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- Validation of next-generation sequencing for the diagnosis of hereditary breast and ovarian cancerPublication . Theisen, Patrícia; Silva, Catarina; Pereira-Caetano, Iris; Rodrigues, Pedro; Isidro, Glória; Vieira, Luís; Gonçalves, JoãoIntroduction: Molecular diagnosis of hereditary breast and ovarian cancer (HBOC) has been mostly based on the identification of germline inactivating mutations in the high-penetrant genes BRCA1 and BRCA2. Although several other HBOC susceptibility genes have been identified, mutations in any of those are rare, rendering sequential genetic testing with standard methodologies time consuming and expensive. Next-generation sequencing (NGS) gene panels allow the simultaneous sequencing of multiple HBOC susceptibility genes at a lower cost. The aim of this work was to validate the use of an NGS cancer susceptibility gene panel for the identification of mutations previously detected by Sanger sequencing in the BRCA1, BRCA2 and TP53 genes. Methods: 20 samples from patients with personal/family history of breast cancer were sequenced on a MiSeq using the Trusight Cancer Sequencing Panel (Illumina). Bioinformatic analysis of NGS data included the MiSeq Reporter, VariantStudio and Isaac Enrichment tools (Illumina). Results: NGS successfully identified all 204 variants (38 unique, including 2 deletions and a splice variant) previously detected by Sanger sequencing in the BRCA1, BRCA2 and TP53 genes. Until now, no false-negative or false-positive results were obtained. Discussion: These results demonstrate the high analytical sensitivity and specificity obtained with NGS for the detection of sequence variants in 3 HBOC high-penetrant genes. These validation assays open the way to the definition of a clinically useful multigene panel for HBOC susceptibility based on the Trusight Cancer Sequencing Panel. This will allow a comprehensive and cost-effective molecular diagnosis of HBOC with a shorter turnaround time when compared to standard methodologies. In addition, with appropriate genetic counselling and specialized clinical surveillance, families with HBOC will benefit from these new technologies which have high impact in public health.
- Inherited Colorectal Cancer - validation of molecular diagnosis by Next Generation SequencingPublication . Theisen, Patrícia; Rodrigues, Pedro; Silva, Catarina; Pereira-Caetano, Iris; Isidro, Glória; Vieira, Luís; Gonçalves, JoãoIntroduction:Colorectal cancer is the third major cause of cancer related deaths worldwide. Around 5% of these cases are due to Inherited Colorectal Cancer (ICC) associated with highly penetrant single-gene mutations. Conventional molecular analysis of patients with ICC is well established and usually comprises PCR followed by Sanger sequencing of different genes with autosomal dominant inheritance - MLH1, MSH2, MSH6, PMS2 and EPCAM-3’ deletions in Lynch syndrome, APC in Familial Adenomatous Polyposis (FAP), or the study of an autosomal recessive condition with colorectal polyps associated with MUTYH variants. As standard molecular methodologies have high costs and are time consuming, they are progressively being replaced by Next Generation Sequencing (NGS), which allows the analysis of multiple genes simultaneously and with lower costs compared to Sanger sequencing. In order to validate NGS analysis for a set of genes associated with ICC, we performed NGS in 26 DNA samples from patients previously analysed by Sanger sequencing. Methods: NGS was performed using the Trusight Cancer Sequencing Panel and the MiSeq sequencer (Illumina), followed by bioinformatic analysis of the MLH1, MSH2, APC, MUTYH and STK11 genes using the MiSeq Reporter, VariantStudio and Isaac Enrichment tools. Results: Data analysis revealed 77 variants (31 unique, comprising 4 deletions, 1 insertion, 2 indels and 24 single nucleotide variants). Of these, 76 variants were previously identified by Sanger sequencing. NGS produced a false positive result associated with low coverage in STK11 (c.375-49G>A). Discussion: Results obtained by NGS are consistent with Sanger sequencing and showed high analytical sensitivity and specificity. Therefore after this initial validation, with high repeatability, conventional molecular analysis can be replaced by NGS, allowing us to offer the possibility to screen more genes, at lower costs and with a shorter turnaround time.