Bisulfite Conversion Method Research Articles

Hypermethylation and low expression of FOXM1 predisposes women to unexplained recurrent miscarriage by impairing trophoblast stem cell proliferation

Recurrent miscarriage (RM) is a distressing pregnancy complication with an unknown etiology. Increasing evidence indicates the relevance of dysregulation of human trophoblast stem cells (hTSCs), which may play a role in the development of RM. However, the potential molecular regulatory mechanism underlying the initiation and maintenance of hTSCs is yet to be fully elucidated. In this study, we performed data analysis and identified Forkhead box M1 (FOXM1) as a potential factor associated with RM. FOXM1 is a typical transcription factor known for its involvement in various pathophysiological processes, while the precise function of FOXM1 functions in hTSCs and RM remains incompletely understood. Utilizing RNA-seq, CUT&Tag, ChIP-qPCR, and sodium bisulfite conversion methods for methylation analysis, we elucidate the underlying regulatory mechanisms of FOXM1 in hTSCs and its implications in RM. Our findings demonstrate the relative high expression of FOXM1 in proliferating cytotrophoblasts (CTBs) compared to differentiated extravillous cytotrophoblasts (EVTs) and syncytiotrophoblasts (STBs). Besides, we provide evidence supporting a significant correlation between FOXM1 downregulation and the incidence of RM. Furthermore, we demonstrate the significant role of FOXM1 in regulating hTSCs proliferation and cell cycle through the transcriptional regulation of CDKN3, CCNB2, CCNA2, MAD2L1 and CDC25C. Notably, we observed a correlation between the downregulation of FOXM1 in RM and hypermethylation in its promoter region. Collectively, these results provide insights into the impact of FOXM1 on trophoblast regulation and offer a novel perspective on RM.

Evaluation of silica spin‑column and magnetic bead formats for rapid DNA methylation analysis in clinical and point‑of‑care settings.

Late-stage cancers lack effective treatment, underscoring the need for early diagnosis to improve prognosis and decrease mortality rates. Molecular markers, such as DNA methylation, offer promise in early cancer detection. The present study compared commercial kits for analyzing DNA from cervical liquid cytology samples in cancer screening. Rapid bisulfite conversion kits using silica spin-columns and magnetic beads were assessed against standard DNA extraction and bisulfite conversion methods for profiling DNA methylation using quantitative methylation-specific PCR. β-actin amplification indicated the suitability of small sample volumes for methylation studies using either the pellet or supernatant (cell-free DNA) parts. Comparison of Bisulfite Conversion Kit-Whole Cell (Abcam), Methylamp Bisulfite Modification (Epigentek), EpiTect Fast LyseAll Bisulfite Kit (Qiagen GmbH) and EZ DNA Methylation-Direct Kit (Zymo Research Corp.) showed no significant differences in β-actin cycle threshold values. EZ-96 DNA Methylation-Lightning MagPrep (Zymo Research Corp.), a hybrid kit in a 96-well plate format, exhibited swift turnaround time and similar amplification efficiency. Automation with magnetic bead kits increased throughput without compromising amplification efficiency in open PCR systems. Cost analysis favored direct kits over the gold standard manual protocol. This comparison aids in selecting cost-effective DNA methylation diagnostic tests. The present study confirmed comparable kit performance in methylation-based analysis, highlighting the adequacy of cytology samples and the potential of bodily fluids as alternatives for liquid biopsy.

