A 3D Renal Proximal Tubule on Chip Model Phenocopies Lowe Syndrome and Dent II Disease Tubulopathy.

Lowe syndrome and Dent II disease are X-linked monogenetic diseases characterised by a renal reabsorption defect in the proximal tubules and caused by mutations in the OCRL gene, which codes for an inositol-5-phosphatase. The life expectancy of patients suffering from Lowe syndrome is largely reduced because of the development of chronic kidney disease and related complications. There is a need for physiological human in vitro models for Lowe syndrome/Dent II disease to study the underpinning disease mechanisms and to identify and characterise potential drugs and drug targets. Here, we describe a proximal tubule organ on chip model combining a 3D tubule architecture with fluid flow shear stress that phenocopies hallmarks of Lowe syndrome/Dent II disease. We demonstrate the high suitability of our in vitro model for drug target validation. Furthermore, using this model, we demonstrate that proximal tubule cells lacking OCRL expression upregulate markers typical for epithelial-mesenchymal transition (EMT), including the transcription factor SNAI2/Slug, and show increased collagen expression and deposition, which potentially contributes to interstitial fibrosis and disease progression as observed in Lowe syndrome and Dent II disease.

Integrated transcriptomic and epigenomic analysis of ovarian cancer reveals epigenetically silenced GULP1.

Many epigenetically inactivated genes involved in ovarian cancer (OC) development and progression remain to be identified. In this study we undertook an integrated approach that consisted of identification of genome-wide expression patterns of primary OC samples and normal ovarian surface epithelium along with a pharmacologic unmasking strategy using 3 OC and 3 immortalized normal ovarian epithelial cell lines. Our filtering scheme identified 43 OC specific methylated genes and among the 5 top candidates (GULP1, CLIP4, BAMBI, NT5E, TGFß2), we performed extended studies of GULP1. In a training set, we identified GULP1 methylation in 21/61 (34%) of cases with 100% specificity. In an independent cohort, the observed methylation was 40% (146/365) in OC, 12.5% (2/16) in borderline tumors, 11% (2/18) in cystadenoma and 0% (0/13) in normal ovarian epithelium samples. GULP1 methylation was associated with clinicopathological parameters such as stage III/IV (p = 0.001), poorly differentiated grade (p = 0.033), residual disease (p < 0.0003), worse overall (p = 0.02) and disease specific survival (p = 0.01). Depletion of GULP1 in OC cells led to increased pro-survival signaling, inducing survival and colony formation, whereas reconstitution of GULP1 negated these effects, suggesting that GULP1 is required for maintaining cellular growth control.

A high-throughput 3' UTR reporter screening identifies microRNA interactomes of cancer genes.

INTRODUCTION: Despite the established contribution of deregulated microRNA (miRNA) function to carcinogenesis, relatively few miRNA-cancer gene interactions have been validated, making it difficult to appreciate the true complexity of miRNA-cancer gene regulatory networks. RESULTS: In this effort, we identify miRNA interactomes of 17 well-established cancer genes, involved in various cancer types, through a miRNome-wide 3' UTR reporter screening. Using a novel and performant strategy for high-throughput screening data analysis, we identify 390 interactions, quadrupling the size of the known miRNA interactome for the cancer genes under investigation. Clear enrichments of established and predicted interactions underscore the validity of the interactome data set. Interactomes appear to be primarily driven by canonical binding site interactions. Nonetheless, non-canonical binding sites, such as offset 6mer and seed-mismatched or G:U wobble sites, also have regulatory activity, albeit clearly less pronounced. Furthermore, we observe enhanced regulation in the presence of 3' supplementary pairing for both canonical and non-canonical binding sites. CONCLUSIONS: Altogether, the cancer gene-miRNA interactome data set represents a unique resource that will aid in the unraveling of regulatory miRNA networks and the dynamic regulation of key protein-coding cancer genes. In addition, it uncovers aspects of the functional miRNA binding site's architecture and the relative contributions of different binding site types.

Neutralization of IL-17C Reduces Skin Inflammation in Mouse Models of Psoriasis and Atopic Dermatitis.

