The DNA methylation landscape of multiple myeloma shows extensive inter- and intrapatient heterogeneity that fuels transcriptomic variability

AbstractBackgroundCancer evolution depends on epigenetic and genetic diversity. Historically, in multiple myeloma (MM), subclonal diversity and tumor evolution have been investigated mostly from a genetic perspective.ResultsHere, we combined the notions of epipolymorphism and epiallele switching to analyze DNA methylation heterogeneity in MM patients. We show that MM is characterized by the continuous accumulation of stochastic methylation at the promoters of development-related genes. High entropy change is associated with poor outcomes and depends predominantly on partially methylated domains (PMDs). These PMDs, which represent the major source of inter- and intrapatient DNA methylation heterogeneity in MM, are linked to other key epigenetic aberrations, such as CpG island (CGI)/transcription start site (TSS) hypermethylation and H3K27me3 redistribution as well as 3D organization alterations. In addition, transcriptome analysis revealed that intratumor methylation heterogeneity was associated with low-level expression and high variability.ConclusionWe propose that disordered methylation in MM is responsible for high epigenetic and transcriptomic instability allowing tumor cells to adapt to environmental changes by tapping into a pool of evolutionary trajectories.

Download Full-text

The DNA methylation landscape of multiple myeloma shows extensive inter- and intrapatient heterogeneity that fuels transcriptomic variability

Genome Medicine ◽

10.1186/s13073-021-00938-3 ◽

2021 ◽

Vol 13 (1) ◽

Author(s):

Jennifer Derrien ◽

Catherine Guérin-Charbonnel ◽

Victor Gaborit ◽

Loïc Campion ◽

Magali Devic ◽

...

Keyword(s):

Dna Methylation ◽

Multiple Myeloma ◽

Environmental Changes ◽

Cpg Island ◽

Entropy Change ◽

Tumor Evolution ◽

Cancer Evolution ◽

Reduced Representation ◽

Reduced Representation Bisulfite Sequencing ◽

Poor Outcomes

Abstract Background Cancer evolution depends on epigenetic and genetic diversity. Historically, in multiple myeloma (MM), subclonal diversity and tumor evolution have been investigated mostly from a genetic perspective. Methods Here, we performed an analysis of 42 MM samples from 21 patients by using enhanced reduced representation bisulfite sequencing (eRRBS). We combined several metrics of epigenetic heterogeneity to analyze DNA methylation heterogeneity in MM patients. Results We show that MM is characterized by the continuous accumulation of stochastic methylation at the promoters of development-related genes. High combinatorial entropy change is associated with poor outcomes in our pilot study and depends predominantly on partially methylated domains (PMDs). These PMDs, which represent the major source of inter- and intrapatient DNA methylation heterogeneity in MM, are linked to other key epigenetic aberrations, such as CpG island (CGI)/transcription start site (TSS) hypermethylation and H3K27me3 redistribution as well as 3D organization alterations. In addition, transcriptome analysis revealed that intratumor methylation heterogeneity was associated with low-level expression and high variability. Conclusions We propose that disrupted DNA methylation in MM is responsible for high epigenetic and transcriptomic instability allowing tumor cells to adapt to environmental changes by tapping into a pool of evolutionary trajectories.

Download Full-text

Epigenome Chaos: Stochastic and Deterministic DNA Methylation Events Drive Cancer Evolution

Cancers ◽

10.3390/cancers13081800 ◽

2021 ◽

Vol 13 (8) ◽

pp. 1800

Author(s):

Giusi Russo ◽

Alfonso Tramontano ◽

Ilaria Iodice ◽

Lorenzo Chiariotti ◽

Antonio Pezone

Keyword(s):

Dna Methylation ◽

Tumor Heterogeneity ◽

Tumor Evolution ◽

Aberrant Methylation ◽

Epigenetic Alterations ◽

Cancer Evolution ◽

Cancer Types ◽

Chromatin Proteins ◽

Aberrant Dna Methylation ◽

Methylation Patterns

Cancer evolution is associated with genomic instability and epigenetic alterations, which contribute to the inter and intra tumor heterogeneity, making genetic markers not accurate to monitor tumor evolution. Epigenetic changes, aberrant DNA methylation and modifications of chromatin proteins, determine the “epigenome chaos”, which means that the changes of epigenetic traits are randomly generated, but strongly selected by deterministic events. Disordered changes of DNA methylation profiles are the hallmarks of all cancer types, but it is not clear if aberrant methylation is the cause or the consequence of cancer evolution. Critical points to address are the profound epigenetic intra- and inter-tumor heterogeneity and the nature of the heterogeneity of the methylation patterns in each single cell in the tumor population. To analyze the methylation heterogeneity of tumors, new technological and informatic tools have been developed. This review discusses the state of the art of DNA methylation analysis and new approaches to reduce or solve the complexity of methylated alleles in DNA or cell populations.

