GNOSIS: an R Shiny app supporting cancer genomics survival analysis with cBioPortal.

Exploratory analysis of cancer consortia data curated by the cBioPortal repository typically requires advanced programming skills and expertise to identify novel genomic prognostic markers that have the potential for both diagnostic and therapeutic exploitation. We developed GNOSIS (GeNomics explOrer using StatistIcal and Survival analysis in R), an R Shiny App incorporating a range of R packages enabling users to efficiently explore and visualise such clinical and genomic data. GNOSIS provides an intuitive graphical user interface and multiple tab panels supporting a range of functionalities, including data upload and initial exploration, data recoding and subsetting, data visualisations, statistical analysis, mutation analysis and, in particular, survival analysis to identify prognostic markers. GNOSIS also facilitates reproducible research by providing downloadable input logs and R scripts from each session, and so offers an excellent means of supporting clinician-researchers in developing their statistical computing skills.

Exploring breast and prostate cancer RNA-seq derived radiosensitivity with the Genomic Adjusted Radiation Dose (GARD) model.

The use of a 10 gene transcriptional signature as part of the GARD model has been shown to be predictive of radiotherapy benefit for a range of cancers, with the potential to determine an optimal overall dose per patient. We used publicly available RNA-seq transcriptomics data from a luminal B breast cancer patient and from 14 prostate cancer patients to explore the radiosensitivity indices (RSI) and so GARD estimates of both tumour and proximal normal biopsies from each individual. Clear differences of clinical relevance in derived radiobiological properties between tumour and proximal normal tissues were evident for the breast cancer patient, whilst such differences across the prostate cancer cohort were more equivocal. Using the prostate cancer cohort's median tumour predicted GARD value as a threshold for high therapeutic effect for radiotherapy, we found evidence that a higher overall prescribed dose than the widely used 72 Gy/36fx could benefit half of these patients. This exploratory study demonstrates the potential combining the GARD model with sequencing based transcriptomics could have in informing personalised radiotherapeutic practise for both breast and prostate cancer patients.

Loss of MMR and TGFBR2 Increases the Susceptibility to Microbiota-Dependent Inflammation-Associated Colon Cancer.

BACKGROUND AND AIMS: Mutations in DNA mismatch repair (MMR) genes are causative in Lynch syndrome and a significant proportion of sporadic colorectal cancers (CRCs). MMR-deficient (dMMR) CRCs display increased mutation rates, with mutations frequently accumulating at short repetitive DNA sequences throughout the genome (microsatellite instability). The TGFBR2 gene is one of the most frequently mutated genes in dMMR CRCs. Therefore, we generated an animal model to study how the loss of both TGFBR2 signaling impacts dMMR-driven intestinal tumorigenesis in vivo and explore the impact of the gut microbiota. METHODS: We generated VCMsh2/Tgfbr2 mice in which Msh2loxP and Tgfbr2loxP alleles are inactivated by Villin-Cre recombinase in the intestinal epithelium. VCMsh2/Tgfbr2 mice were analyzed for their rate of intestinal cancer development and for the mutational spectra and gene expression profiles of tumors. In addition, we assessed the impact of chemically induced chronic inflammation and gut microbiota composition on colorectal tumorigenesis. RESULTS: VCMsh2/Tgfbr2 mice developed small intestinal adenocarcinomas and CRCs with histopathological features highly similar to CRCs in Lynch syndrome patients. The CRCs in VCMsh2/Tgfbr2 mice were associated with the presence of colitis and displayed genetic and histological features that resembled inflammation-associated CRCs in human patients. The development of CRCs in VCMsh2/Tgfbr2 mice was strongly modulated by the gut microbiota composition, which in turn was impacted by the TGFBR2 status of the tumors. CONCLUSIONS: Our results demonstrate a synergistic interaction between MMR and TGFBR2 inactivation in inflammation-associated colon tumorigenesis and highlight the crucial impact of the gut microbiota on modulating the incidence of inflammation-associated CRCs.

Survival outcomes are associated with genomic instability in luminal breast cancers.

