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GTR Home > Tests > RASOPATHY-RELATED SYNDROME

Methodology

Methodology

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Molecular Genetics
DDeletion/duplication analysis
Multiplex Ligation-dependent Probe Amplification (MLPA)
ESequence analysis of select exons
Bi-directional Sanger Sequence Analysis
CSequence analysis of the entire coding region
Bi-directional Sanger Sequence Analysis

Test development

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Test developed by laboratory (no manufacturer test name)

Test procedure

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DNA is extracted from a patient's sample. An NGS library is prepared and enriched by capture in solution using our custom hereditary cancer sub-exome target panel (ICO-IMPPC_HC_Panel_V2.0), which include the whole coding regions of NF1 (NM_000267.3), SPRED1 (NM_152594.2), PTPN11 (NM_002834), SOS1 (NM_005633), SOS2 (NM_006939.3), SHOC2 (NM_007373), RAF1 (NM_002880), NRAS (NM_002524), HRAS (NM_001130442), KRAS (NM_004985), RRAS (NM_006270.4), RASA1 (NM_002890), RASA2 (NM_001303246.1), CBL (NM_005188), BRAF (NM_004333), MAP2K1 (NM_002755), MAP2K2 (NM_030662), LZTR1 (NM_006767.3), RIT1 (NM_006912.5), A2ML (NM_144670.5) genes among others. Enriched library is sequenced in a Illumina platform and data is bioinformatically analyzed for a primary QC. A dedicated bioinformatic pipeline analysis and a further manual inspection of all genes is performed to detect possible damaging variants, which are validated by Sanger sequencing. Coding regions of 14 genes covered by less than 30 reads per base are also analyzed by Sanger sequencing in case that no mutations were detected. The presence of partial or total gene deletions or duplications are analyzed using the MLPA (Multiplex Ligation-dependent Probe Amplification) technique. Once the mutation is detected, it is confirmed by PCR and Sanger sequencing of the exon involved at DNA level. We also perform haplotype analysis of the NF1 region in patients and families when indicated.

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Confirmation of results

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Mutation is detected DNA level by NGS and validated by an independent technique (Sanger). Splicing mutations that are not previously described are also validated at RNA levels. Studies from patients with mosaicism two independent affected tissues from the same patient are analyzed to detect both first and second hits.

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