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Test Code LAB1920 VANseq Vascular Anomalies Sequencing Panel

Important Note

The test code for ALL of the sequencing panels and single gene sequencing requests listed below is VANseq Vascular Anomalies Sequencing Panel (TEST CODE: LAB1920). 

For electronic order entry in EPIC please use this test code and answer the order questions to make specific selections for sub-panel orders.

Additional Codes

VanSeq Panel*

 Genes
Capillary Malformations EPHB4, GNA11, GNAQ, RASA1
Lymphatic Malformations, Venous Malformations, Arteriovenous Malformations (LM/VM/AVM) ACVRL1, ARAF, BRAF, ELMO2, ENG, EPHB4, GDF2, GLMN, HRAS, KRAS, MAP2K1, MAP3K3, NRAS, PIK3CA, PIK3R1, PTEN, RASA1, TEK (TIE2)
Lymphedema ANGPT2, ARAF, BRAF, CCBE1, CELSR1, DCHS1, EPHB4, FAT4, FLT4, FOXC2, GATA2, GJC2, HGF, HRAS, KIF11, KRAS, MAP2K1, MDFIC, MET, NRAS, PIEZO1, PTPN14, RASA1, SOX18, VEGFC 
Vascular Tumor BRAF, CTNNB1, GNA11, GNA14, GNAQ, HRAS, IDH1, IDH2, KRAS, NRAS
VANseq Expanded ACVRL1, ANGPT2, ARAF, BRAF, CCBE1, CCM2, CELSR1, CTNNB1, DCHS1, ELMO2, ENG, EPHB4, FAT4, FGFR1, FLT4, FOXC2, GATA2, GDF2, GJC2, GLMN, GNA11, GNA14, GNAQ, HGF, HRAS, IDH1, IDH2, KIF11, KRAS, KRIT1, MAP2K1, MAP3K3, MDFIC, MET, NRAS, PDCD10, PDGFRB, PIEZO1, PIK3CA, PIK3R1, PTEN, PTPN14, RASA1, SMAD4, SOX18, TEK, VEGFC

*Detailed descriptions of VANSeq subpanels available here

 

Additional testing options - we offer the following additional options for custom VANseq testing:

  • Single-gene sequencing  When there is strong clinical suspicion, any gene listed on our VANseq Expanded Panel is available as a single-gene sequencing test.

  • Reflex to expanded sequencing Panel  The option to reflex to the expanded Vascular Anomalies Sequencing Panel (VANseq) when the single-gene or the focused panel test is non-diagnostic.
  • Reflex to deletion/duplication analysis  The provider has the option to order reflexive deletion/duplication analysis with each sequencing panel. Note that deletion/duplication analysis cannot be performed on FFPE tissue at this time.

Clinical System Name

VANseq (Vascular Anomalies Seq)

Description

Vascular anomalies are a heterogeneous group of disorders consisting of abnormalities in blood vessel growth and development. The International Society for the Study of Vascular Anomalies (ISSVA, https://www.issva.org/classification) classifies vascular anomalies into two major categories: vascular tumors, and vascular malformations. Vascular malformations are further classified according to the type of vessel affected and the speed of blood flow within the lesion: slow-flow capillary, venous, and lymphatic malformations (CM, VM, and LM) and high-flow arteriovenous malformations (AVMs). Primary lymphedema is also classified as a vascular malformation, though it is clinically and genetically distinct from lymphatic malformations.

 

Vascular anomalies can occur in isolation or as a part of a syndrome. The natural history, prognosis, and treatment of vascular anomalies vary greatly depending on the diagnosis, and genetic testing can be very helpful in making a specific diagnosis. In addition, molecular classification of vascular anomalies is beginning to guide specific pharmacologic treatment decisions.

 

The genetic causes of vascular anomalies are numerous. Anomalies can be localized, multifocal, or age dependent. Pathogenic variants can be inherited or de novo. Somatic mosaicism is a common feature for some vascular anomalies; consequently blood and saliva-derived DNA may be of little diagnostic value in some circumstances, and tissue-based testing may be required.