Breast cancer is the leading cause of cancer related death among women. Breast cancers are generally diagnosed and treated based on clinical and histopathological features, along with subtype classification determined by the Prosigna Breast Cancer Prognostic Gene Signature Assay (also known as PAM50). Currently the copy number alteration (CNA) landscape of the tumour is not considered. We set out to examine the role of genomic instability (GI) in breast cancer survival since CNAs reflect GI and correlate with survival in other cancers. We focused on the 70% of breast cancers classified as luminal and carried out a comprehensive survival and association analysis using Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) data to determine whether CNA Score Quartiles derived from absolute CNA counts are associated with survival. Analysis revealed that patients diagnosed with luminal A breast cancer have a CNA landscape associated with disease specific survival, suggesting that CNA Score can provide a statistically robust prognostic factor. Furthermore, stratification of patients into subtypes based on gene expression has shown that luminal A and B cases overlap, and it is in this region we largely observe luminal A cases with reduced survival outlook. Therefore, luminal A breast cancer patients with quantitatively elevated CNA counts may benefit from more aggressive therapy. This demonstrates how individual genomic landscapes can facilitate personalisation of therapeutic interventions to optimise survival outcomes.

Correction: Septin 9 isoforms promote tumorigenesis in mammary epithelial cells by increasing migration and ECM degradation through metalloproteinase secretion at focal adhesions.

The original version of this Article contained an error in the author affiliations. Vladislav V. Verkhusha was incorrectly associated with the School of Mathematics, Statistics & Applied Mathematics, National University of Ireland Galway, Galway, Ireland. The correct affiliation is Anatomy and Structural Biology, Albert Einstein College of Medicine, Yeshiva University, Bronx, NY, USA.

Septin 9 isoforms promote tumorigenesis in mammary epithelial cells by increasing migration and ECM degradation through metalloproteinase secretion at focal adhesions.

The cytoskeletal interacting protein Septin 9 (SEPT9), a member of the septin gene family, has been proposed to have oncogenic functions. It is a known hot spot of retroviral tagging insertion and a fusion partner of both de novo and therapy-induced mixed lineage leukemia (MLL). Of all septins, SEPT9 holds the strongest link to cancer, especially breast cancer. Murine models of breast cancer frequently exhibit SEPT9 amplification in the form of double minute chromosomes, and about 20% of human breast cancer display genomic amplification and protein over expression at the SEPT9 locus. Yet, a clear mechanism by which SEPT9 elicits tumor-promoting functions is lacking. To obtain unbiased insights on molecular signatures of SEPT9 upregulation in breast tumors, we overexpressed several of its isoforms in breast cancer cell lines. Global transcriptomic profiling supports a role of SEPT9 in invasion. Functional studies reveal that SEPT9 upregulation is sufficient to increase degradation of the extracellular matrix, while SEPT9 downregulation inhibits this process. The degradation pattern is peripheral and associated with focal adhesions (FAs), where it is coupled with increased expression of matrix metalloproteinases (MMPs). SEPT9 overexpression induces MMP upregulation in human tumors and in culture models and promotes MMP3 secretion to the media at FAs. Downregulation of SEPT9 or chemical inhibition of septin filament assembly impairs recruitment of MMP3 to FAs. Our results indicate that SEPT9 promotes upregulation and both trafficking and secretion of MMPs near FAs, thus enhancing migration and invasion of breast cancer cells.

Inflammation-associated DNA methylation patterns in epithelium of ulcerative colitis.

Aberrant DNA methylation patterns have been reported in inflamed tissues and may play a role in disease. We studied DNA methylation and gene expression profiles of purified intestinal epithelial cells from ulcerative colitis patients, comparing inflamed and non-inflamed areas of the colon. We identified 577 differentially methylated sites (false discovery rate <0.2) mapping to 210 genes. From gene expression data from the same epithelial cells, we identified 62 differentially expressed genes with increased expression in the presence of inflammation at prostate cancer susceptibility genes PRAC1 and PRAC2. Four genes showed inverse correlation between methylation and gene expression; ROR1, GXYLT2, FOXA2, and, notably, RARB, a gene previously identified as a tumor suppressor in colorectal adenocarcinoma as well as breast, lung and prostate cancer. We highlight targeted and specific patterns of DNA methylation and gene expression in epithelial cells from inflamed colon, while challenging the importance of epithelial cells in the pathogenesis of chronic inflammation.

Resveratrol Improves Vascular Function and Mitochondrial Number but Not Glucose Metabolism in Older Adults.

