DNA mismatch and damage patterns revealed by single-molecule sequencing.

Mutations accumulate in the genome of every cell of the body throughout life, causing cancer and other diseases1,2. Most mutations begin as nucleotide mismatches or damage in one of the two strands of the DNA before becoming double-strand mutations if unrepaired or misrepaired3,4. However, current DNA-sequencing technologies cannot accurately resolve these initial single-strand events. Here we develop a single-molecule, long-read sequencing method (Hairpin Duplex Enhanced Fidelity sequencing (HiDEF-seq)) that achieves single-molecule fidelity for base substitutions when present in either one or both DNA strands. HiDEF-seq also detects cytosine deamination-a common type of DNA damage-with single-molecule fidelity. We profiled 134 samples from diverse tissues, including from individuals with cancer predisposition syndromes, and derive from them single-strand mismatch and damage signatures. We find correspondences between these single-strand signatures and known double-strand mutational signatures, which resolves the identity of the initiating lesions. Tumours deficient in both mismatch repair and replicative polymerase proofreading show distinct single-strand mismatch patterns compared to samples that are deficient in only polymerase proofreading. We also define a single-strand damage signature for APOBEC3A. In the mitochondrial genome, our findings support a mutagenic mechanism occurring primarily during replication. As double-strand DNA mutations are only the end point of the mutation process, our approach to detect the initiating single-strand events at single-molecule resolution will enable studies of how mutations arise in a variety of contexts, especially in cancer and ageing.

Combined genetic and epigenetic alterations of the TERT promoter affect clinical and biological behavior of bladder cancer.

In urothelial bladder cancer (UBC), risk stratification remains an important unmet need. Limitless self-renewal, governed by TERT expression and telomerase activation, is crucial for cancer progression. Thus, telomerase activation through the interplay of mutations (TERTpMut ) and epigenetic alterations in the TERT promoter may provide further insight into UBC behavior. Here, we investigated the combined effect of TERTpMut and the TERT Hypermethylated Oncological Region (THOR) status on telomerase activation and patient outcome in a UBC international cohort (n = 237). We verified that TERTpMut were frequent (76.8%) and present in all stages and grades of UBC. Hypermethylation of THOR was associated with higher TERT expression and higher-risk disease in nonmuscle invasive bladder cancers (NMIBC). TERTpMut alone predicted disease recurrence (HR: 3.18, 95%CI 1.84 to 5.51, p < 0.0001) but not progression in NMIBC. Combined THORhigh /TERTpMut increased the risk of disease recurrence (HR 5.12, p < 0.0001) and progression (HR 3.92, p = 0.025). Increased THOR hypermethylation doubled the risk of stage progression of both TERTpwt and TERTpMut NMIBC. These results highlight that both mechanisms are common and coexist in bladder cancer and while TERTpMut is an early event in bladder carcinogenesis THOR hypermethylation is a dynamic process that contributes to disease progression. While the absence of alterations comprises an extremely indolent phenotype, the combined genetic and epigenetic alterations of TERT bring additional prognostic value in NMIBC and provide a novel insight into telomere biology in cancer.

Anaplastic astrocytoma with piloid features, a novel molecular class of IDH wildtype glioma with recurrent MAPK pathway, CDKN2A/B and ATRX alterations.

Tumors with histological features of pilocytic astrocytoma (PA), but with increased mitotic activity and additional high-grade features (particularly microvascular proliferation and palisading necrosis) have often been designated anaplastic pilocytic astrocytomas. The status of these tumors as a separate entity has not yet been conclusively demonstrated and molecular features have only been partially characterized. We performed DNA methylation profiling of 102 histologically defined anaplastic pilocytic astrocytomas. T-distributed stochastic neighbor-embedding (t-SNE) and hierarchical clustering analysis of these 102 cases against 158 reference cases from 12 glioma reference classes revealed that a subset of 83 of these tumors share a common DNA methylation profile that is distinct from the reference classes. These 83 tumors were thus denominated DNA methylation class anaplastic astrocytoma with piloid features (MC AAP). The 19 remaining tumors were distributed amongst the reference classes, with additional testing confirming the molecular diagnosis in most cases. Median age of patients with MC AAP was 41.5 years. The most frequent localization was the posterior fossa (74%). Deletions of CDKN2A/B (66/83, 80%), MAPK pathway gene alterations (49/65, 75%, most frequently affecting NF1, followed by BRAF and FGFR1) and mutations of ATRX or loss of ATRX expression (33/74, 45%) were the most common molecular alterations. All tumors were IDH1/2 wildtype. The MGMT promoter was methylated in 38/83 tumors (45%). Outcome analysis confirmed an unfavorable clinical course in comparison to PA, but better than IDH wildtype glioblastoma. In conclusion, we show that a subset of histologically defined anaplastic pilocytic astrocytomas forms a separate DNA methylation cluster, harbors recurrent alterations in MAPK pathway genes in combination with alterations of CDKN2A/B and ATRX, affects patients who are on average older than those diagnosed with PA and has an intermediate clinical outcome.

