Article Comprehensive evaluation of coding region point mutations in microsatellite‐unstable colorectal cancer KONDELIN, Johanna, et al. Reference KONDELIN, Johanna, et al. Comprehensive evaluation of coding region point mutations in microsatellite‐unstable colorectal cancer. EMBO Molecular Medicine, 2018, vol. 10, no. 9, p. e8552 PMID : 30108113 DOI : 10.15252/emmm.201708552 Available at: http://archive-ouverte.unige.ch/unige:120174 Disclaimer: layout of this document may differ from the published version. 1 / 1 Published online: August 14, 2018 Report Comprehensive evaluation of coding region point mutations in microsatellite-unstable colorectal cancer Johanna Kondelin1,2, Kari Salokas3,4, Lilli Saarinen2, Kristian Ovaska2, Heli Rauanheimo1,2, Roosa-Maria Plaketti1,2, Jiri Hamberg1,2, Xiaonan Liu3,4 , Leena Yadav3,4, Alexandra E Gylfe1,2, Tatiana Cajuso1,2, Ulrika A Hänninen1,2, Kimmo Palin1,2, Heikki Ristolainen1,2, Riku Katainen1,2, Eevi Kaasinen1,2, Tomas Tanskanen1,2, Mervi Aavikko1,2, Minna Taipale5, Jussi Taipale1,2,6,7 , Laura Renkonen-Sinisalo8, Anna Lepistö8, Selja Koskensalo9, Jan Böhm10, Jukka-Pekka Mecklin11,12, Halit Ongen13,14,15, Emmanouil T Dermitzakis13,14,15, Outi Kilpivaara1,2, Pia Vahteristo1,2, Mikko Turunen2, Sampsa Hautaniemi2, Sari Tuupanen1,2, Auli Karhu1,2, Niko Välimäki1,2, Markku Varjosalo3,4, Esa Pitkänen1,2 & Lauri A Aaltonen1,2,* Abstract not been linked to CRC before. Two genes, SMARCB1 and STK38L, were also functionally scrutinized, providing evidence of Microsatellite instability (MSI) leads to accumulation of an a tumorigenic role, for SMARCB1 mutations in particular. excessive number of mutations in the genome, mostly small insertions and deletions. MSI colorectal cancers (CRCs), however, Keywords cancer genetics; colorectal cancer; microsatellite instability also contain more point mutations than microsatellite-stable Subject Categories Cancer; Chromatin, Epigenetics, Genomics & Functional (MSS) tumors, yet they have not been as comprehensively stud- Genomics; Systems Medicine ied. To identify candidate driver genes affected by point muta- DOI 10.15252/emmm.201708552 | Received 5 October 2017 | Revised 6 July tions in MSI CRC, we ranked genes based on mutation 2018 | Accepted 9 July 2018 | Published online 14 August 2018 significance while correcting for replication timing and gene EMBO Mol Med (2018) 10:e8552 expression utilizing an algorithm, MutSigCV. Somatic point mutation data from the exome kit-targeted area from 24 exome-sequenced sporadic MSI CRCs and respective normals, Introduction and 12 whole-genome-sequenced sporadic MSI CRCs and respective normals were utilized. The top 73 genes were vali- Colorectal cancer (CRC) is one of the most fatal cancers in Western dated in 93 additional MSI CRCs. The MutSigCV ranking identi- countries leading to death in nearly 50% of the cases (Jemal et al, fied several well-established MSI CRC driver genes and provided 2011). Approximately 15% of CRCs exhibit microsatellite instability additional evidence for previously proposed CRC candidate (MSI), which results from defective DNA mismatch repair (MMR) genes as well as shortlisted genes that have to our knowledge machinery (Boland & Goel, 2010). This is most often the result of 1 Medicum/Department of Medical and Clinical Genetics, University of Helsinki, Helsinki, Finland 2 Genome-Scale Biology Research Program, Research Programs Unit, University of Helsinki, Helsinki, Finland 3 Institute of Biotechnology, University of Helsinki, Helsinki, Finland 4 Helsinki Institute of Life Science, University of Helsinki, Helsinki, Finland 5 Division of Functional Genomics, Department of Medical Biochemistry and Biophysics (MBB), Karolinska Institutet, Stockholm, Sweden 6 Department of Biosciences and Nutrition, Karolinska Institutet, Huddinge, Sweden 7 Science for Life Center, Huddinge, Sweden 8 Department of Surgery, Helsinki University Central Hospital, Hospital District of Helsinki and Uusimaa, Helsinki, Finland 9 The HUCH Gastrointestinal Clinic, Helsinki University Central Hospital, Helsinki, Finland 10 Department of Pathology, Jyväskylä Central Hospital, Jyväskylä, Finland 11 Department of Surgery, Jyväskylä Central Hospital, University of Eastern Finland, Jyväskylä, Finland 12 Department Sport and Health Sciences, University of Jyväskylä, Jyväskylä, Finland 13 Department of Genetic Medicine and Development, University of Geneva Medical School, Geneva, Switzerland 14 Institute for Genetics and Genomics in Geneva (iGE3), University of Geneva, Geneva, Switzerland 15 Swiss Institute of Bioinformatics, Geneva, Switzerland *Corresponding author. Tel: +358 2941 25595; E-mail: [email protected] ª 2018 The Authors. Published under the terms of the CC BY 4.0 license EMBO Molecular Medicine 