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Renal Cancer Sequencing Panel

  • Summary and Pricing
  • Clinical Features and Genetics
  • Citations
  • Methods
  • Ordering/Specimens
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TEST METHODS

Sequencing

Test Code TestCPT Code Copy CPT Codes
1331 BAP1 81479 Add to Order
CDC73 81479
FH 81405
FLCN 81479
MET 81479
MLH1 81292
MSH2 81295
MSH6 81298
PMS2 81317
PTEN 81321
SDHB 81405
SDHC 81405
SDHD 81404
SMARCB1 81479
TP53 81405
TSC1 81406
TSC2 81407
VHL 81404
WT1 81405
Full Panel Price* $2590.00
Test Code Test Total Price CPT Codes Copy CPT Codes
1331 Genes x (19) $2590.00 81292, 81295, 81298, 81317, 81321, 81404(x2), 81405(x5), 81406, 81407, 81479(x5) Add to Order
Pricing Comment

If you would like to order a subset of these genes contact us to discuss pricing.

Targeted Testing

For ordering targeted known variants, please proceed to our Targeted Variants landing page.

Turnaround Time

The great majority of tests are completed within 28 days.

Clinical Sensitivity

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 pathogenic variant 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 pathogenic variant 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). This test is predicted to detect pathogenic PTEN variants in ~80% of patients with Cowden syndrome, ~65% of patients with Bannayan–Riley–Ruvalcaba syndrome and ~20% of patients with Proteus syndrome (Eng 2003). Pathogenic variants can be identified in approximately 85% of individuals with tuberous sclerosis; 15% of individuals with TSC will not have a pathogenic variant identified. Individuals with a TSC pathogenic variant will either have an identifiable TSC2 or TSC1 variant in approximately 60-70% and 30% of cases, respectively (Northrup et al. 1993). Most Wilms tumors have a sporadic cause; germline pathogenic variants in WT1 are responsible for less than 20% cases of isolated Wilms tumors (non-syndromic) (Md Zin et al. 2011). Pathogenic variants in the FH gene will be detected by sequencing in 80-100% of individuals suspected of having hereditary leiomyomatosis and renal cell cancer (Pithukpakorn and Toro 1993). Clinical sensitivity of MET sequencing in hereditary papillary renal cell carcinoma is unknown at this time. Germline pathogenic variants in the SMARCB1 gene have been found in 23-60% of individuals with rhabdoid tumors, with increased rates observed at an earlier age of diagnosis (Frantzen et al. 1993). FLCN causative variants will be detected in approximately 88% of individuals with BHDS by sequencing. Almost half of the individuals with BHDS will have a deletion (c.1285delC) or duplication (c.1285dupC) of a C nucleotide in the polycytosine tract in exon 11 (Toro 1993). Although the majority of Paraganglioma/Pheochromocytoma tumors are sporadic (i.e. non-familial), approximately 13% of all PGL/PCC tumors are caused by germline mutations in known PGL/PCC syndrome genes (Welander et al. 2011). Clinical sensitivity for pathogenic variants is dependent on tumor location. For the SDHB gene, pathogenic variants are detectable in up to 44% of hereditary PGL/PCC cases; pathogenic variants in the SDHC gene are detectable in up to 8% of PGL/PCC hereditary cases; and pathogenic variants for the SDHD gene are detectable in up to 50% of hereditary PGL/PCC cases (Kirmani and Young 2012). Approximately 60% of individuals with hyperparathyroidism-jaw tumor syndrome will have a pathogenic variant in the CDC73 gene (Carpten et al. 2002), and 20-33% of individuals with sporadic parathyroid carcinoma (Shattuck et al. 2003; Masi et al. 2008). CDC73 pathogenic variants are a rare cause of familial isolated hyperparathyroidism, but can been seen in up to 7% of cases (Villablanca 2004; Masi et al. 2008). The clinical sensitivity of BAP1 germline pathogenic variants in patients with BAP1-related tumors is currently unknown.

