Multicenter Optimization and Validation of a 2-Gene mRNA Urine Test for Detection of Clinically Significant Prostate Cancer before Initial Prostate Biopsy.

PURPOSE: A 2-gene, urine based molecular test that combines mRNA biomarkers with clinical factors can risk stratify patients for clinically significant prostate cancer. To ensure the generalizability of assay results we optimized and validated the clinical model for men with serum prostate specific antigen less than 10 ng/ml who were undergoing initial prostate biopsy. MATERIALS AND METHODS: Urine samples were collected from 1,955 men from The Netherlands, France and Germany prior to an initial prostate biopsy and study subjects were divided into training and validation cohorts. Urinary HOXC6 and DLX1 mRNA levels were quantified and RNA results were then combined with other risk factors in a clinical model optimized to detect ISUP (International Society of Urological Pathology) Grade Group 2 or greater prostate cancer in men with prostate specific antigen less than 10 ng/ml. Results in the validation cohort were compared with the PCPTRC (Prostate Cancer Prevention Trial Risk Calculator), version 2.0. RESULTS: The optimal clinical model included urinary HOXC6 and DLX1 mRNA levels, patient age, digital rectal examination and prostate specific antigen density (serum prostate specific antigen/prostate volume). In the 715 validation cohort subjects with prostate specific antigen less than 10 ng/ml the AUC was 0.82 with 89% sensitivity, 53% specificity and 95% negative predictive value. The PCPTRC AUC was 0.70. The full validation cohort of 916 men including all prostate specific antigen levels yielded an AUC of 0.85 with 93% sensitivity, 47% specificity and 95% negative predictive value. The PCPTRC AUC was 0.76. CONCLUSIONS: The 2-gene based urine assay, which is optimized for biopsy naïve patients with serum prostate specific antigen less than 10 ng/ml, demonstrated high sensitivity and negative predictive value to detect clinically significant prostate cancer. These data support using the test to help guide initial prostate biopsy decisions.

Exploratory analysis of the human breast DNA methylation profile upon soymilk exposure.

Upon soy consumption, isoflavone metabolites attain bioactive concentrations in breast tissue possibly affecting health. Though in vitro epigenetic activity of soy metabolites has been described, the in vivo impact on the epigenome is largely unknown. Therefore, in this case-control study, the breast glandular tissue DNA methylome was explored in women undergoing an aesthetic breast reduction. After a run-in phase, 10 generally healthy Belgian or Dutch women received soymilk for 5 days. MethylCap-seq methylation profiles were compared with those of 10 matched controls. Isoflavones and their microbial metabolites were quantified in urine, serum, and glandular breast tissue (liquid chromatography-mass spectrometry) and 17ß-estradiol in glandular breast tissue (immunoassay). Global DNA methylation levels were obtained for 6 cases and 5 controls using liquid chromatography-mass spectrometry. Although lower MethylCap-seq coverages were observed, mass spectrometry results and computational LINE-1 methylation analysis did not provide evidence supporting global methylation alterations upon treatment. At a false discovery rate of 0.05, no differentially methylated loci were identified. Moreover, a set of previously identified loci was specifically tested, but earlier reported results could not be validated. In conclusion, after a 5-day soymilk treatment, no major general epigenetic reprogramming in breast tissue could be found in this exploratory study.

Decoy receptor 1 (DCR1) promoter hypermethylation and response to irinotecan in metastatic colorectal cancer.

