Inherited Colorectal Cancer - validation of molecular diagnosis by Next Generation Sequencing

Theisen, Patrícia; Rodrigues, Pedro; Silva, Catarina; Pereira-Caetano, Iris; Isidro, Glória; Vieira, Luís; Gonçalves, João

http://hdl.handle.net/10400.18/3259

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Authors

Theisen, Patrícia

Rodrigues, Pedro

Silva, Catarina

Pereira-Caetano, Iris

Isidro, Glória

Vieira, Luís

Gonçalves, João

Abstract(s)

Introduction: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.