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Department of Genetics Heersink School of Medicine

Next Gen Sequencing of 17 RASopathy related genes and Deletion/Duplication analysis (RAS-NG)

Acceptable specimen types:

  • Blood (2-3ml EDTA; no time limitations associated with receipt)
  • Saliva (OGR-575 DNA Genotek; kits are provided upon request),
  • DNA (extracted from lymphocyte cells, a minimum of 3μg, O.D. value at 260:280nm ≥1.8)

TURNAROUND TIME

  • 25 working days

Candidates for this test:

  • Patients with clinical features suggestive of either NS, NSML, CFC, NF1, Legius syndrome or Noonan-like syndrome; 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.

TEST DESCRIPTION

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 an extensively customized and optimized set of Agilent HaloPlex capture probes, followed by sequencing of overlapping amplicons within the regions of interest using Illumina sequencing chemistry. Each coding exon plus ~50bp of flanking intronic sequence are simultaneously sequenced. 5’ and 3’ untranslated sequences are not included.he test uses the same approach as detailed above (see: NF1-only by NGS). The average coverage is >2000x 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) for the NF1 gene. For the remainder of the genes, the average coverage is 2000x with >99.5% of the coding region covered at ≥350x and 99.2% 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 14 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.

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.

REQUIRED FORMS

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 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

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

 

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