ORCID Profile
0000-0001-6003-2432
Current Organisations
Uniwersytet Slaski w Katowicach
,
Aarhus Institute of Advanced Studies
,
Aarhus Universitet
,
Aarhus Universitetshospital
,
University of Southampton
,
Pomeranian Medical University
,
Peter MacCallum Cancer Centre
,
The Lundbeck Foundation Centre for Interventional Research in Radiation Oncology (CIRRO)
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Publications
Methylation of the BRCA1 promoter in peripheral blood DNA is associated with triple-negative and medullary breast cancer.
Publisher: Springer Science and Business Media LLC
Date: 07-11-2014
DOI: 10.1007/S10549-014-3179-0
Abstract: It has been proposed that methylation signatures in blood-derived DNA may correlate with cancer risk. In this study, we evaluated whether methylation of the promoter region of the BRCA1 gene detectable in DNA from peripheral blood cells is a risk factor for breast cancer, in particular for tumors with pathologic features characteristic for cancers with BRCA1 gene mutations. We conducted a case-control study of 66 breast cancer cases and 36 unaffected controls. Cases were triple-negative or of medullary histology, or both 30 carried a constitutional BRCA1 mutation and 36 did not carry a mutation. Blood for DNA methylation analysis was taken within three months of diagnosis. Methylation of the promoter of the BRCA1 gene was measured in cases and controls using methylation-sensitive high-resolution melting (MS-HRM). A s le with any detectable level of methylation was considered to be positive. Methylation of the BRCA1 promoter was detected in 15 of 66 cases and in 2 of 36 controls (OR 5.0, p = 0.03). Methylation was present in 15 of 36 women with breast cancer and without germline BRCA1 mutation, but in none of 30 women with breast cancer and a germline mutation (p < 0.01). The association between methylation and breast cancer was restricted to women with no constitutional BRCA1 mutation (OR 12.1, p = 0.0006). Methylation of the promoter of the BRCA1 gene detectable in peripheral blood DNA may be a marker of increased susceptibility to triple-negative or medullary breast cancer.
Combining genetic and epigenetic parameters of the serotonin transporter gene in obsessive-compulsive disorder.
Publisher: Elsevier BV
Date: 2017
DOI: 10.1016/J.JPSYCHIRES.2017.10.010
Abstract: While genetic variants have been reported to be associated with obsessive-compulsive disorder (OCD), the small effect sizes suggest that epigenetic mechanisms such as DNA methylation may also be relevant. The serotonin transporter (SLC6A4) gene has been extensively investigated in relation to OCD, since serotonin reuptake inhibitors are the pharmacological treatment of choice for the disorder. The current study set three questions: Firstly, whether the high expressing loci of the SLC6A4 polymorphisms, 5-HTTLPR + rs25531, rs25532 and rs16965628 are associated with family-based (n = 164 trios) and case-control OCD (n = 186, 152, respectively). This was also examined by a meta-analysis. Secondly, whether DNA methylation and RNA levels of the SLC6A4 differ in saliva and blood of a subset of s les from pediatric and adult OCD patients and matched controls. And lastly, whether morning awakening cortisol levels correlate with the above. A meta-analysis confirmed the association of the L
Biological and methodological aspects of assessment of locus specific de novo methylation in blood.
Publisher: Future Medicine Ltd
Date: 12-2015
DOI: 10.2217/BMM.15.83
Abstract: Aim: Locus-specific methylation in blood differs between in iduals. As those changes may represent de novo methylation, induced by environmental factors, we aimed to evaluate the biological and methodological limitations of detection of methylation in blood. Materials & methods: We used Methylation-Sensitive High Resolution Melting to analyze methylation at 21 gene loci in peripheral blood DNA s les from 203 healthy women. Results: Overall nine of the screened loci displayed marked inter-in idual variation in methylation frequency with methylation levels predominantly around 1%. The methylation of specific loci showed different association with age and reproducibility of detection. Conclusions: Our results allowed benchmarking of both technological and biological limitations that need to be accounted for when evaluating locus specific methylation in blood as potential biomarker.
A new approach to primer design for the control of PCR bias in methylation studies.
Publisher: Springer Science and Business Media LLC
Date: 2008
Oxidative stress, telomere shortening, and DNA methylation in relation to low-to-moderate occupational exposure to welding fumes.
Publisher: Wiley
Date: 27-05-2015
DOI: 10.1002/EM.21958
Limitations and advantages of MS-HRM and bisulfite sequencing for single locus methylation studies.