Abstract 7025: Advancing methylation analysis: TAPS v1.0: A fast, scalable, and non-destructive sequencing method

Abstract In the pursuit of advancing multi-cancer early detection, the exploration of cell-free DNA (cfDNA) through methylation profiling has emerged as a promising avenue. However, the realization of its full potential is impeded by limitations inherent in conventional methodologies, notably the 'gold standard' bisulfite conversion methods. These approaches pose challenges such as DNA destruction and the generation of low-complexity sequences resulting from the conversion of unmethylated cytosines to thymine. TET Assisted Pyridine-Borane Sequencing (TAPS) holds the potential as a non-destructive, rapid, and scalable alternative for base-level methylation analysis. In this context, we introduce TET Assisted Pyridine-Borane Sequencing version 1.0 (TAPS v1.0), showcasing advancements to the technology originally introduced by Schuster-Böckler, Song, and their collaborators. TAPS v1.0 transforms 5mC into dihydrouracil (DHU) through a series of oxidation and reduction reactions. Ultimately, DHU is recognized as dU during PCR resulting in C-to-T conversions at 5mC sites, leaving unmethylated cytosines, the majority of cytosines, unchanged. The nondestructive nature of 5mC conversion yields significantly higher library yield and complexity compared to BS-seq. By leveraging Watchmaker's proprietary polymerase evolution platform, a DNA polymerase tolerant to DHU was selected for library amplification post DHU conversion, enabling unimpeded detection of 5mC, particularly in densely packed CpG repeats. Coupled with an optimized End Repair and A-tailing chemistry preceding TAPS v1.0, clinically relevant cfDNA samples of low quality and/or quantity can be analyzed in methylation detection pipelines. To evaluate the performance of TAPS v1.0, human genomic DNA with control spike-ins featuring varying methylation statuses were employed. TAPS v1.0 exhibited significantly higher final library yield and greater complexity compared to BS-seq. Similar outcomes were observed in clinically relevant cfDNA samples. Citation Format: Josh Haimes, Craig Marshall, Travis Sanders, Sam Vogel, Martin Ranik, Eduard Casas, Doug Wendel, Leo Karamanof, Bjarne Faurholme, Abre de Beer, Lee French, Ross Wadsworth, Kristina Giordia, Eric van der Walt, Brian Kudlow. Advancing methylation analysis: TAPS v1.0: A fast, scalable, and non-destructive sequencing method [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7025.

SP1 Gene Methylation in Head and Neck Squamous Cell Cancer in HPV-Negative Patients.

There is still much to learn about the epigenetic mechanisms controlling gene expression during carcinogenesis. When researching aberrant DNA methylation, active proliferative tumor cells from head and neck squamous cell cancer (HNSCC) can be used as a model. The aim of the study was to investigate the methylation status of CDKN1, CDKN2A, MYC, Smad3, SP1, and UBC genes in tumor tissue (control-normal tissue) in 50 patients (37 men and 13 women) with HPV-negative HNSCC. Methods: Bisulfite conversion methods and methyl-sensitive analysis of high-resolution melting curves were used to quantify the methylation of genes. In all patients and across various subgroups (tongue carcinoma, laryngeal and other types of carcinomas T2, T3, T4 status; age before and after 50 years; smoking and non-smoking), there are consistent differences in the methylation levels in the SP1 gene in tumor DNA compared to normal. Results: The methylation of the SP1 gene in tumor DNA suppresses its expression, hinders HNSCC cell proliferation regulation, and could be a molecular indicator of malignant cell growth. The study of DNA methylation of various genes involved in carcinogenesis is promising because hypermethylated promoters can serve as potential biomarkers of disease.

Bioinformatic analysis for age prediction using epigenetic clocks: Application to fisheries management and conservation biology