Diversity in colorectal cancer biology is associated with variable responses to standard chemotherapy. We aimed to identify and validate DNA hypermethylated genes as predictive biomarkers for irinotecan treatment of metastatic CRC patients. Candidate genes were selected from 389 genes involved in DNA Damage Repair by correlation analyses between gene methylation status and drug response in 32 cell lines. A large series of samples (n=818) from two phase III clinical trials was used to evaluate these candidate genes by correlating methylation status to progression-free survival after treatment with first-line single-agent fluorouracil (Capecitabine or 5-fluorouracil) or combination chemotherapy (Capecitabine or 5-fluorouracil plus irinotecan (CAPIRI/FOLFIRI)). In the discovery (n=185) and initial validation set (n=166), patients with methylated Decoy Receptor 1 (DCR1) did not benefit from CAPIRI over Capecitabine treatment (discovery set: HR=1.2 (95%CI 0.7-1.9, p=0.6), validation set: HR=0.9 (95%CI 0.6-1.4, p=0.5)), whereas patients with unmethylated DCR1 did (discovery set: HR=0.4 (95%CI 0.3-0.6, p=0.00001), validation set: HR=0.5 (95%CI 0.3-0.7, p=0.0008)). These results could not be replicated in the external data set (n=467), where a similar effect size was found in patients with methylated and unmethylated DCR1 for FOLFIRI over 5FU treatment (methylated DCR1: HR=0.7 (95%CI 0.5-0.9, p=0.01), unmethylated DCR1: HR=0.8 (95%CI 0.6-1.2, p=0.4)). In conclusion, DCR1 promoter hypermethylation status is a potential predictive biomarker for response to treatment with irinotecan, when combined with capecitabine. This finding could not be replicated in an external validation set, in which irinotecan was combined with 5FU. These results underline the challenge and importance of extensive clinical evaluation of candidate biomarkers in multiple trials.

Genome-wide DNA Methylation Profiling Reveals Methylation Markers Associated with 3q Gain for Detection of Cervical Precancer and Cancer.

Purpose: Epigenetic host cell changes involved in cervical cancer development following a persistent high-risk human papillomavirus (hrHPV) infection, provide promising markers for the management of hrHPV-positive women. In particular, markers based on DNA methylation of tumor suppressor gene promoters are valuable. These markers ideally identify hrHPV-positive women with precancer (CIN2/3) in need of treatment. Here, we set out to identify biologically relevant methylation markers by genome-wide methylation analysis of both hrHPV-transformed cell lines and cervical tissue specimens.Experimental Design and Results: Genome-wide discovery by next-generation sequencing (NGS) of methyl-binding domain-enriched DNA (MBD-Seq) yielded 20 candidate methylation target genes. Further verification and validation by multiplex-targeted bisulfite NGS and (quantitative) methylation-specific PCR (MSP) resulted in 3 genes (GHSR, SST, and ZIC1) that showed a significant increase in methylation with severity of disease in both tissue specimens and cervical scrapes (P < 0.005). The area under the ROC curve for CIN3 or worse varied between 0.86 and 0.89. Within the group of CIN2/3, methylation levels of all 3 genes increased with duration of lesion existence (P < 0.0005), characterized by duration of preceding hrHPV infection, and were significantly higher in the presence of a 3q gain (P < 0.05) in the corresponding tissue biopsy.Conclusions: By unbiased genome-wide DNA methylation profiling and comprehensive stepwise verification and validation studies using in vitro and patient-derived samples, we identified 3 promising methylation markers (GHSR, SST, and ZIC1) associated with a 3q gain for the detection of cervical (pre)cancer. Clin Cancer Res; 23(14); 3813-22. ©2017 AACR.

Detection of High-grade Prostate Cancer Using a Urinary Molecular Biomarker-Based Risk Score.