Publisher: Informa UK Limited
Date: 07-2010
DOI: 10.1586/ERM.10.46
Abstract: The methylation-sensitive high-resolution melting (MS-HRM) protocol, as described by Wojdacz and Dobrovic, enables detection of a methylated template in an unmethylated background, with sensitivity similar to that of methylation-specific PCR (MSP). Furthermore, MS-HRM-based methylation screening is cost, labor and time efficient in contrast to direct bisulfite sequencing, which, therefore, is unsuitable as a screening method, but is still required to reveal the methylation status of in idual CpG sites. In some experiments, detailed information on the methylation status of in idual CpGs may be of interest for at least a subset of s les from MS-HRM-based methylation screening. For those s les, sequencing-based methodology has to be coupled with the MS-HRM protocol to investigate the methylation status of single CpG sites within the locus of interest. In this article, we review the limitations and advantages of MS-HRM and bisulfite sequencing protocols for single-locus methylation studies. Furthermore, we provide the insights into interpretation of the results obtained when a combination of the protocols is used for single-locus methylation studies.
Quality assessment of DNA derived from up to 30 years old formalin fixed paraffin embedded (FFPE) tissue for PCR-based methylation analysis using SMART-MSP and MS-HRM.
Publisher: Springer Science and Business Media LLC
Date: 12-2009
Abstract: The High Resolution Melting (HRM) technology has recently been introduced as a rapid and robust analysis tool for the detection of DNA methylation. The methylation status of multiple tumor suppressor genes may serve as biomarkers for early cancer diagnostics, for prediction of prognosis and for prediction of response to treatment. Therefore, it is important that methodologies for detection of DNA methylation continue to evolve. Sensitive Melting Analysis after Real Time - Methylation Specific PCR (SMART-MSP) and Methylation Sensitive - High Resolution Melting (MS-HRM) are two methods for single locus DNA methylation detection based on HRM. Here, we have assessed the quality of DNA extracted from up to 30 years old Formalin Fixed Paraffin Embedded (FFPE) tissue for DNA methylation analysis using SMART-MSP and MS-HRM. The quality assessment was performed on DNA extracted from 54 Non-Small Cell Lung Cancer (NSCLC) s les derived from FFPE tissue, collected over 30 years and grouped into five years intervals. For each s le, the methylation levels of the CDKN2A ( p16 ) and RARB promoters were estimated using SMART-MSP and MS-HRM assays designed to assess the methylation status of the same CpG positions. This allowed for a direct comparison of the methylation levels estimated by the two methods for each s le. CDKN2A promoter methylation levels were successfully determined by SMART-MSP and MS-HRM in all 54 s les. Identical methylation estimates were obtained by the two methods in 46 of the s les. The methylation levels of the RARB promoter were successfully determined by SMART-MSP in all s les. When using MS-HRM to assess RARB methylation five s les failed to lify and 15 s les showed a melting profile characteristic for heterogeneous methylation. Twenty-seven of the remaining 34 s les, for which the methylation level could be estimated, gave the same result as observed when using SMART-MSP. MS-HRM and SMART-MSP can be successfully used for single locus methylation studies using DNA derived from up to 30 years old FFPE tissue. Furthermore, it can be expected that MS-HRM and SMART-MSP will provide similar methylation estimates when assays are designed to analyze the same CpG positions.
The transcriptional coregulator MAML1 affects DNA methylation and gene expression patterns in human embryonic kidney cells.
Publisher: Springer Science and Business Media LLC
Date: 08-02-2016
DOI: 10.1007/S11033-016-3946-6
Abstract: Mastermind-like 1 (MAML1) is a transcriptional coregulator that has been associated with early development of many systems such as neuronal, muscular and urogenital. The present study aimed to explore the genome wide effects of MAML1 on DNA methylation and RNA expression in human embryonic kidney cells. Infinium HumanMethylation450 BeadChip Illumina array, methylation-sensitive high-resolution melt technique, Chip Analysis Methylation Pipeline and RNA profiling approaches were used to study MAML1 effects on the epigenome. We found that 11802 CpG sites were differentially methylated in MAML1-expressing cells while only 225 genes were differentially expressed. MAML1 overexpression induced more global differential hypermethylation than hypomethylation changes. In addition, the differentially methylated regions were mapped predominantly to 3'untranslated regions, intragenic regions and gene bodies and to a lesser extent to gene regulatory sequences. Gene ontology analysis revealed that the differentially changed genes (including HOXC11, HTATIP2, SLFN12 and SOX11) are involved in the regulation of urogenital system development, cell adhesion and embryogenesis. This study is the first report that shows the global effect of a single coregulator on DNA methylation and gene expression. Our results stress and support the effects of transcriptional coregulators on the cell methylome.