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Deletion/Duplication Testing via aCGH

Test Code TestIndividual Gene PriceCPT Code Copy CPT Codes
600 BAP1$690.00 81479 Add to Order
CDC73$690.00 81479
FH$690.00 81479
FLCN$690.00 81479
MET$690.00 81479
MLH1$690.00 81294
MSH2$690.00 81297
MSH6$690.00 81300
PMS2$690.00 81319
PTEN$690.00 81323
SDHB$690.00 81479
SDHC$690.00 81479
SDHD$690.00 81479
SMARCB1$690.00 81479
TP53$690.00 81479
TSC1$540.00 81405
TSC2$540.00 81406
VHL$690.00 81403
WT1$690.00 81479
Full Panel Price* $1290.00
Test Code Test Total Price CPT Codes Copy CPT Codes
600 Genes x (19) $1290.00 81294, 81297, 81300, 81319, 81323, 81403, 81405, 81406, 81479(x11) Add to Order
Pricing Comment

# of Genes Ordered

Total Price

1

$690

2

$730

3

$770

4-10

$840

11-30

$1,290

31-100

$1,670

Over 100

Call for quote

Turnaround Time

The great majority of tests are completed within 28 days.

Clinical Sensitivity

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). Deletions of the PTEN gene are found in ~11% of patients with BRRS but not known for other PTEN related disorders (Eng 2003). Individuals with an identifiable TSC pathogenic variant will have either a large deletion or duplication in approximately 6% and 1% of cases in the TSC2 and TSC1 genes, respectively (Northrup et al. 2011). Clinical sensitivity of WT1 deletions are unknown for Wilms Tumor, but have been reported (Schumacher et al. 1997). Gross deletions and duplications of the FH gene will be detected in up to 14% of individuals affected with hereditary leiomyomatosis and renal cell cancer (Pithukpakorn and Toro 1993). Large deletions and duplications in the SMARCB1 gene that usually cannot be detected via sequencing are found in approximately 30% (11/35) affected individuals, with the majority of these being deletions (Eaton et al. 2011). Up to 28% of VHL pathogenic variants involve gross deletions (Frantzen et al. 1993). Large deletions and duplications that usually cannot be detected via sequencing have been reported in the FLCN gene for Birt-Hogg-Dube Syndrome (Benhammou et al. 2011). Deletions have been reported in the SDHB gene in 12% of patients (Cascón et al. 2006) and have also been reported less frequently in the SDHC and SDHD genes (Burnichon et al. 2009). Clinical sensitivity of large rearrangements in the CDC73 gene in CDC-Related disorders is unknown at this time, but gross deletions in CDC73 have been reported (Bricaire et al. 2013; Cascón et al. 2011; Domingues et al. 2012). Clinical sensitivity for BAP1 and MET gene deletions/duplications is currently unknown and have not been reported.

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Clinical Features

Renal cell carcinoma is the most common type of kidney cancer and includes many subtypes such as clear cell, papillary, chromophobe, and oncocytoma accounting for approximately 75%, 12%, 5% and 4% of cases. Although most of these renal tumors occur sporadically, about 3% of renal cell carcinomas (RCC) are considered familial (Maher 2011). Other less common types of kidney cancer which can also be familial, include transitional cell carcinomas, Wilms tumors, and renal sarcomas (http://www.cancer.org/). Individuals with hereditary renal cancers often show earlier ages of renal cancer diagnosis, which are often multifocal or bilateral, and these individuals may also have other physical characteristics which may be associated with specific syndromes (Coleman and Russo 2009; Linehan et al. 2010). The following syndromes that have renal cancer as a clinical feature include Birt-Hogg-Dube Syndrome, CDC73-Related Disorders, Hereditary Leiomyomatosis and Renal Cell Cancer (HLRCC), Hereditary Papillary Renal Cell Carcinoma (HPRCC), Li-Fraumeni Syndrome, Lynch Syndrome, PTEN Hamartoma Tumor Syndrome, Rhabdoid Tumor Predisposition Syndrome, Tuberous Sclerosis Complex, Tumor Predisposition Syndrome, Von Hippel-Lindau Disease, and Wilms Tumor.