A functional human Poly(A) site requires only a potent DSE and an A-rich upstream sequence.

We have analysed the sequences required for cleavage and polyadenylation in the intronless melanocortin 4 receptor (MC4R) pre-mRNA. Unlike other intronless genes, 3'end processing of the MC4R primary transcript is independent of any auxiliary sequence elements and only requires the core poly(A) sequences. Mutation of the AUUAAA hexamer had little effect on MC4R 3'end processing but small changes in the short DSE severely reduced cleavage efficiency. The MC4R poly(A) site requires only the DSE and an A-rich upstream sequence to direct efficient cleavage and polyadenylation. Our observation may be highly relevant for the understanding of how human noncanonical poly(A) sites are recognised. This is supported by a genome-wide analysis of over 10 000 poly(A) sites where we show that many human noncanonical poly(A) signals contain A-rich upstream sequences and tend to have a higher frequency of U and GU nucleotides in their DSE compared with canonical poly(A) signals. The importance of A-rich elements for noncanonical poly(A) site recognition was confirmed by mutational analysis of the human JUNB gene, which contains an A-rich noncanonical poly(A) signal.

THOR is a targetable epigenetic biomarker with clinical implications in breast cancer.

BACKGROUND: Breast cancer (BC) is the most frequently diagnosed cancer and a leading cause of death among women worldwide. Early BC is potentially curable, but the mortality rates still observed among BC patients demonstrate the urgent need of novel and more effective diagnostic and therapeutic options. Limitless self-renewal is a hallmark of cancer, governed by telomere maintenance. In around 95% of BC cases, this process is achieved by telomerase reactivation through upregulation of the human telomerase reverse transcriptase (hTERT). The hypermethylation of a specific region within the hTERT promoter, termed TERT hypermethylated oncological region (THOR) has been associated with increased hTERT expression in cancer. However, its biological role and clinical potential in BC have never been studied to the best of our knowledge. Therefore, we aimed to investigate the role of THOR as a biomarker and explore the functional impact of THOR methylation status in hTERT upregulation in BC. RESULTS: THOR methylation status in BC was assessed by pyrosequencing on discovery and validation cohorts. We found that THOR is significantly hypermethylated in malignant breast tissue when compared to benign tissue (40.23% vs. 12.81%, P < 0.0001), differentiating malignant tumor from normal tissue from the earliest stage of disease. Using a reporter assay, the addition of unmethylated THOR significantly reduced luciferase activity by an average 1.8-fold when compared to the hTERT core promoter alone (P < 0.01). To further investigate its biological impact on hTERT transcription, targeted THOR demethylation was performed using novel technology based on CRISPR-dCas9 system and significant THOR demethylation was achieved. Cells previously demethylated on THOR region did not develop a histologic cancer phenotype in in vivo assays. Additional studies are required to validate these observations and to unravel the causality between THOR hypermethylation and hTERT upregulation in BC. CONCLUSIONS: THOR hypermethylation is an important epigenetic mark in breast tumorigenesis, representing a promising biomarker and therapeutic target in BC. We revealed that THOR acts as a repressive regulatory element of hTERT and that its hypermethylation is a relevant mechanism for hTERT upregulation in BC.

Dual role of allele-specific DNA hypermethylation within the TERT promoter in cancer.

