari et al. 1994. PubMed ID: 7906810). In contrast, patients with attenuated FAP (fewer than 100 colorectal polyps) usually have pathogenic variants at the very 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 pathogenic variants between codons 457 and 1444 (Caspari et al. 1995. PubMed ID: 7795585). 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). The risk of developing pancreatic cancer in individuals with an APC pathogenic variant is <5% (Bartsch et al. 2012. PubMed ID: 22664588).

ATM: Ataxia-telangiectasia is an autosomal recessive disorder that is caused by pathogenic variants 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. PubMed ID: 15279810). Pathogenic variants in ATM result in defective checkpoint cycling. Over 500 private causative variants are described with no common hot spots. In North America, most affected individuals are compound heterozygotes for two ATM pathogenic variants. However, founder ATM pathogenic variants have been observed in several populations (Gatti. 2010. PubMed ID: 20301790). Heterozygous carriers of single pathogenic variants in ATM may be at increased risk for pancreatic cancer (Bartsch et al. 2012. PubMed ID: 22664588; Solomon et al. 2012. PubMed ID: 23187834).

BRCA1/BRCA2: Hereditary breast and ovarian cancer (HBOC) is inherited in an autosomal dominant manner. BRCA1 is a tumor suppressor gene that is involved in cellular processes including DNA damage repair, cell cycle progression, gene transcription, and ubiquitination. BRCA2 is a tumor suppressor gene that along with RAD51 has a large role in DNA repair processes and genome stability. Most pathogenic variants in the BRCA1 and BRCA2 genes are private pathogenic variants 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); the coexistence of more than one founder pathogenic variant has been reported in some Ashkenazi Jewish families. Most BRCA pathogenic variants are inherited from a parent who may or may not have been affected with HBOC due to incomplete penetrance of the pathogenic variant, gender, and other factors (Petrucelli et al. 2013. PubMed ID: 20301425). Most pathogenic variants are predicted to result in truncated BRCA1 or BRCA2 proteins.

CDKN2A/CDK4: 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. The CDKN2A gene is a tumor suppressor gene, and its protein products help regulate cell division and apoptosis (Nelson and Tsao. 2009. PubMed ID: 19095153). CDKN2A encodes two separate but related proteins, p16/INK4a and p14/ARF, by using two different promoters. 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. PubMed ID: 17314970). 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. This 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, which functions to ubiquitinate proteins and target them for degradation. Pathogenic variants 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 pathogenic variants (Lin and Fisher. 2007. PubMed ID: 17314970).

Families with mutated p14/ARF proteins also have an increase in neural system tumors in addition to melanoma (Nelson and Tsao. 2009. PubMed ID: 19095153). CDKN2A causative variants reported to date include mostly missense pathogenic variants; 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 pathogenic variant. The risk of developing pancreatic cancer in melanoma predisposition syndrome is about 17% (Bartsch et al. 2012. PubMed ID: 22664588). Melanoma patients with pathogenic variants in the related gene CDK4 may also have an increased risk for pancreatic cancer (Goldstein et al. 2006. PubMed ID: 17047042; Vasen et al. 2000. PubMed ID: 10956390).

MLH1, MSH2, MSH6, PMS2 and EPCAM: Pathogenic variants in the MLH1, MSH2, MSH6, PMS2 and EPCAM genes cause Lynch syndrome, which can include pancreatic cancer. Most of these genes are involved in mismatch repair. Pathogenic variants result in defective DNA repair, which leads to cancer (Bujanda et al. 2017. PubMed ID: 29151953). The risk of developing pancreatic cancer in Lynch syndrome is <5% (Bartsch et al. 2012. PubMed ID: 22664588). Pancreatic tumors in Lynch syndrome tend to have a medullary appearance and present with microsatellite instability (Grover and Syngal. 2010. PubMed ID: 20727885). These patients tend to have a better prognosis in comparison to individuals with conventional pancreatic ductal adenocarcinomas (Shi et al. 2009. PubMed ID: 19260742).

PALB2: 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. PubMed ID: 23187834). Heterozygous pathogenic variants in the PALB2 gene confer susceptibility to familial pancreatic cancer (Jones et al. 2009. PubMed ID: 19264984; Slater et al. 2010. PubMed ID: 20412113).

PALLD: PALLD encodes a component of the cytoskeleton, which is involved in controlling cell shape and motility. A missense variant in PALLD has been reported to segregate in a family with pancreatic cancer (Pogue-Geile et al. 2006. PubMed ID: 17194196). However, in other familial pancreatic cancer studies PALLD variants were not observed (Klein et al. 2009. PubMed ID: 19336541; Ghiorzo et al. 2012. PubMed ID: 22368299).

STK11: Peutz-Jeghers syndrome (PJS) is caused by heterozygous germline pathogenic variants 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. PubMed ID: 10430928). Second hit pathogenic variants in STK11 ultimately lead to unfettered growth and tumorigenesis. To date, ~100 unique pathogenic variants have been described throughout the STK11 gene (Human Gene Mutation Database). Most (80%) are truncating pathogenic variants (frameshift, nonsense, splice-site, or exonic deletions) that result in early translation termination (Hearle. 2006. PubMed ID: 16707622). The remaining pathogenic variants are missense or in-frame deletions. Large genomic deletions in STK11 have also been described. The lifetime risk for pancreatic cancer can be 36% (Grover and Syngal. 2010. PubMed ID: 20727885), and individuals with STK11 pathogenic variants are predisposed to intraductal papillary mucincous neoplasms (Shi et al. 2009. PubMed ID: 19260742).

