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Next-Gen Sequencing and Deletion/Duplication Analysis of SPRED1 Only (SPD1-NG)

Information for Ordering

{slide=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)


{slide=Turnaround Time}

Average = 30 working days


{slide=Price, CPT codes, and Z code}

$800 (USD – institutional/self-pay price)

CPT: 81405 and 81479

Z code: ZB6AC


{slide=Candidates for Testing}

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


{slide=Specimen Shipping and Handling}

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.


{slide=Required Forms}

Test Requisition Form

Form for Customs



{slide=Disorder Background}

Germline loss-of-function variants in SPRED1, a negative regulator of the RAS-MAPK pathway,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 typicalNF1 associated features (Lisch nodules, bone abnormalities, neurofibromas, optic pathwaygliomas) 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 suchpatients, a correct diagnosis has important implications for prognosis, counseling, and potentialprenatal genetic diagnosis. Based on a cross-sectional study we estimate that patientspresenting sporadically with these pigmentary signs only will carry a variants inthe SPRED1 gene in ~1.3% of cases. When such patients have a family history of CALMs with orwithout 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.


{slide=Test Description}

The DNA-based SPRED1-only by NGS involves sequencing as well as deletion/duplication analysis of the entire coding SPRED1 regions. 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 of the SPRED1-only by NGS panel is >1600x with >98% of the codingregion ≥350x and >99% ≥200X, allowing detection of very low level mosaicism, down to 3-5% variant allele fraction respectively (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 frameshiftscaused by deletion/ duplication up to 112bp.

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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