IL-17C is a functionally distinct member of the IL-17 family that was believed to play a role in the pathogenesis of psoriasis. Here we confirmed that IL-17C is involved in psoriasis and explored potential roles for IL-17C in atopic dermatitis (AD). An anti-IL-17C antibody, MOR106, was generated that potently and selectively binds to human and mouse IL-17C, thereby inhibiting the binding of IL-17C to its IL-17RE receptor. The antibody inhibited cutaneous inflammation in an IL-23-induced psoriatic-like skin inflammation model. In lesional skin of patients with AD, IL-17C expression levels were increased and localized to keratinocytes and infiltrating immune cells. To determine the contribution of IL-17C to AD pathogenesis, MOR106 was tested in two distinct in vivo models. In the calcipotriol-induced AD model, ear skin inflammation, TSLP, and IL-33 protein production in ears was suppressed by MOR106. Consistently, in the flaky tail strain mouse model, spontaneous development of AD-like skin inflammation was reduced by MOR106. Moreover, serum IgE levels, number of mast cells in skin and T helper type 2-related cytokines IL-4 and CCL17 in serum were all reduced. Overall, our results indicate that IL-17C is a central mediator of skin inflammation beyond psoriasis and is relevant in particular in AD.

Stage 4S neuroblastoma tumors show a characteristic DNA methylation portrait.

Stage 4S neuroblastoma (NB) is a special type of NB found in infants with metastases at diagnosis and is associated with an excellent outcome due to its remarkable capacity to undergo spontaneous regression. As genomics have not been able to explain this intriguing clinical presentation, we here aimed at profiling the DNA methylome of stage 4S NB to better understand this phenomenon. To this purpose, differential methylation analyses between International Neuroblastoma Staging System (INSS) stage 4S, stage 4 and stage 1/2 were performed, using methyl-CpG-binding domain (MBD) sequencing data of 14 stage 4S, 14 stage 4, and 13 stage 1/2 primary NB tumors (all MYCN non-amplified in order not to confound results). Stage 4S-specific hyper- and hypomethylated promoters were determined and further characterized for genomic localization and function by cytogenetic band enrichment, gene set enrichment, transcription factor target enrichment and differential RNA expression analyses. We show that specific chromosomal locations are enriched for stage 4S differentially methylated promoters and that stage 4S tumors show characteristic hypermethylation of specific subtelomeric promoters. Furthermore, genes involved in important oncogenic pathways, in neural crest development and differentiation, and in epigenetic processes are differentially methylated and expressed in stage 4S tumors. Based on these findings, we describe new biological mechanisms possibly contributing to the stage 4S-specific tumor biology and spontaneous regression. In conclusion, this study is the first to describe the highly characteristic stage 4S DNA methylome. These findings will open new avenues to further unravel the NB pathology in general and stage 4S disease specifically.

DNA methylation profiling of primary neuroblastoma tumors using methyl-CpG-binding domain sequencing.

Comprehensive genome-wide DNA methylation studies in neuroblastoma (NB), a childhood tumor that originates from precursor cells of the sympathetic nervous system, are scarce. Recently, we profiled the DNA methylome of 102 well-annotated primary NB tumors by methyl-CpG-binding domain (MBD) sequencing, in order to identify prognostic biomarker candidates. In this data descriptor, we give details on how this data set was generated and which bioinformatics analyses were applied during data processing. Through a series of technical validations, we illustrate that the data are of high quality and that the sequenced fragments represent methylated genomic regions. Furthermore, genes previously described to be methylated in NB are confirmed. As such, these MBD sequencing data are a valuable resource to further study the association of NB risk factors with the NB methylome, and offer the opportunity to integrate methylome data with other -omic data sets on the same tumor samples such as gene copy number and gene expression, also publically available.

Methyl-CpG-binding domain sequencing reveals a prognostic methylation signature in neuroblastoma.