 

The Seattle Children's Hospital Molecular Laboratory offers different vascular anomalies panels based on clinical presentation with additional testing options. Detailed descriptions are available here. 

 

Please contact Lab Client Services, 206-987-2617, labclientservices@seattlechildrens.org, if you would like to order reflexive testing after the original testing report has been issued.

Sample Requirements

Samples MUST have two of the following to be accepted as properly labeled: first & last name, outside medical record number, unique accession number, or date of birth.

  • If both frozen tissue and FFPE tissue are available, frozen tissue from an affected site is preferred. Tissue sampled from an affected site is preferred over non-affected tissue.  
  • Please select one sample type for submission. Paired sample testing is NOT accepted unless approved by lab director. 
  • At this time we do NOT offer Cell-free DNA (cfDNA) testing for VANseq
  • For patients who have had a whole blood transfusion, wait 10 days post transfusion to draw blood or collect saliva for genetic testing. No wait time is necessary for blood or saliva collection if the patient received leuko-reduced red cells or plasma. 
  • Please contact Lab Client Services if you would like assistance selecting the most appropriate sample type.
Sample Requirement Important notes
Fresh frozen tissue (-70C)

25-50 mg in a sterile container.

A copy of the pathology report is recommended.

 

Specify tissue source and site on requisition.
Skin biopsy

2-4 mm punch biopsy of skin collected under sterile conditions in a sterile vial, frozen with no media.  

 

Also acceptable, refrigerate with 1-3 mL of tissue transport medium.

DO NOT use formaldehyde, formalin, alcohol, or 5% dextrose.  

 

Specify tissue source and site on requisition.
FFPE (Formalin-Fixed Paraffin-Embedded) Slides or Scrolls
  • 10 freshly cut sections of FFPE tissue, each with a thickness of 10 microns.
  • Also include one slide at 4-micron thickness stained with hematoxylin-and-eosin.

 

To send FFPE scrolls:

  • Use TWO 1.7mL polypropylene microcentrifuge tubes
  • No more than 5 scrolls/tube 

A copy of the pathology report is required.

 

Minimum acceptable tissue area is 10 square millimeters (total of 1 cubic millimeter of tissue). Tissue sections should contain as much lesional tissue as possible.

 

Note that deletion/duplication analysis cannot be performed on FFPE tissue at this time.

To send FFPE slides: 

  • Unstained, non-baked slides
  • Charged or uncharged slides may be used
Extracted DNA

2-5 mcg DNA from tissue or EDTA blood

 

DNA concentration minimum 50 µg/mL (40uL minimum volume); 260/280 ratio 1.70-2.00. 

 Isolation of nucleic acids for clinical testing must be performed in a CLIA-certified laboratory or a laboratory meeting equivalent requirements as determined by the CAP and/or the CMS. 
Saliva

Oragene Dx OGD-575/675 collection kit

 

Contact the lab directly for more information or to obtain a kit - 206-987-2563, ReferenceLabTeam@seattlechildrens.org.

 Manufacturer instructions must be followed. Please review link: How to Collect a Saliva Sample for Genetic Testing (Spanish).
Whole blood

1-3mL in Lavender/EDTA tube

  

 

Processing Instructions

 

Specimen Type Description

Temperature

 Storage instructions
Whole blood EDTA or ACD tube Refrigerate Molecular Genetics box in CPA refrigerator #2
Extracted DNA DNA aliquot tube Refrigerate Molecular Genetics box in CPA refrigerator #2
Fresh frozen tissue Frozen aliquot of 25-50 mg tissue Frozen (-70C) CPA -70 freezer (SNP array/Molecular box)
Skin biopsy Sterile container (no media) Frozen (-70C) CPA -70 freezer (SNP array/Molecular box)
In medium Refrigerate Molecular Genetics box in CPA refrigerator #2
FFPE Blocks, slides or scrolls Block, glass slides or sterile tube Room temp Place in CPA Cytogenetics room temp box with requisition
Saliva OGD-575/675 kit Room temp Place in CPA Cytogenetics room temp box with requisition

Off-site collection: Refrigerate blood samples until ready to ship. Transport blood, DNA, slides or scrolls at room temperature via overnight shipping. Transport fresh frozen tissue on dry ice.

