Non-NF1 RASopathy Panel by Next-Generation Sequencing and Deletion/Duplication Analysis of LZTR1 and SPRED1 (NNP-NG)
Information for Ordering
Acceptable Specimen Types
- Fresh blood sample: 3–6 mL EDTA; no time limitations associated with receipt
- Saliva: OGD-575 DNA Genotek collection kit; kits are provided upon request
- DNA extracted from lymphocyte cells: minimum volume of 25 µL containing 3 µg of DNA; A260/A280 ratio ≥1.8; must be extracted in a CLIA-certified or equivalent laboratory
Turnaround Time
Average turnaround time: 30 working days for blood, saliva, or DNA specimens
Price, CPT Codes, and Z Code
- Institutional/self-pay price: $1,200 USD for blood, saliva, or DNA specimens
- CPT codes: 81442 and 81479 × 2
- Z code: ZB6AD
Candidates for Testing
Patients with clinical features suggestive of Noonan syndrome, Noonan syndrome with multiple lentigines, cardio-facio-cutaneous syndrome, NF1, Legius syndrome, or another Noonan-like syndrome when no variant was previously identified through comprehensive RNA-based NF1 testing with or without SPRED1 testing; patients with a clinical diagnosis of one of these syndromes who previously tested negative for a subset of the genes included in this panel; and patients with a diagnosis of Costello syndrome in whom no HRAS variant was previously identified.
Specimen Shipping and Handling
Please refer to the specimen requirements listed above.
All submitted specimens must be shipped at room temperature. Do not ship specimens on ice.
Specimens must be packaged to prevent breakage. Absorbent material must be included to contain liquids if breakage occurs. Specimens must also be shipped in double watertight containers, such as a specimen pouch placed inside the shipping company’s diagnostic envelope.
To request a collection kit, please complete the Collection Kit Request Form .
Before shipping a specimen, please contact the UAB Medical Genomics Laboratory by email at medgenomics@uabmc.edu or by phone at 205-934-5562. Please provide the shipment date and package tracking number so the laboratory can help ensure receipt.
Required Forms
About
Disorder Background
The RASopathies are a genetically heterogeneous group of disorders caused by variants 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. Variants 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 due to their 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 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. 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.
An individual can carry a variant either:
- Because (s)he inherited the variant from a parent (parent clinically affected or “non-penetrant”), or
- Because the variant arose “de novo” in the egg or sperm from which the individual developed.
Sometimes, the variant occurred “post-zygotically”, i.e. during development and in these individuals the variant may not be present in every cell of the body, typically resulting in a milder phenotype (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, LZTR1, KRAS, SOS1, RAF1, NRAS, BRAF, MAP2K1, CBL, RIT1, RASA2, and SOS2. The SHOC2 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, a SOS1 variant in an individual with CFC (Nystrom AM et al, 2008), PTPN11 mutations 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 17 genes in some individuals eliminates the need to determine which genes to test based on an individual’s clinical signs.
Test Description
The Non-NF1 RASopathy Panel by NGS involves simultaneous sequencing of 17 genes: SPRED1, PTPN11, PPP1CB, BRAF, CBL, HRAS, KRAS, LZTR1, NRAS, MAP2K1, MAP2K2, RAF1, RIT1, RASA2, SHOC2, SOS1, and SOS2. The test uses a 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 approximately 50 bp of flanking intronic sequence is simultaneously sequenced. The 5′ and 3′ untranslated regions are not included.
The average coverage is greater than 1,600×, with more than 98% of the coding region covered at ≥350× and 99% covered at ≥200×. This allows detection of low-level mosaicism down to approximately 3%–5% variant allele fraction, depending on coverage, with 95% confidence. The minimum coverage for additional regions is greater than 30×.
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 LZTR1 and 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. Nevertheless, deletion/duplication analysis through aCGH analysis can be offered as a reflex test through the UAB cytogenetic laboratory (directed by Drs. A. Carroll and F. Mikhail).
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.
View references for this testing .
Other Related Test Options
- Expanded RASopathy panel by Next-Gen Sequencing (RAS-NG)
- Single Gene sequencing for Costello Syndrome (HRAS)
For more information, test requisition forms, or collection kits, please contact the UAB Medical Genomics Laboratory at 205-934-5562 or medgenomics@uabmc.edu.