ABSTRACT
The molecular alterations that occur in cells before cancer is manifest are largely uncharted. Lung carcinoma in situ (CIS) lesions are the pre-invasive precursor to squamous cell carcinoma. Although microscopically identical, their future is in equipoise, with half progressing to invasive cancer and half regressing or remaining static. The cellular basis of this clinical observation is unknown. Here, we profile the genomic, transcriptomic, and epigenomic landscape of CIS in a unique patient cohort with longitudinally monitored pre-invasive disease. Predictive modeling identifies which lesions will progress with remarkable accuracy. We identify progression-specific methylation changes on a background of widespread heterogeneity, alongside a strong chromosomal instability signature. We observed mutations and copy number changes characteristic of cancer and chart their emergence, offering a window into early carcinogenesis. We anticipate that this new understanding of cancer precursor biology will improve early detection, reduce overtreatment, and foster preventative therapies targeting early clonal events in lung cancer.
Subject(s)
Carcinoma in Situ/genetics , Carcinoma, Squamous Cell/genetics , Gene Expression Regulation, Neoplastic , Lung Neoplasms/genetics , Adult , Aged , Aged, 80 and over , Carcinogenesis/genetics , Chromosomal Instability/genetics , Cohort Studies , DNA Copy Number Variations , DNA Methylation/genetics , Disease Progression , Epigenomics , Female , Gene Expression Profiling , Genomics , Humans , Longitudinal Studies , Male , Middle Aged , MutationABSTRACT
Imprinting is the preferential expression of one parental allele over the other. It is controlled primarily through differential methylation of cytosine at CpG dinucleotides. Here we combine 285 methylomes and 11,617 transcriptomes from peripheral blood samples with parent-of-origin phased haplotypes, to produce a new map of imprinted methylation and gene expression patterns across the human genome. We demonstrate how imprinted methylation is a continuous rather than a binary characteristic. We describe at high resolution the parent-of-origin methylation pattern at the 15q11.2 Prader-Willi/Angelman syndrome locus, with nearly confluent stochastic paternal methylation punctuated by 'spikes' of maternal methylation. We find examples of polymorphic imprinted methylation unrelated (at VTRNA2-1 and PARD6G) or related (at CHRNE) to nearby SNP genotypes. We observe RNA isoform-specific imprinted expression patterns suggestive of a methylation-sensitive transcriptional elongation block. Finally, we gain new insights into parent-of-origin-specific effects on phenotypes at the DLK1/MEG3 and GNAS loci.
Subject(s)
DNA Methylation/genetics , Genome, Human , Genomic Imprinting/physiology , Inheritance Patterns/genetics , Parents , Transcriptome/genetics , Angelman Syndrome/genetics , Case-Control Studies , Chromosomes, Human, Pair 15 , Cohort Studies , CpG Islands/genetics , Female , Genetic Loci , Humans , Iceland , Male , Polymorphism, Single Nucleotide , Prader-Willi Syndrome/genetics , Quantitative Trait Loci/geneticsABSTRACT
SUMMARY: The Illumina Infinium HumanMethylationEPIC BeadChip is the new platform for high-throughput DNA methylation analysis, effectively doubling the coverage compared to the older 450 K array. Here we present a significantly updated and improved version of the Bioconductor package ChAMP, which can be used to analyze EPIC and 450k data. Many enhanced functionalities have been added, including correction for cell-type heterogeneity, network analysis and a series of interactive graphical user interfaces. AVAILABILITY AND IMPLEMENTATION: ChAMP is a BioC package available from https://bioconductor.org/packages/release/bioc/html/ChAMP.html. CONTACT: a.teschendorff@ucl.ac.uk or s.beck@ucl.ac.uk or a.feber@ucl.ac.uk. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Subject(s)
DNA Methylation , Oligonucleotide Array Sequence Analysis/methods , SoftwareABSTRACT
Non-CG methylation is an unexplored epigenetic hallmark of pluripotent stem cells. Here we report that a reduction in non-CG methylation is associated with impaired differentiation capacity into endodermal lineages. Genome-wide analysis of 2,670 non-CG sites in a discovery cohort of 25 phenotyped human induced pluripotent stem cell (hiPSC) lines revealed unidirectional loss (Δß=13%, P<7.4 × 10(-4)) of non-CG methylation that correctly identifies endodermal differentiation capacity in 23 out of 25 (92%) hiPSC lines. Translation into a simplified assay of only nine non-CG sites maintains predictive power in the discovery cohort (Δß=23%, P<9.1 × 10(-6)) and correctly identifies endodermal differentiation capacity in nine out of ten pluripotent stem cell lines in an independent replication cohort consisting of hiPSCs reprogrammed from different cell types and different delivery systems, as well as human embryonic stem cell (hESC) lines. This finding infers non-CG methylation at these sites as a biomarker when assessing endodermal differentiation capacity as a readout.
