NEUROFIBROMATOSIS Type 1

Comprehensive Testing from Blood (NFSP1/NF11)                                                           

DESCRIPTION

Mendelian Inheritance in Man number: 162200

Click here for Gene Reviews Clinical Summary.

Neurofibromatosis type 1 is a completely penetrant, autosomal dominant disorder with a frequency of 1/3500 births in all ethnic populations. NF1 is a progressive disorder, characterized by multiple café-au-lait spots, neurofibromas, and Lisch nodules, although additional features may develop.  NF1 is notorious for its variable expression. About 50% of cases are due to new dominant mutations, where neither parent has signs of the disorder. An affected individual has a 50% risk of transmitting NF1 to each offspring, although the degree of severity can differ from person to person, even within the same family. 

INDICATIONS FOR DIRECT TESTING

  • Individuals suspected to have NF1 in presence of only one of the NIH  diagnostic criteria
  • Individuals presenting with an atypical presentation of the disease
  • Individuals who seek confirmation of a clinical diagnosis
  • Individuals who want to prepare for prenatal / pre-implantation diagnosis


TESTING METHODOLOGY

NF1/SPRED1 Combination :

Cominbination NF1/SPRED1 testing (NFSP1)  includes direct sequencing of all coding exons, copy number analysis by MLPA plus RT-PCR and assessment of deep intronic splice mutations through RNA-studies for the NF1 gene. These splice mutations will not be detected if a DNA-based sequencing approach is used. Mutations screened for include truncating mutations (nonsense, frameshift, splicing mutations), missense mutations, multi-exon deletions and total gene deletions.  In certain circumstances when it is impossible for the MGL to receive a fresh blood sample within 60-72 hours of blood draw, the MGL offers a DNA-based sequence analysis using DNA extracted from an EDTA blood sample.  Please note that the comprehensive testing using an RNA/cDNA-based core assay complemented with copy number analysis is more sensitive and specific, and therefore the preferred method [Messiaen and Wimmer 2008, NF1 Mutational Spectrum].  If no clearcut pathogenic mutation is identified after exon by exon direct sequencing, copy number analysis by MLPA is recommended as the next step as 5% of mutations are total gene deletions and 2% are one to multiple exon deletions or duplications. [Wimmer et al. 2006, Genes Chromosomes Cancer]

In addition, patients will receive simultaneous comprehensive SPRED1 analysis. We offer a direct test based on a gDNA based core assay resulting in the full characterization of the SPRED1 mutation. The complete SPRED1 coding region is analyzed by sequencing exons 1 to 7. In addition, DNA copy number analysis by MLPA is performed.  Mutations screened for include truncating mutations (nonsense, frameshift, splicing mutations), missense mutations, multi-exon deletions or duplications and total gene deletions.


NF1 Only: 

We offer a direct test based on a RNA core assay and resulting in the full characterization of the NF1 mutation at the genomic DNA level. From a fresh EDTA blood sample, DNA is extracted directly and a short term phytohemagglutinin-stimulated lymphocyte culture is initiated and used as starting material to extract RNA. The complete NF1 coding region is analyzed by a cascade of complementary mutation detection techniques, including RT-PCR, direct sequencing, microsatellite marker analysis, copy number analysis by MLPA and interphase FISH (if needed), enabling us to identify the mutation in ~95% of non-founder patients fulfilling the NIH diagnostic criteria [Messiaen et al 2000, Messiaen and Wimmer 2005, Wimmer et al 2007, Messiaen and Wimmer, 2008]. 

Comprehensive NF1 testing (NF11) as such includes direct sequencing of all coding exons, copy number analysis by MLPA plus RT-PCR and assessment of deep intronic splice mutations through RNA-studies. These splice mutations will not be detected if a DNA-based sequencing approach is used. Mutations screened for include truncating mutations (nonsense, frameshift, splicing mutations), missense mutations, multi-exon deletions and total gene deletions.  In certain circumstances when it is impossible for the MGL to receive a fresh blood sample within 60-72 hours of blood draw, the MGL offers a DNA-based sequence analysis using DNA extracted from an EDTA blood sample.  Please note that the comprehensive testing using an RNA/cDNA-based core assay complemented with copy number analysis is more sensitive and specific, and therefore the preferred method [Messiaen and Wimmer 2008, NF1 Mutational Spectrum].  If no clearcut pathogenic mutation is identified after exon by exon direct sequencing, copy number analysis by MLPA is recommended as the next step as 5% of mutations are total gene deletions and 2% are one to multiple exon deletions or duplications. [Wimmer et al. 2006, Genes Chromosomes Cancer].


SPECIMEN REQUIREMENTS

We require 10 milliliters of whole blood. Blood samples must be collected in EDTA (purple topped) tubes. For pediatric patients or those for whom venipuncture is very difficult, please send a minimum of 3 ml in EDTA.

Segmental/ mosaic patients: We offer testing on affected tissue for patients believed to have segmental NF1 (cfr NF14C/NF14N).  Comprehensive testing can be performed on either neurofibromas or café-au-lait spot biopsies. 


