Detection of subclonal L1 transductions in colorectal cancer by long-distance inverse-PCR and Nanopore sequencing

Long interspersed nuclear elements-1 (L1s) are a large family of retrotransposons. Retrotransposons are repetitive sequences that are capable of autonomous mobility via a copy-and-paste mechanism. In most copy events, only the L1 sequence is inserted, however, they can also mobilize the flanking non-repetitive region by a process known as 3' transduction. L1 insertions can contribute to genome plasticity and cause potentially tumorigenic genomic instability. However, detecting the activity of a particular source L1 and identifying new insertions stemming from it is a challenging task with current methodological approaches. We developed a long-distance inverse PCR (LDI-PCR) based approach to monitor the mobility of active L1 elements based on their 3' transduction activity. LDI-PCR requires no prior knowledge of the insertion target region. By applying LDI-PCR in conjunction with Nanopore sequencing (Oxford Nanopore Technologies) on one L1 reported to be particularly active in human cancer genomes, we detected 14 out of 15 3' transductions previously identified by whole genome sequencing in two different colorectal tumour samples. In addition we discovered 25 novel highly subclonal insertions. Furthermore, the long sequencing reads produced by LDI-PCR/Nanopore sequencing enabled the identification of both the 5' and 3' junctions and revealed detailed insertion sequence information.Peer reviewe

Colibactin DNA-damage signature indicates mutational impact in colorectal cancer

The mucosal epithelium is a common target of damage by chronic bacterial infections and the accompanying toxins, and most cancers originate from this tissue. We investigated whether colibactin, a potent genotoxin(1) associated with certain strains of Escherichia coli(2), creates a specific DNA-damage signature in infected human colorectal cells. Notably, the genomic contexts of colibactin-induced DNA double-strand breaks were enriched for an AT-rich hexameric sequence motif, associated with distinct DNA-shape characteristics. A survey of somatic mutations at colibactin target sites of several thousand cancer genomes revealed notable enrichment of this motif in colorectal cancers. Moreover, the exact double-strand-break loci corresponded with mutational hot spots in cancer genomes, reminiscent of a trinucleotide signature previously identified in healthy colorectal epithelial cells(3). The present study provides evidence for the etiological role of colibactin in human cancer. Identification of a DNA-damage signature induced by colibactin, a toxin expressed by some strains of Escherichia coli, is enriched in human colorectal cancers.Peer reviewe

No evidence of EMAST in whole genome sequencing data from 248 colorectal cancers

Microsatellite instability (MSI) is caused by defective DNA mismatch repair (MMR), and manifests as accumulation of small insertions and deletions (indels) in short tandem repeats of the genome. Another form of repeat instability, elevated microsatellite alterations at selected tetranucleotide repeats (EMAST), has been suggested to occur in 50% to 60% of colorectal cancer (CRC), of which approximately one quarter are accounted for by MSI. Unlike for MSI, the criteria for defining EMAST is not consensual. EMAST CRCs have been suggested to form a distinct subset of CRCs that has been linked to a higher tumor stage, chronic inflammation, and poor prognosis. EMAST CRCs not exhibiting MSI have been proposed to show instability of di- and trinucleotide repeats in addition to tetranucleotide repeats, but lack instability of mononucleotide repeats. However, previous studies on EMAST have been based on targeted analysis of small sets of marker repeats, often in relatively few samples. To gain insight into tetranucleotide instability on a genome-wide level, we utilized whole genome sequencing data from 227 microsatellite stable (MSS) CRCs, 18 MSI CRCs, 3 POLE-mutated CRCs, and their corresponding normal samples. As expected, we observed tetranucleotide instability in all MSI CRCs, accompanied by instability of mono-, di-, and trinucleotide repeats. Among MSS CRCs, some tumors displayed more microsatellite mutations than others as a continuum, and no distinct subset of tumors with the previously proposed molecular characters of EMAST could be observed. Our results suggest that tetranucleotide repeat mutations in non-MSI CRCs represent stochastic mutation events rather than define a distinct CRC subclass.Peer reviewe

Deficient H2A.Z deposition is associated with genesis of uterine leiomyoma

One in four women suffers from uterine leiomyomas (ULs)-benign tumours of the uterine wall, also known as uterine fibroids-at some point in premenopausal life. ULs can cause excessive bleeding, pain and infertility(1), and are a common cause of hysterectomy(2). They emerge through at least three distinct genetic drivers: mutations in MED12 or FH, or genomic rearrangement of HMGA2(3). Here we created genome-wide datasets, using DNA, RNA, assay for transposase-accessible chromatin (ATAC), chromatin immunoprecipitation (ChIP) and HiC chromatin immunoprecipitation (HiChIP) sequencing of primary tissues to profoundly understand the genesis of UL. We identified somatic mutations in genes encoding six members of the SRCAP histone-loading complex(4), and found that germline mutations in the SRCAP members YEATS4 and ZNHIT1 predispose women to UL. Tumours bearing these mutations showed defective deposition of the histone variant H2A.Z. In ULs, H2A.Z occupancy correlated positively with chromatin accessibility and gene expression, and negatively with DNA methylation, but these correlations were weak in tumours bearing SRCAP complex mutations. In these tumours, open chromatin emerged at transcription start sites where H2A.Z was lost, which was associated with upregulation of genes. Furthermore, YEATS4 defects were associated with abnormal upregulation of bivalent embryonic stem cell genes, as previously shown in mice(5). Our work describes a potential mechanism of tumorigenesis-epigenetic instability caused by deficient H2A.Z deposition-and suggests that ULs arise through an aberrant differentiation program driven by deranged chromatin, emanating from a small number of mutually exclusive driver mutations.Peer reviewe