Subject(s)
Cell Differentiation , DNA Methylation , Endoderm/cytology , Induced Pluripotent Stem Cells/cytology , Biomarkers/metabolism , Cohort Studies , Endoderm/metabolism , Humans , Induced Pluripotent Stem Cells/metabolismSubject(s)
Adaptor Proteins, Signal Transducing/genetics , Neuroendocrine Tumors/genetics , Nuclear Proteins/genetics , Pancreatic Neoplasms/genetics , Adaptor Proteins, Signal Transducing/metabolism , Adult , Aged , Aged, 80 and over , Co-Repressor Proteins , DNA Helicases/genetics , DNA Helicases/metabolism , Epigenomics , Female , Gene Expression Regulation, Neoplastic , Genome-Wide Association Study , Humans , Male , Middle Aged , Molecular Chaperones , Neuroendocrine Tumors/metabolism , Neuroendocrine Tumors/pathology , Nuclear Proteins/metabolism , Pancreatic Neoplasms/metabolism , Pancreatic Neoplasms/pathology , Prognosis , Survival Analysis , X-linked Nuclear Protein , Young AdultABSTRACT
DNA methylation analysis has become an integral part of biomedical research. For high-throughput applications such as epigenome-wide association studies, the Infinium HumanMethylation450 (450K) BeadChip is currently the platform of choice. However, BeadChip processing relies on traditional bisulfite (BS) based protocols which cannot discriminate between 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC). Here, we report the adaptation of the recently developed oxidative bisulfite (oxBS) chemistry to specifically detect both 5mC and 5hmC in a single workflow using 450K BeadChips, termed oxBS-450K. Supported by validation using mass spectrometry and pyrosequencing, we demonstrate reproducible (R(2)>0.99) detection of 5hmC in human brain tissue using the optimised oxBS-450K protocol described here.
Subject(s)
DNA Methylation , Epigenomics/methods , Epigenomics/instrumentation , Humans , Oligonucleotide Array Sequence Analysis/instrumentation , Oligonucleotide Array Sequence Analysis/methods , Reproducibility of Results , Sequence Analysis, DNA/methodsABSTRACT
The Illumina HumanMethylation450 BeadChip has become a popular platform for interrogating DNA methylation in epigenome-wide association studies (EWAS) and related projects as well as resource efforts such as the International Cancer Genome Consortium (ICGC) and the International Human Epigenome Consortium (IHEC). This has resulted in an exponential increase of 450k data in recent years and triggered the development of numerous integrated analysis pipelines and stand-alone packages. This review will introduce and discuss the currently most popular pipelines and packages and is particularly aimed at new 450k users.
Subject(s)
CpG Islands , DNA Methylation , Oligonucleotide Array Sequence Analysis/methods , Epigenomics/methods , Genome , Genome, Human , Humans , SoftwareABSTRACT
Aging is a progressive process that results in the accumulation of intra- and extracellular alterations that in turn contribute to a reduction in health. Age-related changes in DNA methylation have been reported before and may be responsible for aging-induced changes in gene expression, although a causal relationship has yet to be shown. Using genome-wide assays, we analyzed age-induced changes in DNA methylation and their effect on gene expression with and without transient induction with the synthetic transcription modulating agent WY14,643. To demonstrate feasibility of the approach, we isolated peripheral blood mononucleated cells (PBMCs) from five young and five old healthy male volunteers and cultured them with or without WY14,643. Infinium 450K BeadChip and Affymetrix Human Gene 1.1 ST expression array analysis revealed significant differential methylation of at least 5 % (ΔYO > 5 %) at 10,625 CpG sites between young and old subjects, but only a subset of the associated genes were also differentially expressed. Age-related differential methylation of previously reported epigenetic biomarkers of aging including ELOVL2, FHL2, PENK, and KLF14 was confirmed in our study, but these genes did not display an age-related change in gene expression in PBMCs. Bioinformatic analysis revealed that differentially methylated genes that lack an age-related expression change predominantly represent genes involved in carcinogenesis and developmental processes, and expression of most of these genes were silenced in PBMCs. No changes in DNA methylation were found in genes displaying transiently induced changes in gene expression. In conclusion, aging-induced differential methylation often targets developmental genes and occurs mostly without change in gene expression.
Subject(s)
Aging/genetics , Epigenesis, Genetic/genetics , Gene Expression Regulation, Developmental , Leukocytes, Mononuclear/metabolism , RNA/genetics , Adult , Aged , Aging/metabolism , Cells, Cultured , DNA Methylation , Genome, Human , Healthy Volunteers , Humans , Leukocytes, Mononuclear/cytology , Male , Middle AgedABSTRACT
The integration of genomic and epigenomic data is an increasingly popular approach for studying the complex mechanisms driving cancer development. We have developed a method for evaluating both methylation and copy number from high-density DNA methylation arrays. Comparing copy number data from Infinium HumanMethylation450 BeadChips and SNP arrays, we demonstrate that Infinium arrays detect copy number alterations with the sensitivity of SNP platforms. These results show that high-density methylation arrays provide a robust and economic platform for detecting copy number and methylation changes in a single experiment. Our method is available in the ChAMP Bioconductor package: http://www.bioconductor.org/packages/2.13/bioc/html/ChAMP.html.
