Neurofibromatosis Type 1 and Related Disorders via the NF1 Gene

  • Summary and Pricing
  • Clinical Features and Genetics
  • Citations
  • Methods
  • Ordering/Specimens
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NGS Sequencing

Test Code Test Copy GenesIndividual Gene PriceCPT Code Copy CPT Codes
4361 NF1$640.00 81408 Add to Order
Pricing Comment

Our most cost-effective testing approach is NextGen sequencing with Sanger sequencing supplemented as needed to ensure sufficient coverage and to confirm NextGen calls that are pathogenic, likely pathogenic or of uncertain significance. If, however, full gene Sanger sequencing only is desired (for purposes of insurance billing or STAT turnaround time for example), please see link below for Test Code, pricing, and turnaround time information.

The Sanger Sequencing method for this test is NY State approved.

For Sanger Sequencing click here.
Targeted Testing

For ordering targeted known variants, please proceed to our Targeted Variants landing page.

Turnaround Time

The great majority of tests are completed within 26 days.

Clinical Sensitivity

This test will detect pathogenic variants in the NF1 gene in ~ 60 % of patients with NIH clinical diagnostic criteria for neurofibromatosis type 1 (van Minkelen et al. 2014).

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Test Code Test Copy GenesIndividual Gene PriceCPT Code Copy CPT Codes
2057 NF1$540.00 81479 Add to Order
Turnaround Time

The great majority of tests are completed within 20 days.

Clinical Sensitivity

Deletions and duplications of the NF1 gene represent approximately 5% of cases (Friedman 2014).

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Clinical Features

Neurofibromatosis type 1 (NF1) is characterized by cutaneous neurofibromas, café-au-lait spots, iris hamartoma (Lisch nodules) and freckling of axillary and inguinal regions. These features usually become apparent during puberty. Additional features include plexiform neurofibromas, central nervous system gliomas, including optic glioma, macrocephaly, scoliosis, pseudoarthritis, overgrowth and learning difficulties (Riccardi 1993). There is extensive clinical variability between individuals in age of onset, tumor burden, and disease progression. NF1 is panethnic and affects 1 in 3,000 people (Rasmussen and Friedman 2000).

Neurofibromatosis-Noonan Syndrome patients (NFNS) present with clinical features characteristic of both neurofibromatosis type 1 (NF1) and Noonan syndrome (NS). The NF1 features include café-au-lait spots, freckling, neurofibromas, hamartoma of the iris, optic gliomas, and other MRI findings, such as unidentified bright objects (UBO). The NS features include facial anomalies such as hypertelorism, low-set ears, short stature, congenital heart defects, primary pulmonary valve stenosis, webbed neck and thoracic abnormalities. In NFNS patients, features common to both NF1 and NS include macrocephaly, scoliosis, mental retardation or learning difficulties (Allanson et al.1985; De Luca et al. 2005).

Spinal Neurofibromatosis (SNF) is characterized by multiple bilateral spinal neurofibromas and other tumors of the central nervous system, with few or no other clinical features of NF1.  Symptoms usually begin in adulthood (Kluwe et al. 2003; Quintáns et al. 2011).

Features of Watson Syndrome (WS) overlap with those of NF1 and Noonan syndrome. WS is characterized by café-au-lait spots, pulmonary valvular stenosis, low intelligence and short stature (Watson et al. 1967).  WS is also referred to as Pulmonic Stenosis with Café au Lait Spots.


NF1 is caused by loss-of-function mutations in the NF1 gene.  It is inherited as an autosomal dominant trait in about half of cases, and is caused by de novo mutations in the other half.  Over 2,000 NF1 germline variants have been reported and include all types. Large deletions account for ~ 5% of patients with NF1 and are usually associated with a severe phenotype (Friedman, 2014). Gross insertions and complex rearrangements are rare. Nearly all de novo mutations occur in the paternal chromosomes (Jadayel et al.1990), with the exception of large deletions, which occur in maternal chromosomes (Lázaro et al. 1996; Upadhyaya et al. 1998). Parental germline mosaicism has been reported (Lázaro et al. 1994).

