Gut Online First, published on February 9, 2017 as 10.1136/gutjnl-2016-313334 Colon ORIGINAL ARTICLE MED12 is recurrently mutated in Middle Eastern Gut: first published as 10.1136/gutjnl-2016-313334 on 9 February 2017. Downloaded from colorectal cancer Abdul K Siraj,1 Tariq Masoodi,1 Rong Bu,1 Poyil Pratheeshkumar,1 Nasser Al-Sanea,2 Luai H Ashari,2 Alaa Abduljabbar,2 Samar Alhomoud,2 Fouad Al-Dayel,3 Fowzan S Alkuraya,4,5 Khawla S Al-Kuraya1 ▸ Additional material is ABSTRACT published online only. To view Objective Colorectal cancer (CRC) is a common cancer Significance of this study please visit the journal online (http://dx.doi.org/10.1136/ and a leading cause of cancer deaths. Previous studies gutjnl-2016-313334). have identified a number of key steps in the evolution of CRC but our knowledge of driver mutations in CRC 1Human Cancer Genomic What is already known on this subject? Research, King Faisal Specialist remains incomplete. Recognising the potential of ▸ Colorectal cancer is known to develop as a Hospital and Research Centre, studying different human populations to reveal novel multistep process involving driver mutations. Riyadh, Saudi Arabia insights in disease pathogenesis, we conducted genomic ▸ 2 MED12 mutations are known to drive several Department of Surgery and analysis of CRC in Saudi patients. tumour types. Colorectal Section, King Faisal Specialist Hospital and Design In the discovery phase of the study, we conducted whole genome sequencing of tumour and What are the new findings? Research Centre, Riyadh, ▸ Saudi Arabia corresponding germline DNA in 27 patients with CRC. We show that MED12 is recurrently mutated in 3Department of Pathology and In addition to known driver mutations, we identified colorectal cancer, uniformly in the active Laboratory Medicine, King three MED12 somatic mutations. In the replication (unmethylated) X chromosome. Faisal Specialist Hospital and ▸ We also show that MED12 deficiency confers Research Centre, Riyadh, phase, we employed a next-generation sequencing Saudi Arabia approach to capture and sequence MED12 and other resistance to chemotherapy at the tissue culture 4Department of Genetics, King candidate genes in a larger sample of 400 patients with level. Faisal Specialist Hospital and fi CRC and con rmed the enrichment for recurrent MED12 How might it impact on clinical practice in Research Centre, Riyadh, mutations. Saudi Arabia the foreseeable future? 5 Department of Anatomy and Results In order to gain insight into a plausible ▸ MED12 mutations identified through genomic Cell Biology, College of biological mechanism for the potential role of MED12 sequencing of colorectal cancer may predict Medicine, Alfaisal University, mutations in CRC, we studied CRC cell lines that differ resistance to chemotherapy. Riyadh, Saudi Arabia substantially in the expression level of MED12, and http://gut.bmj.com/ Correspondence to found the latter to be correlated inversely with Dr Khawla S Al-Kuraya, Human transforming growth factor (TGF)-β signalling and Cancer Genomic Research, directly with apoptosis in response to chemotherapeutic King Faisal Specialist Hospital agents. Importantly, these correlations were replicated transformation of intestinal epithelium into invasive and Research Centre, Research when MED12 expression was experimentally adenocarcinoma through a slow multistage muta- Centre at KFNCCC, MBC#98- 56 16, P.O. Box 3354, Riyadh manipulated. tional process. Efforts to identify mutations in tumour-suppressor genes and oncogenes that play a 11211, Saudi Arabia; Conclusions Our data expand the recently described on October 1, 2021 by guest. Protected copyright. [email protected] role of MED12 as a tumour suppressor in other cancers key role in the initial stages of CRC tumourigenesis to include CRC, and suggest TGF-β signalling as a were limited in scope due to technical challenges, Received 31 October 2016 although early attempts to sequence large sets of Revised 5 January 2017 potential mediator of this effect. genes using Sanger sequencing have provided valu- Accepted 12 January 2017 – able insights.7 9 Recent advances in sequencing technology have propelled the implementation of genomic analysis of CRC, which greatly expanded INTRODUCTION the repertoire of driver mutations, both at the base Colorectal cancer (CRC) is a very common cancer pair (bp) and chromosomal scale.10 that is only second to breast cancer in women, and MED12 encodes a member of Mediator, a multi- lung and prostate cancer in men, and is the second protein complex involved in the transcriptional leading cause of cancer death in Western coun- regulation of many genes by mediating the inter- tries.12In the USA alone, it is estimated that action of RNA polymerase with various transcrip- 134 000 persons will be found to have CRC and tional factors.11 Germline mutations in this X that 49 000 will die from it. These alarming linked gene are known to cause intellectual disabil- figures, however, are at least 40% lower than the ity in various syndromic forms.12 This was