Next-Generation Sequencing and Deletion/Duplication Analysis of NF2 Only (NF2-NG)
Information for Ordering
Acceptable Specimen Types- Fresh blood sample (3-6 ml EDTA; no time limitations associated with receipt)
- Saliva (OGR-575 DNA Genotek; kits are provided upon request)
- DNA (extracted from lymphocyte cells; a minimum volume of 25μL at 3μg; O.D. of 260:280nm ≥1.8; must be extracted in a CLIA or equivalent certified lab)
- A minimum of 2 anatomically distinct tumor samples is suggested, however, a single tumor can be provided. Tumor specimens should contain at least 70% pure tumor content and >80% nucleated cells.
- Flash frozen tumor sent on dry ice
- Fresh tumor or affected tissue biopsy, immersed in sterile culture media (PBS/RPMI)
- Tumor block - should have a surface area ≥5mm squared or the specimen contains at least 3-6 loose paraffin curls (no slides) that are 30-50 microns thick
Average = 30 working days for blood, saliva, or DNA
Average = 50 working days for fresh/frozen tumor or tumor block
$800 for blood, saliva, or DNA (USD – institutional/self-pay)
$1,500 for fresh/frozen tumor or tumor block (USD – institutional/self-pay)
CPT: 81406 and 81405
Z code: ZB6AA
Candidates for Testing
Patients with bilateral vestibular schwannomas with or without other typical NF2- associated features (congenial cataracts, ependymoma, facial weakness, etc).
Patients with one or more features associated with NF2 with or without a family history of the condition and/or no variant was identified by blood based testing (for tumor based testing)
Please find the specimen requirement specifications above.
All submitted specimens must be sent at room temperature. DO NOT ship on ice.
Specimens must be packaged to prevent breakage, and absorbent material must be included in the package to absorb liquids in the event that breakage occurs. Also, the package must be shipped in double watertight containers (e.g. a specimen pouch + the shipping company’s diagnostic envelope).
To request a sample collection kit, please click here or email medgenomics@uabmc.edu to complete the specimen request form.
Click here for the Fresh/Frozen Tumor Submission Checklist
Please contact the MGL (via email at medgenomics@uabmc.edu, or via phone at 205-934-5562) prior to sample shipment and provide us with the date of shipment and tracking number of the package so that we can better ensure receipt of the samples.
About
Disorder BackgroundNF2-related schwannomatosis is an autosomal dominant disorder characterized by bilateral vestibular schwannomas with associated symptoms of tinnitus, hearing loss and balance dysfunction. Almost all affected individuals develop bilateral vestibular schwannomas by 30 years of age. Affected individuals may also develop schwannomas of other cranial and peripheral nerves, meningiomas, and ependymomas. NF2-related schwannomatosis is an autosomal dominant disorder with a frequency of 1:33-40,000 births in all populations. About 50% of patients are due to a de novo variant, where neither parent has signs of the disorder. The offspring of an affected individual have a 50% risk of inheriting the altered NF2 gene. NF2 is the only gene in which pathogenic variants are known to cause NF2-related schwannomatosis.
The NF2-only by NGS involves sequencing as well as deletion/duplication analysis of the entire coding NF2 region using MLPA. The test uses an extensively customized and optimized set of Agilent HaloPlex capture probes, followed by sequencing of overlapping amplicons within the regions of interest using 300bp paired-end Illumina sequencing chemistry. Each coding exon plus ~50bp of flanking intronic sequence are simultaneously sequenced. 5’ and 3’ untranslated sequences are not included. Sanger sequencing will be utilized as needed.
The average coverage is >1600x with 100% of the NF2 coding region ≥350x. The This allows for detection of very low-level mosaicism by sequencing (as low as 3% of the alleles in 100% of the coding region with >95% confidence). Variant and copy number calls are made using a unique bioinformatics pipeline detecting all types of variants including single nucleotide substitutions, indels, and frameshifts caused by deletion/ duplication up to 112bp. Deletion/duplication analysis for NF2 is included in this test, as such variants are a part of the variant spectrum for these conditions.
Variant detection rate in leukocytes is >90% in non-founder NF2 patients. Variants detected include truncating (nonsense, frameshift, splicing variants), missense variants, multi-exon deletions or duplications and total gene deletions.
In about 25-30% of founders (simplex cases, patients with unaffected parents), variants are not detected in blood lymphocytes as a result of somatic mosaicism. Only variants with mosaicism levels greater than 10% can be detected in lymphocyte DNA (Evans et al, 2007). Identification of the majority of mosaic variants requires testing of tumor tissue (Evans et al, 2007). As RNA is most often degraded in available tumor material, a DNA-based comprehensive analysis is applied.
REFERENCES available here.
Other related test options:
- Meningiomatosis/Multiple Meningioma Panel by Next-Gen Sequencing (MEN-NG)
- Schwannomatosis/Multiple Schwannoma Panel by Next-Gen Sequencing (SCH-NG)
For more information, test requisition forms, or sample collection and mailing kits, please call: 205-934-5562.
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