Accurate assessment of neuroblastoma outcome prediction remains challenging. Therefore, this study aims at establishing novel prognostic tumor DNA methylation biomarkers. In total, 396 low- and high-risk primary tumors were analyzed, of which 87 were profiled using methyl-CpG-binding domain (MBD) sequencing for differential methylation analysis between prognostic patient groups. Subsequently, methylation-specific PCR (MSP) assays were developed for 78 top-ranking differentially methylated regions and tested on two independent cohorts of 132 and 177 samples, respectively. Further, a new statistical framework was used to identify a robust set of MSP assays of which the methylation score (i.e. the percentage of methylated assays) allows accurate outcome prediction. Survival analyses were performed on the individual target level, as well as on the combined multimarker signature. As a result of the differential DNA methylation assessment by MBD sequencing, 58 of the 78 MSP assays were designed in regions previously unexplored in neuroblastoma, and 36 are located in non-promoter or non-coding regions. In total, 5 individual MSP assays (located in CCDC177, NXPH1, lnc-MRPL3-2, lnc-TREX1-1 and one on a region from chromosome 8 with no further annotation) predict event-free survival and 4 additional assays (located in SPRED3, TNFAIP2, NPM2 and CYYR1) also predict overall survival. Furthermore, a robust 58-marker methylation signature predicting overall and event-free survival was established. In conclusion, this study encompasses the largest DNA methylation biomarker study in neuroblastoma so far. We identified and independently validated several novel prognostic biomarkers, as well as a prognostic 58-marker methylation signature.

Hidden Genetic Variation in LCA9-Associated Congenital Blindness Explained by 5'UTR Mutations and Copy-Number Variations of NMNAT1.

Leber congenital amaurosis (LCA) is a severe autosomal-recessive retinal dystrophy leading to congenital blindness. A recently identified LCA gene is NMNAT1, located in the LCA9 locus. Although most mutations in blindness genes are coding variations, there is accumulating evidence for hidden noncoding defects or structural variations (SVs). The starting point of this study was an LCA9-associated consanguineous family in which no coding mutations were found in the LCA9 region. Exploring the untranslated regions of NMNAT1 revealed a novel homozygous 5'UTR variant, c.-70A>T. Moreover, an adjacent 5'UTR variant, c.-69C>T, was identified in a second consanguineous family displaying a similar phenotype. Both 5'UTR variants resulted in decreased NMNAT1 mRNA abundance in patients' lymphocytes, and caused decreased luciferase activity in human retinal pigment epithelial RPE-1 cells. Second, we unraveled pseudohomozygosity of a coding NMNAT1 mutation in two unrelated LCA patients by the identification of two distinct heterozygous partial NMNAT1 deletions. Molecular characterization of the breakpoint junctions revealed a complex Alu-rich genomic architecture. Our study uncovered hidden genetic variation in NMNAT1-associated LCA and emphasized a shift from coding to noncoding regulatory mutations and repeat-mediated SVs in the molecular pathogenesis of heterogeneous recessive disorders such as hereditary blindness.

Genome wide expression profiling of p53 regulated miRNAs in neuroblastoma.

Restoration of the antitumor activity of p53 could offer a promising approach for the treatment of neuroblastoma. MicroRNAs (miRNAs) are important mediators of p53 activity, but their role in the p53 response has not yet been comprehensively addressed in neuroblastoma. Therefore, we set out to characterize alterations in miRNA expression that are induced by p53 activation in neuroblastoma cells. Genome-wide miRNA expression analysis showed that miR-34a-5p, miR-182-5p, miR-203a, miR-222-3p, and miR-432-5p are upregulated following nutlin-3 treatment in a p53 dependent manner. The function of miR-182-5p, miR-203a, miR-222-3p, and miR-432-5p was analyzed by ectopic overexpression of miRNA mimics. We observed that these p53-regulated miRNAs inhibit the proliferation of neuroblastoma cells to varying degrees, with the most profound growth inhibition recorded for miR-182-5p. Overexpression of miR-182-5p promoted apoptosis in some neuroblastoma cell lines and induced neuronal differentiation of NGP cells. Using Chromatin Immunoprecipitation-qPCR (ChIP-qPCR), we did not observe direct binding of p53 to MIR182, MIR203, MIR222, and MIR432 in neuroblastoma cells. Taken together, our findings yield new insights in the network of p53-regulated miRNAs in neuroblastoma.

miRBase Tracker: keeping track of microRNA annotation changes.