Discordant pattern of BRCA1 gene epimutation in blood between mothers and daughters.
Publisher: BMJ
Date: 15-04-2015
DOI: 10.1136/JCLINPATH-2015-202982
Abstract: Methylation of the promoter of BRCA1 gene in peripheral blood (epimutation) has been associated with increased risk for breast cancer. Some studies have indicated that this epimutation is of constitutional origin and hence it could potentially be transmitted across generations. We used methylation sensitive high resolution melting technique to measure methylation of BRCA1 promoter in blood s les from 226 healthy women from the Andean region in Salta province, northern Argentina. In total 29 (13%) of the women showed detectable methylation of this gene. The analyses of mother-daughter pairs in this study, showed discordant methylation of BRCA1 between generations, with mothers tested positive for BRCA1 methylation in blood having daughters without signs of BRCA1 methylation, and vice versa. Our results show that the BRCA1 epimutation is unlikely transmitted from mother to daughters and hence may be a consequence of environmental exposure.
DNA methylation profiles of elderly individuals subjected to indentured childhood labor and trauma.
Publisher: Springer Science and Business Media LLC
Date: 27-02-2017
Methylation-sensitive high resolution melting (MS-HRM): a new approach for sensitive and high-throughput assessment of methylation.
Publisher: Oxford University Press (OUP)
Date: 03-2007
DOI: 10.1093/NAR/GKM013
Prenatal lead exposure is associated with decreased cord blood DNA methylation of the glycoprotein VI gene involved in platelet activation and thrombus formation.
Publisher: Oxford University Press (OUP)
Date: 2015
DOI: 10.1093/EEP/DVV007
Melting curve assays for DNA methylation analysis.
Publisher: Humana Press
Date: 2009
DOI: 10.1007/978-1-59745-522-0_17
Abstract: The ability of sodium bisulfite to modify cytosines in a methylation-dependent manner allows the conservation of DNA methylation information during PCR lification. PCR products lified from bisulfite-modified DNA have significantly different base compositions according to whether they originate from methylated or unmethylated variants of the target template. Different base compositions give rise to different thermal properties of the PCR products. Hence, melting analysis of lification products in methylation studies allows the determination of whether the PCR products originate from methylated or unmethylated templates. Here, we briefly review recent advances in methodologies based on melting analyses of PCR products derived from bisulfite-modified templates and provide a methodology for methylation-sensitive high-resolution melting.
Challenges for the application of DNA methylation biomarkers in molecular diagnostic testing for cancer.
Publisher: Informa UK Limited
Date: 04-2013
DOI: 10.1586/ERM.13.9
Abstract: Aberrant DNA methylation is ubiquitous in human cancer and has been shown to occur early during carcinogenesis, thus providing attractive potential biomarkers for the early detection of cancer. The introduction of genome-wide DNA methylation analysis comparing tumor and nonmalignant tissues resulted in the discovery of many regions that undergo aberrant methylation during carcinogenesis. Those regions can potentially be used as biomarkers for cancer detection. However, a biomarker will be useful for screening or early detection of cancer only if it can be detected in a noninvasive or minimally invasive fashion without tissue biopsy. The authors discuss the challenges in translating DNA methylation biomarkers to cancer diagnosis - including obstacles in assay development, tissue-specific methylation load on tumor suppressor genes, detecting markers with sufficient sensitivity and specificity in the periphery, and ways in which these obstacles can be overcome.
Comprehensive genome methylation analysis in bladder cancer: identification and validation of novel methylated genes and application of these as urinary tumor markers.