Epigenetic clocks are accurate tools for age prediction and are of great interest for fisheries management and conservation biology. Here, we review the necessary computational steps and tools in order to build an epigenetic clock in any species focusing on fish. Currently, a bisulfite conversion method which allows the distinction of methylated and unmethylated cytosines is the recommended method to be performed at single nucleotide resolution. Typically, reduced representation bisulfite sequencing methods provide enough coverage of CpGs to select from for age prediction while the exact implemented method depends on the specific objectives and cost of the study. Sequenced reads are controlled for their quality, aligned to either a reference or a deduced genome and methylation levels of CpGs are extracted. Methylation values are obtained in biological samples of fish that cover the widest age range possible. Using these datasets, machine learning statistical procedures and, in particular, penalized regressions, are applied in order to identify a set of CpGs the methylation of which in combination is enough to accurately predict age. Training and test datasets are used to build the optimal model or “epigenetic clock”, which can then be used to predict age in independent samples. Once a set of CpGs is robustly identified to predict age in a given species, DNA methylation in only a small number of CpGs is necessary, thus, sequencing efforts including data and money resources can be adjusted to interrogate a small number of CpGs in a high number of samples. Implementation of this molecular resource in routine evaluations of fish population structure is expected to increase in the years to come due to high accuracy, robustness and decreasing costs of sequencing. In the context of overexploited fish stocks, as well as endangered fish species, accurate age prediction with easy-to-use tools is much needed for improved fish populations management and conservation.

The role of clopidogrel resistance-related genetic and epigenetic factors in major adverse cardiovascular events among patients with acute coronary syndrome after percutaneous coronary intervention.

Despite patients with acute coronary syndrome (ACS) undergoing percutaneous coronary intervention (PCI) and receiving clopidogrel therapy, some patients still experience major adverse cardiovascular events (MACEs). Clopidogrel resistance, which may be regulated by genetic and epigenetic factors, may play a role in MACEs. This study aimed to determine the association between genetic (CYP2C19 and P2Y12 polymorphisms) and epigenetic (DNA methylation of CYP2C19 and P2Y12 and miRNA-26a expression) factors and their effects on MACEs among post-PCI patients. Post-PCI patients who received a standard dosage of clopidogrel at Harapan Kita Hospital between September 2018 and June 2020 were included in this study. MACEs were observed in patients within 1 year after PCI. Platelet aggregation was assessed using light transmission aggregometry (LTA). DNA methylation of CYP2C19 and P2Y12 was assessed using the bisulfite conversion method. CYP2C19 and P2Y12 polymorphisms and miRNA-26a expression were evaluated using quantitative real-time polymerase chain reaction (qRT-PCR). Among a total of 201 subjects, 49.8% were clopidogrel-resistant, and 14.9% experienced MACEs within 1 year after PCI (death was 7.5%). Hypomethylation of CYP2C19 (p = 0.037) and miRNA-26a upregulation (p = 0.020) were associated with clopidogrel resistance. CYP2C19*2/*3 polymorphisms (p = 0.047) were associated with MACEs in 1 year. This study demonstrated that hypomethylation of CYP2C19 and miRNA-26a upregulation increased the risk of clopidogrel resistance in post-PCI patients, but there was no correlation between clopidogrel resistance and MACEs. However, CYP2C19*2/*3 polymorphisms were the factors that predicted MACEs within 1 year.

Carboplatin Plus Vincristine as an Alternative Chemotherapeutic Scheme in Patients With Glioblastoma.

Background and objectiveAlternative chemotherapy regimens, including cisplatin, carmustine, or other agents, have been shown to be effective; however, the use of carboplatin plus vincristine (C/V) has not been studied before. In this study, we aimed to determine the survival rates in patients treated with C/V, by comparing our findings with treatments based on temozolomide (TMZ), and to explore a possible relationship with the methylation status of the methylguanine methyltransferase (MGMT) promoter in patients with glioblastoma (GB).MethodsA retrospective cohort study was conducted involving 45 surgically treated patients diagnosed with GB. Fresh tissue samples were examined by the DNA bisulfite conversion method to determine methylation status. After surgery, different chemotherapy regimens were employed as adjuvants. Follow-up of participants was performed as outpatients at three-month intervals to determine overall survival (OS), by comparing the use of TMZ versus C/V.ResultsMGMT promoter methylation status could only be determined in 35 samples; 20 patients received adjuvant chemotherapy, of which 14 were treated with C/V and six with TMZ-based schemes. The median OS (mOS) was eight months (range: 1-24 months). OS was 57.25% at six months, 48.7% at 12 months, and 28.5% at 24 months. In the TMZ group, an OS of 83% was observed at 24 months. In the C/V group, OS was 71.4% at six months, 57.1% at 12 months, and 35.7% at 24 months. Patients who did not receive adjuvant chemotherapy treatment had the lowest survival rates with an OS of 39.9% at six months, 26.6% at 12 months, and 19.9% ​​at 24 months.ConclusionsBased on our findings, C/V offers an accessible and effective alternative treatment when the TMZ-based scheme is not accessible, providing higher rates of OS compared to patients without chemotherapy management. The methylation status of the MGMT promoter is a significant prognostic factor, resulting in higher survival rates among patients when it is methylated.