Aberrant activation of telomerase in human cancer is achieved by various alterations within the TERT promoter, including cancer-specific DNA hypermethylation of the TERT hypermethylated oncological region (THOR). However, the impact of allele-specific DNA methylation within the TERT promoter on gene transcription remains incompletely understood. Using allele-specific next-generation sequencing, we screened a large cohort of normal and tumor tissues (n = 652) from 10 cancer types and identified that differential allelic methylation (DAM) of THOR is restricted to cancerous tissue and commonly observed in major cancer types. THOR-DAM was more common in adult cancers, which develop through multiple stages over time, than in childhood brain tumors. Furthermore, THOR-DAM was especially enriched in tumors harboring the activating TERT promoter mutations (TPMs). Functional studies revealed that allele-specific gene expression of TERT requires hypomethylation of the core promoter, both in TPM and TERT WT cancers. However, the expressing allele with hypomethylated core TERT promoter universally exhibits hypermethylation of THOR, while the nonexpressing alleles are either hypermethylated or hypomethylated throughout the promoter. Together, our findings suggest a dual role for allele-specific DNA methylation within the TERT promoter in the regulation of TERT expression in cancer.

Two G-rich regulatory elements located adjacent to and 440 nucleotides downstream of the core poly(A) site of the intronless melanocortin receptor 1 gene are critical for efficient 3' end processing.

Cleavage and polyadenylation is an essential processing reaction required for the maturation of pre-mRNAs into stable, export- and translation-competent mature mRNA molecules. This reaction requires the assembly of a multimeric protein complex onto a bipartite core sequence element consisting of an AAUAAA hexamer and a GU/U-rich downstream sequence element. In this study we have analyzed 3' end processing of the human melanocortin 1 receptor gene (MC1R). The MC1R gene is an intron-free transcription unit, and its poly(A) site lacks a defined U/GU-rich element. We describe two G-rich sequence elements that are critical for efficient cleavage at the MC1R poly(A) site. The first element is located 30 nucleotides downstream of the cleavage site and acts as an essential closely positioned enhancer. The second G-rich region is positioned more than 440 nucleotides downstream of the MC1R processing site and is instrumental for optimal processing efficiency. Both G-rich sequences contain clusters of heterogeneous nuclear ribonucleoprotein binding motifs and act together to enhance cleavage at the MC1R poly(A) site.

DNA methylation of the TERT promoter and its impact on human cancer.

Telomere maintenance is a hallmark of human cancer that enables replicative immortality. Most cancer cells acquire telomere maintenance by telomerase activation through expression of telomerase reverse transcriptase (TERT), a rate-limiting component of the telomerase holoenzyme. Although multiple cancer-specific genetic alterations such as gain of TERT copy number and recurrent TERT promoter mutations (TPM) have been identified, the majority of cancers still express TERT via unknown mechanisms. In the last decade, DNA methylation of the TERT promoter emerged as a putative epigenetic regulatory mechanism of telomerase activation in cancer. Here, we comparatively discuss studies that investigated the DNA methylation landscape of the TERT promoter. We further review the biological and clinical impacts of TERT promoter hypermethylation in cancer and provide insight into future applications of this phenomenon.

DNA hypermethylation within TERT promoter upregulates TERT expression in cancer.

Replicative immortality is a hallmark of cancer cells governed by telomere maintenance. Approximately 90% of human cancers maintain their telomeres by activating telomerase, driven by the transcriptional upregulation of telomerase reverse transcriptase (TERT). Although TERT promoter mutations (TPMs) are a major cancer-associated genetic mechanism of TERT upregulation, many cancers exhibit TERT upregulation without TPMs. In this study, we describe the TERT hypermethylated oncological region (THOR), a 433-bp genomic region encompassing 52 CpG sites located immediately upstream of the TERT core promoter, as a cancer-associated epigenetic mechanism of TERT upregulation. Unmethylated THOR repressed TERT promoter activity regardless of TPM status, and hypermethylation of THOR counteracted this repressive function. THOR methylation analysis in 1,352 human tumors revealed frequent (>45%) cancer-associated DNA hypermethylation in 9 of 11 (82%) tumor types screened. Additionally, THOR hypermethylation, either independently or along with TPMs, accounted for how approximately 90% of human cancers can aberrantly activate telomerase. Thus, we propose that THOR hypermethylation is a prevalent telomerase-activating mechanism in cancer that can act independently of or in conjunction with TPMs, further supporting the utility of THOR hypermethylation as a prognostic biomarker.