Publisher: American Association for Cancer Research (AACR)
Date: 09-2011
DOI: 10.1158/1078-0432.CCR-10-2659
Abstract: Purpose: Epigenetic alterations are common and can now be addressed in a parallel fashion. We investigated the methylation in bladder cancer with respect to location in genome, consistency, variation in metachronous tumors, impact on transcripts, chromosomal location, and usefulness as urinary markers. Experimental Design: A microarray assay was utilized to analyze methylation in 56 s les. Independent validation was conducted in 63 s les by a PCR-based method and bisulfite sequencing. The methylation levels in 174 urine specimens were quantified. Transcript levels were analyzed using expression microarrays and pathways were analyzed using dedicated software. Results: Global methylation patterns were established within and outside CpG islands. We validated methylation of the eight tumor markers genes ZNF154 (P & 0.0001), HOXA9 (P & 0.0001), POU4F2 (P & 0.0001), EOMES (P = 0.0005), ACOT11 (P = 0.0001), PCDHGA12 (P = 0.0001), CA3 (P = 0.0002), and PTGDR (P = 0.0110), the candidate marker of disease progression TBX4 (P & 0.04), and other genes with stage-specific methylation. The methylation of metachronous tumors was stable and targeted to certain pathways. The correlation to expression was not stringent. Chromosome 21 showed most differential methylation (P & 0.0001) and specifically hypomethylation of keratins, which together with keratin-like proteins were epigenetically regulated. In DNA from voided urine, we detected differential methylation of ZNF154 (P & 0.0001), POU4F2 (P & 0.0001), HOXA9 (P & 0.0001), and EOMES (P & 0.0001), achieving 84% sensitivity and 96% specificity. Conclusions: We initiated a detailed mapping of the methylome in metachronous bladder cancer. Novel genes with tumor, chromosome, as well as pathway-specific differential methylation in bladder cancer were identified. The methylated genes were promising cancer markers for early detection of bladder cancer. Clin Cancer Res 17(17) 5582–92. ©2011 AACR.
ChAMP: 450k Chip Analysis Methylation Pipeline
Publisher: Oxford University Press (OUP)
Date: 12-12-2013
DOI: 10.1093/BIOINFORMATICS/BTT684
Abstract: 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 Contact: tiffany.morris@ucl.ac.uk
The limitations of locus specific methylation qualification and quantification in clinical material.
Publisher: Frontiers Media SA
Date: 2012
Clinical significance of DNA methylation in chronic lymphocytic leukemia patients: results from 3 UK clinical trials.
Publisher: American Society of Hematology
Date: 21-08-2019
DOI: 10.1182/BLOODADVANCES.2019000237
Abstract: Chronic lymphocytic leukemia patients with mutated immunoglobulin heavy-chain genes (IGHV-M), particularly those lacking poor-risk genomic lesions, often respond well to chemoimmunotherapy (CIT). DNA methylation profiling can sub ide early-stage patients into naive B-cell–like CLL (n-CLL), memory B-cell–like CLL (m-CLL), and intermediate CLL (i-CLL), with differing times to first treatment and overall survival. However, whether DNA methylation can identify patients destined to respond favorably to CIT has not been ascertained. We classified treatment-naive patients (n = 605) from 3 UK chemo and CIT clinical trials into the 3 epigenetic subgroups, using pyrosequencing and microarray analysis, and performed expansive survival analysis. The n-CLL, i-CLL, and m-CLL signatures were found in 80% (n = 245/305), 17% (53/305), and 2% (7/305) of IGHV-unmutated (IGHV-U) cases, respectively, and in 9%, (19/216), 50% (108/216), and 41% (89/216) of IGHV-M cases, respectively. Multivariate Cox proportional analysis identified m-CLL as an independent prognostic factor for overall survival (hazard ratio [HR], 0.46 95% confidence interval [CI], 0.24-0.87 P = .018) in CLL4, and for progression-free survival (HR, 0.25 95% CI, 0.10-0.57 P = .002) in ARCTIC and ADMIRE patients. The analysis of epigenetic subgroups in patients entered into 3 first-line UK CLL trials identifies m-CLL as an independent marker of prolonged survival and may aid in the identification of patients destined to demonstrate prolonged survival after CIT.
Exposure to arsenic and intra-chromosomal instability in blood.
Publisher: Oxford University Press (OUP)
Date: 2014
DOI: 10.1039/C4MT00128A
Abstract: The 450k Chip Analysis Methylation Pipeline (ChAMP) is a novel Illumina Infinium HumanMethylation450 BeadChip data processing algorithm that allows the analysis of copy number alterations (CNAs).
Gene silencing of Nox4 by CpG island methylation during hepatocarcinogenesis in rats.
