Expanded NF1 RASopathy Panel by Next-Gen Sequencing and Deletion/Duplication Analysis of NF1 and SPRED1 (RAS-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)

25 working days (RUSH option: 15 working days for additional $600 USD)

$1,500 (USD – institutional/self-pay price)

CPT: 81442 and 81479 (x2)

Z code: ZB6A6

Patients with clinical features suggestive of either NS, NSML, CFC, NF1, Legius syndrome or Noonan-like syndrome with no mutation previously found by comprehensive RNA-based NF1+/- SPRED1 testing; patients with a clinical diagnosis of any of these syndromes that previously tested negative in a subset of the genes included in this panel; patients with a diagnosis of Costello syndrome but no HRAS mutation previously identified

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

The RASopathies are a genetically heterogeneous group of disorders caused by mutations in the genes involved in the Ras-MAPK pathway. As a group, the RASopathies are one of the largest groups of malformation syndromes known, affecting ~1:1,000 and include Neurofibromatosis type 1, Legius syndrome, Noonan syndrome, cardio-facio-cutaneous (CFC) syndrome, Noonan Syndrome with Multiple Lentigines (NSML/LEOPARD) and Costello syndrome. Mutations in NF1 and SPRED1 are typically loss-of-function mutations and include the full spectrum of nonsense, missense, splice, frameshift, insertion-deletion, and copy number changes. Mutations in the other rasopathy genes are typically missense mutations or an in-frame deletion/insertion of an amino acid.
The Ras/MAPK pathway can have a profound deleterious effect on development as it plays a key role in differentiation, growth, senescence, and dysregulation. Clinical features of the RASopathies include short stature; cardiovascular defects; cutaneous and pigmentary findings; characteristic facies; skeletal and neurocognitive delays as well as a predisposition to neoplasia, both benign and malignant. The disorders have variable expressivity (individuals with the same disorder may show differing features and severity of symptoms, even within the same family). Some of the genes/mutations are not fully penetrant; therefore an individual may carry a mutation but not show any or only few signs of the syndrome. Moreover, features can change/progress with age, which makes it difficult to make an accurate clinical diagnosis. The RASopathies are inherited in an autosomal dominant manner. A parent who carries a mutated gene has a 50% chance of passing it on to every child, regardless of gender. 

An individual can carry a mutation either:

  1. Because (s)he inherited the mutation from a parent (parent clinically affected or “non-penetrant”), or
  2. Because the mutation arose “de novo” in the egg or sperm from which the individual developed.

Sometimes, the mutation occurred “post-zygotically”, i.e. during development and in these individuals the mutation may not be present in every cell of the body, typically resulting in a milder phenotype due to mosaicism.

Noonan syndrome (NS), Noonan Syndrome with Multiple Lentigines (NSML, aka LEOPARD) and Noonan syndrome with “loose anagen hair” are autosomal dominant disorders affecting ~1:1,000-2,000 individuals. Patients present with craniofacial features and a variable clinical phenotype including congenital heart defects, reduced growth, bleeding disorders (NS), and variable degrees of neurocognitive delay. Patients with NSML also have multiple lentigines, genital abnormalities and sensorineural deafness. Patients with NS also have an increased cancer predisposition. Genes associated with NS and NSML are PTPN11, KRAS, SOS1, RAF1, NRAS, BRAF, MAP2K1, CBL, RIT1, RASA2, and SOS2. TheSHOC2 gene is associated with NS with “loose anagen hair” or sparse slow growing hair.

Cardio-Facio-Cutaneous syndrome (CFC) is a rare condition with genetic and phenotypic overlap with NS. Clinical features include craniofacial features similar to those found in NS, neurocognitive delay, failure to thrive, congenital heart defects, epilepsy and a wide range of ectodermal manifestations. Four genes have been associated with CFC: BRAF, MAP2K1, MAP2K2 and KRAS.

Costello syndrome (CS), caused by activating HRAS mutations, is a very rare condition with the following key features: coarse facial features, severe feeding difficulty, mild to moderate intellectual disability, relative macrocephaly and short stature, high incidence of cardiac abnormalities and malignancy. Differentiation of CS from other rasopathies, particularly CFC may be difficult especially early in life.

Some individuals with a clinical diagnosis of one of the RASopathies have been found to carry a mutation in a gene that was not considered to be consistent with their clinical diagnosis. Examples include BRAF variants reported in individuals with a clinical diagnosis of Noonan syndrome, aSOS1 variant in an individual with CFC (Nystrom AM et al, 2008), PTPN11mutations in individuals with paraspinal neurofibromas (Conboy E. et al, 2015), and an NF1 missense mutation in patients with Noonan-like features and no neurofibromas (Rojnueangnit K et al, 2015). In addition, some genes are associated with more than one syndrome (PTPN11, KRAS, BRAF, RAF1, and NF1). Therefore, the comprehensive approach of simultaneously testing all 16 genes in some individuals eliminates the need to determine which genes to test based on an individual’s clinical signs.

The Expanded NF1-Rasopathy panel by NGS involves the simultaneous sequencing of 17 genes: NF1, SPRED1, PTPN11, PPP1CB, BRAF, CBL, HRAS, KRAS, NRAS, MAP2K1, MAP2K2, RAF1, RIT1, RASA2, SHOC2, SOS1 and SOS2. The test uses the same approach as detailed previously (see: NF1-only by NGS). The average coverage is >1800x with >99.8% of the NF1 coding region ≥350x and 100% ≥200x, allowing detection of very low level mosaicism, down to 3-5% MAF respectively (regions covered by ≥350x respectively ≥200x) with 95% confidence. For the remainder of the genes, the average coverage is 1800x with >99.4% of the coding region covered at ≥350x and 99.85% covered at 200x. The minimum coverage for any additional areas is >30x. Variant and copy number calls are made using a unique bioinformatics pipeline detecting all types of mutations including single nucleotide substitutions, indels, and frameshifts caused by deletion/ duplication up to 112bp. Deletion/duplication analysis for NF1/SPRED1 is included in this test, as such mutations are a part of the mutation spectrum for these conditions. Deletion/duplication analysis for the other 15 genes on this panel is not offered as current empirical and biological evidence is not sufficient to allow the conclusion that an altered copy number of these genes is a mechanism critical for the phenotype associated with the Rasopathies.

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 >8,100 NF1-mutation-positive individuals carrying 1 out of >3,100 different unique NF1 mutations identified in the UAB MGL cohort. Included in the NGS assay are the regions covering >65 different deep intronic splice mutations (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 mutations 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. This panel has not yet been validated to identify deletions/duplications >112bp and <1 exon, but such mutations have not yet been found in the UAB cohort, and therefore are likely very rare. The panel has been validated for the detection of germline (heterozygous) single-exon deletions/duplications as well as multi-exon deletions/duplications, however mosaic single-exon deletion/duplications validation is still pending. 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). Detection of Alu/LINE insertions, identified in 0.25% of patients from the UAB NF1-positive cohort, has not yet been validated using the current NGS approach.

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. Free of charge evaluation by FISH analysis on 200 interphase chromosomes is 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.

← Return to NF1, Legius Syndrome, and RASopathies