Download Full-text

Tissue Factor Pathway Inhibitor 2 (TFPI2) Is Commonly Methylated in Multiple Myeloma

Blood ◽

10.1182/blood.v120.21.4617.4617 ◽

2012 ◽

Vol 120 (21) ◽

pp. 4617-4617

Author(s):

Eleftheria Hatzimichael ◽

Aggeliki Dasoula ◽

Valentinos Kounnis ◽

Andreas Katsenos ◽

Tim Crook ◽

...

Keyword(s):

Dna Methylation ◽

Multiple Myeloma ◽

Cell Lines ◽

Genomic Dna ◽

Tissue Factor Pathway Inhibitor ◽

Cpg Island ◽

Methylation Status ◽

Tissue Factor Pathway ◽

Beta 2 ◽

Beta 2 Microglobulin

Abstract Abstract 4617 Background: Tissue Factor Pathway Inhibitor 2 (TFPI2) is a member of the Kunitz-type serine proteinase inhibitor family that regulates extracellular tissue homeostasis by inhibiting matrix metalloproteinases. Transcriptional silencing of TFPI2 due to DNA methylation of the CpG island of its promoter has been reported in several tumors and has been suggested to facilitate tumor cell invasion and angiogenesis. We investigated the DNA methylation status of TFPI2 in multiple myeloma (MM) patients as part of a program of epigenetic profiling of multiple myeloma. Methods: Genomic DNA extracted from two human MM cell lines (U-266 and RPMI-8226) and bone marrow aspirate samples from a well chracterized series of MM patients was modified by sodium bisulphite using the EZ DNA methylation kit, ZymoResearch. Bisulfite modified DNA was used as a template for Methylation Specific PCR with primers specific for unmethylated and methylated alleles. Control methylated (CpG Genome™ Universal Methylated, Chemicon International) and unmethylated genomic DNA was included in each experiment. Chi-square test, logistic regression analysis and unpaired t-test were used to investigate associations between gene methylation and ISS stage, presence of extramedullary disease, bone disease, anemia (hemoglobin ≤10 mg/dL), renal failure, serum albumin and beta (2)-microglobulin level. Kaplan-Meier curves were used to estimate the probabilities of survival and the Log-rank test to assess the statistical significance of differences in event rates. Results: Forty six patients with MM (26 male, 20 female; mean age 64) and two human MM cell lines were studied. According to the International Staging System (ISS) 21 patients were classified as stage I, 10 stage II and 15 as stage III disease. Methylation in the CpG island of the promoter of TFPI2 was detected in 30 patients (65%, 95%CI= 52,6–78,7%) and in both cell lines (100%). Patients with methylated TFPI2 had significantly elevated beta 2 microglobulin levels (5553 vs 3787 mg/L mean values, p=0.0002) but we did not detect a statistically significant difference in overall survival among patients with methylated versus non methylated TFPI2. No other relevant correlations were detected. Conclusion: TFPI2 is commonly methylated in MM patients. Transcriptional silencing of TFPI2 may play a role in the mechanism of myelomatogenesis and its methylation status warrants further evaluation as a diagnostic co-marker for this disease. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Epigenetic silencing of LPP/miR-28 in multiple myeloma

Journal of Clinical Pathology ◽

10.1136/jclinpath-2017-204501 ◽

2017 ◽

Vol 71 (3) ◽

pp. 253-258 ◽

Cited By ~ 6

Author(s):

Zhenhai Li ◽

Kwan Yeung Wong ◽

Godfrey Chi-fung Chan ◽

Chor Sang Chim

Keyword(s):