Publisher: The Company of Biologists
Date: 2017
DOI: 10.1242/BIO.020370
Abstract: The association between the downregulation of genes and DNA methylation in their CpG islands has been extensively studied as a mechanism that favors carcinogenesis. The objective of this study was to analyze the methylation of a set of genes selected based on their microarray expression profiles during the process of hepatocarcinogenesis. Rats were sacrificed at: 24 Hours, 7, 11, 16 and 30 days and 5, 9, 12 and 18 months post-treatment. We evaluated the methylation status in the CpG islands of four deregulated genes (Casp3, Cldn1, Pex11a and Nox4) using methylation-sensitive high-resolution melting technology for the s les obtained from different stages of hepatocarcinogenesis. We did not observe methylation in Casp3, Cldn1 or Pex11a. However, Nox4 exhibited altered methylation patterns, reaching a maximum of 10%, even during the early stages of hepatocarcinogenesis. We observed downregulation of mRNA and protein of Nox4 (97.5% and 40%, respectively) after the first carcinogenic stimulus relative to the untreated s les. Our results suggest that Nox4 downregulation is associated with DNA methylation of the CpG Island in its promoter. We propose that methylation is a mechanism that can silence the expression of Nox4, which could contribute to the acquisition of neoplastic characteristics during hepatocarcinogenesis in rats.
Methylation of cancer related genes in tumor and peripheral blood DNA from the same breast cancer patient as two independent events.
Publisher: Springer Science and Business Media LLC
Date: 30-11-2011
The influence of DNA degradation in formalin-fixed, paraffin-embedded (FFPE) tissue on locus-specific methylation assessment by MS-HRM.
Publisher: Elsevier BV
Date: 12-2015
DOI: 10.1016/J.YEXMP.2015.11.007
Abstract: Readily accessible formalin-fixed paraffin embedded (FFPE) tissues are a highly valuable source of genetic material for molecular analyses in both research and in vitro diagnostics but frequently genetic material in those s les is highly degraded. With locus-specific methylation changes being widely investigated for use as biomarkers in various aspects of clinical disease management, we aimed to evaluate to what extent standard laboratory procedures can approximate the quality of the DNA extracted from FFPE s les prior to methylation analyses. DNA quality in 107 FFPE non-small cell lung cancer (NSCLC) s les was evaluated using spectrophotometry and gel electrophoresis. Subsequently, the quality assessment results were correlated with the results of locus specific methylation assessment with methylation sensitive high resolution melting (MS-HRM). The correlation of template quality with PCR lification performance and HRM based methylation detection indicated a significant influence of DNA quality on PCR lification but not on methylation assessment. In conclusion, standard laboratory procedures fairly well approximate DNA degradation of FFPE s les and DNA degradation does not seem to considerably affect locus-specific methylation assessment by MS-HRM.
BRCA1 promoter methylation in peripheral blood is associated with the risk of triple‐negative breast cancer
Publisher: Wiley
Date: 29-09-2019
DOI: 10.1002/IJC.32655
Abstract: Methylation of the promoter of the BRCA1 gene in DNA derived from peripheral blood cells is a possible risk factor for breast cancer. It is not clear if this association is restricted to certain types of breast cancer or is a general phenomenon. We evaluated BRCA1 methylation status in peripheral blood cells from 942 breast cancer patients and from 500 controls. We also assessed methylation status in 262 paraffin-embedded breast cancer tissues. Methylation status was assessed using methylation-sensitive high-resolution melting and was categorized as positive or negative. BRCA1 methylation in peripheral blood cells was strongly associated with the risk of triple-negative breast cancer (TNBC) (odds ratio [OR] 4.70 95% confidence interval [CI]: 3.13-7.07 p < 0.001), but not of estrogen-receptor positive breast cancer (OR 0.80 95% CI: 0.46-1.42 p = 0.46). Methylation was also overrepresented among patients with high-grade cancers (OR 4.53 95% CI: 2.91-7.05 p < 0.001) and medullary cancers (OR 3.08 95% CI: 1.38-6.88 p = 0.006). Moreover, we detected a significant concordance of BRCA1 promoter methylation in peripheral blood and paired tumor tissue (p < 0.001). We found that BRCA1 promoter methylation in peripheral blood cells is associated with approximately five times greater risk of TNBC. We propose that BRCA1 methylation in blood-derived DNA could be a novel biomarker of increased breast cancer susceptibility, in particular for triple-negative tumors.
Short history of 5-methylcytosine: from discovery to clinical applications
Publisher: BMJ
Date: 11-01-2021
DOI: 10.1136/JCLINPATH-2020-206922
Abstract: Covalent modifications of nucleotides in genetic material have been known from the beginning of the last century. Currently, one of those modifications referred to as DNA methylation, is impacting personalised medicine both as a treatment target and a biomarker source for clinical disease management. In this short review, we describe landmark discoveries that led to the elucidation of the DNA methylation importance in the cell’s physiology and clarification of its role as one of the major processes in disease pathology. We also describe turning points in the development of methodologies to study this modification, which ultimately resulted in the development of in-vitro diagnostic kits targeting disease related DNA methylation changes as biomarkers.