The vast majority of NFNS cases are sporadic, although several families transmitting the trait in an autosomal dominant manner have been reported (Quattrin et al.1987; Abuelo et al. 1988; Colley et al. 1996). Heterozygous mutations in the NF1 gene were reported in patients with NFNS, including sporadic and familial cases (Baralle et al. 2003; De Luca et al. 2005; Stevenson et al. 2006; Huffmeier et al. 2006). These data led the authors to suggest that NFNS represents an allelic variation of NF1. At least 10 different pathogenic variants in the NF1 gene were detected in patients with NFNS.  About half of these are missense, the other half are small deletions or insertions that are predicted to result either in frameshift or in-frame deletions.  The majority of the NFNS-causing variants are clustered in exons 27 to 35. Four of the NFNS-causing pathogenic variants were found in patients with classic NF1; these pathogenic variants, however, were outside of exons 27-35. 

WS is also caused by autosomal dominant variants in the NF1 gene (Tassabehji et al. 1993).  To date, one large deletion and one small in-frame duplication have been reported (Tassabehji et al. 1993; HGMD).

NF1 encodes the neurofibromin protein, a negative regulator of the RAS/MAPK pathway.

Testing Strategy

For this NextGen test, the full coding regions plus ~10 bp of non-coding DNA flanking each exon are sequenced for the gene listed below. Sequencing is accomplished by capturing specific regions with an optimized solution-based hybridization kit, followed by massively parallel sequencing of the captured DNA fragments. Additional Sanger sequencing is performed for any regions not captured or with insufficient number of sequence reads. All pathogenic, likely pathogenic, or variants of uncertain significance are confirmed by Sanger sequencing.

Indications for Test

Patients with NF1, NFNS, SNF, and WS are candidates.


Official Gene Symbol OMIM ID
NF1 613113
Inheritance Abbreviation
Autosomal Dominant AD
Autosomal Recessive AR
X-Linked XL
Mitochondrial MT


