Diagnosis; Mutation Confirmation

Establish or confirm diagnosis

Guidance for management

Lifestyle planning

Predictive risk information for patient and/or family members

Reproductive decision-making

Mainly cases fulfilling Tuberous sclerosis clinical diagnsotic criteria where it is needed a genetic confirmation (complete study) or in cases where the analysis is needed to detect the presence of a previously identified mutation in a family (direct study)

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 TSC1 (LRG_486) and TSC2 (LRG_487) genes among others. Enriched library is sequenced in a Illumina platform … 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 TSC1 (LRG_486) and TSC2 (LRG_487) 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 TSC1 and TSC2 is performed to detect possible damaging variants, which are validated by Sanger sequencing. Regions of TSC1 and TSC2 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 NF1 gene deletions or duplications are analyzed using the MLPA (Multiplex Ligation-dependent Probe Amplification) technique. Since this NGS-based strategy do not allow to detect deep intronic mutations, for those cases that meet tuberous sclerosis clinical criteria and no mutation were found, if necessary we would request patient's blood to perform an RNA-based analysis of both genes. In these cases a short puromycin-treated lymphocyte culture is performed and the coding region of TSC1 and TSC2 genes (cDNA) (NM_000368.4 and NM_000548.3) is amplified by PCR. Those products that seem to have a splicing alteration are analyzed by Sanger sequencing. Once the mutation is detected, it is confirmed by PCR and Sanger sequencing of the exon involved at DNA level. View more

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.

Tests performed
Entire test performed in-house

95% (n=20)

Deep intronic mutations, copy number alterations and gross rearrangements, mosaicism

Is proficiency testing performed for this test? Help
No

Method used for proficiency testing: Help
Intra-Laboratory

Software used to interpret novel variations Help
Polyphen, SIFT, PROVEAN, Condel, HSF, NNSplice, SpliceAIDs,