Methylation-sensitive high-resolution melting in the context of legislative requirements for validation of analytical procedures for diagnostic applications.
Publisher: Informa UK Limited
Date: 2012
DOI: 10.1586/ERM.11.88
Abstract: Temperature gradient was first used to identify the methylation status of the DNA sequence over 10 years ago however, the initially published protocol was shown to have poor analytical sensitivity. Recent developments in the field of DNA melting technologies, combined with the identification of procedures to overcome the sensitivity issues in the PCR-based methylation detection applications, led to the development of the methylation-sensitive high-resolution melting (MS-HRM) protocol. This protocol allows for highly sensitive detection of methylation levels in a labor- and cost-efficient fashion. Moreover, it enables investigation of methylation status of imprinted loci as well as identification of heterogeneous methylation. The MS-HRM technology is being increasingly applied in research laboratories and has a potential for future application in diagnostic settings. The focus of this article is to describe the development of the HRM technology for methylation analyses and evaluate the diagnostic applicability of the MS-HRM technology.
Transcriptomics and methylomics of CD4-positive T cells in arsenic-exposed women.
Publisher: Springer Science and Business Media LLC
Date: 12-11-2016
Techniques used in studies of age-related DNA methylation changes.
Publisher: Wiley
Date: 05-2006
Abstract: Epigenetic modification of CpG islands (CGIs) in promoter regions is an important regulatory mechanism of gene expression in eukaryotic cells. Hypermethylation of CGIs may silence a gene, whereas hypomethylation of previously methylated CGIs allows gene expression. The pattern of methylation is cell-type-specific and established during development of the organisms. Changes in the methylation pattern have been found in all cancer forms and in aging cells. The epigenetic-related alternations of gene expression status may significantly contribute to the initiation and maintenance of malignant growth. Cancer incidence increases dramatically with age and correlates strongly with age-related methylation changes. Many techniques have been developed to analyze the genome-wide methylation content and the methylation status of specific loci. The majority of methylation screening protocols utilizes methylation-sensitive endonuclease digestion or bisulfite treatment of the template followed by subsequent PCR lification of a specific sequence. All methods either examine only one specific DNA sequence at a time, or provide limited genomic information on the screened sequences. The principle of our new approach is to combine methylation-sensitive enzyme digestion with the comparative genomic hybridization (CGH) technique to develop an array-based method to screen the entire genome for changes of methylation pattern. The new technique will serve as an efficient tool in understanding the nature of epigenetic changes and their significance to the aging process and cancer development.
Lamin B1 regulates somatic mutations and progression of B-cell malignancies.
Publisher: Springer Science and Business Media LLC
Date: 14-08-2017
DOI: 10.1038/LEU.2017.255
Methylation-sensitive high-resolution melting.
Publisher: Springer Science and Business Media LLC
Date: 20-11-2008
Abstract: The base composition of PCR products derived from sodium bisulfite-modified templates is methylation dependent. Hence, methylated and unmethylated, PCR products show different melting profiles when subjected to thermal denaturation. The methylation-sensitive high-resolution melting (MS-HRM) protocol is based on the comparison of the melting profiles of PCR products from unknown s les with profiles specific for PCR products derived from methylated and unmethylated control DNAs. The protocol consists of PCR lification of bisulfite-modified DNA with primers designed to proportionally lify both methylated and unmethylated templates and subsequent high-resolution melting analysis of the PCR product. The MS-HRM protocol allows in-tube determination of the methylation status of the locus of interest following sodium bisulfite modification of template DNA in less than 3 h. Here, we provide a protocol for MS-HRM, which enables highly sensitive, labor- and cost-efficient single-locus methylation studies on the basis of DNA high-resolution melting technology.
No difference in the frequency of locus-specific methylation in the peripheral blood DNA of women diagnosed with breast cancer and age-matched controls.
Publisher: Future Medicine Ltd
Date: 12-2011
DOI: 10.2217/FON.11.123
Abstract: There is no doubt that aberrant somatic (tumor-specific) methylation significantly contributes to the carcinogenic process. However, the question of the relevance of methylation pattern changes, acquired by in iduals during their development and lifetime or inherited through the germline, to the pathology of different diseases, remains open. Recently, a number of studies addressed the question of the prevalence of aberrant methylation of cancer-related genes in peripheral blood leukocyte (PBL) DNA and indicated a strong possibility that the presence of constitutional methylation of different genes might predispose for cancer development. Here, we have used the methlyation-sensitive high-resolution melting approach to examine the methylation status of the BRCA1, BRCA2, APC, RASSF1A and RARβ2 genes in PBLs of a group of women diagnosed with breast cancer, and an age-matched control group with no signs of breast cancer. No significant differences in the frequency of methylation of the above genes were found between cases and controls in our study. Hence, testing for the presence of methylation of cancer-related genes in PBL DNA from women diagnosed with sporadic breast cancer and classified for testing without any pathological or clinical selection criteria does not seem to have clinical applicability.
