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1.  Brave new epigenomes: the dawn of epigenetic engineering 
Genome Medicine  2015;7(1):59.
Editorial summary
New methods for epigenome editing now make it possible to manipulate the epigenome in living cells with unprecedented specificity and efficiency. These ground-breaking approaches are beginning to yield novel insights into the function of individual chromatin marks in the context of cellular phenotype.
PMCID: PMC4472160  PMID: 26089986
2.  Comparative methylome analysis identifies new tumour subtypes and biomarkers for transformation of nephrogenic rests into Wilms tumour 
Genome Medicine  2015;7(1):11.
Wilms tumours (WTs) are characterised by several hallmarks that suggest epimutations such as aberrant DNA methylation are involved in tumour progression: loss of imprinting at 11p15, lack of recurrent mutations and formation of nephrogenic rests (NRs), which are lesions of retained undifferentiated embryonic tissue that can give rise to WTs.
To identify such epimutations, we performed a comprehensive methylome analysis on 20 matched trios of micro-dissected WTs, NRs and surrounding normal kidneys (NKs) using Illumina Infinium HumanMethylation450 Bead Chips and functionally validated findings using RNA sequencing.
Comparison of NRs with NK revealed prominent tissue biomarkers: 629 differentially methylated regions, of which 55% were hypermethylated and enriched for domains that are bivalent in embryonic stem cells and for genes expressed during development (P = 2.49 × 10-5). Comparison of WTs with NRs revealed two WT subgroups; group-2 WTs and NRs were epigenetically indistinguishable whereas group-1 WTs showed an increase in methylation variability, hypomethylation of renal development genes, hypermethylation and relative loss of expression of cell adhesion genes and known and potential new WT tumour suppressor genes (CASP8, H19, MIR195, RB1 and TSPAN32) and was strongly associated with bilateral disease (P = 0.032). Comparison of WTs and NRs to embryonic kidney highlighted the significance of polycomb target methylation in Wilms tumourigenesis.
Methylation levels vary during cancer evolution. We have described biomarkers related to WT evolution from its precursor NRs which may be useful to differentiate between these tissues for patients with bilateral disease.
Electronic supplementary material
The online version of this article (doi:10.1186/s13073-015-0136-4) contains supplementary material, which is available to authorized users.
PMCID: PMC4354990  PMID: 25763109
3.  C2c: turning cancer into chronic disease 
Genome Medicine  2014;6(5):38.
PMCID: PMC4062053  PMID: 24944585
4.  Human-specific epigenetic variation in the immunological Leukotriene B4 Receptor (LTB4R/BLT1) implicated in common inflammatory diseases 
Genome Medicine  2014;6(3):19.
Common human diseases are caused by the complex interplay of genetic susceptibility as well as environmental factors. Due to the environment’s influence on the epigenome, and therefore genome function, as well as conversely the genome’s facilitative effect on the epigenome, analysis of this level of regulation may increase our knowledge of disease pathogenesis.
In order to identify human-specific epigenetic influences, we have performed a novel genome-wide DNA methylation analysis comparing human, chimpanzee and rhesus macaque.
We have identified that the immunological Leukotriene B4 receptor (LTB4R, BLT1 receptor) is the most epigenetically divergent human gene in peripheral blood in comparison with other primates. This difference is due to the co-ordinated active state of human-specific hypomethylation in the promoter and human-specific increased gene body methylation. This gene is significant in innate immunity and the LTB4/LTB4R pathway is involved in the pathogenesis of the spectrum of human inflammatory diseases. This finding was confirmed by additional neutrophil-only DNA methylome and lymphoblastoid H3K4me3 chromatin comparative data. Additionally we show through functional analysis that this receptor has increased expression and a higher response to the LTB4 ligand in human versus rhesus macaque peripheral blood mononuclear cells. Genome-wide we also find human species-specific differentially methylated regions (human s-DMRs) are more prevalent in CpG island shores than within the islands themselves, and within the latter are associated with the CTCF motif.
This result further emphasises the exclusive nature of the human immunological system, its divergent adaptation even from very closely related primates, and the power of comparative epigenomics to identify and understand human uniqueness.
PMCID: PMC4062055  PMID: 24598577
5.  Targeted next-generation sequencing of head and neck squamous cell carcinoma identifies novel genetic alterations in HPV+ and HPV- tumors 
Genome Medicine  2013;5(5):49.
Human papillomavirus positive (HPV+) head and neck squamous cell carcinoma (HNSCC) is an emerging disease, representing a distinct clinical and epidemiological entity. Understanding the genetic basis of this specific subtype of cancer could allow therapeutic targeting of affected pathways for a stratified medicine approach.
Twenty HPV+ and 20 HPV- laser-capture microdissected oropharyngeal carcinomas were used for paired-end sequencing of hybrid-captured DNA, targeting 3,230 exons in 182 genes often mutated in cancer. Copy number alteration (CNA) profiling, Sequenom MassArray sequencing and immunohistochemistry were used to further validate findings.
