RASopathy NGS Panel

Overview

RASopathy NGS Panel (RAS-NG)

Establish or confirm diagnosis

Not provided

The Expanded NF1-Rasopathy panel by NGS involves the 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 as detailed previously (see: NF1-only by NGS). The average coverage is ~2000x with >98.5% of the coding region ≥350x and >99.4% ≥200x, allowing detection of very low-level mosaicism, down to 3-5% variant allele fraction with 95% confidence. 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 variants including single nucleotide substitutions, indels, and frameshifts caused by deletion/ duplication up to 112bp. Deletion/duplication analysis for NF1, SPRED1, and LZTR1 is included in this test, as such variants are a part of the variant 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-variant-positive individuals carrying 1 out of >3,100 different unique NF1 variants identified in the UAB MGL cohort. Included in the NGS assay are the regions covering >65 different deep intronic splice variants (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 variants 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 variants 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 using orthogonal methods as needed. 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 of this novel variant. Relevant family members of a proband with a (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 in such families. 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 free of charge in such cases. 000 Additional information regarding the specific details needed for test submission can be found on our website

• Clinical Testing/Confirmation of Mutations Identified Previously
• Custom Deletion/Duplication Testing
• Result interpretation