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Department of Genetics Heersink School of Medicine

RNA-based NF1/SPRED1 Testing on Cultured from Affected Tissues (NF14N/NF14C)

Information for Ordering

Acceptable Specimen Types
  •   A minimum of 2, 3-5mm anatomically distinct café-au-lait spot punch biopsies submitted in our provided culture media. Call, email, or see website to request a media collection kit
  •   A minimum of 2 anatomically distinct fresh neurofibroma biopsies submitted in our provided culture media. Call, email, or see website to request a media collection kit
  •   A blood (3-6ml whole blood in EDTA) specimen may be provided for free-of-charge targeted testing of any suspected germline (1st hit pathogenic variant) identified during biopsy based testing



Turnaround Time

Average = 120 working days


Price, CPT codes, and Z code:}

$2,600 for NF1-only* (USD – institutional/self-pay);

CPT: 88233, 81408 and 81479

Z code: ZB6AG for CALs, ZB67X for neurofibromas

*For CAL biopsies when necessary, added reflex to SPRED1 testing: $3,200 (USD total cost of testing – institutional/self-pay); additional CPT codes 81405 and 81404


Candidates for Testing

Patients suspected to have segmental NF1, with symptoms restricted to a defined area of the body; sporadic patients who have (mild) non-localized symptoms of NF1 and in whom no NF1 variant was identified in the blood lymphocytes and may have disease due to a postzygotic mutation; reflex testing for familial or sporadic patients with a first hit pathogenic variant refractory to detection by RNA- or DNA/NGS assay

Specimen Shipping and Handling

Please find specimen requirement specifications above.

Contact us to request a media collection kit or set up a time to discuss your patient prior to taking biopsy/biopsies in your patient, so we can provide individualized advice and ship out appropriate collection/transport media and forms prior to the procedure.

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 instructions for collecting and shipping biopsy specimens (CAL-spots) for NF1 Testing

Click here for instructions for collecting and shipping neurofibromas for NF1 Testing

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.


Required Forms

Test Requisition Form

Form for Customs


About

Disorder Background

The NF1 gene, cloned in 1990, was the first gene within the Ras-MAPK pathway shown to be associated with an autosomal dominant disorder, Neurofibromatosis type I (NF1). NF1 affects ~1/3000 individuals worldwide, with half of the patients being sporadic. NF1 is notorious for its phenotypic variability and is a progressive disorder with more signs developing with time. Although the NIH criteria enables clinicians to make a diagnosis in the majority of classically affected cases, diagnostic criteria are not met until a given age is reached. Atypical presentations also exist with patients not yet fulfilling NIH criteria by adulthood. The variant spectrum of NF1 is very complex and includes a wealth of unusual splice variants affecting exonic sequences as well as deep intronic variants resulting in exonization of intronic sequences at the mRNA level.

Germline loss-of-function variants in SPRED1, a negative regulator of the RAS-MAPK pathway, cause a neurofibromatosis type 1-like phenotype, first described in 2007 (Legius syndrome). Patients present with multiple café-au-lait spots with or without skinfold freckling. Other typical NF1 associated features (Lisch nodules, bone abnormalities, neurofibromas, optic pathway gliomas) are systematically absent. However, in some individuals Noonan-like features have been reported. In individuals with CALMs with or without freckling and no other specific distinguishing features, the NIH criteria cannot reliably distinguish NF1 from Legius syndrome. In such patients, a correct diagnosis has important implications for prognosis, counseling, and potential prenatal genetic diagnosis. Based on a cross-sectional study we estimate that patients presenting sporadically with these pigmentary signs only will carry a variants in the SPRED1 gene in ~1.3% of cases. When such patients have a family history of CALMs with or without freckling and no additional NF1-related criteria, a SPRED1 variants will be found in ~19% of cases. SPRED1 is a member of the SPROUTY/SPRED family of proteins that act as negative regulators of RAS-RAF interaction and mitogen-activated protein kinase (MAPK) signaling.

Test Description

The RNA-based NF1/SPRED1 testing on cultured cells from affected tissues is offered starting from biopsies of café-au-lait macules (CALM) and/or neurofibromas. Melanocytes cultured from CALMs and Schwann cells cultured from neurofibromas are the starting material to extract RNA. The complete NF1 coding region is analyzed by a cascade of complementary variant detection techniques, including RT-PCR, direct sequencing of cDNA fragments, microsatellite marker analysis, and copy number analysis by MLPA.
RNA-based NF1 testing allows finding deep intronic splice variants through their observed effect on splicing. These splice variants would not be detected if a “simple” exon-by-exon DNA-based (NGS/Sanger) sequencing approach is used. During the >15 years we have offered comprehensive RNA-based NF1 testing, we have identified >65 different locations harboring deep intronic splice variants; together they account for 2.5% of all pathogenic variants identified in the NF1 UAB cohort. Please note that all known deep intronic splice variants have been incorporated in the customized UAB NGS available assays. 
In addition, for patients with only pigmentary features (CALMs with/without skinfold freckling but no neurofibromas), and no NF1 variants found in the melanocytes (no first or second hit pathogenic variants), the SPRED1 gene is analyzed as a reflex test (sequencing and deletion/duplication analysis), as these patients may have mosaic Legius syndrome. As a result of this test, if features are NF1 or SPRED1-related, a common first hit is identified in both biopsies and a different second hit is identified in every anatomically different biopsy evaluated. If no variants are identified despite full analysis on 2 biopsies with successful cultures, (mosaic) NF1/Legius syndrome is very unlikely (<0.2%).

REFERENCES available here.


Other related test options:


For more information, test requisition forms, or sample collection and mailing kits, please call: 205-934-5562.

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