BACKGROUND: Resveratrol, a plant-derived polyphenol, has been reported to improve glucose metabolism and vascular function and to extend life span in animal models, but studies in humans have been inconclusive. METHODS: In a randomized, double-blind crossover study, we treated older glucose-intolerant adults (n = 30) with resveratrol (2-3 g/daily) or placebo, each for 6 weeks. A standard mixed-meal test was used to assess insulin sensitivity (Matsuda index) and secretion (C-peptide deconvolution) and vascular function by reactive hyperemia peripheral arterial tonometry. Skeletal muscle samples were obtained for gene expression using RNA-Seq analysis and to assess mitochondrial morphology. RESULTS: There were no changes in glucose tolerance, insulin sensitivity, weight, blood pressure, or lipid profile following resveratrol treatment. Fasting reactive hyperemia index improved with resveratrol (2.02 ± 0.2 vs 1.76 ± 0.02, p = .002). RNA-Seq analysis yielded 140 differentially expressed transcripts (corrected p-value ≤ .05), predominantly associated with mitochondrial genes and noncoding RNA. Ingenuity Pathway Analysis confirmed that mitochondrial dysfunction (p = 2.77 × 10-12) and oxidative phosphorylation (p = 1.41 × 10-11) were the most significantly perturbed pathways. Mitochondrial number, but not size, was increased. CONCLUSIONS: Resveratrol treatment of older adults with impaired glucose regulation may have beneficial effects on vascular function, but not glucose metabolism or insulin sensitivity. Changes in gene expression suggest effects similar to those observed with caloric restriction, which has been shown to increase life and health span in animal models, although its significance for humans is uncertain. Future human studies should address the appropriate dose range and low bioavailability of resveratrol.

LPA receptor activity is basal specific and coincident with early pregnancy and involution during mammary gland postnatal development.

During pregnancy, luminal and basal epithelial cells of the adult mammary gland proliferate and differentiate resulting in remodeling of the adult gland. While pathways that control this process have been characterized in the gland as a whole, the contribution of specific cell subtypes, in particular the basal compartment, remains largely unknown. Basal cells provide structural and contractile support, however they also orchestrate the communication between the stroma and the luminal compartment at all developmental stages. Using RNA-seq, we show that basal cells are extraordinarily transcriptionally dynamic throughout pregnancy when compared to luminal cells. We identified gene expression changes that define specific basal functions acquired during development that led to the identification of novel markers. Enrichment analysis of gene sets from 24 mouse models for breast cancer pinpoint to a potential new function for insulin-like growth factor 1 (Igf1r) in the basal epithelium during lactogenesis. We establish that ß-catenin signaling is activated in basal cells during early pregnancy, and demonstrate that this activity is mediated by lysophosphatidic acid receptor 3 (Lpar3). These findings identify novel pathways active during functional maturation of the adult mammary gland.

Differential DNA methylation patterns of homeobox genes in proximal and distal colon epithelial cells.

Region and cell-type specific differences in the molecular make up of colon epithelial cells have been reported. Those differences may underlie the region-specific characteristics of common colon epithelial diseases such as colorectal cancer and inflammatory bowel disease. DNA methylation is a cell-type specific epigenetic mark, essential for transcriptional regulation, silencing of repetitive DNA and genomic imprinting. Little is known about any region-specific variations in methylation patterns in human colon epithelial cells. Using purified epithelial cells and whole biopsies (n= 19) from human subjects, we generated epigenome-wide DNA methylation data (using the HELP-tagging assay), comparing the methylation signatures of the proximal and distal colon. We identified a total of 125 differentially methylated sites (DMS) mapping to transcription start sites of protein-coding genes, most notably several members of the homeobox (HOX) family of genes. Patterns of differential methylation were validated with MassArray EpiTYPER. We also examined DNA methylation in whole biopsies, applying a computational technique to deconvolve variation in methylation within cell types and variation in cell-type composition across biopsies. Including inferred epithelial proportions as a covariate in differential methylation analysis applied to the whole biopsies resulted in greater overlap with the results obtained from purified epithelial cells compared with when the covariate was not included. Results obtained from both approaches highlight region-specific methylation patterns of HOX genes in colonic epithelium. Regional variation in methylation patterns has implications for the study of diseases that exhibit regional expression patterns in the human colon, such as inflammatory bowel disease and colorectal cancer.

Clinical and molecular significance of microvascular inflammation in transplant kidney biopsies.