MS-HRM assay identifies high levels of epigenetic heterogeneity in human immortalized cell lines.
Publisher: Elsevier BV
Date: 04-2015
DOI: 10.1016/J.GENE.2015.01.063
Abstract: Immortalized cell lines are widely used in genetic and epigenetic studies, from exploration of basic molecular pathways to evaluation of disease-specific cellular properties. They are also used in biotechnology, e.g., in drug toxicity tests and vaccine production. Cellular and genetic uniformity is the main feature of immortalized cell lines and it has been particularly advantageous in functional genomic research, which has in recent years been expanded to include epigenetic mechanisms of gene expression regulation. Using the MS-HRM technique, we demonstrated heterogeneity in locus-specific methylation patterns in different cell cultures of four human cell lines: HEK293, HEK293T, LCL and DU145. Our results show that some human immortalized cell lines consist of cells that differ in the methylation status of specific loci, i.e., that they are epigenetically heterogeneous. We show that even two cultures of the same cell line obtained from different laboratories can differ in the methylation status of the specific loci. The results indicated that epigenetic uniformity of the cell lines cannot be assumed in experiments which utilize cell cultures and that the methylation status of the specific loci in the immortalized cell lines should be re-characterized and carefully profiled before epigenetic studies are performed.
Methylation biomarker development in the context of the EU regulations for clinical use of in-vitro diagnostic devices.
Publisher: Informa UK Limited
Date: 17-05-2019
Rapid detection of methylation change at H19 in human imprinting disorders using methylation-sensitive high-resolution melting.
Publisher: Hindawi Limited
Date: 10-2008
DOI: 10.1002/HUMU.20779
Abstract: Beckwith Wiedemann syndrome (BWS) and Russell Silver syndrome (RS) are growth disorders with opposing epimutations affecting the H19/IGF2 imprinting center at 11p15.5. Overgrowth and tumor risk in BWS is caused by aberrant expression of the paternally expressed, imprinted IGF2 gene, occurring as a consequence of mosaic hypermethylation within the imprinting center, or to mosaic paternal uniparental disomy (UPD). RS is characterized by severe intrauterine growth retardation (IUGR). A subset of RS cases were recently shown to have mosaic hypomethylation within the H19/IGF2 imprinting center, predicted to silence paternally expressed IGF2 in early development. Molecular diagnosis for BWS and RS involves methylation analysis of the H19 locus, enabling discrimination of allelic methylation patterns. In this study, methylation-sensitive high-resolution melting analysis (MS-HRM) was used to analyze methylation within the intergenic region of the H19 locus. A total of 36 s les comprising normal control (11), BWS (19), and RS (six) DNA were analyzed in a blinded study and scored as hypermethylated, normal, or hypomethylated. Results were compared with those derived by methylation-sensitive Southern blotting using the restriction enzymes Rsa I and Hpa II. A total of 100% concordance was obtained for the Southern blotting and MS-HRM scores. A total of three s les with paternal duplication affecting the H19/IGF2 region were scored as equivocal by both methods however, 33 out of 36 (92%) the s les were unambiguously scored as being hypermethylated, hypomethylated, or normally methylated using MS-HRM. We conclude that MS-HRM is a rapid, cost-effective, and sensitive method for screening mosaic methylation changes at the H19 locus in BWS and RS.
Chronic lymphocytic leukemia patients with heterogeneously or fully methylated LPL promotor display longer time to treatment.
Publisher: Future Medicine Ltd
Date: 09-2018
Abstract: Aim: We investigated whether DNA methylation regulates expression of LPL and PI3K complex genes in chronic lymphocytic leukemia (CLL) and evaluated the prognostic significance of LPL promoter methylation in CLL patients. Patients & methods: Methylation of LPL promoter was assessed in 112 patients using methylation-sensitive high-resolution melting (MS-HRM).Results: Patients with a fully or heterogeneously methylated LPL promoter had significantly longer median time to treatment (p 0.001) and 75% lower (hazard ratio: 0.25 95% CI: 0.15–0.42 p 0.001) risk of requirement for treatment as opposed to patients with nonmethylated promoter. Multivariate modeling confirmed independent prognostic value of these findings. Conclusion: Chronic lymphocytic leukemia patients with a fully or heterogeneously methylated LPL gene promoter display indolent disease course and acquisition of heterogeneous methylation of LPL promoter is insufficient to induce gene expression.