TRANSPORT

If specimen is from clinics within UAB or Kirklin Clinic, please call 934-5562 for pick-up.

IMPORTANT!
Blood specimens must be kept at room temperature and received within 60-72 hours of collection.

1. DO NOT SHIP ON ICE.
2. Be sure that the shipping air bill is marked “Priority”, either Domestic or International.
3. 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 companies Diagnostic Envelope). You can use our collection kits, which we will send to physicians directly upon request.
4.   Please contact us (Email – medgenomics@uabmc.edu, Phone – 205-934-5562) prior to sample shipment and provide us with the date of shipment and the tracking number of the package, so that we can better ensure receipt of the samples within the 60-72-hour window. Please include the form for customs. This is especially important for samples sent from outside the US.


TURN AROUND TIME

Normal service: 25 working days

RUSH testing: 15 working days 


CPT CODES AND PRICES

Please find the most up to date prices and CPT codes for our testing services under the "Prices" tab of this website.


REQUIRED FORMS

NF1 Test Requisition including the phenotypic data form or NF1 Test Requisition-         

Seqmental NF1 including the phenotypic data form.

Form for customs (International shipment)

Note: Detailed and accurate completion of this document is necessary for reporting purposes. The Medical Genomics Laboratory issues its clinical reports based on the demographic data provided by the referring institution on the lab requisition form. It is the responsibility of the referring institution to provide accurate information. If an amended report is necessary due to inaccurate or illegible documentation, additional reports will be drafted with charge.

Requests for Molecular Genetic testing for NF1/SPRED1 will not be accepted for the following reasons:

  • No label (patients full name and date of collection) on the specimens
  • No referring physician’s or genetic counselor’s names and addresses
  • No billing information 
  • No phenotypic checklist

 

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

 

REFERENCES

Gutmann DH, Aylsworth A, Carey JC, Korf B, Marks J, Pyeritz RE, Rubenstein A, Viskochil D. (1997) The diagnostic evaluation and multidisciplinary management of neurofibromatosis 1 and neurofibromatosis 2. JAMA. Jul 2; 278(1): 51-7. Review. (pubmed)

Huson, SM; Hughes, RAC: The Neurofibromatoses: a Pathogenetic and Clinical Overview. London: Chapman & Hall Medical, 1994.  (pubmed)

Maertens O, De Schepper S, Vandesompele J, Brems H, Heyns I, Janssens S, Speleman F, Legius E, Messiaen L (2007) Molecular dissection of isolated disease features in mosaic neurofibromatosis type 1. Am J Hum Genet 81(2):243-251. (pubmed)

Messiaen LM, Callens T, Mortier G, Beysen D, Vandenbroucke I, Van Roy N, Speleman F, De Paepe A (2000) Exhaustive mutation analysis of the NF1 gene allows identification of 95% of mutations and reveals a high frequency of unusual splicing defects. Hum Mutat.15(6):541-55. (pubmed)

Messiaen LM and Wimmer K (2008) NF1 Mutational Spectrum, in Kaufmann D (ed): Neurofibromatoses. Monogr Hum Genet. Basel, Karger, Vol 16:63-77. (pubmed)

Messiaen L, Vogt J, Bengesser K, Fu C, Mikhail F, Serra E, Garcia-Linares C, Cooper DN, Lazaro C, Kehrer-Sawatzki H (2011) Mosaic type-1 NF1 microdeletions as a cause of both generalized and segmental neurofibromatosis type-1 (NF1).  Hum Mutat. 32(2):213-9.  (pubmed)

Messiaen L and Wimmer K (2005) Pitfalls of automated comparative sequence analysis as a single platform for routine clinical testing for NF1. J. Med. Genet. 42(5): e25. (pubmed)

Upadhyaya M, Huson SM, Davies M, Thomas N, Chuzhanova N, Giovannini S, Evans DG, Howard E, Kerr B, Griffiths S, Consoli C, Side L, Adams D, Pierpont M, Hachen R, Barnicoat A, Li H, Wallace P, Van Biervliet JP, Stevenson D, Viskochil D, Baralle D, Haan E, Riccardi V, Turnpenny P, Lazaro C, Messiaen L (2007) An absence of cutaneous neurofibromas associated with a 3-bp inframe deletion in exon 17 of the NF1 gene (c.2970-2972delAAT): evidence of a clinically significant NF1 genotype-phenotype correlation. Am J Hum Genet 80(1):140-51. (pubmed)

Wimmer K,  Roca X, Beiglbock H, Callens T, Etzler J, Rao A, Krainer A, Fonatsch C, Messiaen L (2007) Extensive in silico analysis of NF1 splicing defects uncovers determinants for splicing outcome upon 5’ splice-site disruption. Hum Mutat. 28(6): 599-612. (pubmed)

Wimmer K, Yao S, Claes K, Kehrer-Sawatzki H, Tinschert S, De Raedt T, Legius E, Callens T, Beiglböck H, Maertens O, Messiaen L (2006)  Spectrum of single- and multiexon NF1 copy number changes in a cohort of 1,100 unselected NF1 patients. Genes Chromosomes Cancer 45(3): 265-276.  (pubmed)