Since 2002, information on individual microRNAs (miRNAs), such as reference names and sequences, has been stored in miRBase, the reference database for miRNA annotation. As a result of progressive insights into the miRNome and its complexity, miRBase underwent addition and deletion of miRNA records, changes in annotated miRNA sequences and adoption of more complex naming schemes over time. Unfortunately, miRBase does not allow straightforward assessment of these ongoing miRNA annotation changes, which has resulted in substantial ambiguity regarding miRNA identity and sequence in public literature, in target prediction databases and in content on various commercially available analytical platforms. As a result, correct interpretation, comparison and integration of miRNA study results are compromised, which we demonstrate here by assessing the impact of ignoring sequence annotation changes. To address this problem, we developed miRBase Tracker (www.mirbasetracker.org), an easy-to-use online database that keeps track of all historical and current miRNA annotation present in the miRBase database. Three basic functionalities allow researchers to keep their miRNA annotation up-to-date, reannotate analytical miRNA platforms and link published results with outdated annotation to the latest miRBase release. We expect miRBase Tracker to increase the transparency and annotation accuracy in the field of miRNA research. DATABASE URL: www.mirbasetracker.org.

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.

Genome-wide promoter methylation analysis in neuroblastoma identifies prognostic methylation biomarkers.

BACKGROUND: Accurate outcome prediction in neuroblastoma, which is necessary to enable the optimal choice of risk-related therapy, remains a challenge. To improve neuroblastoma patient stratification, this study aimed to identify prognostic tumor DNA methylation biomarkers. RESULTS: To identify genes silenced by promoter methylation, we first applied two independent genome-wide methylation screening methodologies to eight neuroblastoma cell lines. Specifically, we used re-expression profiling upon 5-aza-2'-deoxycytidine (DAC) treatment and massively parallel sequencing after capturing with a methyl-CpG-binding domain (MBD-seq). Putative methylation markers were selected from DAC-upregulated genes through a literature search and an upfront methylation-specific PCR on 20 primary neuroblastoma tumors, as well as through MBD- seq in combination with publicly available neuroblastoma tumor gene expression data. This yielded 43 candidate biomarkers that were subsequently tested by high-throughput methylation-specific PCR on an independent cohort of 89 primary neuroblastoma tumors that had been selected for risk classification and survival. Based on this analysis, methylation of KRT19, FAS, PRPH, CNR1, QPCT, HIST1H3C, ACSS3 and GRB10 was found to be associated with at least one of the classical risk factors, namely age, stage or MYCN status. Importantly, HIST1H3C and GNAS methylation was associated with overall and/or event-free survival. CONCLUSIONS: This study combines two genome-wide methylation discovery methodologies and is the most extensive validation study in neuroblastoma performed thus far. We identified several novel prognostic DNA methylation markers and provide a basis for the development of a DNA methylation-based prognostic classifier in neuroblastoma.

The microRNA body map: dissecting microRNA function through integrative genomics.

While a growing body of evidence implicates regulatory miRNA modules in various aspects of human disease and development, insights into specific miRNA function remain limited. Here, we present an innovative approach to elucidate tissue-specific miRNA functions that goes beyond miRNA target prediction and expression correlation. This approach is based on a multi-level integration of corresponding miRNA and mRNA gene expression levels, miRNA target prediction, transcription factor target prediction and mechanistic models of gene network regulation. Predicted miRNA functions were either validated experimentally or compared to published data. The predicted miRNA functions are accessible in the miRNA bodymap, an interactive online compendium and mining tool of high-dimensional newly generated and published miRNA expression profiles. The miRNA bodymap enables prioritization of candidate miRNAs based on their expression pattern or functional annotation across tissue or disease subgroup. The miRNA bodymap project provides users with a single one-stop data-mining solution and has great potential to become a community resource.

Neuroblastoma epigenetics: from candidate gene approaches to genome-wide screenings.

Neuroblastoma (NB) is a childhood tumor originating from sympathetic nervous system cells. Although recently new insights into genes involved in NB have emerged, the molecular basis of neuroblastoma development and progression still remains poorly understood. The best-characterized genetic alterations include amplification of the proto-oncogene MYCN, ALK activating mutations or amplification, gain of chromosome arm 17q and losses of 1p, 3p, and 11q. Epigenetic alterations have been described as well: caspase-8 (CASP8) and RAS-association domain family 1 isoform A (RASSF1A) DNA-methylation are important events for the development and progression of neuroblastoma. In total, there are about 75 genes described as epigenetically affected in NB cell lines and/or NB primary samples. These epigenetic alterations were either found using a candidate gene approach or based on the analysis of genome-wide screening techniques. This review gives an extensive overview of all epigenetic changes described in NB as of today, with a main focus on both prognostic use and the potential of genome-wide techniques to find epigenetic prognostic biomarkers in NB. We summarize the key findings so far and the state-of-the-art of the upcoming methods at a unique time frame in the transition towards combined genome wide chromatin immune-precipitation (ChIP) and DNA sequencing techniques.