A pilot investigation on DNA methylation modifications associated with complex posttraumatic symptoms in elderly traumatized in childhood.
Publisher: Springer Science and Business Media LLC
Date: 12-2017
Identification and characterization of locus-specific methylation patterns within novel loci undergoing hypermethylation during breast cancer pathogenesis.
Publisher: Springer Science and Business Media LLC
Date: 02-2014
DOI: 10.1186/BCR3612
Methylation levels assessment with Methylation-Sensitive High-Resolution Melting (MS-HRM)
Publisher: Public Library of Science (PLoS)
Date: 06-09-2022
DOI: 10.1371/JOURNAL.PONE.0273058
Abstract: Testing for disease-related DNA methylation changes provides clinically relevant information in personalized patient care. Methylation-Sensitive High-Resolution Melting (MS-HRM) is a method used for measuring methylation changes and has already been used in diagnostic settings. This method utilizes one set of primers that initiate the lification of both methylated and non-methylated templates. Therefore, the quantification of the methylation levels using MS-HRM is h ered by the PCR bias phenomenon. Some approaches have been proposed to calculate the methylation level of s les using the high-resolution melting (HRM) curves. However, limitations of the methylation calculation using MS-HRM have not been evaluated systematically and comprehensively. We used the Area Under the Curve (AUC), a derivative of the HRM curves, and least square approximation (LSA) to establish a procedure that allowed us to infer methylation levels in an MS-HRM experiment and assess the limitations of that procedure for the assays’ specific methylation level measurement. The developed procedure allowed, with certain limitations, estimation of the methylation levels using HRM curves.
Current methylation screening methods.
Publisher: Future Medicine Ltd
Date: 12-2009
DOI: 10.2217/EPI.09.32
Identification and validation of highly frequent CpG island hypermethylation in colorectal adenomas and carcinomas.
Publisher: Wiley
Date: 04-2011
DOI: 10.1002/IJC.25951
Abstract: In our study, whole-genome methylation arrays were applied to identify novel genes with tumor specific DNA methylation of promoter CpG islands in pre-malignant and malignant colorectal lesions. Using a combination of Illumina HumanMethylation27 beadchips, Methylation-Sensitive High Resolution Melting (MS-HRM) analysis, and Exon arrays (Affymetrix) the DNA methylation pattern of ∼14,000 genes and their transcript levels were investigated in six normal mucosas, six adenomas and 30 MSI and MSS carcinomas. Sixty eight genes with tumor-specific hypermethylation were identified (p < 0.005). Identified hypermethylated sites were validated in an independent s le set of eight normal mucosas, 12 adenomas, 40 MSS and nine MSI cancer s les. The methylation patterns of 15 selected genes, hypermethylated in adenomas and carcinomas (FLI1, ST6GALNAC5, TWIST1, ADHFE1, JAM2, IRF4, CNRIP1, NRG1 and EYA4), in carcinomas only (ABHD9, AOX1 and RERG), or in MSI but not MSS carcinomas (RAMP2, DSC3 and MLH1) were validated using MS-HRM. Four of these genes (MLH1, AOX1, EYA4 and TWIST1) had previously been reported to be hypermethylated in CRC. Eleven genes, not previously known to be affected by CRC specific hypermethylation, were identified and validated. Inverse correlation to gene expression was observed for six of the 15 genes with Spearman correlation coefficients ranging from -0.39 to -0.60. For six of these genes the altered methylation patterns had a profound transcriptional association, indicating that methylation of these genes may play a direct regulatory role. The hypermethylation changes often occurred already in adenomas, indicating that they may be used as biomarkers for early detection of CRC.
Genomic disruption of the histone methyltransferase SETD2 in chronic lymphocytic leukaemia.
Publisher: Springer Science and Business Media LLC
Date: 20-05-2016
DOI: 10.1038/LEU.2016.134
Related Organisations
University Of Southampton
Location: United Kingdom of Great Britain and Northern Ireland
The Lundbeck Foundation Centre For Interventional Research In Radiation Oncology (CIRRO)
Location: Denmark
Related Funding Activities
No related grants have been discovered for Tomasz K Wojdacz.