Tissue specific patterns of methylated cytosine residues vary with age, can be altered by environmental factors, and are often abnormal in human disease yet the cellular consequences of DNA ...methylation are incompletely understood. Although the bodies of highly expressed genes are often extensively methylated in plants, the relationship between intragenic methylation and expression is less clear in mammalian cells. We performed genome-wide analyses of DNA methylation and gene expression to determine how the pattern of intragenic methylation correlates with transcription and to assess the relationship between methylation of exonic and intronic portions of the gene body. We found that dense exonic methylation is far more common than previously recognized or expected statistically, yet first exons are relatively spared compared to more downstream exons and introns. Dense methylation surrounding the transcription start site (TSS) is uncoupled from methylation within more downstream regions suggesting that there are at least two classes of intragenic methylation. Whereas methylation surrounding the TSS is tightly linked to transcriptional silencing, methylation of more downstream regions is unassociated with the magnitude of gene expression. Notably, we found that DNA methylation downstream of the TSS, in the region of the first exon, is much more tightly linked to transcriptional silencing than is methylation in the upstream promoter region. These data provide direct evidence that DNA methylation is interpreted dissimilarly in different regions of the gene body and suggest that first exon methylation blocks transcript initiation, or vice versa. Our data also show that once initiated, downstream methylation is not a significant impediment to polymerase extension. Thus, the consequences of most intragenic DNA methylation must extend beyond the modulation of transcription magnitude.Sequencing data and gene expression microarray data have been submitted to the GEO online database (accession number SRA012081.1). Supporting information including expanded methods and ten additional figures in support of the manuscript is provided.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The seminal importance of DNA sequencing to the life sciences, biotechnology and medicine has driven the search for more scalable and lower-cost solutions. Here we describe a DNA sequencing ...technology in which scalable, low-cost semiconductor manufacturing techniques are used to make an integrated circuit able to directly perform non-optical DNA sequencing of genomes. Sequence data are obtained by directly sensing the ions produced by template-directed DNA polymerase synthesis using all-natural nucleotides on this massively parallel semiconductor-sensing device or ion chip. The ion chip contains ion-sensitive, field-effect transistor-based sensors in perfect register with 1.2 million wells, which provide confinement and allow parallel, simultaneous detection of independent sequencing reactions. Use of the most widely used technology for constructing integrated circuits, the complementary metal-oxide semiconductor (CMOS) process, allows for low-cost, large-scale production and scaling of the device to higher densities and larger array sizes. We show the performance of the system by sequencing three bacterial genomes, its robustness and scalability by producing ion chips with up to 10 times as many sensors and sequencing a human genome.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Abstract
Survival rates for patients with muscle-invasive bladder cancer have not improved in the past 20 years, and new therapies are imperative. Intratumor heterogeneity can complicate molecular ...profiling attempts to optimize therapy for cancers harboring several actionable tumor subclones. To develop personalized treatment strategies, there is a need for assays to measure intratumor heterogeneity in bladder cancers.
We conducted a pilot study of two muscle invasive high-grade transitional cell carcinoma cases. We used a comprehensive cancer panel (Thermo Fisher) covering >400 cancer genes to analyze distinct tumor loci and matched normal tissues. Based on the identified somatic mutations, we designed a bladder-specific panel to (1) validate our results with increased coverage, and (2) analyze liquid biopsy samples.
Using the comprehensive cancer panel, we sequenced 6 tumor loci to an average sequencing depth of approximately 100x. We detected intratumor heterogeneity in both patients: By applying a combination of frequency-based (minor allele frequency >10%) and probabilistic (probability of difference between observed frequencies due to sampling) filters, we identified 44 credible somatic SNVs, including mutations that were not shared among all three loci. We used these SNVs to design a custom amplicon panel covering 42 SNVs across 38 genes that is suitable for highly fragmented DNA. The custom panel was used to validate the SNVs in the same tumor regions and in liquid biopsy samples from plasma and urine (approximate coverage 6,000x). In both cases, we identified private mutations reported in The Cancer Genome Atlas Urothelial Bladder Carcinoma (TCGA-BLCA) data collection, reflecting tumor evolution. Liquid biopsy samples from urine revealed all trunk mutations but only 1 out of 5 private mutations.
We conclude that tumor evolution can affect distinct loci within bladder tumors, which may not be fully represented in liquid biopsy samples. These results suggest the need for analyzing multiple tumor regions to identify all actionable driver mutations. In the future, we plan to apply our assay to additional foci and patients in order to identify optimal bladder tumor sampling strategies.
Citation Format: Katherin Patsch, Naim Matasci, Anjana Soundararajan, John Nicoll, Jonathan Katz, Antonio Sanchez, Erika Feierstein, Christina Van Loy, Zhao Xu, David B. Agus, Mitchell E. Gross, Daniel Ruderman. Bladder cancer tumor heterogeneity: development of a system-level mutation assay abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3934. doi:10.1158/1538-7445.AM2017-3934
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