Methods for analysis of specific DNA methylation status.

Methylation of CpG dinucleotides plays a crucial role in the regulation of gene expression and therefore in the development of different pathologies. Aberrant methylation has been associated to the majority of the diseases, including cancer, neurodegenerative, cardiovascular and autoimmune disorders. Analysis of DNA methylation patterns is crucial to understand the underlying molecular mechanism of these diseases. Moreover, DNA methylation patterns could be used as biomarker for clinical management, such as diagnosis, prognosis and treatment response. Nowadays, a variety of high throughput methods for DNA methylation have been developed to analyze the methylation status of a high number of CpGs at once or even the whole genome. However, identification of specific methylation patterns at specific loci is essential for validation and also as a tool for diagnosis. In this review, we describe the most commonly used approaches to evaluate specific DNA methylation. There are three main groups of techniques that allow the identification of specific regions that are differentially methylated: bisulfite conversion-based methods, restriction enzyme-based approaches, and affinity enrichment-based assays. In the first group, specific restriction enzymes recognize and cleave unmethylated DNA, leaving methylated sequences intact. Bisulfite conversion methods are the most popular approach to distinguish methylated and unmethylated DNA. Unmethylated cytosines are deaminated to uracil by sodium bisulfite treatment, while the methyl cytosines remain unconverted. In the last group, proteins with methylation binding domains or antibodies against methyl cytosines are used to recognize methylated DNA. In this review, we provide the theoretical basis and the framework of each technique as well as the analysis of their strength and the weaknesses.

DNA Methylation of the Keratin 18 Gene Regulates Brain Metastasis of Triple Negative Breast Cancer

Background IntroductionBreast cancer patients with brain metastasis have a poor prognosis and current chemotherapeutic agents are largely ineffective, which led us to seek new chemotherapeutic agents for the treatment. Our preliminary results showed the hypomethylating agent azacitidine (abbreviated as AZA) is effective in treating brain metastasis breast cancer using in vitro cell and in vivo preclinical mouse models. In the present study, we explored the molecular mechanism of action of AZA for this treatment. Keratin 18 is a member of the cytokeratin family proteins (epithelial marker). We observed that the keratin 18 gene is present in both parental regular MDA‐MB‐231 triple negative breast cancer cells (“231”) and their brain metastasized counterpart cells (“231Br”). However, mRNA and protein levels of keratin 18 are significantly decreased in 231Br cells compared to 231 cells.HypothesisBased on our preliminary results, we hypothesize that decreased expression of the keratin 18 gene in 231Br cells is due to DNA hypermethylation, which contributes to the brain metastatic characteristics and sensitivity to AZA treatment in these cells.Research Method and ResultsThe study by Umezawa et al. showed that DNA methylation of intron 1 in the keratin 18 gene is important in regulating its expression. In our study, we compared the DNA methylation status of the intron 1 region (737 bp) of the keratin 18 gene between the regular 231 and brain metastasized 231Br breast cancer cells.We found that: (1) The DNA sequence of intron 1 of the keratin 18 gene is identical between 231 and 231Br cells. We did not detect DNA mutation or deletion of the intron 1 region of the keratin 18 gene between both cell lines, suggesting the decreased expression of keratin 18 in 231Br cells may due to DNA hypermethylation. (2) The DNA methylation status is different in intron 1 of the keratin 18 gene between 231 and 231Br cells. We fully sequenced and compared the DNA methylation of the intron 1 region of the keratin 18 gene between both cell lines by using the bisulfite conversion method. In this method, treating DNA with bisulfite chemically modifies non‐methylated cytosines into uracil (appears as thymine in DNA sequencing results), but methylated cytosines remain unchanged. We found three cytosines were converted into uracil in 231 cells while they remained unchanged in 231Br cells, suggesting the DNA methylation status of the keratin 18 gene is different between both cell lines.ConclusionIn this study, we found that the keratin 18 gene is hypermethylated in brain metastasized breast cancer cells, which is important in regulating its expression and response to hypomethylating agent treatment. Based on our findings, the hypermethylated keratin 18 gene can be a potential drug target used for the development of novel targeted therapy drugs in treating patients with brain metastasis breast cancer.Support or Funding InformationThe research was supported by the National Institute of General Medical Sciences of NIH (Award Number 5U54GM104942‐03) and WVSOM intramural fund

