RASopathy NGS Panel

Clinical Information

This panel consists of 23 genes associated with various RASopathy disorders. Multisystem effects of alterations in the RAS-MAPK pathway lead to physiological and anatomical abnormalities including dysmorphic features, heart defects, short stature, skin lesions, and intellectual disability. Conditions associated with variants in the RAS-MAPK pathway include:

Cardio-facio-cutaneous syndrome – Features of CFC syndrome include macrocephaly, cardiac abnormalities, short neck, ectodermal changes, and variable degrees of intellectual disability.

Costello syndrome – This condition is characterized by intellectual disability, failure to thrive, coarse facial features, loose skin, hyperextensibility, short stature, and heart defects. Papillomatous skin lesions in the oral, nasal, and anal regions are common.

LEOPARD syndrome – This disorder, also known as multiple lentigines syndrome, involves lentigines (pigmented skin lesions), cardiac rhythm abnormalities, hypertelorism, pulmonic stenosis, growth retardation, and deafness. Intellectual disability is common.

Noonan syndrome – Features of Noonan syndrome include short stature, a broad or webbed neck, low-set nipples, heart defects, and mild intellectual disability in approximately 1/3 of patients.

Neurofibromatosis – Individuals with Neurofibromatosis 1 (NF1) have two or more of the following findings: six or more cafe au laits spots, two or more neurofibromas or one plexiform neuroma, axial or inguinal freckling, Lisch nodules, optic glioma, specific skeletal findings, or an affected first-degree relative. This condition is relatively common, and it demonstrates a wide range of expressivity, even within the same family. Neurofibromatosis 2 (NF2) is a rare disorder characterized by acoustic neuromas and vestibular schwannomas that may affect hearing and balance. New variants are responsible for approximately half of all cases.

Schwannomatosis – This condition includes non-vestibular schwannomas that are often associated with significant degrees of pain. Scwhannomas may be widespread or limited to a specific area of the body.

Legius syndrome – The phenotype of Legius syndrome may resemble that of Neurofibromatosis 1 with the exception of cafe au laits spots. Other dysmorphic features may or may not be present. This disorder is caused by variants in SPRED1.

Technical Information

This panel is performed by Next Generation Sequencing and covers the coding regions of the listed genes and the flanking intronic sequences. Promoter, 3′ untranslated sequences, and deep intronic sequences are also covered, but only known disease-causing variants in these regions will be reported. Variants identified on the panel are confirmed with Sanger sequencing if they do not meet certain quality thresholds.

Large deletions and duplications (CNVs) affecting the genes of the panel can be detected; however, due to defined settings in the analysis software, CNVs smaller than 2-kb may not be identified (for example, some small exonic level copy number changes may not be identified). Please note that certain types of genetic alterations including trinucleotide repeat expansions, methylation abnormalities, and balanced rearrangements (e.g., inversions, reciprocal translocations) may not be detected by the current analysis. Please note that alterations in NF1 that are detectable only at the cDNA level may not be identified by this testing.

Specimen Requirements

• The preferred sample type is 3-4 ml of peripheral blood collected in an EDTA (purple top) tube. Extracted DNA and saliva are also accepted for this test. Blood and saliva kits are available by request.
• Send approximately 5µg of extracted DNA at a requested concentration of 90-130 ng/µl.
• If saliva is submitted, and the extracted DNA is below quality control, then you will be contacted to submit a blood sample or the panel can be completed on an exome backbone. Saliva samples must be submitted in an approved saliva kit.

Transport Instructions

• The blood specimen should be kept at room temperature and delivered via overnight shipping. If shipment is delayed by one or two days, the specimen should be refrigerated and shipped at room temperature. Do not freeze the specimen. Samples collected on Friday can be safely designated for Monday delivery.
• Extracted DNA should be sent at room temperature via overnight delivery.
• Saliva is stable at room temperature and can be delivered via overnight or ground shipping.

Prenatal Testing Information

Prenatal diagnosis can be requested when there are ultrasound findings suggestive of a diagnosis. Additional fees for cell culture and maternal cell contamination may apply. Maternal cell contamination studies are required for all prenatal molecular tests.

Prenatal Specimen Requirements

• Amniotic Fluid: Direct amniotic fluid will be accepted for analysis given there is sufficient volume for back-up culture to be established. A back-up culture at a reference lab OR Greenwood lab is required. Additional charges may apply if cell culture is required after receipt of the sample. If sending direct fluid for molecular analysis only, 10-20 ml of amniotic fluid is requested. Chromosome studies will require an additional 10-15 ml of fluid.
• Chorionic Villi Sample (CVS): 10-50 mg of chorionic villi is requested in a sterile tube with CVS media. Direct testing on CVS is not available; an additional 1-3 weeks may be needed for cultures to grow.
• Cultured amniocytes/CVS: 2x T25 confluent flasks
• A maternal sample is required, and accepted sample types include blood, extracted DNA, and saliva. Please refer to the specimen requirements above for more information.

Prenatal Transport Instructions

• Amniotic fluid should be kept at room temperature; do not freeze or refrigerate. Specimen should be sent by courier or overnight mail to arrive at the laboratory the next day. FedEx is preferred.
• CVS and cultured amniocytes should be kept at room temperature; do not freeze or refrigerate. Specimen should be sent by courier or overnight mail to arrive at the laboratory the next day. FedEx is preferred.
• Please refer to the transport instructions above for sending the maternal sample.