title: Next-generation sequencing for genetic testing of familial colorectal cancer syndromes.
abstract: BACKGROUND
  Genetic screening in families with high risk to develop colorectal cancer (CRC) prevents incurable disease and permits personalized therapeutic and follow-up strategies. The advancement of next-generation sequencing (NGS) technologies has revolutionized the throughput of DNA sequencing.
  
  
  METHODS
  A series of 16 probands for either familial adenomatous polyposis (FAP; 8 cases) or hereditary nonpolyposis colorectal cancer (HNPCC; 8 cases) were investigated for intragenic mutations in five CRC familial syndromes-associated genes (APC, MUTYH, MLH1, MSH2, MSH6) applying both a custom multigene Ion AmpliSeq NGS panel and conventional Sanger sequencing.
  
  
  RESULTS
  Fourteen pathogenic variants were detected in 13/16 FAP/HNPCC probands (81.3 %); one FAP proband presented two co-existing pathogenic variants, one in APC and one in MUTYH. Thirteen of these 14 pathogenic variants were detected by both NGS and Sanger, while one MSH2 mutation (L280FfsX3) was identified only by Sanger sequencing. This is due to a limitation of the NGS approach in resolving sequences close or within homopolymeric stretches of DNA. To evaluate the performance of our NGS custom panel we assessed its capability to resolve the DNA sequences corresponding to 2225 pathogenic variants reported in the COSMIC database for APC, MUTYH, MLH1, MSH2, MSH6. Our NGS custom panel resolves the sequences where 2108 (94.7 %) of these variants occur. The remaining 117 mutations reside inside or in close proximity to homopolymer stretches; of these 27 (1.2 %) are imprecisely identified by the software but can be resolved by visual inspection of the region, while the remaining 90 variants (4.0 %) are blind spots. In summary, our custom panel would miss 4 % (90/2225) of pathogenic variants that would need a small set of Sanger sequencing reactions to be solved.
  
  
  CONCLUSIONS
  The multiplex NGS approach has the advantage of analyzing multiple genes in multiple samples simultaneously, requiring only a reduced number of Sanger sequences to resolve homopolymeric DNA regions not adequately assessed by NGS. The implementation of NGS approaches in routine diagnostics of familial CRC is cost-effective and significantly reduces diagnostic turnaround times.
authors: ARC-Net Research Centre, University and Hospital Trust of Verona, Verona, Italy.
authors: Michele
authors: M
authors: Simbolo
authors: ARC-Net Research Centre, University and Hospital Trust of Verona, Verona, Italy.
authors: Andrea
authors: A
authors: Mafficini
authors: Department of Surgery, Oncology and Gastroenterology, University of Padua, Padua, Italy ; Nano Inspired Biomedicine, Institute of Pediatric Research, Città della Speranza, Padua, Italy.
authors: Marco
authors: M
authors: Agostini
authors: Department of Surgery, General Surgery A, University of Verona, Verona, Italy.
authors: Corrado
authors: C
authors: Pedrazzani
authors: Department of Surgery, Oncology and Gastroenterology, University of Padua, Padua, Italy ; Nano Inspired Biomedicine, Institute of Pediatric Research, Città della Speranza, Padua, Italy.
authors: Chiara
authors: C
authors: Bedin
authors: Department of Surgery, Oncology and Gastroenterology, University of Padua, Padua, Italy.
authors: Emanuele D
authors: ED
authors: Urso
authors: Department of Surgery, Oncology and Gastroenterology, University of Padua, Padua, Italy.
authors: Donato
authors: D
authors: Nitti
authors: Department of Pathology and Diagnostics, University and Hospital Trust of Verona, Verona, Italy.
authors: Giona
authors: G
authors: Turri
authors: Department of Pathology and Diagnostics, University and Hospital Trust of Verona, Verona, Italy.
authors: Maria
authors: M
authors: Scardoni
authors: ARC-Net Research Centre, University and Hospital Trust of Verona, Verona, Italy ; ARC-Net Research Centre, Department of Pathology & Diagnostics, University of Verona, Policlinico GB Rossi, Piazzale L.A. Scuro, 10, Verona, Italy.
authors: Matteo
authors: M
authors: Fassan
authors: ARC-Net Research Centre, University and Hospital Trust of Verona, Verona, Italy ; Department of Pathology and Diagnostics, University and Hospital Trust of Verona, Verona, Italy ; ARC-Net Research Centre, Department of Pathology & Diagnostics, University of Verona, Policlinico GB Rossi, Piazzale L.A. Scuro, 10, Verona, Italy.
authors: Aldo
authors: A
authors: Scarpa
keywords: Colorectal adenocarcinoma; FAP; HNPCC; Next generation sequencing