Subject(s)
DNA Copy Number Variations/genetics , DNA Methylation/genetics , Oligonucleotide Array Sequence Analysis/methods , Algorithms , Genome, Human , Humans , Polymorphism, Single Nucleotide , SoftwareABSTRACT
BACKGROUND: Maternal undernutrition leads to an increased risk of metabolic disorders in offspring including obesity and insulin resistance, thought to be due to a programmed thrifty phenotype which is inappropriate for a subsequent richer nutritional environment. In a rat model, both male and female offspring of undernourished mothers are programmed to become obese, however postnatal leptin treatment gives discordant results between males and females. Leptin treatment is able to rescue the adverse programming effects in the female offspring of undernourished mothers, but not in their male offspring. Additionally, in these rats, postnatal leptin treatment of offspring from normally-nourished mothers programmes their male offspring to develop obesity in later life, while there is no comparable effect in their female offspring. RESULTS: We show by microarray analysis of the female liver transcriptome that both maternal undernutrition and postnatal leptin treatment independently induce a similar thrifty transcriptional programme affecting carbohydrate metabolism, amino acid metabolism and oxidative stress genes. Paradoxically, however, the combination of both stimuli restores a more normal transcriptional environment. This demonstrates that "leptin reversal" is a global phenomenon affecting all genes involved in fetal programming by maternal undernourishment and leptin treatment. The thrifty transcriptional programme was associated with pro-inflammatory markers and downregulation of adaptive immune mediators, particularly MHC class I genes, suggesting a deficit in antigen presentation in these offspring. CONCLUSIONS: We propose a revised model of developmental programming reconciling the male and female observations, in which there are two competing programmes which collectively drive liver transcription. The first element is a thrifty metabolic phenotype induced by early life growth restriction independently of leptin levels. The second is a homeostatic set point calibrated in response to postnatal leptin surge, which is able to over-ride the metabolic programme. This "calibration model" for the postnatal leptin surge, if applicable in humans, may have implications for understanding responses to catch-up growth in infants. Additionally, the identification of an antigen presentation deficit associated with metabolic thriftiness may relate to a previously observed correlation between birth season (a proxy for gestational undernutrition) and infectious disease mortality in rural African communities.
Subject(s)
Fetal Nutrition Disorders/genetics , Leptin/pharmacology , Liver/drug effects , Amino Acids/metabolism , Animals , Carbohydrate Metabolism/genetics , Diet , Disease Models, Animal , Female , Fetal Development , Fetal Nutrition Disorders/metabolism , Fetal Nutrition Disorders/pathology , Histocompatibility Antigens Class I/genetics , Histocompatibility Antigens Class I/metabolism , Inflammation Mediators/metabolism , Liver/metabolism , Male , Obesity/metabolism , Obesity/pathology , Oxidative Stress/genetics , Phenotype , Pregnancy , Rats , Rats, Wistar , Transcriptome/drug effectsABSTRACT
UNLABELLED: The Illumina Infinium HumanMethylation450 BeadChip is a new platform for high-throughput DNA methylation analysis. Several methods for normalization and processing of these data have been published recently. Here we present an integrated analysis pipeline offering a choice of the most popular normalization methods while also introducing new methods for calling differentially methylated regions and detecting copy number aberrations. AVAILABILITY AND IMPLEMENTATION: ChAMP is implemented as a Bioconductor package in R. The package and the vignette can be downloaded at bioconductor.org
Subject(s)
DNA Methylation , Oligonucleotide Array Sequence Analysis/methods , Software , DNA Copy Number VariationsABSTRACT
A link has been established between prenatal nutrition and the development of metabolic and cardiovascular diseases later in life, a process referred to as developmental programming. It has been suggested that the trajectory of development is shifted by alterations in the maternal nutritional state leading to changes in developmental plasticity, in part underpinned by epigenetic changes in gene regulation. However, to date, only candidate gene approaches have been used to assess expression and molecular changes in the offspring of maternally undernourished animals. Furthermore, most work has focused on animals at an age where the programmed phenotype is already manifest and little is known about changes in gene expression in the offspring prior to development of obesity and related metabolic disorders. Gene expression profiles of liver, retroperitoneal white adipose fat, and biceps femoris skeletal muscle tissue from young adult male rats (55 days old) in which nutritional status had been manipulated in utero by maternal undernutrition (UN) were compared to the profiles of offspring of ad libitum fed mothers serving as the control group (AD) (8 offspring/group). The expression profiles were determined using the Illumina RatRef-12 BeadChip. No significant changes in expression were identified for skeletal muscle or white adipose tissue. However, studies of liver tissue showed 249 differentially expressed genes (143 up regulated, 106 down regulated). Although the animals at day 55 have yet to develop obesity they already show biochemical abnormalities and by day 110 express a phenotype characterized by increased adiposity and altered insulin sensitivity. An analysis of pathways affected suggests that intrauterine programming of UN animals to favor fat as an energy source results in mitochondrial dysfunction which initially affects the postnatal hepatic function and subsequently, via the resultant metabolic changes in other organs leads to the evolution of a phenotype similar to that of the metabolic syndrome.