BACKGROUND: To reduce overdiagnosis and overtreatment, a test is urgently needed to detect clinically significant prostate cancer (PCa). OBJECTIVE: To develop a multimodal model, incorporating previously identified messenger RNA (mRNA) biomarkers and traditional risk factors that could be used to identify patients with high-grade PCa (Gleason score ≥7) on prostate biopsy. DESIGN, SETTING, AND PARTICIPANTS: In two prospective multicenter studies, urine was collected for mRNA profiling after digital rectal examination (DRE) and prior to prostate biopsy. The multimodal risk score was developed on a first cohort (n=519) and subsequently validated clinically in an independent cohort (n=386). OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: The mRNA levels were measured using reverse transcription quantitative polymerase chain reaction. Logistic regression was used to model patient risk and combine risk factors. Models were compared using the area under the curve (AUC) of the receiver operating characteristic, and clinical utility was evaluated with a decision curve analysis (DCA). RESULTS AND LIMITATIONS: HOXC6 and DLX1 mRNA levels were shown to be good predictors for the detection of high-grade PCa. The multimodal approach reached an overall AUC of 0.90 (95% confidence interval [CI], 0.85-0.95) in the validation cohort (AUC 0.86 in the training cohort), with the mRNA signature, prostate-specific antigen (PSA) density, and previous cancer-negative prostate biopsies as the strongest, most significant components, in addition to nonsignificant model contributions of PSA, age, and family history. For another model, which included DRE as an additional risk factor, an AUC of 0.86 (95% CI, 0.80-0.92) was obtained (AUC 0.90 in the training cohort). Both models were successfully validated, with no significant change in AUC in the validation cohort, and DCA indicated a strong net benefit and the best reduction in unnecessary biopsies compared with other clinical decision-making tools, such as the Prostate Cancer Prevention Trial risk calculator and the PCA3 assay. CONCLUSIONS: The risk score based on the mRNA liquid biopsy assay combined with traditional clinical risk factors identified men at risk of harboring high-grade PCa and resulted in a better patient risk stratification compared with current methods in clinical practice. Therefore, the risk score could reduce the number of unnecessary prostate biopsies. PATIENT SUMMARY: This study evaluated a novel urine-based assay that could be used as a noninvasive diagnostic aid for high-grade prostate cancer (PCa). When results of this assay are combined with traditional clinical risk factors, risk stratification for high-grade PCa and biopsy decision making are improved.

WRN Promoter CpG Island Hypermethylation Does Not Predict More Favorable Outcomes for Patients with Metastatic Colorectal Cancer Treated with Irinotecan-Based Therapy.

PURPOSE: WRN promoter CpG island hypermethylation in colorectal cancer has been reported to increase sensitivity to irinotecan-based therapies. We aimed to characterize methylation of the WRN promoter, determine the effect of WRN promoter hypermethylation upon expression, and validate a previous report that WRN promoter hypermethylation predicts improved outcomes for patients with metastatic colorectal cancer (mCRC) treated with irinotecan-based therapy. EXPERIMENTAL DESIGN: WRN methylation status was assessed using methylation-specific PCR and bisulfite sequencing assays. WRN expression was determined using qRT-PCR and Western blotting. WRN methylation status was correlated with overall survival (OS) and progression-free survival (PFS) in 183 patients with mCRC. Among these patients, 90 received capecitabine monotherapy as first-line therapy, and 93 received capecitabine plus irinotecan (CAPIRI) therapy as part of the CAIRO phase III clinical trial. RESULTS: WRN mRNA and WRN protein expression levels were low in colorectal cancer cell lines and in primary colorectal cancer and were largely independent of WRN methylation status. Patients with methylated WRN colorectal cancer had a shorter OS compared with patients who had unmethylated WRN colorectal cancer (HR = 1.6; 95% confidence interval [CI], 1.2-2.2; P = 0.003). Patients with unmethylated WRN showed a significantly longer PFS when treated with CAPIRI compared with capecitabine alone (HR = 0.48; 95% CI, 0.32-0.70; P = 0.0001). In contrast, patients did not benefit from adding irinotecan to capecitabine when WRN was methylated (HR = 1.1; 95% CI, 0.69-1.77; P = 0.7). CONCLUSIONS: WRN expression is largely independent of WRN promoter hypermethylation in colorectal cancer. Moreover, we could not validate the previous finding that WRN promoter hypermethylation predicts improved clinical outcomes of mCRC treated with irinotecan-based therapy and found instead the opposite result. Clin Cancer Res; 22(18); 4612-22. ©2016 AACR.

Dynamic epigenetic changes to VHL occur with sunitinib in metastatic clear cell renal cancer.