Eleven Candidate Susceptibility Genes for Common Familial Colorectal Cancer

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Retrotransposon insertions can initiate colorectal cancer and are associated with poor survival

Genomic instability pathways in colorectal cancer (CRC) have been extensively studied, but the role of retrotransposition in colorectal carcinogenesis remains poorly understood. Although retrotransposons are usually repressed, they become active in several human cancers, in particular those of the gastrointestinal tract. Here we characterize retro-transposon insertions in 202 colorectal tumor whole genomes and investigate their associations with molecular and clinical characteristics. We find highly variable retrotransposon activity among tumors and identify recurrent insertions in 15 known cancer genes. In approximately 1% of the cases we identify insertions in APC, likely to be tumor-initiating events. Insertions are positively associated with the CpG island methylator phenotype and the genomic fraction of allelic imbalance. Clinically, high number of insertions is independently associated with poor disease-specific survival.Peer reviewe

Mendelian randomisation implicates hyperlipidaemia as a risk factor for colorectal cancer.

While elevated blood cholesterol has been associated with an increased risk of colorectal cancer (CRC) in observational studies, causality is uncertain. Here we apply a Mendelian randomisation (MR) analysis to examine the potential causal relationship between lipid traits and CRC risk. We used single nucleotide polymorphisms (SNPs) associated with blood levels of total cholesterol (TC), triglyceride (TG), low-density lipoprotein (LDL), and high-density lipoprotein (HDL) as instrumental variables (IV). We calculated MR estimates for each risk factor with CRC using SNP-CRC associations from 9,254 cases and 18,386 controls. Genetically predicted higher TC was associated with an elevated risk of CRC (odds ratios (OR) per unit SD increase = 1.46, 95% confidence interval [CI]: 1.20-1.79, P=1.68x10−4). The pooled ORs for LDL, HDL, and TG were 1.05 (95% CI: 0.92-1.18, P=0.49), 0.94 (95% CI: 0.84-1.05, P= 0.27), and 0.98 (95% CI: 0.85-1.12, P=0.75) respectively. A genetic risk score for 3-hydoxy-3-methylglutaryl-coenzyme A reductase (HMGCR) to mimic the effects of statin therapy was associated with a reduced CRC risk (OR=0.69, 95% CI: 0.49-0.99, P=0.046). This study supports a causal relationship between higher levels of TC with CRC risk, and a further rationale for implementing public health strategies to reduce the prevalence of hyperlipidaemia. This article is protected by copyright. All rights reserved

Discovery of potential causative mutations in human coding and noncoding genome with the interactive software BasePlayer

Next-generation sequencing (NGS) is routinely applied in life sciences and clinical practice, but interpretation of the massive quantities of genomic data produced has become a critical challenge. The genome-wide mutation analyses enabled by NGS have had a revolutionary impact in revealing the predisposing and driving DNA alterations behind a multitude of disorders. The workflow to identify causative mutations from NGS data, for example in cancer and rare diseases, commonly involves phases such as quality filtering, case-control comparison, genome annotation, and visual validation, which require multiple processing steps and usage of various tools and scripts. To this end, we have introduced an interactive and user-friendly multi-platform-compatible software, BasePlayer, which allows scientists, regardless of bioinformatics training, to carry out variant analysis in disease genetics settings. A genome-wide scan of regulatory regions for mutation clusters can be carried out with a desktop computer in -10 min with a dataset of 3 million somatic variants in 200 whole-genome-sequenced (WGS) cancers.Peer reviewe

Deciphering colorectal cancer genetics through multi-omic analysis of 100,204 cases and 154,587 controls of European and east Asian ancestries

In the version of this article initially published, the author affiliations incorrectly listed “Candiolo Cancer Institute FPO-IRCCS, Candiolo (TO), Italy” as “Candiolo Cancer Institute, Candiolo, Italy.” The change has been made to the HTML and PDF versions of the article

Variation at 2q35 (PNKD and TMBIM1) influences colorectal cancer risk and identifies a pleiotropic effect with inflammatory bowel disease

To identify new risk loci for colorectal cancer (CRC), we conducted a meta-analysis of seven genome-wide association studies (GWAS) with independent replication, totalling 13 656 CRC cases and 21 667 controls of European ancestry. The combined analysis identified a new risk association for CRC at 2q35 marked by rs992157 (P = 3.15 x 10(-8), odds ratio = 1.10, 95% confidence interval = 1.06-1.13), which is intronic to PNKD (paroxysmal non-kinesigenic dyskinesia) and TMBIM1 (transmembrane BAX inhibitor motif containing 1). Intriguingly this susceptibility single-nucleotide polymorphism (SNP) is in strong linkage disequilibrium (r(2) = 0.90, D' = 0.96) with the previously discovered GWAS SNP rs2382817 for inflammatory bowel disease (IBD). Following on from this observation we examined for pleiotropy, or shared genetic susceptibility, between CRC and the 200 established IBD risk loci, identifying an additional 11 significant associations (false discovery rate [FDR]) <0.05). Our findings provide further insight into the biological basis of inherited genetic susceptibility to CRC, and identify risk factors that may influence the development of both CRC and IBD.Peer reviewe