Label-Free and Immobilization-Free Electrochemical Magnetobiosensor for Sensitive Detection of 5-Hydroxymethylcytosine in Genomic DNA.

DNA 5-hydroxymethylcytosine (5-hmC) is an important epigenetic biomarker for tumorigenesis, and the loss of 5-hmC levels is associated with leukemia and melanoma cancers. However, it is a great challenge to discriminate 5-hmC from 5-methylcytosine (5-mC) using the conventional bisulfite conversion methods. Herein, we report a label-free and immobilization-free electrochemical magnetobiosensor for sensitive quantification of 5-hmC in genomic DNA based on a dual signal amplification strategy coupled with terminal deoxynucleotidyl transferase (TDT) enzymatic amplification and Ru(III) redox cycling. This screen-printed carbon electrode (SPCE)-based electrochemical magnetobiosensor shows distinct advantages of having low cost and simple fabrication and being label-free, immobilization-free, PCR-free, and radioactive-free. It exhibits high sensitivity with a detection limit of as low as 9.06 fM and a large dynamic range from 0.01 to 1000 pM. Importantly, this biosensor can discriminate 5-hmC from cytosine and 5-mC, and it can successfully detect 5-hmC in live cells.

An optimized rapid bisulfite conversion method with high recovery of cell-free DNA

BackgroundMethylation analysis of cell-free DNA is a encouraging tool for tumor diagnosis, monitoring and prognosis. Sensitivity of methylation analysis is a very important matter due to the tiny amounts of cell-free DNA available in plasma. Most current methods of DNA methylation analysis are based on the difference of bisulfite-mediated deamination of cytosine between cytosine and 5-methylcytosine. However, the recovery of bisulfite-converted DNA based on current methods is very poor for the methylation analysis of cell-free DNA.ResultsWe optimized a rapid method for the crucial steps of bisulfite conversion with high recovery of cell-free DNA. A rapid deamination step and alkaline desulfonation was combined with the purification of DNA on a silica column. The conversion efficiency and recovery of bisulfite-treated DNA was investigated by the droplet digital PCR. The optimization of the reaction results in complete cytosine conversion in 30 min at 70 °C and about 65% of recovery of bisulfite-treated cell-free DNA, which is higher than current methods.ConclusionsThe method allows high recovery from low levels of bisulfite-treated cell-free DNA, enhancing the analysis sensitivity of methylation detection from cell-free DNA.

