Expanded NF1 RASopathy Panel by Next-Generation Sequencing and Deletion/Duplication Analysis of LZTR1, NF1, and SPRED1 (RAS-NG)

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Information for Ordering

Acceptable Specimen Types

• Blood: 2–3 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 of 3 µg with an A₂₆₀/A₂₈₀ ratio ≥1.8
• Flash-frozen tumor shipped on dry ice
• Fresh tumor or affected-tissue biopsy immersed in sterile culture media, such as PBS or RPMI

Turnaround Time

• Blood, saliva, or DNA: Average of 30 working days
• Fresh or frozen tumor: Average of 50 working days

Price, CPT Codes, and Z Code

• Institutional/self-pay price for blood, saliva, or DNA: $1,500 USD
• Institutional/self-pay price for fresh or frozen tumor: $2,500 USD
• CPT codes: 81442 and 81479 × 3
• Z code: ZB6A6

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; 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.

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 specimen type, shipment date, and package tracking number so the laboratory can help ensure proper and timely receipt.

Required Forms

• Test Requisition Form

About

Disorder Background

The RASopathies are a genetically heterogeneous group of disorders caused by variants in genes involved in the RAS/MAPK pathway. As a group, the RASopathies are among the largest groups of malformation syndromes known, affecting approximately 1 in 1,000 individuals. They include neurofibromatosis type 1, Legius syndrome, Noonan syndrome, cardio-facio-cutaneous syndrome, Noonan syndrome with multiple lentigines, and Costello syndrome.

Variants in NF1 and SPRED1 are typically loss-of-function variants and include nonsense, missense, splice, frameshift, insertion-deletion, and copy-number changes. Variants in other RASopathy genes are typically missense variants or in-frame deletions or insertions involving one or more amino acids.

Dysregulation of the RAS/MAPK pathway can have profound effects on development because of its key role in differentiation, growth, senescence, and cellular signaling. Clinical features of the RASopathies include short stature, cardiovascular defects, cutaneous and pigmentary findings, characteristic facial features, skeletal abnormalities, neurocognitive delays, and a predisposition to benign and malignant neoplasia.

The RASopathies are generally inherited in an autosomal dominant manner. A parent who carries a disease-associated variant has a 50% chance of passing it to each child, regardless of sex. These disorders demonstrate variable expressivity, meaning individuals with the same disorder may show different features and symptom severity, even within the same family. Some variants are not fully penetrant, and an individual may carry a variant while showing few or no signs of the syndrome. Clinical features may also change or progress with age, making an accurate clinical diagnosis difficult.

An individual may carry a variant because:

1. The variant was inherited from a parent who may be clinically affected or nonpenetrant; or
2. The variant arose de novo in the egg or sperm from which the individual developed.

In some individuals, the variant occurs postzygotically during development. In these cases, the variant may not be present in every cell of the body and may result in a milder phenotype due to mosaicism.

Noonan syndrome, Noonan syndrome with multiple lentigines, and Noonan syndrome with loose anagen hair are autosomal dominant disorders affecting approximately 1 in 1,000 to 2,000 individuals. Patients may present with characteristic craniofacial features, congenital heart defects, reduced growth, bleeding disorders, and variable degrees of neurocognitive delay. Patients with Noonan syndrome with multiple lentigines may also have multiple lentigines, genital abnormalities, and sensorineural hearing loss. Patients with Noonan syndrome may also have an increased cancer predisposition.

Genes associated with Noonan syndrome and Noonan syndrome with multiple lentigines include PTPN11, LZTR1, KRAS, SOS1, RAF1, NRAS, BRAF, MAP2K1, CBL, RIT1, RASA2, and SOS2. The SHOC2 gene is associated with Noonan syndrome with loose anagen hair or sparse, slow-growing hair.

Cardio-facio-cutaneous syndrome is a rare condition with genetic and phenotypic overlap with Noonan syndrome. Clinical features include craniofacial findings similar to those associated with Noonan syndrome, neurocognitive delay, failure to thrive, congenital heart defects, epilepsy, and a wide range of ectodermal manifestations. Genes associated with cardio-facio-cutaneous syndrome include BRAF, MAP2K1, MAP2K2, and KRAS.