corro- mid-1980s thanks in large part to the implementa- borated by mouse data that show a strict To cite: Siraj AK, tion of preventive screening programmes that were requirement for MED12 level in normal gastrula- Masoodi T, Bu R, et al. Gut 13 Published Online First: conceived on the basis of improved understanding tion and neurulation. More recently, somatic [please include Day Month of CRC pathogenesis.34 mutations in MED12 have been found to play a sig- Year] doi:10.1136/gutjnl- The seminal proposal by Vogelstein et al in 1988 nificant role in the aetiology of uterine leiomyoma, 2016-313334 laid the ground for the contemporary view of the a finding that was reproduced in several cohorts.14 Siraj AK, et al. Gut 2017;0:1–9. doi:10.1136/gutjnl-2016-313334 1 Copyright Article author (or their employer) 2017. Produced by BMJ Publishing Group Ltd (& BSG) under licence. Colon Subsequently, driver MED12 mutations were identified in other DNA isolation tumours including adrenocortical carcinoma, prostate cancer DNA isolation from peripheral blood, fresh frozen and – and breast fibroadenoma.15 17 Interestingly, the distribution of paraffin-embedded tissues was performed using Gentra DNA Gut: first published as 10.1136/gutjnl-2016-313334 on 9 February 2017. Downloaded from MED12 mutations differs in different types of tumour. In isolation kit (Gentra, Minneapolis, Minnesota, USA) following uterine leiomyoma, most of MED12 mutations were identified the manufacturer’s recommendations. in exon 2 and lead to the activation of WNT pathway genes through interaction with β-catenin, while in prostate cancer Whole genome sequencing MED12 mutations were mainly detected in the non-N-terminal Whole genome sequencing was performed on 27 patients with region and seem to influence p53 and androgen signalling their corresponding normal blood. Paired-end libraries of 16 18–21 pathway. approximately 500-bp inserts were sequenced as 150-bp In this study, we implemented a hypothesis-free genomics paired-end reads. Raw sequencing data were processed by an approach in our search for relevant somatic mutations in CRC ultrafast Isaac DNA sequence aligner designed to align next- in Saudi Arabia, a country with a particularly high incidence of generation sequencing data with low-error rates using human 22 CRC with an unusually young age of onset. This allowed us genome V.19. The Isaac Variant Caller was used to call single- MED12 to identify recurrent somatic mutations suggesting a nucleotide variants (SNVs) and small indels from the bam potential role in CRC pathogenesis. Furthermore, functional files.24 SnpEff was used for annotating the variants with analysis of relevant cell lines supports a previously reported role dbSNP138, dbSNP142, 1000 Genomes project and ESP6500 MED12 fi 23 25 of de ciency in mediating drug resistance. database. We excluded common single-nucleotide polymorph- isms (SNPs) with minor allele frequency of >0.001 as recorded MATERIALS AND METHODS in either dbSNP, National Heart, Lung, and Blood Institute Human subjects exome sequencing project, 1000 Genomes and our in-house A total of 427 patient samples diagnosed with CRC at the King data from exome sequencing of over 730 normal samples. Faisal Specialist Hospital and Research Centre were collected Non-coding variants, synonymous variants and variants present from Department of Pathology. Detailed clinicopathological in highly repetitive regions were excluded for further analysis. data were noted from case records and summarised in table 1. All tissue and blood samples were obtained from patients with Candidate gene capture and sequencing approval from Institutional Review Board of the hospital. Recurrently mutated genes (mutated in at least three samples) Informed consent was obtained from the patients from whom from the 27 whole genome cases were selected for capture fresh cancer tissues were harvested for the study. For the study, sequencing using SureSelect Target Enrichment Kit on Illumina waiver of consent was obtained for archived paraffin tissue HiSeq 2500 Sequencer. Fastq files were aligned to the human blocks from Research Advisory Council (RAC) under project reference genome hg19 using burrows-wheeler aligner (BWA).26 RAC#2140 005. Local realignment, PCR duplicates and base quality recalibration was performed using genome analysis toolkit (GATK) and Picard-tools.27 SNVs and small indels were called using GATK. fi The identi ed variants were annotated using SnpEff. Similar http://gut.bmj.com/ Table 1 Clinicopathological variables for the patient cohort filters as applied to whole genome samples were used to get (n=427) final mutation list. Age Median 57.0 PCR and Sanger sequencing Range (IQR) 47.0–68.0 Primer 3 software was used to design the primers for the par- fi Gender ticular variants identi ed by whole exome or capture sequen- cing. PCR and Sanger sequencing were performed as described on October 1, 2021 by guest. Protected copyright. Male 225 (52.7) 28 Female 202 (47.3) previously. Reference sequences were downloaded from Status GenBank.