Genetics

The renal cancer next generation sequencing panel assesses genes that have been shown to be causative when mutated for disorders that have renal cancer as a clinical feature.  For instance, VHL pathogenic variants cause Von Hippel-Lindau Disease, which is associated with renal clear cell carcinoma. The VHL gene is a tumor suppressor, and its protein product requires inactivation of both alleles at the cellular level leading to abnormal activation of genes involved in hypoxia (Maher et al. 2011).

MET pathogenic variants cause HPRCC, which is associated with type 1 papillary renal tumors. Pathogenic variants in the MET oncogene lead to ligand-independent activation of the tyrosine kinase domain of the protein, which leads to constitutive activation of the hepatocyte growth factor (HGF)/c- Met pathway (Coleman and Russo. 2009).

FH pathogenic variants are responsible for HLRCC and are associated with type 2 papillary renal tumors. It is thought that FH pathogenic variants result in a loss of function leading to increases in cellular fumurate. This increase causes decreased hypoxia-inducible factor (HIF) degradation and overexpression of genes further downstream in the HIF pathway leading to tumor formation (Pithukpakorn and Toro. 2010; Badeloe et al. 2009).

CDC73 pathogenic variants cause CDC73-Related disorders (e.g. parathyroid carcinoma) is associated with papillary RCC, hamartomas, and Wilms tumor). CDC73 encodes parafibromin, which is a tumor suppressor that is involved in regulating the cell cycle and gene expression (Bradley et al. 2006; Masi et al. 2008).

Pathogenic variants in the SDHB, SBHC, SDHD genes cause hereditary paraganglioma and pheochromocytoma syndrome. FLCN pathogenic variants cause Birt-Hogg-Dube syndrome. Both are associated with variable types of renal cancers. The tested SDH genes are nuclear genes, which encode three of the four subunits of the mitochondrial enzyme succinate dehydrogenase (SDH). The FLCN gene is a putative tumor suppressor that acts downstream of rapamycin (mTOR) and adenosine monophosphate-activated protein kinase (AMPK), and may have a role in the modulation of energy/nutrient sensing and signaling pathways (Hartnan 2009).

Pathogenic variants in the TSC1 and TSC2 genes cause Tuberous Sclerosis, which is associated with renal cell carcinoma and angiomylipoma. These genes encode tumor suppressors that are involved in cellular proliferation and act through multiple signaling pathways (e.g. mTOR/AKT pathways) (Orlova et al.  2010).

Pathogenic variants in the MLH1, MSH2, MSH6, and PMS2 genes cause Lynch Syndrome and are associated with transitional cell carcinomas of the renal pelvis and ureter. These genes are involved in mismatch repair, and pathogenic variants result in defective DNA repair leading to cancer.

SMARCB1 pathogenic variants cause Rhabdoid Tumor Predisposition syndrome and predisposes individuals to renal and extrarenal malignant rhabdoid tumors.  SMARCB1 encodes a tumor suppressor that functions as a member of  the human ATP-dependent SWI/SNF complex, which has a role in epigenetic modification by regulating gene transcription and DNA repair (Reisman et al. 2009).  

Pathogenic variants in the WT1 gene cause Wilms tumor. WT1 encodes a tumor suppressor that interacts with the Wnt/beta-catenin signaling pathway and many other downstream targets of cellular growth, differentiation and apoptosis (Md Zin et al. 2011).