BACKGROUND: Genetic intratumoral heterogeneity (ITH) hinders biomarker development in metastatic clear cell renal cancer (mccRCC). Epigenetic relative to genetic ITH or the presence of consistent epigenetic changes following targeted therapy in mccRCC have not been evaluated. The aim of this study was to determine methylome/genetic ITH and to evaluate specific epigenetic and genetic changes associated with sunitinib therapy. PATIENTS AND METHODS: Multi-region DNA sampling performed on sequential frozen pairs of primary tumor tissue from 14 metastatic ccRCC patients, in the Upfront Sunitinib (SU011248) Therapy Followed by Surgery in Patients with Metastatic Renal Cancer: a Pilot Phase II Study (SuMR; ClinicalTrials.gov identifier: NCT01024205), at presentation (biopsy) and after 3-cycles of 50mg sunitinib (nephrectomy). Untreated biopsy and nephrectomy samples before and after renal artery ligation were controls. Ion Proton sequencing of 48 key ccRCC genes, and MethylCap-seq DNA methylation analysis was performed, data was analysed using the statistical computing environment R. RESULTS: Unsupervised hierarchical clustering revealed complete methylome clustering of biopsy and three nephrectomy samples for each patient (14/14 patients). For mutational status, untreated biopsy and all treated nephrectomy samples clustered together in 8/13 (61.5%) patients. The only methylation target significantly altered following sunitinib therapy was VHL promoter region 7896829 which was hypermethylated with treatment (FDR=0.077, P<0.001) and consistent for all patients (pre-treatment 50% patients had VHL mutations, 14% patients VHL hypermethylation). Renal artery ligation did not affect this result. No significant differences in driver or private mutation count was found with sunitinib treatment. CONCLUSIONS: Demonstration of relative methylome homogeneity and consistent VHL hypermethylation, after sunitinib, may overcome the hurdle of ITH present at other molecular levels for biomarker research.

Epigenetic effects of casein-derived opioid peptides in SH-SY5Y human neuroblastoma cells.

BACKGROUND: Casein-free, gluten-free diets have been reported to mitigate some of the inflammatory gastrointestinal and behavioral traits associated with autism, but the mechanism for this palliative effect has not been elucidated. We recently showed that the opioid peptide beta-casomorphin-7, derived from bovine (bBCM7) milk, decreases cysteine uptake, lowers levels of the antioxidant glutathione (GSH) and decreases the methyl donor S-adenosylmethionine (SAM) in both Caco-2 human GI epithelial cells and SH-SY5Y human neuroblastoma cells. While human breast milk can also release a similar peptide (hBCM-7), the bBCM7 and hBCM-7 vary greatly in potency; as the bBCM-7 is highly potent and similar to morphine in it's effects. Since SAM is required for DNA methylation, we wanted to further investigate the epigenetic effects of these food-derived opioid peptides. In the current study the main objective was to characterize functional pathways and key genes responding to DNA methylation effects of food-derived opioid peptides. METHODS: SH-SY5Y neuroblastoma cells were treated with 1 µM hBCM7 and bBCM7 and RNA and DNA were isolated after 4 h with or without treatment. Transcriptional changes were assessed using a microarray approach and CpG methylation status was analyzed at 450,000 CpG sites. Functional implications from both endpoints were evaluated via Ingenuity Pathway Analysis 4.0 and KEGG pathway analysis was performed to identify biological interactions between transcripts that were significantly altered at DNA methylation or transcriptional levels (p < 0.05, FDR <0.1). RESULTS: Here we show that hBCM7 and bBCM7, as well as morphine, cause epigenetic changes affecting gene pathways related to gastrointestinal disease and inflammation. These epigenetic consequences exhibited the same potency order as opiate inhibition of cysteine uptake insofar as hBCM7 was less potent than bBCM7, which was less potent than morphine. CONCLUSION: Our findings indicate that epigenetic effects of milk-derived opiate peptides may contribute to GI dysfunction and inflammation in sensitive individuals. While the current study was performed using SH-SY5Y neuronal cellular models, similar actions on other cells types might combine to cause symptoms of intolerance. These actions may provide a potential contributing mechanism for the beneficial effects of a casein-free diet in alleviating gastrointestinal symptoms in neurological conditions including autism and other conditions. Lastly, our study also contributes to the evolving awareness of a "gut-brain connection".

