Next-Gen Sequencing and Deletion/Duplication Analysis of NF1 and SPRED1 (NFSP-NG)

Information for Ordering

• 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)

30 working days 

$1,100 (USD- institutional/self-pay price)

CPT: 81408, 81405, and 81479 (x2)

Z code: ZB6A8

Patients with multiple CALMs with/without skinfold freckling and no other typical NF1 features (Lisch nodules, bone abnormalities, neurofibromas, optic pathway gliomas)

Please find 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.

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

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 patients Noonan-like features are present.

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 NF1 gene in ~43% of cases and 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, an NF1 variants will be found in ~73% of cases and in the SPRED1 gene 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.


The DNA-based NF1/SPRED1-only by NGS involves sequencing as well as deletion/duplication analysis of the entire coding NF1 region plus the alternatively spliced exons 9br, 23a and 48a (67 exons total), as well as sequencing and deletion/duplication analysis for SPRED1. 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. The average coverage is >1600x with >98% of the NF1 coding region ≥350x and 99% ≥200x, allowing detection of very low level mosaicism, down to 3-5% variant allele fraction (regions covered by ≥350x and ≥200x respectively) 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.

Based on >15 years of experience with comprehensive RNA-based NF1 testing, we designed the customized and optimized NGS NF1-component of the assay to comprise all regions encountered through analysis of >15,000 unrelated individuals including >8,000 NF1-variant-positive individuals carrying 1 out of >3,100 different unique NF1 variants identified in the UAB MGL cohort. Included in the NGS assay are the regions covering >65 different deep intronic splice variants (which reside beyond the +/-50 intronic base pairs that flank all exons). Validation of the full panel included, besides substitutions (missense, nonsense, splice variants), the most challenging variants such as insertions/deletions/duplications of 1-112bp (~25% of the UAB NF1 cohort) and one-to-multiple exon deletions/duplications (~2.8% of the UAB NF1 cohort). The analytical sensitivity of our NGS testing approach was 100% for substitutions as well as insertion/deletions up to 112bp. The panel has been validated for the detection of germline (heterozygous) single-exon deletions/duplications as well as multi-exon deletions/duplications. Single exon deletions/duplications are present in ~0.45% of NF1-positive patients from the UAB cohort with 9% of these individuals being mosaic (~0.045% of all in the UAB NF1-positive cohort). 

With the largest dataset of NF1 genotypes matched with phenotypes, any genotype-phenotype correlations identified will be reported in real time.

Confirmatory testing of reportable variants is performed by Sanger sequencing or other orthogonal methods.

For novel NF1 variants of unknown significance, we offer free of charge targeted RNA-based testing to assess the effect of the variant on splicing and enhance the correct classification/ interpretation.

Relevant family members of a proband with any (novel or previously identified) variant of unknown significance are offered free of charge targeted analysis as long as accurate phenotypic data are provided by a health care professional to enhance the interpretation. There is no limitation to the number of relatives that can be tested free of charge.

Mosaicism is often present in sporadic patients with an NF1 microdeletion and has important repercussions for counseling. Evaluation by FISH analysis on 200 interphase chromosomes can be offered in such cases.

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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