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Neurofibromatosis Type 1 and Related Disorders via NF1 Gene Sequencing with CNV Detection

  • Summary and Pricing
  • Clinical Features and Genetics
  • Citations
  • Methods
  • Ordering/Specimens
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TEST METHODS

Sequencing and CNV

Test Code Test Copy GenesPriceCPT Code Copy CPT Codes
4361 NF1$640 81408,81479 Add to Order

New York State Approved Test

Pricing Comments

Deletion and duplication testing for NF1 is performed using NGS, but CNVs detected in this gene are usually confirmed via multiplex ligation-dependent probe amplification (MLPA). Please see limitations for CNV detection via NGS.

This test is also offered via our exome backbone with CNV detection (click here). The exome-based test may be higher priced, but permits reflex to the entire exome or to any other set of clinically relevant genes.

Targeted Testing

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

Turnaround Time

The great majority of tests are completed within 20 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).

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.

Genetics

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 Next Generation Sequencing (NGS) test, 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 regions not captured or with insufficient number of sequence reads.

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.

Copy number variants (CNVs) are also detected from NGS data. We utilize a CNV calling algorithm that compares mean read depth and distribution for each target in the test sample against multiple matched controls. Neighboring target read depth and distribution and zygosity of any variants within each target region are used to reinforce CNV calls. All CNVs are confirmed using another technology such as aCGH, MLPA, or PCR before they are reported.

This test provides full coverage of all coding exons of the NF1 gene, plus ~10 bases of flanking noncoding DNA. We define full coverage as >20X NGS reads or Sanger sequencing.

Deletion and duplication testing for NF1 is performed using NGS, but CNVs detected in this gene are usually confirmed via multiplex ligation-dependent probe amplification (MLPA). Please see limitations for CNV detection via NGS.

Indications for Test

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

Gene

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

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CONTACTS

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Citations
  • 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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TEST METHODS

Sequencing and CNV Detection via NextGen Sequencing using PG-Select Capture Probes

Test Procedure

NextGen Sequencing

We use a combination of Next Generation Sequencing (NGS) and Sanger sequencing technologies to cover the full coding regions of the listed genes plus ~10 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.

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 Variants

Human Genome Variation Society (HGVS) recommendations are used to describe sequence variants (http://www.hgvs.org).  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.

Deletion and Duplication Testing via NGS

Copy number variants (CNVs) such as deletions and duplications are detected from next generation sequencing data. We utilize a CNV calling algorithm that compares mean read depth and distribution for each target in the test sample against multiple matched controls. Neighboring target read depth and distribution, and zygosity of any variants within each target region are used to reinforce CNV calls. All CNVs are confirmed using another technology such as PCR, aCGH or MLPA before they are reported.
Analytical Validity

NextGen Sequencing

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.

Deletion and Duplication Testing via NGS
 
In general, sensitivity for single, double, or triple exon CNVs is ~80% and for CNVs of four exon size or larger is close to 100%, but may vary from gene-to-gene based on exon size, depth of coverage, and characteristics of the region.
Analytical Limitations

NextGen Sequencing

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 ~10 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.

Deletion and Duplication Testing via NGS
 
This CNV calling algorithm used in this test detects most deletions and duplications; however aberrations in a small percentage of regions may not be accurately detected due to sequence paralogy (e.g. pseudogenes, segmental duplications), sequence properties, deletion/duplication size (e.g. single vs. two or more exons), and inadequate coverage. 
 
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.
 
In nearly all cases, our ability to determine the exact copy number change within a targeted gene is limited. In particular, when we find copy excess within a targeted gene, we cannot be certain that the region is duplicated, triplicated etc. In many duplication cases, we are unable to determine the genomic location or the orientation of the duplicated segment with respect to the gene. In particular, we often cannot determine if the duplicated segment is inserted in tandem within the gene or inserted elsewhere in the genome. Similarly, we may not be able to determine the orientation of the duplicated segment (direct or inverted), and whether it will disrupt the open reading frame of the given gene.
 
Our ability to detect CNVs due to somatic mosaicism is limited.
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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.
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.

SPECIMEN TYPES
WHOLE BLOOD

(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.

DNA

(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.

CELL CULTURE

(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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