Next-Generation Sequencing and Deletion/Duplication Analysis of NF2 Only (NF2-NG)

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Information for Ordering

Acceptable Specimen Types

• Fresh blood sample: 3–6 mL EDTA; no time limitations associated with receipt
• Saliva: OGD-575 DNA Genotek collection kit; kits are provided upon request
• DNA extracted from lymphocyte cells: minimum volume of 25 µL containing 3 µg of DNA; A₂₆₀/A₂₈₀ ratio ≥1.8; must be extracted in a CLIA-certified or equivalent laboratory
• Tumor specimens: A minimum of two anatomically distinct tumor specimens is recommended; however, a single tumor specimen may be submitted. Tumor specimens should contain at least 70% tumor content and greater than 80% nucleated cells.
  • Flash-frozen tumor shipped on dry ice
  • Fresh tumor or affected-tissue biopsy immersed in sterile culture media, such as PBS or RPMI
  • Formalin-fixed, paraffin-embedded tumor block with a surface area of at least 5 mm², or three to six loose paraffin curls measuring 30–50 µm thick; do not submit slides

Turnaround Time

• Blood, saliva, or DNA: Average of 30 working days
• Fresh or frozen tumor or tumor block: Average of 50 working days

Price, CPT Codes, and Z Code

• Institutional/self-pay price for blood, saliva, or DNA: $800 USD
• Institutional/self-pay price for fresh or frozen tumor or tumor block: $1,500 USD
• CPT codes: 81406 and 81405
• Z code: ZB6AA

Candidates for Testing

Patients with bilateral vestibular schwannomas, with or without other typical features of NF2-related schwannomatosis, such as congenital cataracts, ependymomas, or facial weakness.

Testing may also be appropriate for patients with one or more features associated with NF2-related schwannomatosis, with or without a family history of the condition. Tumor-based testing may be considered when no variant is identified through blood-based testing.

Specimen Shipping and Handling

Please refer to the specimen requirements listed above.

Specimens must be packaged to prevent breakage. Absorbent material must be included to contain liquids if breakage occurs. Specimens must also be shipped in double watertight containers, such as a specimen pouch placed inside the shipping company’s diagnostic envelope.

To request a collection kit, please complete the Collection Kit Request Form .

Please complete the Fresh/Frozen Tumor Submission Checklist when submitting tumor specimens.

Before shipping a specimen, please contact the UAB Medical Genomics Laboratory by email at medgenomics@uabmc.edu or by phone at 205-934-5562. Please provide the specimen type, shipment date, and package tracking number so the laboratory can help ensure proper and timely receipt.

Required Forms

• Test Requisition Form

About

Disorder Background

NF2-related schwannomatosis is an autosomal dominant disorder characterized by bilateral vestibular schwannomas with associated symptoms of tinnitus, hearing loss, and balance dysfunction. Nearly all affected individuals develop bilateral vestibular schwannomas by 30 years of age. Individuals may also develop schwannomas of other cranial and peripheral nerves, meningiomas, and ependymomas.

NF2-related schwannomatosis has an estimated frequency of approximately 1 in 33,000–40,000 births across all populations. Approximately 50% of affected individuals have a de novo pathogenic variant, meaning neither parent shows signs of the disorder. The offspring of an affected individual have a 50% chance of inheriting the altered NF2 gene. NF2 is currently the only gene known to cause NF2-related schwannomatosis.

Test Description

The DNA-based NF2-only by NGS assay includes sequencing and deletion/duplication analysis of the entire coding region of NF2 using MLPA. The test utilizes extensively customized Agilent HaloPlex capture probes followed by 300 bp paired-end Illumina sequencing. Each coding exon and approximately 50 bp of flanking intronic sequence are analyzed. The 5′ and 3′ untranslated regions are not included. Sanger sequencing is performed as needed.

The average coverage is >1600× with 100% of the NF2 coding region covered at ≥350×. This allows detection of very low-level mosaicism (down to approximately a 3% variant allele fraction across the entire coding region with >95% confidence). Variant and copy number calls are generated using a validated bioinformatics pipeline capable of detecting single nucleotide variants, insertions, deletions, and duplication events up to 112 bp. Deletion/duplication analysis of NF2 is included because these alterations are an established component of the disease spectrum.

The variant detection rate in leukocyte DNA is greater than 90% in non-founder patients with NF2-related schwannomatosis. Variants detected include nonsense, frameshift, splice-site, missense, multi-exon deletion/duplication, and whole-gene deletion variants.

Approximately 25–30% of founder (simplex) patients with unaffected parents have mosaic pathogenic variants that are not detectable in blood lymphocytes. Blood-based testing generally detects mosaic variants only when the variant allele fraction exceeds approximately 10% (Evans et al., 2007). Identification of lower-level mosaic variants often requires analysis of tumor tissue (Evans et al., 2007). Because RNA is frequently degraded in available tumor specimens, comprehensive DNA-based analysis is performed for tumor testing.

REFERENCES available here.

Other Related Test Options

• Meningiomatosis / Multiple Meningioma Panel by Next-Generation Sequencing (MEN-NG)
• Schwannomatosis / Multiple Schwannoma Panel by Next-Generation Sequencing (SCH-NG)

For more information, test requisition forms, or collection kits, please contact the UAB Medical Genomics Laboratory at 205-934-5562 or medgenomics@uabmc.edu.

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