Jasmonate response of the Nicotiana tabacum agglutinin promoter in Arabidopsis thaliana.

NICTABA is a carbohydrate-binding protein (also called lectin) that is expressed in several Nicotiana species after treatment with jasmonates and insect herbivory. Analyses with tobacco lines overexpressing the NICTABA gene as well as lines with reduced lectin expression have shown the entomotoxic effect of NICTABA against Lepidopteran larvae, suggesting a role of the lectin in plant defense. Until now, little is known with respect to the upstream regulatory mechanisms that are controlling the expression of inducible plant lectins. Using Arabidopsis thaliana plants stably expressing a promoter-ß-glucuronidase (GUS) fusion construct, it was shown that jasmonate treatment influenced the NICTABA promoter activity. A strong GUS staining pattern was detected in very young tissues (the apical and root meristems, the cotyledons and the first true leaves), but the promoter activity decreased when plants were getting older. NICTABA was also expressed at low concentrations in tobacco roots and expression levels increased after cold treatment. The data presented confirm a jasmonate-dependent response of the promoter sequence of the tobacco lectin gene in Arabidopsis. These new jasmonate-responsive tobacco promoter sequences can be used as new tools in the study of jasmonate signalling related to plant development and defense.

Epigenetic databases and computational methodologies in the analysis of epigenetic datasets.

Epigenetics research is one of the emerging research fields in biomedical research. During the last few decades, a collection of useful tools (both to design the experiments and to analyze the results) and databases are developed. This review chapter discusses basic tools which are used to detect CpG islands and the Transcription Start Site (TSS) and discusses experimental design and analysis, mainly of DNA-methylation experiments. During the last years, an enormous amount of experimental data had been generated and published. Therefore, we describe some epigenetic databases, with a special focus on DNA methylation and cancer. Some general cancer databases are discussed as well, as they might reveal the link between the results from epigenetic experiments and their biological influence on the development or progression of cancer. Next, some novel computational approaches in epigenetics are discussed, for instance used to predict the methylation state of a promoter in certain circumstances. To show a possible data analysis strategy of an epigenetic dataset in cancer research, there is a showcase where a DNA-methylation dataset, generated on colorectal cancer samples, is analyzed. This demonstrates how a DNA-methylation dataset might look like and the different steps in a possible analysis strategy and how to interpret the results.

The N-myc downstream regulated gene (NDRG) family: diverse functions, multiple applications.

The N-myc downstream regulated gene (NDRG) family of proteins consists of 4 members, NDRG1-4, which are well conserved through evolution. The first member to be discovered and responsible for the family name was NDRG1, because its expression is repressed by the proto-oncogenes MYCN and MYC. All family members are characterized by an α/ß hydrolase-fold motif; however, the precise molecular and cellular function of these family members has not been fully elucidated. Although the exact function of NDRG family members has not been clearly elucidated, emerging evidence suggests that mutations in these genes are associated with diverse neurological and electrophysiological syndromes. In addition, aberrant expression as well as tumor suppressor and oncogenic functions affecting key hallmarks of carcinogenesis such as cell proliferation, differentiation, migration, invasion, and stress response have been reported for several of the NDRG proteins. In this review, we summarize the current literature on the NDRG family members concerning their structure, origin, and tissue distribution. In addition, we review the current knowledge regarding the regulation and signaling of the NDRG family members in development and normal physiology. Finally, their role in disease and potential clinical applications (their role as detection or prognostic markers) are discussed.

Proteins with an Euonymus lectin-like domain are ubiquitous in Embryophyta.

