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HelpTest Code LAB1821 Cerebral Cavernous Malformations (CCM) Sequencing Panel
Clinical System Name
Cerebral Cavernous Malformations Seq (CCM Seq)
Description
Cerebral cavernous malformations (CCMs) are collections of abnormal slow-flow capillaries in the central nervous system (brain and spinal cord) that are enlarged and irregular in structure. These capillaries have abnormally thin walls and lack support tissues such as elastic fibers, making them prone to leakage. The number of CCMs in an individual can increase with age and lesions can increase or decrease in size. Many individuals (20-50%) are asymptomatic, whereas others present with neurologic symptoms, such as seizures (40%-70%), headaches (10%-30%), and even fatal cerebral hemorrhage. CCMs can be sporadic or inherited; the latter referred to as familial CCM.
Sporadic CCM (sCCM) can be caused by somatic "activating" (gain-of-function) point mutations in MAP3K3 and/or PIK3CA. Or alternatively, sCCM can be caused by somatic loss-of-function mutations in the CCM genes CCM2, KRIT1, and PDCD10. Due to the mosaic nature of these variants, tissue-based testing of resected cavernoma tissue is recommended, if available. Testing blood or saliva-derived genomic DNA may be of little diagnostic value.
Familial CCM (fCCM) is an autosomal dominant disorder caused by a heterozygous pathogenic variant in one of three genes (CCM2, KRIT1, and PDCD10). Heterozygous pathogenic sequencing variants in these genes account for approximately 75% of individuals with familial CCMs. The use of deletion/duplication analysis of these 3 genes increases the detection rate by approximately 20%.
The Seattle Children's Hospital Molecular Laboratory offers different options for custom CCM testing:
- Cerebral Cavernous Malformations (CCM) Panel:
A 5 gene panel which includes CCM2, KRIT1, MAP3K3, PDCD10, PIK3CA
- Additional testing options - we offer the following additional options for custom CCM testing:
- Reflex to deletion/duplication analysis The provider has the option to order reflexive deletion/duplication analysis with each sequencing panel. (Please note this is NOT available for FFPE samples)
- Reflex to VANseq Expanded Panel The option to reflex to the Vascular Anomalies Expanded Sequencing Panel (VANseq) ± deletion/duplication analysis when the CCM Panel is non-diagnostic.
Please contact LabGC@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 this test
- 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. |
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| FFPE (Formalin-Fixed Paraffin-Embedded) Slides or Scrolls |
|
To send FFPE scrolls:
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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. |
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To send FFPE slides:
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| 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 |
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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) |
| 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 >7 days may be submitted to be assessed by our lab for acceptability for testing.
Availability
| STAT | Performed | TAT |
|---|---|---|
| Contact lab | Monday - Friday | 4-8 weeks |
Performing Laboratory
Seattle Children's Laboratory
Department
Department: Molecular Genetics Laboratory
Phone: 206-987-3872
Lab Client Services: 206-987-2617
Lab Genetic Counselor: LabGC@seattlechildrens.org
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.
Reported gene set:
CCM Panel: CCM2, KRIT1, MAP3K3, PDCD10, PIK3CA
VANseq Expanded Panel: 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
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 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
The diagnosis of familial CCM is established in an individual with either or both of the following criteria
- Multiple CCMs, or one CCM and at least one other family member with one or more CCMs
-
A heterozygous pathogenic variant in KRIT1, CCM2, or PDCD10 identified by molecular genetic testing
Approximately 20% to 50% of individuals with CCMs are asymptomatic throughout their lives. CCMs can present at any age, including infants and children. The majority of cases are evident between the second and fifth decade of life, with the mean age of presentation around 37 years of age. Clinically affected individuals most often present with seizures (40%-70%), focal neurologic deficits (35%-50%), nonspecific headaches (10%-30%), and cerebral hemorrhage (32%).
Approximately 95% of individuals with familial CCM will have a heterozygous pathogenic variant identified in one of three genes (CCM2, KRIT1, and PDCD10). Gene-targeted deletion/duplication testing should be considered when the CCM Sequencing Panel is non-diagnostic. Timely identification of the genetic cause of CCM can aid clinicians in making an accurate diagnosis and direct clinical management.
In approximately 5% of individuals with multiple lesions or a positive family history, no pathogenic variant is identified in any of the 3 genes associated with familial CCM.
When affected tissue is available, mutations in one of the 5 genes (KRIT1, CCM2, PDCD10, PIK3CA or MAP3K3) are detected in the majority (over 60%) of samples.
More recently, tissue-restricted activating mutations in PIK3CA have also been detected in resected cavernoma tissue, both in familial and sporadic CCM. The presence of a PIK3CA mutation may have prognostic value in determining the frequency of follow-up imaging.
Special Instructions
Link to: CCM GeneReviews