Dna Methylation ◽

Multiple Myeloma ◽

Cpg Island ◽

Myeloma Cell ◽

Tumour Suppressor ◽

Host Gene ◽

Healthy Controls ◽

Promoter Dna Methylation ◽

Promoter Dna ◽

Partially Methylated

AimsmiR-28-5- is a tumour suppressor microRNA implicated in cancers. As a CpG island is absent in miR-28-5- but present in its host gene, LPP (LIM domain containing preferred translocation partner in lipoma), we hypothesized that miR-28-5p is epigenetically silenced by promoter DNA methylation of its host gene in multiple myeloma.MethodsMethylation-specific PCR, verified by quantitative bisulfite pyrosequencing, was employed to study methylation of LPP/miR-28 in healthy controls (n=10), human myeloma cell lines (HMCLs) (n=15), and primary myeloma marrow samples at diagnosis (n=49) and at relapse (n=18). Quantitative reverse transcription PCR was used to investigate expression of miR-28-5p, LPP and CCND1.ResultsLPP/miR-28 was completely unmethylated in all healthy controls and 12 (80%) HMCLs, but partially methylated in three (20%) HMCLs. Methylation of LPP/miR-28 correlated with low expression of miR-285p (p=0.012) and LPP (p=0.037) in HMCLs. In RPMI-8226R cells, in which LPP/miR-28 was partially methylated, 5-AzadC treatment led to demethylation of LPP/miR-28 and re-expression of both miR-28-5p (p=0.0007) and LPP (p=0.0007), whereas continuous culture without 5-AzadC restored LPP/miR-28 methylation and reduced expression of both miR-28-5p (p=0.0013) and LPP (p=0.0025). Moreover, a known miR-28-5p target, CCND1, was expressed at higher levels in HMCLs with LPP/miR-28 methylation than those without, consistent with a tumour suppressor role of miR-28-5p in myeloma. However, in primary samples, LPP/miR-28 was methylated in two (4.1%) at diagnosis, whereas none at relapse.ConclusionsThis is the first report of epigenetic regulation of the intronic miR-28-5p expression by promoter DNA methylation of its host gene, hence warrants further study in different cancers.

Download Full-text

Maternal anxiety and depression in pregnancy and DNA methylation of the NR3C1 glucocorticoid receptor gene

Epigenomics ◽

10.2217/epi-2020-0022 ◽

2020 ◽

Author(s):

Alexandra E Dereix ◽

Rachel Ledyard ◽

Allyson M Redhunt ◽

Tessa R Bloomquist ◽

Kasey JM Brennan ◽

...

Keyword(s):

Dna Methylation ◽

Depressive Symptoms ◽

Trait Anxiety ◽

Cpg Island ◽

Depression Scale ◽

Maternal Anxiety ◽

Anxiety And Depression ◽

Pregnancy Anxiety ◽

Glucocorticoid Receptor Gene ◽

Island Shore

Aim: To quantify associations of anxiety and depression during pregnancy with differential cord blood DNA methylation of the glucorticoid receptor ( NR3C1). Materials & methods: Pregnancy anxiety, trait anxiety and depressive symptoms were collected using the Pregnancy Related Anxiety Scale, State-Trait Anxiety Index and Edinburgh Postnatal Depression Scale, respectively. NR3C1 methylation was determined at four methylation sites. Results: DNA methylation of CpG 1 in the NR3C1 CpG island shore was higher in infants born to women with high pregnancy anxiety (β 2.54, 95% CI: 0.49–4.58) and trait anxiety (β 1.68, 95% CI: 0.14–3.22). No significant association was found between depressive symptoms and NR3C1 methylation. Conclusion: We found that maternal anxiety was associated with increased NR3C1 CpG island shore methylation.

Download Full-text

Integrated analysis of DNA methylation profile in HLA-G gene and imaging in coronary heart disease

European Heart Journal ◽

10.1093/ehjci/ehaa946.1255 ◽

2020 ◽

Vol 41 (Supplement_2) ◽

Author(s):

G Benincasa ◽

C Schiano ◽

T Infante ◽

M Franzese ◽

R Casale ◽

...

Keyword(s):