BACKGROUND: Cloning of the Euonymus lectin led to the discovery of a novel domain that also occurs in some stress-induced plant proteins. The distribution and the diversity of proteins with an Euonymus lectin (EUL) domain were investigated using detailed analysis of sequences in publicly accessible genome and transcriptome databases. RESULTS: Comprehensive in silico analyses indicate that the recently identified Euonymus europaeus lectin domain represents a conserved structural unit of a novel family of putative carbohydrate-binding proteins, which will further be referred to as the Euonymus lectin (EUL) family. The EUL domain is widespread among plants. Analysis of retrieved sequences revealed that some sequences consist of a single EUL domain linked to an unrelated N-terminal domain whereas others comprise two in tandem arrayed EUL domains. A new classification system for these lectins is proposed based on the overall domain architecture. Evolutionary relationships among the sequences with EUL domains are discussed. CONCLUSION: The identification of the EUL family provides the first evidence for the occurrence in terrestrial plants of a highly conserved plant specific domain. The widespread distribution of the EUL domain strikingly contrasts the more limited or even narrow distribution of most other lectin domains found in plants. The apparent omnipresence of the EUL domain is indicative for a universal role of this lectin domain in plants. Although there is unambiguous evidence that several EUL domains possess carbohydrate-binding activity further research is required to corroborate the carbohydrate-binding properties of different members of the EUL family.

Critical evaluation of the use of bioinformatics as a theoretical tool to find high-potential sources of ACE inhibitory peptides.

A bioinformatics analysis to screen for high-potential sources of angiotensin converting enzyme (ACE) inhibitory peptides was conducted in the area of insect muscle proteins. Vertebrate muscle proteins are reported as good sources of ACE inhibitory peptides, while the research on invertebrate muscle proteins is limited. A phylogenetic tree constructed with actin sequences of both vertebrate and invertebrate species indicated a high homology. Furthermore, a quantitative in silico ACE inhibition analysis suggested that actin proteins of invertebrates have potentials as new sources of ACE inhibitory peptides. On one insect, Bombyx mori, a more detailed in silico analysis was done followed by a small experimental study. The in silico analysis indicated B. mori as a high-potential source of ACE inhibitory peptides and this was supported by the ACE inhibitory activity of the partially purified actin preparation. In conclusion, in food science, in silico analysis can be used as fast initial screening tool to look for high-potential sources of ACE inhibitory peptides and other peptidic bioactivities.

Discovery of DNA methylation markers in cervical cancer using relaxation ranking.

BACKGROUND: To discover cancer specific DNA methylation markers, large-scale screening methods are widely used. The pharmacological unmasking expression microarray approach is an elegant method to enrich for genes that are silenced and re-expressed during functional reversal of DNA methylation upon treatment with demethylation agents. However, such experiments are performed in in vitro (cancer) cell lines, mostly with poor relevance when extrapolating to primary cancers. To overcome this problem, we incorporated data from primary cancer samples in the experimental design. A strategy to combine and rank data from these different data sources is essential to minimize the experimental work in the validation steps. AIM: To apply a new relaxation ranking algorithm to enrich DNA methylation markers in cervical cancer. RESULTS: The application of a new sorting methodology allowed us to sort high-throughput microarray data from both cervical cancer cell lines and primary cervical cancer samples. The performance of the sorting was analyzed in silico. Pathway and gene ontology analysis was performed on the top-selection and gives a strong indication that the ranking methodology is able to enrich towards genes that might be methylated. Terms like regulation of progression through cell cycle, positive regulation of programmed cell death as well as organ development and embryonic development are overrepresented. Combined with the highly enriched number of imprinted and X-chromosome located genes, and increased prevalence of known methylation markers selected from cervical (the highest-ranking known gene is CCNA1) as well as from other cancer types, the use of the ranking algorithm seems to be powerful in enriching towards methylated genes.Verification of the DNA methylation state of the 10 highest-ranking genes revealed that 7/9 (78%) gene promoters showed DNA methylation in cervical carcinomas. Of these 7 genes, 3 (SST, HTRA3 and NPTX1) are not methylated in normal cervix tissue. CONCLUSION: The application of this new relaxation ranking methodology allowed us to significantly enrich towards methylation genes in cancer. This enrichment is both shown in silico and by experimental validation, and revealed novel methylation markers as proof-of-concept that might be useful in early cancer detection in cervical scrapings.