BAP1 is a tumor suppressor gene that encodes a deubiquitinating enzyme containing numerous functional domains, including the ubiquitin C-terminal hydrolase (UCH) domain, a host cell factor-1 (HCF-1) binding domain and binding domains for BRCA1 and BARD1. BAP1 has been functionally implicated in numerous biologic processes, including chromatin dynamics, DNA damage response, and regulation of the cell cycle and cell growth (Goldstein et al. 2011).


Lastly, TP53 and PTEN pathogenic variants cause Li-Fraumeni syndrome and PTEN Hamartoma syndrome, respectively. These genes encode for tumor suppressors and pathogenic variants have been associated with renal cell carcinoma.

See individual gene test descriptions for additional information.

Testing Strategy

For this NGS panel, the full coding regions, plus ~20 bp 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. 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.

Indications for Test

This test is suitable for individuals with a clinical history of familial renal cancers. 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 renal tumors that are bilateral, multifocal, recurrent, or early onset (e.g. less than 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.

Genes

Official Gene Symbol OMIM ID
BAP1 603089
CDC73 607393
FH 136850
FLCN 607273
MET 164860
MLH1 120436
MSH2 609309
MSH6 600678
PMS2 600259
PTEN 601728
SDHB 185470
SDHC 602413
SDHD 602690
SMARCB1 601607
TP53 191170
TSC1 605284
TSC2 191092
VHL 608537
WT1 607102
Inheritance Abbreviation
Autosomal Dominant AD
Autosomal Recessive AR
X-Linked XL
Mitochondrial MT

Diseases

Name Inheritance OMIM ID
Bannayan-Riley-Ruvalcaba Syndrome AD 153480
Carcinoid Tumors, Intestinal AD 114900
Cowden Disease AD 158350
Cowden syndrome 3 AD 615106
Drash Syndrome AD 194080
Frasier Syndrome AD 136680
Hereditary Leiomyomatosis And Renal Cell Cancer AD 150800
Hereditary Nonpolyposis Colorectal Cancer Type 4 AD 614337
Hereditary Nonpolyposis Colorectal Cancer Type 5 AD 614350
Li-Fraumeni Syndrome AD 151623
Lynch Syndrome I AD 120435
Lynch Syndrome II AD 609310
Paraganglioma And Gastric Stromal Sarcoma AD 606864
Paragangliomas 1 AD 168000
Paragangliomas 3 AD 605373
Paragangliomas 4 AD 115310
Parathyroid Carcinoma AD 608266
Pheochromocytoma AD 171300
Renal Cell Carcinoma, Nonpapillary AD 144700
Renal Cell Carcinoma, Papillary, 1 AD 605074
Rhabdoid Tumor Predisposition Syndrome 1 AD 609322
Tuberous Sclerosis 1 AD 191100
Tuberous Sclerosis 2 AD 613254
Tumor Predisposition Syndrome AD 614327
Von Hippel-Lindau Syndrome AD 193300
Wilms' Tumor AD 194070