HPV+ and HPV- oropharyngeal carcinomas cluster into two distinct subgroups. TP53 mutations are detected in 100% of HPV negative cases and abrogation of the G1/S checkpoint by CDKN2A/B deletion and/or CCND1 amplification occurs in the majority of HPV- tumors.
These findings strongly support a causal role for HPV, acting via p53 and RB pathway inhibition, in the pathogenesis of a subset of oropharyngeal cancers and suggest that studies of CDK inhibitors in HPV- disease may be warranted. Mutation and copy number alteration of PI3 kinase (PI3K) pathway components appears particularly prevalent in HPV+ tumors and assessment of these alterations may aid in the interpretation of current clinical trials of PI3K, AKT, and mTOR inhibitors in HNSCC.
PMCID: PMC4064312  PMID: 23718828
6.  Identification and functional validation of HPV-mediated hypermethylation in head and neck squamous cell carcinoma 
Genome Medicine  2013;5(2):15.
Human papillomavirus-positive (HPV+) head and neck squamous cell carcinoma (HNSCC) represents a distinct clinical and epidemiological condition compared with HPV-negative (HPV-) HNSCC. To test the possible involvement of epigenetic modulation by HPV in HNSCC, we conducted a genome-wide DNA-methylation analysis.
Using laser-capture microdissection of 42 formalin-fixed paraffin wax-embedded (FFPE) HNSCCs, we generated DNA-methylation profiles of 18 HPV+ and 14 HPV- samples, using Infinium 450 k BeadArray technology. Methylation data were validated in two sets of independent HPV+/HPV- HNSCC samples (fresh-frozen samples and cell lines) using two independent methods (Infinium 450 k and whole-genome methylated DNA immunoprecipitation sequencing (MeDIP-seq)). For the functional analysis, an HPV- HNSCC cell line was transduced with lentiviral constructs containing the two HPV oncogenes (E6 and E7), and effects on methylation were assayed using the Infinium 450 k technology.
Results and discussion
Unsupervised clustering over the methylation variable positions (MVPs) with greatest variation showed that samples segregated in accordance with HPV status, but also that HPV+ tumors are heterogeneous. MVPs were significantly enriched at transcriptional start sites, leading to the identification of a candidate CpG island methylator phenotype in a sub-group of the HPV+ tumors. Supervised analysis identified a strong preponderance (87%) of MVPs towards hypermethylation in HPV+ HNSCC. Meta-analysis of our HNSCC and publicly available methylation data in cervical and lung cancers confirmed the observed DNA-methylation signature to be HPV-specific and tissue-independent. Grouping of MVPs into functionally more significant differentially methylated regions identified 43 hypermethylated promoter DMRs, including for three cadherins of the Polycomb group target genes. Integration with independent expression data showed strong negative correlation, especially for the cadherin gene-family members. Combinatorial ectopic expression of the two HPV oncogenes (E6 and E7) in an HPV- HNSCC cell line partially phenocopied the hypermethylation signature seen in HPV+ HNSCC tumors, and established E6 as the main viral effector gene.
Our data establish that archival FFPE tissue is very suitable for this type of methylome analysis, and suggest that HPV modulates the HNSCC epigenome through hypermethylation of Polycomb repressive complex 2 target genes such as cadherins, which are implicated in tumor progression and metastasis.
PMCID: PMC3706778  PMID: 23419152
7.  Advances in the identification and analysis of allele-specific expression 
Genome Medicine  2009;1(5):56.
Allele-specific expression (ASE) is essential for normal development and many cellular processes but, if impaired, can result in disease. ASE is a feature of organisms with genomes consisting of more than one set of homologous chromosomes. The higher the number of chromosome sets (ploidy) per cell, the higher the potential complexity of ASE. Humans, for instance, are diploid (except germ cells, which are haploid), resulting in multiple possible expression states in time and space for each set of alleles. ASE is invoked and modulated by both genetic and epigenetic changes, affecting the underlying DNA sequence or chromatin of each allele, respectively. Although numerous methods have been developed to assay ASE, they usually require RNA to be available and are dependent upon genetic polymorphisms (such as single nucleotide polymorphisms (SNPs)) to differentiate between allelic transcripts. The rapid convergence to second-generation sequencing as the method of choice to examine genomic, epigenomic and transcriptomic data enables an integrated and more general approach to define and predict ASE, independent of SNPs. This 'Omni-Seq' approach has the potential to advance our understanding of the biology and pathophysiology of ASE-mediated processes by elucidating subtle combinatorial effects, leading to the accurate delineation of sub-phenotypes with consequential benefit for improved insight into disease etiology.
PMCID: PMC2689448  PMID: 19490587

Results 1-7 (7)