Although RNA-guided genome editing via the CRISPR-Cas9 system is now widely used in biomedical research, genome-wide target specificities of Cas9 nucleases remain controversial. Here we present Digenome-seq, in vitro Cas9-digested whole-genome sequencing, to profile genome-wide Cas9 off-target effects in human cells. This in vitro digest yields sequence reads with the same 5' ends at cleavage sites that can be computationally identified. We validated off-target sites at which insertions or deletions were induced with frequencies below 0.1%, near the detection limit of targeted deep sequencing. We also showed that Cas9 nucleases can be highly specific, inducing off-target mutations at merely several, rather than thousands of, sites in the entire genome and that Cas9 off-target effects can be avoided by replacing 'promiscuous' single guide RNAs (sgRNAs) with modified sgRNAs. Digenome-seq is a robust, sensitive, unbiased and cost-effective method for profiling genome-wide off-target effects of programmable nucleases including Cas9.
Anaplastic thyroid cancer (ATC) and advanced differentiated thyroid cancers (DTCs) show fatal outcomes, unlike DTCs. Here, we demonstrate mutational landscape of 27 ATCs and 86 advanced DTCs by massively-parallel DNA sequencing, and transcriptome of 13 ATCs and 12 advanced DTCs were profiled by RNA sequencing. TERT , AKT1 , PIK3CA , and EIF1AX were frequently co-mutated with driver genes ( BRAF V600E and RAS ) in advanced DTCs as well as ATC, but tumor suppressors (e.g., TP53 and CDKN2A ) were predominantly altered in ATC. CDKN2A loss was significantly associated with poor disease-specific survival in patients with ATC or advanced DTCs, and up-regulation of CD274 (PD-L1) and PDCD1LG2 (PD-L2). Transcriptome analysis revealed a fourth molecular subtype of thyroid cancer (TC), ATC-like, which hardly reflects the molecular signatures in DTC. Furthermore, the activation of JAK-STAT signaling pathway could be a potential druggable target in RAS -positive ATC. Our findings provide insights for precision medicine in patients with advanced TCs.
Extranodal NK/T-cell lymphoma nasal type (ENKL) is a rare type of non-Hodgkin lymphoma that more frequently occurs in East Asia and Latin America. Even though its molecular background has been discussed in the last few years, the current knowledge does not explain the disease pathogenesis in most cases of ENKL. Here, we performed multiple types of next-generation sequencing on 34 ENKL samples, including wholeexome sequencing (9 cancer tissues and 4 cancer cell lines), targeted sequencing (21 cancer tissues), and RNA sequencing (3 cancer tissues and 4 cancer cell lines). Mutations were found most frequently in 3 genes, STAT3, BCOR, and MLL2 (which were present in 9, 7, and 6 cancer samples, respectively), whereas there were only 2 cases of JAK3 mutation. In total, JAK/STAT pathway-and histone modification-related genes accounted for 55.9% and 38.2% of cancer samples, respectively, and their involvement in ENKL pathogenesis was also supported by gene expression analysis. In addition, we provided 177 genes upregulated only in cancer tissues, which appear to be linked with angiocentric and angiodestructive growth of ENKL. In this study, we propose several novel driver genes of ENKL, and show that these genes and their functional groups may be future therapeutic targets of this disease.
Several recurrent mutations and epigenetic changes have been identified in advanced gastric cancer, but the genetic alterations associated with early gastric carcinogenesis and malignant transformation remain unclear. We investigated the genomic and transcriptomic landscape of adenomas with low-grade dysplasia (LGD) and high-grade dysplasia (HGD), and intestinal-type early gastric cancer (EGC). The results were validated in an independent cohort that included EGCs directly adjacent to adenoma (EGC-adenomas) that were in the process of malignant transformation, and de novo EGCs that do not seem to have been derived from adenoma. The expression patterns clearly divided into normal, LGD, and EGC, whereas those of HGD overlapped with LGD or EGC. These results suggest that HGD is the critical stage determining malignant transformation. We found that genes related to focal adhesion and extracellular matrix receptor interaction pathways were upregulated as LGD progressed to EGC, whereas canonical Wnt signalling and peroxisome proliferator-activated receptor (PPAR) signalling pathway genes were downregulated in EGC. Genomic alterations such as somatic mutation, gene fusion and copy number variation increased gradually from LGD to EGC. APC mutations were present in 67% of LGDs, 58% of HGDs, and 18% of EGCs. RNF43 mutations were present only in HGD and EGC, and TP53 mutations were present only in EGC. In a validation cohort, RNF43 mutations were present in 35.2% of EGC-adenomas, but in only 8.6% of de novo EGCs. This is the first study to investigate the genomic and transcriptomic landscape of multistep gastric carcinogenesis. We investigated important alterations and their related pathways in each step as tumours progressed from LGD to HGD and eventually to EGC. We suggest that mutations and downregulation of RNF43 may play a critical role in the transition from adenoma to carcinoma. Given these findings and Wnt dependency in tumours with RNF43 mutation, intestinal-type gastric cancer or adenoma with RNF43 mutation might represent a promising indication for Wnt-targeted agents. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Intestinal-type gastric carcinoma exhibits a multistep carcinogenic sequence from adenoma to carcinoma with a gradual increase in genomic alterations. But the roles of microRNAs (miRNA) in this multistage cascade are not fully explored. To identify differentially expressed miRNA (DEM) during early gastric carcinogenesis, we performed miRNA microarray profiling with 24 gastric cancers and precursor lesions (7 early gastric cancer [EGC], 3 adenomas with high-grade dysplasia, 4 adenomas with low-grade dysplasia, and 10 adjacent normal tissues). Alterations in the expression of 132 miRNA were detected; these were categorized into three groups based on their expression patterns. Of these, 42 miRNAs were aberrantly expressed in EGC. Five miRNA (miR-26a, miR-375, miR-574-3p, miR-145, and miR-15b) showed decreased expression since adenoma. Expression of two miRNA, miR-200C and miR-29a, was down-regulated in EGCs compared to normal mucosa or adenomas. Six miRNA (miR-601, miR-107, miR-18a, miR-370, miR-300, and miR-96) showed increased expression in gastric cancer compared to normal or adenoma samples. Five representative miRNAs were further validated with RT-qPCR in independent 77 samples. Taken together, these results suggest that the dysregulated miRNA show alterations at the early stages of gastric tumorigenesis and may be used as a candidate biomarker.
BackgroundGenome-wide association studies have been used extensively to identify genetic variants linked to metabolic syndrome (MetS), but most of them have been conducted in non-Asian populations. This study aimed to evaluate the association between MetS and previously studied single nucleotide polymorphisms (SNPs), and their interaction with health-related behavior in Korean men.MethodsSeventeen SNPs were genotyped and their association with MetS and its components was tested in 1193 men who enrolled in the study at Seoul National University Hospital.ResultsWe found that rs662799 near APOA5 and rs769450 in APOE had significant association with MetS and its components. The SNP rs662799 was associated with increased risk of MetS, elevated triglyceride (TG) and low levels of high-density lipoprotein, while rs769450 was associated with a decreased risk of TG. The SNPs showed interactions between alcohol drinking and physical activity, and TG levels in Korean men.ConclusionsWe have identified the genetic association and environmental interaction for MetS in Korean men. These results suggest that a strategy of prevention and treatment should be tailored to personal genotype and the population.Electronic supplementary materialThe online version of this article (doi:10.1186/s12944-015-0111-5) contains supplementary material, which is available to authorized users.
To identify epigenetically regulated genes involved in the pathogenesis of Alzheimer’s disease (AD) we analyzed global mRNA expression and methylation profiles in amyloid precursor protein (APP)-Swedish mutant-expressing AD model cells, H4-sw and selected heme oxygenase-1 (HMOX1), which is associated with pathological features of AD such as neurofibrillary tangles and senile plaques. We examined the epigenetic regulatory mechanism of HMOX1 and its application as a diagnostic and prognostic biomarker for AD. Our results show that HMOX1 mRNA and protein expression was approximately 12.2-fold and 7.9-fold increased in H4-sw cells, respectively. Increased HMOX1 expression was also detected in the brain, particularly the hippocampus, of AD model transgenic mice. However, the methylation of specific CpG sites within its promoter, particularly at CpG located −374 was significantly decreased in H4-sw cells. Treatment of neuroglioma cells with the demethylating agent 5-aza-2′-deoxycytidine resulted in reduced methylation of HMOX1 promoter accompanied by enhanced HMOX1 expression strongly supporting DNA methylation-dependent transcriptional regulation of HMOX1. Toxic Aβ-induced aberrant hypomethylation of HMOX1 at −374 promoter CpG site was correlated with increased HMOX1expression. In addition to neuroglioma cells, we also found Aβ-induced epigenetic regulation of HMOX1 in human T lymphocyte Jurkat cells. We evaluated DNA methylation status of HMOX1 at −374 promoter CpG site in blood samples from AD patients, patients with mild cognitive impairment (MCI), and control individuals using quantitative methylation-specific polymerase chain reaction. We observed lower methylation of HMOX1 at the −374 promoter CpG site in AD patients compared to MCI and control individuals, and a correlation between Mini-Mental State Examination score and demethylation level. Receiver operating characteristics analysis revealed good discrimination of AD patients from MCI patients and control individuals. Our findings suggest that the methylation status of HMOX1 at a specific promoter CpG site is related to AD progression.
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