Related Tests

Name
CDC73-Related Disorders via the CDC73 Gene
Birt-Hogg-Dube Syndrome via the FLCN Gene
Cancer Sequencing and Deletion/Duplication Panel
Coffin-Siris Syndrome Sequencing Panel
Colorectal Cancer Sequencing And Deletion/Duplication Panel
Hereditary Breast and Ovarian Cancer Syndrome - HBOC EXPANDED Sequencing and Deletion/Duplication Panel
Hereditary Breast and Ovarian Cancer Syndrome - HBOC HIGH RISK Sequencing and Deletion/Duplication Panel
Hereditary Leiomyomatosis and Renal Cell Cancer or Fumarase Deficiency via the FH Gene
Hereditary Myelodysplastic Syndrome (MDS) / Acute Myeloid Leukemia (AML) Sequencing Panel
Hereditary Papillary Renal Cell Carcinoma via the MET Gene
Hereditary Paraganglioma-Pheochromocytoma Syndrome Sequencing Panel
Hereditary Paraganglioma-Pheochromocytoma Syndrome via the SDHB Gene
Hereditary Paraganglioma-Pheochromocytoma Syndrome via the SDHC Gene
Hereditary Paraganglioma-Pheochromocytoma Syndrome via the SDHD Gene
Li-Fraumeni Syndrome via the TP53 Gene
Lynch Syndrome Sequencing and Deletion/Duplication Panel
Lynch Syndrome via the MLH1 Gene
Lynch Syndrome via the MSH2 Gene
Lynch Syndrome via the PMS2 Gene
Lynch Syndrome via the MSH6 Gene
Mitochondrial Complex II Deficiency Sequencing Panel
Pancreatic Cancer Sequencing Panel
PTEN Hamartoma Tumor Syndrome via the PTEN Gene
Rhabdoid Tumor Predisposition Syndrome via the SMARCB1 Gene
Schwannomatosis Sequencing Panel
Steroid-Resistant Nephrotic syndrome via the WT1 Gene
Tuberous Sclerosis Complex Sequencing and Deletion/Duplication Panel
Tuberous Sclerosis Complex via the TSC1 Gene
Tuberous Sclerosis Complex via the TSC1 Gene
Tuberous Sclerosis Complex via the TSC2 Gene
Tuberous Sclerosis Complex via the TSC2 Gene
Tumor Predisposition Syndrome, Uveal Melanoma and Mesothelioma via the BAP1 Gene
Von Hippel-Lindau Disease via the VHL Gene
Wilms Tumor via the WT1 Gene

CONTACTS

Genetic Counselors
Geneticist
Citations
  • American Cancer Society.
  • Badeloe S., Frank J. 2009. European Journal of Dermatology : Ejd. 19: 545-51. PubMed ID: 19939761
  • Benhammou J.N. et al. 2011. Genes, Chromosomes & Cancer. 50: 466-77. PubMed ID: 21412933
  • Bradley K. J. et al. 2006. Clinical Endocrinology. 64: 299-306. PubMed ID: 16487440
  • Bricaire L. et al. 2013. The Journal of Clinical Endocrinology and Metabolism. 98: E403-8. PubMed ID: 23293331
  • Burnichon N. et al. 2009. The Journal of Clinical Endocrinology and Metabolism. 94: 2817-27. PubMed ID: 19454582
  • Carpten J.D. et al. 2002. Nature Genetics. 32: 676-80. PubMed ID: 12434154
  • Cascón A. et al. 2011. Genes, Chromosomes & Cancer. 50: 922-9. PubMed ID: 21837707
  • Cascón Alberto et al. 2006. Genes, Chromosomes and Cancer. 45: 213-219. PubMed ID: 16258955
  • Coleman J.A., Russo P. 2009. Current Opinion in Urology. 19: 478-85. PubMed ID: 19584731
  • Domingues R. et al. 2012. Clinical Endocrinology. 76: 33-8. PubMed ID: 21790700
  • Eaton K.W. et al. 2011. Pediatric Blood & Cancer. 56: 7-15. PubMed ID: 21108436
  • Eng C. 2003. Human Mutation. 22: 183-98. PubMed ID: 12938083
  • Frantzen C. et al. 1993. Von Hippel-Lindau Disease. 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: 20301636
  • Goldstein AM. 2011. Nature Genetics. 43: 925-6. PubMed ID: 21956388
  • Hartman TR. et al. 2009. Oncogene. 28: 1594-604. PubMed ID: 19234517
  • Kirmani S, Young W.F. 2012. Hereditary Paraganglioma-Pheochromocytoma Syndromes. 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: 20301715
  • Kohlmann W, Gruber S.B. 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
  • Linehan W.M. et al. 2010. Nature Reviews. Urology. 7: 277-85. PubMed ID: 20448661
  • Maher ER. 2011. Nephron. Experimental Nephrology. 118: e21-6. PubMed ID: 21071978
  • Maher ER. et al. 2011. European Journal of Human Genetics : Ejhg. 19: 617-23. PubMed ID: 21386872
  • Masi G. et al. 2008. Endocrine Related Cancer. 15: 1115-1126. PubMed ID: 18755853
  • Md Zin R. et al. 2011. Pathology. 43: 302-12. PubMed ID: 21516053
  • Northrup H. et al. 2011. Tuberous Sclerosis Complex. 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: 20301399
  • Orlova K.A., Crino P.B. 2010. Annals of the New York Academy of Sciences. 1184: 87-105. PubMed ID: 20146692
  • Peltomäki P., Vasen H. 2004. Disease Markers. 20: 269-76. PubMed ID: 15528792
  • Pithukpakorn M., Toro J.R. 1993. Hereditary Leiomyomatosis and Renal Cell 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: 20301430
  • Reisman D. et al. 2009. Oncogene. 28: 1653-68. PubMed ID: 19234488
  • Schneider K. et al. 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
  • Schumacher V. et al. 1997. Proceedings of the National Academy of Sciences of the United States of America. 94: 3972-7. PubMed ID: 9108089
  • Shattuck Trisha M. et al. 2003. New England Journal of Medicine. 349: 1722–1729. PubMed ID: 14585940
  • Syngal S. et al. 2000. Journal of Medical Genetics. 37: 641-5. PubMed ID: 10978352
  • Toro J.R. 1993. Birt-Hogg-Dubé 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: 20301695
  • Villablanca A. 2004. Journal of Medical Genetics. 41: 32e. PubMed ID: 14985403
  • Welander J. et al. 2011. Endocrine Related Cancer. 18: R253-R276. PubMed ID: 22041710
  • Wimmer Katharina, Etzler Julia. 2008. Human Genetics. 124: 105-122. PubMed ID: 18709565
Order Kits
TEST METHODS