Dna Methylation ◽

Coronary Heart Disease ◽

Heart Disease ◽

Cpg Island ◽

Methylation Profile ◽

Integrated Analysis ◽

Funding Source ◽

Relevant Parameter ◽

Cardiac Computed Tomography Angiography ◽

Dna Methylation Profile

Abstract Aims Immune endothelial inflammation, underlie coronary heart disease (CHD) related phenotypes, could provide new insight into the pathobiology of the disease. We investigated DNA methylation level of the unique CpG island of HLA-G gene in CHD patients and evaluated the correlation with cardiac computed tomography angiography (CCTA) features. Methods Thirty-two patients that underwent CCTA for suspected CHD were enrolled for this study. Obstructive CHD group included fourteen patients, in which there was a stenosis greater than or equal to 50% in one or more of the major coronary arteries detected; whereas subjects with Calcium (Ca) Score=0, uninjured coronaries and with no obstructive CHD were considered as control subjects (Ctrls) (n=18). For both groups, DNA methylation profile of the whole 5'UTR-CpG island of HLA-G was measured. The plasma soluble HLA-G (sHLA-G) levels were detected in all subjects by specific ELISA assay. Statistical analysis was performed using R software. Results For the first time, our study reported that 1) a significant hypomethylation characterized three specific fragments (B, C and F) of the 5'UTR-CpG island (p=0.05) of HLA-G gene in CHD patients compared to Ctrl group; 2) hypomethylation level of one specific fragment positively correlated with coronary Ca score, a relevant parameter of CCTA (p<0.05) between two groups. Conclusions Our results showed that reduced levels of circulating HLA-G molecules could derive from epigenetic marks inducing hypomethylation of specific regions into 5'UTR-CpG island of HLA-G gene in CHD patients with obstructive coronary stenosis vs non critical stenosis group. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): Italian Minister of Health

Download Full-text

Identification of Putative Virulence Genes by DNA Methylation Studies in the Cereal Pathogen Fusarium graminearum

Cells ◽

10.3390/cells10051192 ◽

2021 ◽

Vol 10 (5) ◽

pp. 1192

Author(s):

Francesco Tini ◽

Giovanni Beccari ◽

Gianpiero Marconi ◽

Andrea Porceddu ◽

Micheal Sulyok ◽

...

Keyword(s):

Dna Methylation ◽

Fusarium Graminearum ◽

Environmental Changes ◽

Virulent Strain ◽

Fungal Virulence ◽

Wheat Seedlings ◽

Wide Range ◽

Conidia Production ◽

Natural Hosts

DNA methylation mediates organisms’ adaptations to environmental changes in a wide range of species. We investigated if a such a strategy is also adopted by Fusarium graminearum in regulating virulence toward its natural hosts. A virulent strain of this fungus was consecutively sub-cultured for 50 times (once a week) on potato dextrose agar. To assess the effect of subculturing on virulence, wheat seedlings and heads (cv. A416) were inoculated with subcultures (SC) 1, 23, and 50. SC50 was also used to re-infect (three times) wheat heads (SC50×3) to restore virulence. In vitro conidia production, colonies growth and secondary metabolites production were also determined for SC1, SC23, SC50, and SC50×3. Seedling stem base and head assays revealed a virulence decline of all subcultures, whereas virulence was restored in SC50×3. The same trend was observed in conidia production. The DNA isolated from SC50 and SC50×3 was subject to a methylation content-sensitive enzyme and double-digest, restriction-site-associated DNA technique (ddRAD-MCSeEd). DNA methylation analysis indicated 1024 genes, whose methylation levels changed in response to the inoculation on a healthy host after subculturing. Several of these genes are already known to be involved in virulence by functional analysis. These results demonstrate that the physiological shifts following sub-culturing have an impact on genomic DNA methylation levels and suggest that the ddRAD-MCSeEd approach can be an important tool for detecting genes potentially related to fungal virulence.

Download Full-text

Individuals Diagnosed with Binge-Eating Disorder Have DNA Hypomethylated Sites in Genes of the Metabolic System: A Pilot Study

Nutrients ◽

10.3390/nu13051413 ◽

2021 ◽

Vol 13 (5) ◽

pp. 1413

Author(s):

Mariana Lizbeth Rodríguez-López ◽

José Jaime Martínez-Magaña ◽

David Ruiz-Ramos ◽

Ana Rosa García ◽

Laura Gonzalez ◽

...

Keyword(s):

Dna Methylation ◽

Eating Disorder ◽

Protein Kinase ◽

Binge Eating ◽

Binge Eating Disorder ◽

Environmental Changes ◽

Diagnostic Category ◽

The Body ◽

Metabolic System ◽

Genome Wide

Binge-eating disorder, recently accepted as a diagnostic category, is differentiated from bulimia nervosa in that the former shows the presence of binge-eating episodes and the absence of compensatory behavior. Epigenetics is a conjunct of mechanisms (like DNA methylation) that regulate gene expression, which are dependent on environmental changes. Analysis of DNA methylation in eating disorders shows that it is reduced. The present study aimed to analyze the genome-wide DNA methylation differences between individuals diagnosed with BED and BN. A total of 46 individuals were analyzed using the Infinium Methylation EPIC array. We found 11 differentially methylated sites between BED- and BN-diagnosed individuals, with genome-wide significance. Most of the associations were found in genes related to metabolic processes (ST3GAL4, PRKAG2, and FRK), which are hypomethylated genes in BED. Cg04781532, located in the body of the PRKAG2 gene (protein kinase AMP-activated non-catalytic subunit gamma 2), was hypomethylated in individuals with BED. Agonists of PRKAG2, which is the subunit of AMPK (AMP-activated protein kinase), are proposed to treat obesity, BED, and BN. The present study contributes important insights into the effect that BED could have on PRKAG2 activation.