Genetic Counselors
  • Abuelo DN, Meryash DL. 1988. Neurofibromatosis with fully expressed Noonan syndrome. Am. J. Med. Genet. 29: 937–941. PubMed ID: 3135755
  • Allanson JE, Hall JG, Allen MI Van. 1985. Noonan phenotype associated with neurofibromatosis. Am. J. Med. Genet. 21: 457–462. PubMed ID: 2411134
  • Baralle D, Mattocks C, Kalidas K, Elmslie F, Whittaker J, Lees M, Ragge N, Patton MA, Winter RM, ffrench-Constant C. 2003. Different mutations in the NF1 gene are associated with Neurofibromatosis-Noonan syndrome (NFNS). Am. J. Med. Genet. A 119A: 1–8. PubMed ID: 12707950
  • Colley A, Donnai D, Evans DG. 1996. Neurofibromatosis/Noonan phenotype: a variable feature of type 1 neurofibromatosis. Clin. Genet. 49: 59–64. PubMed ID: 8740913
  • De Luca A, Bottillo I, Sarkozy A, Carta C, Neri C, Bellacchio E, Schirinzi A, Conti E, Zampino G, Battaglia A, Majore S, Rinaldi MM, Carella M, Marino B, Pizzuti A, Digilio MC, Tartaglia M, Dallapiccola B. 2005. NF1 Gene Mutations Represent the Major Molecular Event Underlying Neurofibromatosis-Noonan Syndrome. Am J Hum Genet 77: 1092-1101. PubMed ID: 16380919
  • Friedman JM. 2014. Neurofibromatosis 1. In: Pagon RA, Adam MP, Ardinger HH, Wallace SE, Amemiya A, Bean LJ, Bird TD, Dolan CR, Fong C-T, Smith RJ, and Stephens K, editors. GeneReviews(®), Seattle (WA): University of Washington, Seattle. PubMed ID: 20301288
  • HGMD (Human Gene Mutation Database)
  • Hüffmeier U, Zenker M, Hoyer J, Fahsold R, Rauch A. 2006. A variable combination of features of Noonan syndrome and neurofibromatosis type I are caused by mutations in the NF1 gene. Am. J. Med. Genet. A 140: 2749–2756. PubMed ID: 17103458
  • Jadayel D, Fain P, Upadhyaya M, Ponder MA, Huson SM, Carey J, Fryer A, Mathew CG, Barker DF, Ponder BA. 1990. Paternal origin of new mutations in von Recklinghausen neurofibromatosis. Nature 343: 558–559. PubMed ID: 2105472
  • Kluwe L, Tatagiba M, Fünsterer C, Mautner V-F. 2003. NF1 mutations and clinical spectrum in patients with spinal neurofibromas. J. Med. Genet. 40: 368–371. PubMed ID: 12746402
  • Lázaro C, Gaona A, Ainsworth P, Tenconi R, Vidaud D, Kruyer H, Ars E, Volpini V, Estivill X. 1996. Sex differences in mutational rate and mutational mechanism in the NF1 gene in neurofibromatosis type 1 patients. Hum. Genet. 98: 696–699. PubMed ID: 8931703
  • Lázaro C, Ravella A, Gaona A, Volpini V, Estivill X. 1994. Neurofibromatosis type 1 due to germ-line mosaicism in a clinically normal father. N. Engl. J. Med. 331: 1403–1407. PubMed ID: 7969279
  • Quattrin T, McPherson E, Putnam T. 1987. Vertical transmission of the neurofibromatosis/Noonan syndrome. Am. J. Med. Genet. 26: 645–649. PubMed ID: 3105315
  • Quintáns B, Pardo J, Campos B, Barros F, Volpini V, Carracedo á, Sobrido MJ. 2011. Neurofibromatosis without Neurofibromas: Confirmation of a Genotype-Phenotype Correlation and Implications for Genetic Testing. Case Reports in Neurology 3: 86–90. PubMed ID: 21532985
  • Rasmussen SA, Friedman JM. 2000. NF1 gene and neurofibromatosis 1. American Journal of Epidemiology 151: 33–40. PubMed ID: 10625171
  • Riccardi VM. 1993. Genotype, malleotype, phenotype, and randomness: lessons from neurofibromatosis-1 (NF-1). American journal of human genetics 53: 301. PubMed ID: 8328448
  • Stevenson DA, Viskochil DH, Rope AF, Carey JC. 2006. Clinical and molecular aspects of an informative family with neurofibromatosis type 1 and Noonan phenotype. Clin. Genet. 69: 246–253. PubMed ID: 16542390
  • Tassabehji M, Strachan T, Sharland M, Colley A, Donnai D, Harris R, Thakker N. 1993. Tandem duplication within a neurofibromatosis type 1 (NF1) gene exon in a family with features of Watson syndrome and Noonan syndrome. American journal of human genetics 53: 90-95. PubMed ID: 8317503
  • Upadhyaya M, Ruggieri M, Maynard J, Osborn M, Hartog C, Mudd S, Penttinen M, Cordeiro I, Ponder M, Ponder BA, Krawczak M, Cooper DN. 1998. Gross deletions of the neurofibromatosis type 1 (NF1) gene are predominantly of maternal origin and commonly associated with a learning disability, dysmorphic features and developmental delay. Hum. Genet. 102: 591–597. PubMed ID: 9654211
  • van Minkelen R. et al. 2014. Clinical Genetics. 85: 318-327. PubMed ID: 23656349
  • Watson GH. 1967. Pulmonary stenosis, café-au-lait spots, and dull intelligence. Archives of disease in childhood 42: 303-307. PubMed ID: 6025371
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NextGen Sequencing using PG-Select Capture Probes

Test Procedure

We use a combination of Next Generation Sequencing (NGS) and Sanger sequencing technologies to cover the full coding regions of the listed genes plus ~20 bases of non-coding DNA flanking each exon.  As required, genomic DNA is extracted from the patient specimen.  For NGS, patient DNA corresponding to these regions is captured using an optimized set of DNA hybridization probes.  Captured DNA is sequenced using Illumina’s Reversible Dye Terminator (RDT) platform (Illumina, San Diego, CA, USA).  Regions with insufficient coverage by NGS are covered by Sanger sequencing.  All pathogenic, likely pathogenic, or variants of uncertain significance are confirmed by Sanger sequencing.

For Sanger sequencing, Polymerase Chain Reaction (PCR) is used to amplify targeted regions.  After purification of the PCR products, cycle sequencing is carried out using the ABI Big Dye Terminator v.3.0 kit.  PCR products are resolved by electrophoresis on an ABI 3730xl capillary sequencer.  In nearly all cases, cycle sequencing is performed separately in both the forward and reverse directions.

Patient DNA sequence is aligned to the genomic reference sequence for the indicated gene region(s). All differences from the reference sequences (sequence variants) are assigned to one of five interpretation categories, listed below, per ACMG Guidelines (Richards et al. 2015).

(1) Pathogenic Variants
(2) Likely Pathogenic Variants
(3) Variants of Uncertain Significance
(4) Likely Benign Variants
(5) Benign, Common Variants

Human Genome Variation Society (HGVS) recommendations are used to describe sequence variants (  Rare variants and undocumented variants are nearly always classified as likely benign if there is no indication that they alter protein sequence or disrupt splicing.