NextGen Sequencing

Test Procedure

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.

Analytical Validity

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.   

Analytical Limitations

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

Test Procedure

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.

Analytical Validity

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.

Analytical Limitations

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.

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
  • The first four pages of the requisition form must accompany all specimens.
  • Billing information is on the third and fourth pages.
  • Specimen and shipping instructions are listed on the fifth and sixth pages.
  • All testing must be ordered by a qualified healthcare provider.

SPECIMEN TYPES
WHOLE BLOOD

(Delivery accepted Monday - Saturday)

  • Collect 3-5 ml (5 ml preferred) of whole blood in EDTA (purple top tube) or ACD (yellow top tube). For Test #500-DNA Banking only, collect 10-20 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 good for up to 48 hours.
  • If refrigerated, blood specimen is good for up to one week.
  • Label the tube with the patient name, date of birth and/or ID number.

DNA

(Delivery accepted Monday - Saturday)

  • NextGen Sequencing Tests: Send in screw cap tube at least 10 µg of purified DNA at a concentration of at least 50 µg/ml
  • Sanger Sequencing Tests: Send in a screw cap tube at least 15 µg of purified DNA at a concentration of at least 20 µg/ml. For tests involving the sequencing of more than three genes, send an additional 5 µg DNA per gene. DNA may be shipped at room temperature.
  • Deletion/Duplication via aCGH: Send in screw cap tube at least 1 µg of purified DNA at a concentration of at least 100 µg/ml.
  • Whole-Genome Chromosomal Microarray: Collect at least 5 µg of DNA in TE (10 mM Tris-cl pH 8.0, 1mM EDTA), dissolved in 200 µl at a concentration of at least 100 ng/ul (indicate concentration on tube label). DNA extracted using a column-based method (Qiagen) or bead-based technology is preferred.

CELL CULTURE

(Delivery accepted Monday - Thursday)

  • PreventionGenetics should be notified in advance of arrival of a cell culture.
  • Ship at least two T25 flasks of confluent cells.
  • Label the flasks with the patient name, date of birth, and/or ID number.
  • We do not culture cells.