Download Full-text

Exploring Differentially Methylated Genes in Vulvar Squamous Cell Carcinoma

Cancers ◽

10.3390/cancers13143580 ◽

2021 ◽

Vol 13 (14) ◽

pp. 3580

Author(s):

Shatavisha Dasgupta ◽

Patricia C. Ewing-Graham ◽

Sigrid M. A. Swagemakers ◽

Thierry P. P. van den Bosch ◽

Peggy N. Atmodimedjo ◽

...

Keyword(s):

Dna Methylation ◽

Squamous Cell Carcinoma ◽

Cell Carcinoma ◽

Squamous Cell ◽

Dna Sequences ◽

Cpg Island ◽

Epigenetic Modification ◽

Vulvar Squamous Cell Carcinoma ◽

Differentially Methylated Genes ◽

Methylation Profiling

DNA methylation is the most widely studied mechanism of epigenetic modification, which can influence gene expression without alterations in DNA sequences. Aberrations in DNA methylation are known to play a role in carcinogenesis, and methylation profiling has enabled the identification of biomarkers of potential clinical interest for several cancers. For vulvar squamous cell carcinoma (VSCC), however, methylation profiling remains an under-studied area. We sought to identify differentially methylated genes (DMGs) in VSCC, by performing Infinium MethylationEPIC BeadChip (Illumina) array sequencing, on a set of primary VSCC (n = 18), and normal vulvar tissue from women with no history of vulvar (pre)malignancies (n = 6). Using a false-discovery rate of 0.05, beta-difference (Δβ) of ± 0.5, and CpG-island probes as cut-offs, 199 DMGs (195 hyper-methylated, 4 hypo-methylated) were identified for VSCC. Most of the hyper-methylated genes were found to be involved in transcription regulator activity, indicating that disruption of this process plays a vital role in VSCC development. The majority of VSCCs harbored amplifications of chromosomes 3, 8, and 9. We identified a set of DMGs in this exploratory, hypothesis-generating study, which we hope will facilitate epigenetic profiling of VSCCs. Prognostic relevance of these DMGs deserves further exploration in larger cohorts of VSCC and its precursor lesions.

Download Full-text

DNA Methylation of Human Choline Kinase Alpha Promoter-Associated CpG Islands in MCF-7 Cells

Genes ◽

10.3390/genes12060853 ◽

2021 ◽

Vol 12 (6) ◽

pp. 853

Author(s):

Siti Aisyah Faten Mohamed Sa’dom ◽

Sweta Raikundalia ◽

Shaharum Shamsuddin ◽

Wei Cun See Too ◽

Ling Ling Few

Keyword(s):

Dna Methylation ◽

Transcription Factor ◽

Binding Site ◽

Promoter Activity ◽

Cytosine Methylation ◽

Cpg Island ◽

Cpg Islands ◽

Site Directed Mutagenesis ◽

Choline Kinase ◽

Mcf 7

Choline kinase (CK) is the enzyme catalyzing the first reaction in CDP-choline pathway for the biosynthesis of phosphatidylcholine. Higher expression of the α isozyme of CK has been implicated in carcinogenesis, and inhibition or downregulation of CKα (CHKA) is a promising anticancer approach. This study aimed to investigate the regulation of CKα expression by DNA methylation of the CpG islands found on the promoter of this gene in MCF-7 cells. Four CpG islands have been predicted in the 2000 bp promoter region of ckα (chka) gene. Six CpG island deletion mutants were constructed using PCR site-directed mutagenesis method and cloned into pGL4.10 vectors for promoter activity assays. Deletion of CpG4C region located between –225 and –56 significantly increased the promoter activity by 4-fold, indicating the presence of important repressive transcription factor binding site. The promoter activity of methylated full-length promoter was significantly lower than the methylated CpG4C deletion mutant by 16-fold. The results show that DNA methylation of CpG4C promotes the binding of the transcription factor that suppresses the promoter activity. Electrophoretic mobility shift assay analysis showed that cytosine methylation at MZF1 binding site in CpG4C increased the binding of putative MZF1 in nuclear extract. In conclusion, the results suggest that DNA methylation decreased the promoter activity by promoting the binding of putative MZF1 transcription factor at CpG4C region of the ckα gene promoter.

Download Full-text