Stability

Specimen Type Temperature Time
Tissue - Fresh Frozen -70 C years
Tissue - FFPE Slides RT years
Whole blood RT 3-5 d
Whole blood 2 - 8 C

7 d

Saliva fresh or extracted from ORAgene Dx OGD-575/675

 refrigerated or room temp up to 2 weeks
Extracted DNA RT, refrig or frozen  stable

Note: Whole blood samples > 7days may be submitted to be assessed by our lab for acceptability for testing.

Availability

STAT Performed TAT
Contact lab Monday - Friday 8 weeks

Performing Laboratory

Seattle Children's Laboratory

Department

Department:  Molecular Genetics Laboratory

Phone: 206-987-3872

Lab Client Services: 206-987-2617

CPT Codes

81479

Methodology

Method: Next Generation Sequencing technology using an Illumina NextSeq instrument. Target region includes coding exons and a minimum of 25 bp of flanking intron boundaries of the genes tested. Target enrichment performed using a custom Integrated DNA Technologies (IDT) panel. Deletion/duplication analysis is performed with ThermoFisher’s CytoScan XON array, which provides exon-level resolution for detection of copy number variants.

 

Average coverage >1,000x, depth of coverage for all target regions is at least 20x. Recurrent hotspot variants can be detected at lower levels of mosaicism (≥1%) but sensitivity will be affected by DNA quality and quantity.

 

See Description above for reported gene set of each panel
 

Limitations:

NGS: This method can detect single nucleotide variants, small deletions, and small insertions in the regions targeted. Some exons cannot be efficiently captured due to sequence homology or sequence properties. This method will not detect large insertions and deletions, complex indels, structural variants, or copy number variants. Variants located outside of targeted regions will not be detected. Mosaic variants present at <20% may not be reliably detected, and detection sensitivity of mosaic variants is dependent on the nature of the variant. Recurrent hotspot variants can be detected at lower levels of mosaicism (≥1%) but sensitivity will be affected by DNA quality and quantity.

 

Exon array: Analysis is limited to the targeted regions. When a copy number change is detected that extends into flanking genes, the genomic coordinates of the full variant will be reported. Copy number variants outside of the targeted genes are not reported. Regions of homozygosity are not routinely reported. In rare cases, exonic copy number variants that encompass a genomic interval under 500 bp may not be detected. The sensitivity of detection of mosaic copy number variants has not been evaluated.

Reference Range

Interpretive report will be provided. Variants are not reported if they are considered benign or likely benign.

Clinical Utility

Genetic testing of individuals with vascular anomalies can be helpful in confirming a specific diagnosis. Since the prognosis and treatment of these conditions varies greatly, and they are a relatively rare group of conditions, genetic testing may guide the management of these patients. In addition, molecular classification of vascular anomalies is beginning to guide specific pharmacologic treatment decisions. Note that due to the mosaic nature of some of these conditions, blood and saliva-based genetic tests may be of limited clinical utility. Genetic counseling is recommended.