Genome-wide DNA methylation detection by MethylCap-seq and Infinium HumanMethylation450 BeadChips: an independent large-scale comparison.

Two cost-efficient genome-scale methodologies to assess DNA-methylation are MethylCap-seq and Illumina's Infinium HumanMethylation450 BeadChips (HM450). Objective information regarding the best-suited methodology for a specific research question is scant. Therefore, we performed a large-scale evaluation on a set of 70 brain tissue samples, i.e. 65 glioblastoma and 5 non-tumoral tissues. As MethylCap-seq coverages were limited, we focused on the inherent capacity of the methodology to detect methylated loci rather than a quantitative analysis. MethylCap-seq and HM450 data were dichotomized and performances were compared using a gold standard free Bayesian modelling procedure. While conditional specificity was adequate for both approaches, conditional sensitivity was systematically higher for HM450. In addition, genome-wide characteristics were compared, revealing that HM450 probes identified substantially fewer regions compared to MethylCap-seq. Although results indicated that the latter method can detect more potentially relevant DNA-methylation, this did not translate into the discovery of more differentially methylated loci between tumours and controls compared to HM450. Our results therefore indicate that both methodologies are complementary, with a higher sensitivity for HM450 and a far larger genome-wide coverage for MethylCap-seq, but also that a more comprehensive character does not automatically imply more significant results in biomarker studies.

ViVar: a comprehensive platform for the analysis and visualization of structural genomic variation.

Structural genomic variations play an important role in human disease and phenotypic diversity. With the rise of high-throughput sequencing tools, mate-pair/paired-end/single-read sequencing has become an important technique for the detection and exploration of structural variation. Several analysis tools exist to handle different parts and aspects of such sequencing based structural variation analyses pipelines. A comprehensive analysis platform to handle all steps, from processing the sequencing data, to the discovery and visualization of structural variants, is missing. The ViVar platform is built to handle the discovery of structural variants, from Depth Of Coverage analysis, aberrant read pair clustering to split read analysis. ViVar provides you with powerful visualization options, enables easy reporting of results and better usability and data management. The platform facilitates the processing, analysis and visualization, of structural variation based on massive parallel sequencing data, enabling the rapid identification of disease loci or genes. ViVar allows you to scale your analysis with your work load over multiple (cloud) servers, has user access control to keep your data safe and is easy expandable as analysis techniques advance. URL: https://www.cmgg.be/vivar/

Strategies for validation and testing of DNA methylation biomarkers.

DNA methylation is a stable covalent epigenetic modification of primarily CpG dinucleotides that has recently gained considerable attention for its use as a biomarker in different clinical settings, including disease diagnosis, prognosis and therapeutic response prediction. Although the advent of genome-wide DNA methylation profiling in primary disease tissue has provided a manifold resource for biomarker development, only a tiny fraction of DNA methylation-based assays have reached clinical testing. Here, we provide a critical overview of different analytical methods that are suitable for biomarker validation, including general study design considerations, which might help to streamline epigenetic marker development. Furthermore, we highlight some of the recent marker validation studies and established markers that are currently commercially available for assisting in clinical management of different cancers.

SNP-guided identification of monoallelic DNA-methylation events from enrichment-based sequencing data.