TP53: Li-Fraumeni syndrome (LFS) is inherited in an autosomal dominant manner and caused by heterozygous germline pathogenic variants in the TP53 gene (Malkin et. al. 1990. PubMed ID: 1978757; Srivastava et al. 1990. PubMed ID: 2259385). TP53 encodes the often studied cellular tumor p53 antigen (Soussi. 2010. PubMed ID: 20930848). 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). 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 lifetime risk of developing cancer for carriers of TP53 pathogenic variants has been estimated to be 73% for men and nearly 100% for women (Chompret et al. 2000. PubMed ID: 10864200). Li-Fraumeni syndrome is responsible for multiple cancers, including pancreatic cancer.

VHL: Heterozygous VHL pathogenic variants cause Von Hippel-Lindau disease, which is associated with a specific type of pancreatic cancer (pancreatic neuroendocrine tumor). The VHL gene is a tumor suppressor gene that plays a role in transcriptional regulation, post-transcriptional gene expression, extracellular matrix formation, apoptosis, and ubiquitinylation (Roberts and Ohh. 2008. PubMed ID: 18043261).

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 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 analysis of multiple genes simultaneously. Individuals with or without a family history of pancreatic tumors that are early onset (<50 years) could 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.

Genes

Official Gene Symbol OMIM ID
APC 611731
ATM 607585
BRCA1 113705
BRCA2 600185
CDK4 123829
CDKN2A 600160
EPCAM 185535
MLH1 120436
MSH2 609309
MSH6 600678
PALB2 610355
PALLD 608092
PMS2 600259
STK11 602216
TP53 191170
VHL 608537
Inheritance Abbreviation
Autosomal Dominant AD
Autosomal Recessive AR
X-Linked XL
Mitochondrial MT

Diseases

Name Inheritance OMIM ID
Adenomatous Polyposis Coli AD 175100
Ataxia-Telangiectasia Syndrome AR 208900
Breast-Ovarian Cancer, Familial 1 AD 604370
Breast-Ovarian Cancer, Familial 2 AD 612555
Choroid Plexus Papilloma AD 260500
Familial Cancer Of Breast AD 114480
Fanconi Anemia, Complementation Group N AR 610832
Hereditary Nonpolyposis Colorectal Cancer Type 4 AD 614337
Hereditary Nonpolyposis Colorectal Cancer Type 5 AD 614350
Hereditary Nonpolyposis Colorectal Cancer Type 8 AD 613244
Li-Fraumeni Syndrome AD 151623
Lynch Syndrome I AD 120435
Lynch Syndrome II AD 609310
Melanoma, Cutaneous Malignant 2 AD 155601
Melanoma, Cutaneous Malignant 3 AD 609048
Melanoma-Pancreatic Cancer Syndrome AD 606719
Muir-Torre Syndrome AD 158320
Pancreatic Cancer AD 260350
Pancreatic Cancer 1 AD 606856
Pancreatic Cancer 2 AD 613347
Pancreatic Cancer 3 AD 613348
Pancreatic Cancer 4 AD 614320
Peutz-Jeghers Syndrome AD 175200
Turcot Syndrome AR 276300
Von Hippel-Lindau Syndrome AD 193300

Related Test

Name
PGxome®

CONTACTS

Genetic Counselors
Geneticist
Citations
  • Axilbund and Wiley. 2012. PubMed ID: 22846737
  • Bartsch et al. 2012. PubMed ID: 22664588
  • Béroud and Soussi. 1996. PubMed ID: 8594558
  • Bunanda et al. 2017. PubMed ID: 29151953
  • Caspari et al. 1994. PubMed ID: 7906810
  • Caspari et al. 1995. PubMed ID: 7795585
  • Chompret et al. 2000. PubMed ID: 10864200
  • Das and Early. 2017. PubMed ID: 28879469
  • Frayling et al. 1998. PubMed ID: 9724771
  • Gatti. 2010. PubMed ID: 20301790
  • Ghiorzo et al. 2012. PubMed ID: 22368299
  • Goldstein et al. 2006. PubMed ID: 17047042
  • Grover and Syngal. 2010. PubMed ID: 20727885
  • Hearle. 2006. PubMed ID: 16707622
  • Heinimann et al. 2001. PubMed ID: 11606402
  • Human Gene Mutation Database (Bio-base).
  • Jones et al. 2009. PubMed ID: 19264984
  • Kastrinos et al. 2009. PubMed ID: 19861671
  • Klein et al. 2009. PubMed ID: 19336541
  • Lin and Fisher. 2007. PubMed ID: 17314970
  • Malkin et.al. 1990. PubMed ID: 1978757
  • Matsubayashi et al. 2017. PubMed ID: 28246467
  • Matsubayashi. 2011. PubMed ID: 21847571
  • Nagase and Nakamura. 1993. PubMed ID: 8111410
  • Nelson and Tsao. 2009. PubMed ID: 19095153
  • Petrucelli et al. 2013. PubMed ID: 20301425
  • Pogue-Geile et al. 2006. PubMed ID: 17194196
  • Roberts and Ohh. 2008. PubMed ID: 18043261
  • Rosa et al. 2009. PubMed ID: 19279422
  • Shi et al. 2009. PubMed ID: 19260742
  • Slater et al. 2010. PubMed ID: 20412113
  • Solomon et al. 2012. PubMed ID: 23187834
  • Soravia et al. 1998. PubMed ID: 9585611
  • Soussi. 2010. PubMed ID: 20930848
  • Srivastava et al. 1990. PubMed ID: 2259385
  • Taylor et al. 2004. PubMed ID: 15279810
  • Tiainen et al. 1999. PubMed ID: 10430928
  • Vasen et al. 2000. PubMed ID: 10956390
  • Young et al. 1998. PubMed ID: 9603437
  • Zhen et al. 2015. PubMed ID: 25356972
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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