 

A list of conditions for which genetic testing may be considered includes:

◾Blue rubber bleb (Bean) nevus syndrome

◾Capillary malformation-arteriovenous malformation syndrome

◾Cerebral cavernous malformations

◾CLAPO syndrome (capillary malformation lower lip, lymphatic malformation of the face, asymmetry of face and limbs, and overgrowth)

◾CLOVES syndrome (congenital lipomatous overgrowth with vascular anomalies, epidermal nevus and skeletal anomalies)

◾Congenital hemangioma (rapidly involuting and non-involuting)

◾Cowden syndrome

◾FAVA (fibroadipose vascular anomaly)

◾GATA2 related syndromes: Dendritic Cell, Monocyte, B and NK lymphoid deficiency (DCML); Emberger syndrome

◾Glomuvenous malformations

◾Gorham Stout Disease

◾Hennekam syndrome

◾Intraosseous vascular malformation

◾Isolated arteriovenous malformation

◾Isolated capillary malformation

◾Isolated lymphatic malformations

◾Isolated venous malformation

◾Juvenile nasopharyngeal angiofibroma

◾Klippel-Trenaunay syndrome

◾Lymphedema distichiasis

◾Lymphedema with hypotrichosis and telangiectasia

◾Lymphedema-choanal atresia

◾Megalencephaly with Capillary Malformation (MCAP)

◾Meige lymphedema

◾Microcephaly with lymphedema and chorioretinal dysplasia (MLCRD)

◾Milroy disease or Nonne-Milroy syndrome

◾Multiple cutaneous and mucosal venous malformations

◾Parkes-Weber syndrome

◾PTEN hamartoma tumor syndrome (PHTS)

◾Spindle cell hemangioma/Maffuci syndrome

◾Sturge-Weber syndrome

◾Tufted angioma/kaposiform hemangioendothelioma

◾Verrucous venous malformation

 

Special Instructions

Link to: SCH VANseq Panel Descriptions

Other References:

  • Luks VL et al. Lymphatic and other vascular malformative/overgrowth disorders are caused by somatic mutations in PIK3CA. J Pediatr. 2015 Apr;166(4):1048-54.e1-5, PMID: 25681199.
  • Revencu N et al. RASA1 mosaic mutations in patients with capillary malformation-arteriovenous malformation. J Med Genet. 2019 Jul 12. pii: jmedgenet-2019-106024, PMID: 31300548.
  • Queisser A, Boon LM, Vikkula M. Etiology and Genetics of Congenital Vascular Lesions. Otolaryngol Clin North Am. 2018 Feb;51(1):41-53, PMID: 29217067.
  • Boon LM, Vikkula M. Multiple Cutaneous and Mucosal Venous Malformations. 2008 Sep 18 [Updated 2018 May 17]. In: Adam MP, Ardinger HH, Pagon RA, et al., editors. GeneReviews® [Internet]. Seattle (WA): University of Washington, Seattle; 1993-2019. Available from: https://www.ncbi.nlm.nih.gov/books/NBK1967/
  • Bayrak-Toydemir P, Stevenson D. RASA1-Related Disorders. 2011 Feb 22 [Updated 2016 Oct 6]. In: Adam MP, Ardinger HH, Pagon RA, et al., editors. GeneReviews® [Internet]. Seattle (WA): University of Washington, Seattle; 1993-2019. Available from: https://www.ncbi.nlm.nih.gov/books/NBK52764/
  • Al-Olabi L et al. Mosaic RAS/MAPK variants cause sporadic vascular malformations which respond to targeted therapy. J Clin Invest. 2018 Apr 2;128(4):1496-1508, PMID: 29461977.

  • Tan WH et al. The spectrum of vascular anomalies in patients with PTEN mutations: implications for diagnosis and management. J Med Genet. 2007 Sep;44(9):594-602, PMID: 17526801.

  • Amyere M et al. Germline Loss-of-Function Mutations in EPHB4 Cause a Second Form of Capillary Malformation-Arteriovenous Malformation (CM-AVM2) Deregulating RAS-MAPK Signaling. Circulation. 2017 Sep 12;136(11):1037-1048, PMID: 28687708.

  • Nikolaev SI et al. Somatic Activating KRAS Mutations in Arteriovenous Malformations of the Brain. N Engl J Med. 2018 Jan 18;378(3):250-261, PMID: 29298116.

Requisition

Molecular VANseq & SpotSeq Requisition

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