Analytical Validity

As of March 2016, 6.36 Mb of sequence (83 genes, 1557 exons) generated in our lab was compared between Sanger and NextGen methodologies. We detected no differences between the two methods. The comparison involved 6400 total sequence variants (differences from the reference sequences). Of these, 6144 were nucleotide substitutions and 256 were insertions or deletions. About 65% of the variants were heterozygous and 35% homozygous. The insertions and deletions ranged in length from 1 to over 100 nucleotides.

In silico validation of insertions and deletions in 20 replicates of 5 genes was also performed. The validation included insertions and deletions of lengths between 1 and 100 nucleotides. Insertions tested in silico: 2200 between 1 and 5 nucleotides, 625 between 6 and 10 nucleotides, 29 between 11 and 20 nucleotides, 25 between 21 and 49 nucleotides, and 23 at or greater than 50 nucleotides, with the largest at 98 nucleotides. All insertions were detected. Deletions tested in silico: 1813 between 1 and 5 nucleotides, 97 between 6 and 10 nucleotides, 32 between 11 and 20 nucleotides, 20 between 21 and 49 nucleotides, and 39 at or greater than 50 nucleotides, with the largest at 96 nucleotides. All deletions less than 50 nucleotides in length were detected, 13 greater than 50 nucleotides in length were missed. Our standard NextGen sequence variant calling algorithms are generally not capable of detecting insertions (duplications) or heterozygous deletions greater than 100 nucleotides. Large homozygous deletions appear to be detectable.   

Analytical Limitations

Interpretation of the test results is limited by the information that is currently available.  Better interpretation should be possible in the future as more data and knowledge about human genetics and this specific disorder are accumulated.

When Sanger sequencing does not reveal any difference from the reference sequence, or when a sequence variant is homozygous, we cannot be certain that we were able to detect both patient alleles.  Occasionally, a patient may carry an allele which does not amplify, due to a large deletion or insertion.   In these cases, the report will contain no information about the second allele.  Our Sanger and NGS Sequencing tests are generally not capable of detecting Copy Number Variants (CNVs).

We sequence all coding exons for each given transcript, plus ~20 bp of flanking non-coding DNA for each exon.  Test reports contain no information about other portions of the gene, such as regulatory domains, deep intronic regions or any currently uncharacterized alternative exons.

In most cases, we are unable to determine the phase of sequence variants.  In particular, when we find two likely causative mutations for recessive disorders, we cannot be certain that the mutations are on different alleles.

Our ability to detect minor sequence variants due to somatic mosaicism is limited.  Sequence variants that are present in less than 50% of the patient’s nucleated cells may not be detected.

Runs of mononucleotide repeats (eg (A)n or (T)n) with n >8 in the reference sequence are generally not analyzed because of strand slippage during PCR.

Unless otherwise indicated, DNA sequence data is obtained from a specific cell-type (usually leukocytes from whole blood).   Test reports contain no information about the DNA sequence in other cell-types.

We cannot be certain that the reference sequences are correct.

Rare, low probability interpretations of sequencing results, such as for example the occurrence of de novo mutations in recessive disorders, are generally not included in the reports.

We have confidence in our ability to track a specimen once it has been received by PreventionGenetics.  However, we take no responsibility for any specimen labeling errors that occur before the sample arrives at PreventionGenetics.

Multiplex Ligation-Dependent Probe Amplification Assay

Test Procedure

As required, genomic DNA (gDNA) is extracted from the patient specimen. gDNA extracted from blood samples/submitted DNA from the patient is denatured and hybridized to MLPA probes specific to exonic or intronic regions of a particular gene(s). Each probe consists of two adjacent sequences that once hybridized to patient/reference DNA are ligated into a single probe.  Fluorescently labeled PCR is then used to amplify each ligated probe using a common PCR primer set. The amplicon is then visualized during fragment separation using a capillary electrophoresis instrument. The relative peak height of each amplified probe from the patient’s sample is compared to four internal negative control results to determine relative copy number.  For each patient sample the data for only the gene(s) of interest is analyzed and reported.

Analytical Validity

MLPA enables the detection of relatively small deletion and amplification mutations within a single exon of a given gene or deletion and amplification mutations encompassing the entire gene. PreventionGenetics has established and verified this test’s accuracy and precision.