The diagnostic criteria for antibody-mediated rejection (AMR) are continuously evolving. Here we investigated the clinical and molecular significance of different Banff microvascular inflammation (MVI) scores in transplant kidney biopsies. A total of 356 patients with clinically indicated kidney transplant biopsies were classified into three groups based on MVI scores of 0, 1, 2, or more for Groups 1-3, respectively. Gene expression profiles were assessed using arrays on a representative subset of 93 patients. The incidence of donor-specific anti-HLA antibodies was increased from 25% in Group 1 to 36% in Group 2 and to 54% in Group 3. Acute and chronic AMR were significantly more frequent in Group 3 (15% and 35%) compared with the Group 2 (3% and 15%) and Group 1 (0% and 5%), respectively. Gene expression profiles showed increased interferon-γ and rejection-induced, cytotoxic and regulatory T-cell, natural killer cell-associated and donor-specific antibody (DSA)-selective transcripts in Group 3 compared with Groups 1 and 2. There was no significant difference in gene expression profiles between the Groups 1 and 2. Increased intragraft expression of DSA-selective transcripts was found in the biopsies of C4d- Group 3 patients. Thus, an MVI score of 2 or more was significantly associated with a histological diagnosis of acute and chronic antibody-mediated rejection. Hence, increased intragraft DSA-selective gene transcripts may be used as molecular markers for AMR, especially in C4d- biopsies.

Deregulation of the Ras-Erk Signaling Axis Modulates the Enhancer Landscape.

Unrestrained receptor tyrosine kinase (RTK) signaling and epigenetic deregulation are root causes of tumorigenesis. We establish linkage between these processes by demonstrating that aberrant RTK signaling unleashed by oncogenic HRas(G12V) or loss of negative feedback through Sprouty gene deletion remodels histone modifications associated with active typical and super-enhancers. However, although both lesions disrupt the Ras-Erk axis, the expression programs, enhancer signatures, and transcription factor networks modulated upon HRas(G12V) transformation or Sprouty deletion are largely distinct. Oncogenic HRas(G12V) elevates histone 3 lysine 27 acetylation (H3K27ac) levels at enhancers near the transcription factor Gata4 and the kinase Prkcb, as well as their expression levels. We show that Gata4 is necessary for the aberrant gene expression and H3K27ac marking at enhancers, and Prkcb is required for the oncogenic effects of HRas(G12V)-driven cells. Taken together, our findings demonstrate that dynamic reprogramming of the cellular enhancer landscape is a major effect of oncogenic RTK signaling.

Integrative Metabolic Signatures for Hepatic Radiation Injury.

BACKGROUND: Radiation-induced liver disease (RILD) is a dose-limiting factor in curative radiation therapy (RT) for liver cancers, making early detection of radiation-associated liver injury absolutely essential for medical intervention. A metabolomic approach was used to determine metabolic signatures that could serve as biomarkers for early detection of RILD in mice. METHODS: Anesthetized C57BL/6 mice received 0, 10 or 50 Gy Whole Liver Irradiation (WLI) and were contrasted to mice, which received 10 Gy whole body irradiation (WBI). Liver and plasma samples were collected at 24 hours after irradiation. The samples were processed using Gas Chromatography/Mass Spectrometry and Liquid Chromatography/Mass Spectrometry. RESULTS: Twenty four hours after WLI, 407 metabolites were detected in liver samples while 347 metabolites were detected in plasma. Plasma metabolites associated with 50 Gy WLI included several amino acids, purine and pyrimidine metabolites, microbial metabolites, and most prominently bradykinin and 3-indoxyl-sulfate. Liver metabolites associated with 50 Gy WLI included pentose phosphate, purine, and pyrimidine metabolites in liver. Plasma biomarkers in common between WLI and WBI were enriched in microbial metabolites such as 3 indoxyl sulfate, indole-3-lactic acid, phenyllactic acid, pipecolic acid, hippuric acid, and markers of DNA damage such as 2-deoxyuridine. Metabolites associated with tryptophan and indoles may reflect radiation-induced gut microbiome effects. Predominant liver biomarkers in common between WBI and WLI were amino acids, sugars, TCA metabolites (fumarate), fatty acids (lineolate, n-hexadecanoic acid) and DNA damage markers (uridine). CONCLUSIONS: We identified a set of metabolomic markers that may prove useful as plasma biomarkers of RILD and WBI. Pathway analysis also suggested that the unique metabolic changes observed after liver irradiation was an integrative response of the intestine, liver and kidney.

Clinical, Histological, and Molecular Markers Associated With Allograft Loss in Transplant Glomerulopathy Patients.

