Familial Mutation, Targeted Sequencing (TVARFAM) Print

WSLH Department: Cytogenetics
WSLH Test Code: 893M53
Availability: Monday-Saturday
Turn-around Time: 30 days
Recommended Uses: Familial variant testing
Additional Tests Performed:
Patient Preparations:
Specimen Requirements: Blood: 4 ml (preferred) - 2 ml (minimum) whole blood collected in EDTA vacuum type tube.

Dried Blood Spot: At least one circle completely filled and dried on Whatman FTA™ cards.

Saliva: Isohelix™ Saliva Collection kit used according to manufacturer instructions.

Buccal: Stabilized buccal swabs collected with SafeCollect kit (Zymo) according to manufacturer instructions.

Fresh/Frozen tissue: Sample size at least 0.3 cm cubed

Skin biopsies: 1-2mm punch biopsy

DNA: 3-5 ug DNA in TE buffer at a concentration of 50-100 ng/uL. For best results, DNA should be treated with RNAse. DNA must be extracted in a CLIA-certified Laboratory OR a laboratory meeting equivalent requirements as determined by the CAP and/or the CMS.
Specimen Handling & Transport: Store and transport specimens at room temperature (may transport with coolant during hot, >85 degrees Fahrenheit weather). DO NOT FREEZE blood. It is recommended that the laboratory receive the specimen within 48 hours of collection.
Follow collection kit procedure.
Collection Kit/Container:
Collection Instructions: Blood: Draw blood using aseptic techniques into a sterile EDTA vacuum type tube(s). Invert tube(s) to mix. If using larger tubes, draw to full volume to avoid over-treatment with anticoagulant.

Saliva/Buccal: Collect according to manufacturer instructions.

Fresh/frozen tissue: Tissue that is pale tan to brown should be avoided if possible as this indicates necrosis. A 0.3-0.5cm cubed section of each tissue type should be collected using aseptic procedures. Place the specimen in a sterile tissue vial containing transport media. If multiple tissues are sent, please place the placenta in one vial and the other tissue(s) in a separate vial to minimize contamination of the tissues.

For skin biopsies: Skin should be cleaned using alcohol, do not use iodine or betadine as these will compromise the cell growth in culture. Do a 1-2mm punch biopsy that goes full depth through the epidermis into the sub-cutaneous fat. Place the specimen in a sterile tissue vial containing transport media.

DNA extracted from peripheral blood, cord blood, buccal swab, saliva, fresh and frozen tissue (postnatal), dried blood spots. We DO NOT accept DNA from Formalin-Fixed Paraffin-Embedded (FFPE) Tissue. Label with the patient name plus a second identifier. DNA concentration and volume must also be provided on the specimen label.
Unacceptable Conditions: Blood that is clotted or hemolyzed is not acceptable. Blood must not be frozen.
Requisition Form: Cytogenetics Lab Genetic Diagnosis Form #131
Required Information: Documentation of the specific familial variant(s) is required and must be provided with the specimen in order to perform this test. If the original testing in the family was performed by another laboratory (not WSLH), please provide a copy of the original test report. A positive familial control may be needed. Familial testing of variants of uncertain clinical significance (VUS) may be performed on a case by case basis. Consultation with the laboratory is required prior to ordering this test. Laboratory regulations require the following minimum information to be provided on the requisition form for a specimen to be accepted for testing: Patient name or unique identifier; date and time of collection, patient date of birth and sex, specimen type/site of collection, test request(s), reason for referral (detailed phenotype information), clinician name and UPIN/NPI, and address for reporting results. Please be certain that name/identifier on the form matches that on the specimen label.
Results Include: Reports contain:
-gene names
-disease association
-inheritance (ex. Autosomal recessive, autosomal dominant)
-zygosity (homozygous, heterozygous)
-variant classification
Limitations: Mutations may be present below the limit of detection.
Rare diagnostic errors can occur due to primer site mutations. Mutations, other than the one(s) specifically requested, were not evaluated.
Additional Tests Recommended:
Additional Comments: Additional variant analysis available upon request
Methodology: DNA is extracted from the specimen and targeted variant testing is performed by Sanger sequencing. The region surrounding the variant of interest is amplified by PCR and sequenced in both the forward and reverse directions. Sequences are aligned to a reference sequence (GRCh37/hg19) to identify variants. Each variant is evaluated using databases (e.g., ClinVar, 1000 Genomes, NHLBI GO Exome Sequencing Project, etc), published literature, clinical correlation, segregation analysis, and predicted functional or splicing impact (using computational tools such as PolyPhen, SIFT, etc). Variant classification follows ACMG guidelines (Richards et al 2015, PMID:25741868).
Includes: Familial variant testing should be ordered if a pathogenic or likely pathogenic variant was previously identified in the family. Familial variant testing may also be ordered for individuals who wish to have clinical laboratory confirmation of pathogenic or likely pathogenic variants identified through research laboratories.
CPT Code: 81403
Price: For pricing information, please call 608-262-0402.
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