Analytical Limitations

Interpretation of the test results is limited by the information that is currently available. Better interpretation should be possible in the future as more data and knowledge about human genetics and this specific disorder are accumulated.

Only the indicated gene or genes were analyzed. Test reports contain no information about other regions of the genome, including genes that are not requested, and genes that are not targeted. This test does not provide any information about deletions or duplications within repetitive elements.

Balanced translocations or inversions within a targeted gene, or large unbalanced translocations or inversions that extend beyond the genomic location of a targeted gene are not detected.

We cannot determine if the duplicated segment is inserted in tandem within the gene or inserted elsewhere in the genome. Similarly, we cannot determine the orientation of the duplicated segment (direct or inverted), and whether it will disrupt the open reading frame of the given gene.

For a single probe deletion or duplication we will compare MLPA results to sequencing results to ensure that no single nucleotide polymorphisms are underlying the specific probe, which may affect probe hybridization.

Any partial exonic deletions and duplications outside the probe binding sequence cannot be detected.

Impurities in the test and reference DNA samples can increase the chance of false positive or negative results. Where possible similar DNA extraction methods between test and reference samples are ideal for relative copy number analysis.

Our ability to detect minor copy number change, due for example to somatic mosaicism may be limited. 

Unless otherwise indicated, MLPA results are based on DNA isolated from a specific tissue (usually leukocytes). Test reports contain no information about copy number changes in other tissues. 

We cannot be certain that the reference sequence(s) are correct. Exons, for example, may be misidentified.

We have confidence in our ability to track a specimen once it has been received by PreventionGenetics. However, we take no responsibility for any specimen labeling errors that occur before the sample arrives at PreventionGenetics. 

Normal findings within a targeted gene do not rule out the clinical diagnosis of a genetic disease.

Genetic counseling to help explain test results to the patients and to discuss reproductive or medical options is recommended.

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Ordering Options

myPrevent - Online Ordering
  • The test can be added to your online orders in the Summary and Pricing section.
  • Once the test has been added log in to myPrevent to fill out an online requisition form.
  • A completed requisition form must accompany all specimens.
  • Billing information along with specimen and shipping instructions are within the requisition form.
  • All testing must be ordered by a qualified healthcare provider.


(Delivery accepted Monday - Saturday)

  • Collect 3 ml -5 ml (5 ml preferred) of whole blood in EDTA (purple top tube) or ACD (yellow top tube). For Test #500-DNA Banking only, collect 10 ml -20 ml of whole blood.
  • For small babies, we require a minimum of 1 ml of blood.
  • Only one blood tube is required for multiple tests.
  • Ship blood tubes at room temperature in an insulated container. Do not freeze blood.
  • During hot weather, include a frozen ice pack in the shipping container. Place a paper towel or other thin material between the ice pack and the blood tube.
  • In cold weather, include an unfrozen ice pack in the shipping container as insulation.
  • At room temperature, blood specimen is stable for up to 48 hours.
  • If refrigerated, blood specimen is stable for up to one week.
  • Label the tube with the patient name, date of birth and/or ID number.


(Delivery accepted Monday - Saturday)

  • Send in screw cap tube at least 5 µg -10 µg of purified DNA at a concentration of at least 20 µg/ml for NGS and Sanger tests and at least 5 µg of purified DNA at a concentration of at least 100 µg/ml for gene-centric aCGH, MLPA, and CMA tests, minimum 2 µg for limited specimens.
  • For requests requiring more than one test, send an additional 5 µg DNA per test ordered when possible.
  • DNA may be shipped at room temperature.
  • Label the tube with the composition of the solute, DNA concentration as well as the patient’s name, date of birth, and/or ID number.
  • We only accept genomic DNA for testing. We do NOT accept products of whole genome amplification reactions or other amplification reactions.


(Delivery preferred Monday - Thursday)

  • PreventionGenetics should be notified in advance of arrival of a cell culture.
  • Culture and send at least two T25 flasks of confluent cells.
  • Some panels may require additional flasks (dependent on size of genes, amount of Sanger sequencing required, etc.). Multiple test requests may also require additional flasks. Please contact us for details.
  • Send specimens in insulated, shatterproof container overnight.
  • Cell cultures may be shipped at room temperature or refrigerated.
  • Label the flasks with the patient name, date of birth, and/or ID number.
  • We strongly recommend maintaining a local back-up culture. We do not culture cells.
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