BACKGROUND: We aimed to investigate the clinical, histopathological, and molecular factors associated with allograft loss in transplant glomerulopathy (TGP) patients. METHODS: Of the 525 patients who underwent clinically indicated kidney biopsies, 52 (10%) had diagnosis of TGP. Gene expression profiles of 28 TGP and 11 normal transplant kidney biopsy samples were analyzed by Affymetrix HuGene 1.0 ST expression arrays. RESULTS: Over a median follow up of 23 months (1-46 months) after the diagnosis of TGP by biopsy, 17 patients (32%) lost their allografts at a median of 16 months (1-44 months). There was no difference between the 2 groups in terms of any demographic variables, serum creatinine, panel reactive antibody levels, donor-specific antibody frequency, or mean fluorescence intensity values. Patients who lost their allograft had a significantly higher median spot protein to creatinine ratio 2.81 (1.20-6.00) compared to no graft loss patients 1.16 (0.15-2.53), (P < 0.01), and a trends toward a higher mean chronic glomerulopathy (cg) score (1.65 ± 0.93 vs 1.11 ± 0.93) (P = 0.05). There was also no difference in microvascular inflammation or any other Banff injury scores between the 2 groups. Although 117 gene transcripts were upregulated in both groups, 86 and 57 were upregulated in graft loss and functioning allograft groups, respectively. There were significantly increased levels of intragraft endothelial cell-associated transcripts, gene transcripts associated with complement cascade, interleukins and their receptors and granulysin in graft loss patients compared to patients with a functioning allograft. CONCLUSION: Our results demonstrate differential intragraft gene expression profiles in TGP patients with allograft loss.

Intestinal microbiota-derived metabolomic blood plasma markers for prior radiation injury.

PURPOSE: Assessing whole-body radiation injury and absorbed dose is essential for remediation efforts following accidental or deliberate exposure in medical, industrial, military, or terrorist incidents. We hypothesize that variations in specific metabolite concentrations extracted from blood plasma would correlate with whole-body radiation injury and dose. METHODS AND MATERIALS: Groups of C57BL/6 mice (n=12 per group) were exposed to 0, 2, 4, 8, and 10.4 Gy of whole-body gamma radiation. At 24 hours after treatment, all animals were euthanized, and both plasma and liver biopsy samples were obtained, the latter being used to identify a distinct hepatic radiation injury response within plasma. A semiquantitative, untargeted metabolite/lipid profile was developed using gas chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry, which identified 354 biochemical compounds. A second set of C57BL/6 mice (n=6 per group) were used to assess a subset of identified plasma markers beyond 24 hours. RESULTS: We identified a cohort of 37 biochemical compounds in plasma that yielded the optimal separation of the irradiated sample groups, with the most correlated metabolites associated with pyrimidine (positively correlated) and tryptophan (negatively correlated) metabolism. The latter were predominantly associated with indole compounds, and there was evidence that these were also correlated between liver and plasma. No evidence of saturation as a function of dose was observed, as has been noted for studies involving metabolite analysis of urine. CONCLUSIONS: Plasma profiling of specific metabolites related to pyrimidine and tryptophan pathways can be used to differentiate whole-body radiation injury and dose response. As the tryptophan-associated indole compounds have their origin in the intestinal microbiome and subsequently the liver, these metabolites particularly represent an attractive marker for radiation injury within blood plasma.

The meta-epigenomic structure of purified human stem cell populations is defined at cis-regulatory sequences.

The mechanism and significance of epigenetic variability in the same cell type between healthy individuals are not clear. Here we purify human CD34+ haematopoietic stem and progenitor cells (HSPCs) from different individuals and find that there is increased variability of DNA methylation at loci with properties of promoters and enhancers. The variability is especially enriched at candidate enhancers near genes transitioning between silent and expressed states, and encoding proteins with leukocyte differentiation properties. Our findings of increased variability at loci with intermediate DNA methylation values, at candidate 'poised' enhancers and at genes involved in HSPC lineage commitment suggest that CD34+ cell subtype heterogeneity between individuals is a major mechanism for the variability observed. Epigenomic studies performed on cell populations, even when purified, are testing collections of epigenomes, or meta-epigenomes. Our findings show that meta-epigenomic approaches to data analysis can provide insights into cell subpopulation structure.

Increased intragraft rejection-associated gene transcripts in patients with donor-specific antibodies and normal biopsies.