Costello syndrome, caused by activating HRAS variants, is a rare condition associated with coarse facial features, severe feeding difficulties, mild to moderate intellectual disability, relative macrocephaly, short stature, cardiac abnormalities, and an increased risk of malignancy. Differentiating Costello syndrome from other RASopathies, particularly cardio-facio-cutaneous syndrome, may be difficult early in life.

Some individuals with a clinical diagnosis of a RASopathy have been found to carry a variant in a gene not traditionally associated with their clinical diagnosis. Examples include BRAF variants in individuals with a clinical diagnosis of Noonan syndrome, a SOS1 variant in an individual with cardio-facio-cutaneous syndrome, PTPN11 variants in individuals with paraspinal neurofibromas, and an NF1 missense variant in patients with Noonan-like features and no neurofibromas.

Some genes are associated with more than one syndrome, including PTPN11, KRAS, BRAF, RAF1, and NF1. Therefore, simultaneously testing all 18 genes may eliminate the need to determine which individual genes to test based solely on a patient’s clinical features.

Test Description

The Expanded NF1-RASopathy Panel by NGS involves simultaneous sequencing of 18 genes: NF1, SPRED1, LZTR1, PTPN11, PPP1CB, BRAF, CBL, HRAS, KRAS, NRAS, MAP2K1, MAP2K2, RAF1, RIT1, RASA2, SHOC2, SOS1, and SOS2.

The test uses the same approach described for NF1-only testing by next-generation sequencing. The average coverage is approximately 1,600×, with more than 98% of the coding region covered at ≥350× and more than 99% covered at ≥200×. This permits detection of low-level mosaicism down to approximately 3%–5% variant allele fraction with 95% confidence. The minimum coverage for additional regions is greater than 30×.

Variant and copy-number calls are made using a specialized bioinformatics pipeline that detects single-nucleotide substitutions, insertions, deletions, and frameshifts caused by deletions or duplications up to 112 bp. Deletion/duplication analysis of NF1, SPRED1, and LZTR1 is included because copy-number variants are part of the disease-associated variant spectrum for these genes. Deletion/duplication analysis is not offered for the other 15 genes because current empirical and biological evidence is insufficient to establish altered copy number as a critical disease mechanism for those genes.

Based on more than 15 years of experience with comprehensive RNA-based NF1 testing, the customized and optimized NGS NF1 component includes regions identified through analysis of more than 15,000 unrelated individuals, including more than 8,000 NF1-variant-positive individuals carrying more than 3,100 unique NF1 variants identified in the UAB MGL cohort.

The NGS assay includes regions covering more than 65 deep intronic splice variants located beyond the approximately 50 intronic base pairs flanking each exon. Validation included substitutions, splice variants, insertions, deletions, duplications of 1–112 bp, and single- and multi-exon deletions and duplications.

The analytical sensitivity of the NGS approach was 100% for substitutions and insertions or deletions up to 112 bp. The panel has been validated for the detection of germline heterozygous single-exon and multi-exon deletions and duplications.

With the largest dataset of NF1 genotypes matched with phenotypes, applicable genotype-phenotype correlations will be reported.

Confirmatory testing of reportable variants is performed by Sanger sequencing or other orthogonal methods.

For novel NF1 variants of uncertain significance, targeted RNA-based testing may be offered at no charge to assess the effect of the variant on splicing and support accurate classification and interpretation.

Relevant family members of a proband with a novel or previously identified variant of uncertain significance may be offered targeted analysis at no charge when accurate phenotypic information is provided by a healthcare professional.

Mosaicism is often present in sporadic patients with an NF1 microdeletion and may have important implications for genetic counseling. Evaluation by FISH analysis may be offered in these cases.

View references for this testing .

Other Related Test Options

• Next-Generation Sequencing and Deletion/Duplication Analysis of NF1 Only (NF1-NG)
• Next-Generation Sequencing and Deletion/Duplication Analysis of NF1 and SPRED1 (NFSP-NG)
• Next-Generation Sequencing of HRAS Only for Costello Syndrome (CST-NG)

For more information, test requisition forms, or collection kits, please contact the UAB Medical Genomics Laboratory at 205-934-5562 or medgenomics@uabmc.edu.

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