Monoallelic gene expression is typically initiated early in the development of an organism. Dysregulation of monoallelic gene expression has already been linked to several non-Mendelian inherited genetic disorders. In humans, DNA-methylation is deemed to be an important regulator of monoallelic gene expression, but only few examples are known. One important reason is that current, cost-affordable truly genome-wide methods to assess DNA-methylation are based on sequencing post-enrichment. Here, we present a new methodology based on classical population genetic theory, i.e. the Hardy-Weinberg theorem, that combines methylomic data from MethylCap-seq with associated SNP profiles to identify monoallelically methylated loci. Applied on 334 MethylCap-seq samples of very diverse origin, this resulted in the identification of 80 genomic regions featured by monoallelic DNA-methylation. Of these 80 loci, 49 are located in genic regions of which 25 have already been linked to imprinting. Further analysis revealed statistically significant enrichment of these loci in promoter regions, further establishing the relevance and usefulness of the method. Additional validation was done using both 14 whole-genome bisulfite sequencing data sets and 16 mRNA-seq data sets. Importantly, the developed approach can be easily applied to other enrichment-based sequencing technologies, like the ChIP-seq-based identification of monoallelic histone modifications.

Food-derived opioid peptides inhibit cysteine uptake with redox and epigenetic consequences.

Dietary interventions like gluten-free and casein-free diets have been reported to improve intestinal, autoimmune and neurological symptoms in patients with a variety of conditions; however, the underlying mechanism of benefit for such diets remains unclear. Epigenetic programming, including CpG methylation and histone modifications, occurring during early postnatal development can influence the risk of disease in later life, and such programming may be modulated by nutritional factors such as milk and wheat, especially during the transition from a solely milk-based diet to one that includes other forms of nutrition. The hydrolytic digestion of casein (a major milk protein) and gliadin (a wheat-derived protein) releases peptides with opioid activity, and in the present study, we demonstrate that these food-derived proline-rich opioid peptides modulate cysteine uptake in cultured human neuronal and gastrointestinal (GI) epithelial cells via activation of opioid receptors. Decreases in cysteine uptake were associated with changes in the intracellular antioxidant glutathione and the methyl donor S-adenosylmethionine. Bovine and human casein-derived opioid peptides increased genome-wide DNA methylation in the transcription start site region with a potency order similar to their inhibition of cysteine uptake. Altered expression of genes involved in redox and methylation homeostasis was also observed. These results illustrate the potential of milk- and wheat-derived peptides to exert antioxidant and epigenetic changes that may be particularly important during the postnatal transition from placental to GI nutrition. Differences between peptides derived from human and bovine milk may contribute to developmental differences between breastfed and formula-fed infants. Restricted antioxidant capacity, caused by wheat- and milk-derived opioid peptides, may predispose susceptible individuals to inflammation and systemic oxidation, partly explaining the benefits of gluten-free or casein-free diets.

Next-generation technologies and data analytical approaches for epigenomics.

Epigenetics refers to the collection of heritable features that modulate the genome-environment interaction without being encoded in the actual DNA sequence. While being mitotically and sometimes even meiotically transmitted, epigenetic traits often demonstrate extensive flexibility. This allows cells to acquire diverse gene expression patterns during differentiation, but also to adapt to a changing environment. However, epigenetic alterations are not always beneficial to the organism, as they are, for example, frequently identified in human diseases such as cancer. Accurate and cost-efficient genome-scale profiling of epigenetic features is thus of major importance to pinpoint these "epimutations," for example, to monitor the epigenetic impact of environmental exposure. Over the last decade, the field of epigenetics has been revolutionized by several innovative "epigenomics" technologies exactly addressing this need. In this review, we discuss and compare widely used next-generation methods to assess DNA methylation and hydroxymethylation, noncoding RNA expression, histone modifications, and nucleosome positioning. Although recent methods are typically based on "second-generation" sequencing, we also pay attention to still commonly used array- and PCR-based methods, and look forward to the additional advantages of single-molecule sequencing. As the current bottleneck in epigenomics research is the analysis rather than generation of data, the basic difficulties and problem-solving strategies regarding data preprocessing and statistical analysis are introduced for the different technologies. Finally, we also consider the complications associated with epigenomic studies of species with yet unsequenced genomes and possible solutions.

Staphylococcal enterotoxin B influences the DNA methylation pattern in nasal polyp tissue: a preliminary study.