10:e8552 | 2018 1 of 20 Published online: August 14, 2018 EMBO Molecular Medicine Candidate genes based on point mutations Johanna Kondelin et al hypermethylation of the promoter of MLH1, one of the central genes To continue on our previous studies where candidate MSI CRC involved in MMR. oncogenes were identified based on mutation hot spots in a smaller It is estimated that approximately 90% of CRCs are sporadic, dataset (Gylfe et al, 2013; Tuupanen et al, 2014), we repeated the whereas the remaining 10% arise due to inherited predisposition hot spot analysis in this dataset of 36 exome- or whole-genome- (Bogaert & Prenen, 2014). The most common form of inherited sequenced sporadic MSI CRCs and corresponding normals. Hence, predisposition is Lynch syndrome, where the individual inherits a genes containing mutation hot spots in these somatic point mutation germline mutation in one of the MMR genes (MLH1, MSH2, MSH6, data were detected (Figs 1 and 2). From this set of hot spots, the 90 PMS2) and is therefore highly predisposed to CRC and endometrial novel hot spots as well as seven previously studied hot spots were cancer (Boland & Goel, 2010). In addition to CRC, MSI is also re-sequenced in the validation set of 93 additional MSI CRCs. From observed in approximately 15% of sporadic endometrial and gastric this effort, seven new candidate oncogenes emerged (CORIN, cancers (Hamelin et al, 2008). MSI CRCs arise through a distinct KLHL6, PCDHB16, PLEKHG1, PROS1, SPP2, and TROAP). To our genetic pathway as compared to microsatellite-stable (MSS) CRCs knowledge, this study represents the first effort to uncover driver (Boland & Goel, 2010). The defective MMR machinery results in the point mutations in MSI CRC utilizing deep sequencing of a large set accumulation of an excessive number of mutations in the genome. of tumors for validation. Most of the mutations are small insertions and deletions (indels) that target short nucleotide repeats, microsatellites. Genes that provide growth advantage to cells via loss-of-function mutations in Results microsatellites, or MSI target genes (Duval & Hamelin, 2002), have been extensively studied and numerous genes have been published In order to identify new candidates for driver genes affected by point as candidate targets and thus putative tumor suppressors (Alhopuro mutations in MSI CRC, we analyzed sequencing data from a discov- et al, 2012; Kondelin et al, 2017). MSI tumors also contain an order ery set of 36 exome- or whole-genome-sequenced MSI CRCs and of magnitude more point mutations than MSS tumors (Boland & respective normals. MutSigCV analysis was performed on the Goel, 2010), yet to date the point mutations in MSI CRCs have been somatic single-nucleotide variation (SNV) data to identify the genes mostly overlooked. Only few genes with causative point mutations most likely to display an excess of point mutations due to selection, have been identified in this tumor type. Most of these have been and the resulting top 73 genes were further validated by MiSeq flagged by missense mutation hot spots (e.g., BRAF, KRAS, CTNNB1, sequencing in a validation set of 93 additional MSI CRCs. Next, a and PIK3CA), a mutation pattern typical of oncogenes (Fearon, new algorithm, OncodriveFML, had become available during the 2011). study and was utilized on the somatic SNV data from the MiSeq In our past efforts, we have identified candidate oncogenes sequencing to identify the most likely candidates for previously with missense mutation hot spots based on next-generation unknown CRC-driving genes. Of these, SMARCB1 and STK38L were sequencing (NGS) data from a small discovery set of MSI CRCs further validated in functional experiments. The analysis workflow (Gylfe et al, 2013; Tuupanen et al, 2014). To our knowledge, is summarized in Fig 1. however, only few studies have attempted to systematically char- In addition, to continue on our previous efforts (Gylfe et al, acterize the full landscape of coding point mutations in MSI CRC 2013; Tuupanen et al, 2014), we performed an analysis on genes in order to identify new driver genes (Cancer Genome Atlas containing somatic mutation hot spots—mutations residing in either Network 2012; Seshagiri et al, 2012; Kim et al, 2013; Cortes- the same or two adjacent codons, or two bases flanking an exon– Ciriano et al, 2017). intron boundary—in at least two samples. Ninety-seven hot spots In the past few years, NGS has been largely accepted into both from 94 genes were selected for further validation in the set of 93 research and clinical use, and numerous mutations—both somatic additional MSI CRCs. The analysis workflow is summarized in and germline—have been reported to contribute to disease. There Fig 2. is, however, debate on which of the genes and