We investigated why some donor-specific antibody-positive patients do not develop antibody-mediated rejection. Of 71 donor-specific antibody-positive patients, 46 had diagnosis of antibody-mediated rejection and 25 had normal biopsies. Fifty donor-specific antibody-negative patients with normal biopsies were used as a control group. A subgroup of 61 patients with available biopsy and 64 with blood samples were analyzed by microarrays. Both donor-specific antibody-positive/antibody-mediated rejection-positive and negative biopsies showed increased expression of gene transcripts associated with cytotoxic T cells, natural killer cells, macrophages, interferon-gamma, and rejection compared to donor-specific antibody-negative biopsies. Regulatory T-cell transcripts were upregulated in donor-specific antibody-positive/antibody-mediated rejection-positive and B-cell transcripts in donor-specific antibody-positive/antibody-mediated rejection-negative biopsies. Whole-blood gene expression analysis showed increased immune activity in only donor-specific antibody-positive/antibody-mediated rejection-positive but not negative patients. During a median follow-up of 36 months, 4 donor-specific antibody-positive/antibody-mediated rejection-negative patients developed antibody-mediated rejection, 12 continued to have donor-specific antibody, but 9 lost their donor-specific antibody. Gene expression profiles did not predict the development of antibody-mediated rejection or the persistence of donor-specific antibody. Thus, donor-specific antibody-positive/antibody-mediated rejection-negative patients had increased rejection-associated gene transcripts in their allografts despite no histologic findings of rejection but not in their blood. This was found in both biopsy and blood samples of donor-specific antibody-positive/antibody-mediated rejection-positive patients.

Genomics of BK viremia in kidney transplant recipients.

BACKGROUND: This study aimed to investigate global gene expression profiles of BK viremia and nephropathy (BKVN) samples using microarrays to investigate the immunologic response to BK virus. METHODS: Patients were monitored for BK viremia in the blood monthly for 6 months, then at 9 and 12 months after kidney transplantation. BKVN and normal transplant kidney biopsy samples, and whole blood samples of patients with and without BK viremia were analyzed by Affymetrix Human Gene 1.0 ST Arrays. RESULTS: During a mean follow-up of 917±325 days, 61 of the 289 patients (21%) developed BK viremia at a median 149 (27, 1,113) days after transplantation with a median peak PCR titers of 35,900 (1,000, 2,677,000). The only significant risk factor for development of BK viremia was induction with anti-thymocyte globulin (P=0.03). Only four patients developed BKVN (1.3%). Pathogenesis-based transcript analysis revealed a significant increased expression of interferon-gamma and rejection induced (GRIT), quantitative cytotoxic T-cell (QCAT), quantitative constitutive and alternate macrophage, B-cell and natural killer cell-associated transcripts (NKAT), indicating an active inflammatory immune response in BKVN biopsies (n=3) compared to normal transplant kidney biopsies with (n=3) and without BK viremia (n=11). The whole blood gene expression profiles of 19 BK viremia patients revealed significant increased expression of GRIT, QCAT, and NKAT compared to 14 patients without viremia. CONCLUSIONS: The results showed increased activity of cytotoxic T cells and natural killer cells in BKVN and viremia samples resembling acute rejection and suggested the involvement of both adaptive and innate immunity.

Gene-set analysis is severely biased when applied to genome-wide methylation data.

MOTIVATION: DNA methylation is an epigenetic mark that can stably repress gene expression. Because of its biological and clinical significance, several methods have been developed to compare genome-wide patterns of methylation between groups of samples. The application of gene set analysis to identify relevant groups of genes that are enriched for differentially methylated genes is often a major component of the analysis of these data. This can be used, for example, to identify processes or pathways that are perturbed in disease development. We show that gene-set analysis, as it is typically applied to genome-wide methylation assays, is severely biased as a result of differences in the numbers of CpG sites associated with different classes of genes and gene promoters. RESULTS: We demonstrate this bias using published data from a study of differential CpG island methylation in lung cancer and a dataset we generated to study methylation changes in patients with long-standing ulcerative colitis. We show that several of the gene sets that seem enriched would also be identified with randomized data. We suggest two existing approaches that can be adapted to correct the bias. Accounting for the bias in the lung cancer and ulcerative colitis datasets provides novel biological insights into the role of methylation in cancer development and chronic inflammation, respectively. Our results have significant implications for many previous genome-wide methylation studies that have drawn conclusions on the basis of such strongly biased analysis. CONTACT: cathal.seoighe@nuigalway.ie SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.