Staphylococcal enterotoxins may influence the pro-inflammatory pattern of chronic sinus diseases via epigenetic events. This work intended to investigate the potential of staphylococcal enterotoxin B (SEB) to induce changes in the DNA methylation pattern. Nasal polyp tissue explants were cultured in the presence and absence of SEB; genomic DNA was then isolated and used for whole genome methylation analysis. Results showed that SEB stimulation altered the methylation pattern of gene regions when compared with non stimulated tissue. Data enrichment analysis highlighted two genes: the IKBKB and STAT-5B, both playing a crucial role in T- cell maturation/activation and immune response.

Combining in silico prediction and ribosome profiling in a genome-wide search for novel putatively coding sORFs.

BACKGROUND: It was long assumed that proteins are at least 100 amino acids (AAs) long. Moreover, the detection of short translation products (e.g. coded from small Open Reading Frames, sORFs) is very difficult as the short length makes it hard to distinguish true coding ORFs from ORFs occurring by chance. Nevertheless, over the past few years many such non-canonical genes (with ORFs < 100 AAs) have been discovered in different organisms like Arabidopsis thaliana, Saccharomyces cerevisiae, and Drosophila melanogaster. Thanks to advances in sequencing, bioinformatics and computing power, it is now possible to scan the genome in unprecedented scrutiny, for example in a search of this type of small ORFs. RESULTS: Using bioinformatics methods, we performed a systematic search for putatively functional sORFs in the Mus musculus genome. A genome-wide scan detected all sORFs which were subsequently analyzed for their coding potential, based on evolutionary conservation at the AA level, and ranked using a Support Vector Machine (SVM) learning model. The ranked sORFs are finally overlapped with ribosome profiling data, hinting to sORF translation. All candidates are visually inspected using an in-house developed genome browser. In this way dozens of highly conserved sORFs, targeted by ribosomes were identified in the mouse genome, putatively encoding micropeptides. CONCLUSION: Our combined genome-wide approach leads to the prediction of a comprehensive but manageable set of putatively coding sORFs, a very important first step towards the identification of a new class of bioactive peptides, called micropeptides.

Transcriptional analysis through RNA sequencing of giant cells induced by Meloidogyne graminicola in rice roots.

One of the reasons for the progressive yield decline observed in aerobic rice production is the rapid build-up of populations of the rice root knot nematode Meloidogyne graminicola. These nematodes induce specialized feeding cells inside root tissue, called giant cells. By injecting effectors in and sipping metabolites out of these cells, they reprogramme normal cell development and deprive the plant of its nutrients. In this research we have studied the transcriptome of giant cells in rice, after isolation of these cells by laser-capture microdissection. The expression profiles revealed a general induction of primary metabolism inside the giant cells. Although the roots were shielded from light induction, we detected a remarkable induction of genes involved in chloroplast biogenesis and tetrapyrrole synthesis. The presence of chloroplast-like structures inside these dark-grown cells was confirmed by confocal microscopy. On the other hand, genes involved in secondary metabolism and more specifically, the majority of defence-related genes were strongly suppressed in the giant cells. In addition, significant induction of transcripts involved in epigenetic processes was detected inside these cells 7 days after infection.

Methylation-specific digital karyotyping of HPV16E6E7-expressing human keratinocytes identifies novel methylation events in cervical carcinogenesis.

Transformation of epithelial cells by high-risk human papillomavirus (hrHPV) types can lead to anogenital carcinomas, particularly cervical cancer, and oropharyngeal cancers. This process is associated with DNA methylation alterations, often affecting tumour suppressor gene expression. This study aimed to comprehensively unravel genome-wide DNA methylation events linked to a transforming hrHPV-infection, which is driven by deregulated expression of the viral oncogenes E6 and E7 in dividing cells. Primary human keratinocytes transduced with HPV16E6E7 and their untransduced counterparts were subjected to methylation-specific digital karyotyping (MSDK) to screen for genome-wide DNA-methylation changes at different stages of HPV-induced transformation. Integration of the obtained methylation profiles with genome-wide gene expression patterns of cervical carcinomas identified 34 genes with increased methylation in HPV-transformed cells and reduced expression in cervical carcinomas. For 12 genes (CLIC3, CREB3L1, FAM19A4, LFNG, LHX1, MRC2, NKX2-8, NPTX-1, PHACTR3, PRDM14, SOST and TNFSF13) specific methylation in HPV-containing cell lines was confirmed by semi-quantitative methylation-specific PCR. Subsequent analysis of FAM19A4, LHX1, NKX2-8, NPTX-1, PHACTR3 and PRDM14 in cervical tissue specimens showed increasing methylation levels for all genes with disease progression. All six genes were frequently methylated in cervical carcinomas, with highest frequencies (up to 100%) seen for FAM19A4, PHACTR3 and PRDM14. Analysis of hrHPV-positive cervical scrapes revealed significantly increased methylation levels of the latter three genes in women with high-grade cervical disease compared to controls. In conclusion, MSDK analysis of HPV16-transduced keratinocytes at different stages of HPV-induced transformation resulted in the identification of novel DNA methylation events, involving FAM19A4, LHX1, NKX2-8, PHACTR3 and PRDM14 genes in cervical carcinogenesis. These genes may provide promising triage markers to assess the presence of (pre)cancerous cervical lesions in hrHPV-positive women.

Quality evaluation of methyl binding domain based kits for enrichment DNA-methylation sequencing.

DNA-methylation is an important epigenetic feature in health and disease. Methylated sequence capturing by Methyl Binding Domain (MBD) based enrichment followed by second-generation sequencing provides the best combination of sensitivity and cost-efficiency for genome-wide DNA-methylation profiling. However, existing implementations are numerous, and quality control and optimization require expensive external validation. Therefore, this study has two aims: 1) to identify a best performing kit for MBD-based enrichment using independent validation data, and 2) to evaluate whether quality evaluation can also be performed solely based on the characteristics of the generated sequences. Five commercially available kits for MBD enrichment were combined with Illumina GAIIx sequencing for three cell lines (HCT15, DU145, PC3). Reduced representation bisulfite sequencing data (all three cell lines) and publicly available Illumina Infinium BeadChip data (DU145 and PC3) were used for benchmarking. Consistent large-scale differences in yield, sensitivity and specificity between the different kits could be identified, with Diagenode's MethylCap kit as overall best performing kit under the tested conditions. This kit could also be identified with the Fragment CpG-plot, which summarizes the CpG content of the captured fragments, implying that the latter can be used as a tool to monitor data quality. In conclusion, there are major quality differences between kits for MBD-based capturing of methylated DNA, with the MethylCap kit performing best under the used settings. The Fragment CpG-plot is able to monitor data quality based on inherent sequence data characteristics, and is therefore a cost-efficient tool for experimental optimization, but also to monitor quality throughout routine applications.

Transcriptional reprogramming by root knot and migratory nematode infection in rice.

Rice is one of the most important staple crops worldwide, but its yield is compromised by different pathogens, including plant-parasitic nematodes. In this study we have characterized specific and general responses of rice (Oryza sativa) roots challenged with two endoparasitic nematodes with very different modes of action. Local transcriptional changes in rice roots upon root knot (Meloidogyne graminicola) and root rot nematode (RRN, Hirschmanniella oryzae) infection were studied at two time points (3 and 7 d after infection, dai), using mRNA-seq. Our results confirm that root knot nematodes (RKNs), which feed as sedentary endoparasites, stimulate metabolic pathways in the root, and enhance nutrient transport towards the induced root gall. The migratory RRNs, on the other hand, induce programmed cell death and oxidative stress, and obstruct the normal metabolic activity of the root. While RRN infection causes up-regulation of biotic stress-related genes early in the infection, the sedentary RKNs suppress the local defense pathways (e.g. salicylic acid and ethylene pathways). Interestingly, hormone pathways mainly involved in plant development were strongly induced (gibberellin) or repressed (cytokinin) at 3 dai. These results uncover previously unrecognized nematode-induced expression profiles related to their specific infection strategy.