Comparative analysis of 12 different kits for bisulfite conversion of circulating cell-free DNA

ABSTRACTBlood circulating cell-free DNA (cfDNA) is becoming popular in the search of promising predictive and prognostic biomarkers. Among these biomarkers, cfDNA methylation markers have especially gained considerable attention. A significant challenge in the utilization of cfDNA methylation markers is the limited amount of cfDNA available for analyses; reportedly, bisulfite conversion (BSC) reduce cfDNA amounts even further. Nevertheless, few efforts have focused on ensuring high cfDNA conversion efficiency and recovery after BSC. To compare cfDNA recovery of different BSC methods, we compared 12 different commercially available BSC kits. We tested whether DNA recovery was affected by the molecular weight and/or quantity of input DNA. We also tested BSC efficiency for each kit. We found that recovery varied for DNA fragments of different lengths: certain kits recovered short fragments better than others, and only 3 kits recovered DNA fragments of <100 bp well. In contrast, DNA input amount did not seem to affect DNA recovery: for quantities spanning between 820 and ∼25,000 genome equivalents per BSC, a linear relation was found between input and recovery amount. Overall, mean recovery ranged between 9 and 32%, with BSC efficiency of 97–99.9%. When plasma cfDNA was used as input for BSC, recovery varied from 22% for the poorest and 66% for the best performing kits, while conversion efficiency ranged from 96 to 100% among different kits. In conclusion, clear performance differences exist between commercially available BSC kits, both in terms of DNA recovery and conversion efficiency. The choice of BSC kit can substantially impact the amount of converted cfDNA available for downstream analysis, which is critical in a cfDNA methylation marker setting.

Abstract 3804: Comprehensive comparison of bisulfite conversion kits: A guide for optimal sensitivity and specificity of circulating cell-free DNA methylation-based biomarkers

Abstract Analysis of circulating cell-free DNA (cfDNA) is a promising approach for non-invasive detection of human cancer. The approach has wide-spread applications including early detection of disease in asymptomatic individuals, residual disease detection and disease monitoring. A challenge for the clinical utilization of cfDNA is the small fragment size of cfDNA and the limited amount of cfDNA present in blood. For detection of circulating tumor DNA (ctDNA) highly sensitive methods for cfDNA detection e.g. targeted sequencing and digital droplet PCR (ddPCR) target tumor specific mutations or DNA methylation alterations. Detection of methylation-based biomarkers requires that cfDNA is bisulfite converted prior to biomarker detection, which is known to be associated with some loss of cfDNA. Yet, surprisingly few efforts have focused on ensuring high cfDNA bisulfite conversion (BSC) efficiency and -recovery, and few commercial kits are especially designed for this purpose. In this study we aimed to find a BSC method suited for cfDNA methylation biomarker research, with high BS conversion efficiency and minimum loss of cfDNA, in order to increase the amount of cfDNA template available for downstream analysis. We studied 13 different methods for BSC and examined both the recovery and the BSC efficiency using quantitative real-time PCR (qPCR), polyacrylamide gel electrophoresis (PAGE) and ddPCR. A significant variation in DNA recovery was observed between the 13 methods tested using both high and low molecular weight DNA templates. Especially five kits performed consistently well on all DNA templates tested, as evaluated by qPCR and PAGE. As the lengths of cfDNA is 146-167 base pairs, only methods that are able to recover fragments of this lengths is suitable for cfDNA pipelines. Results showed that few methods enabled recovery of DNA fragments ≤ 150 base pairs. However, three kits showed a higher amount of 150 base pair fragments compared to the other methods tested. In a clinical setting it is expected that the target cfDNA compose only a minor fraction of the total cfDNA in plasma. Thus, the five best performing kits were tested using ddPCR on serial dilutions of leucocyte DNA fragmented to ~ 150bp to mimic cfDNA. The best performing kit had a recovery of 69% of the initial input compared to only 13% for the poorest performing kit. Collectively, our comprehensive comparison hereby guide which BS conversion methods are suited for cfDNA methylation-based biomarker pipelines. Citation Format: Sarah Ø. Jensen, Mai-Britt W. Ørntoft, Jesper B. Bramsen, Torben F. Ørntoft, Claus L. Andersen. Comprehensive comparison of bisulfite conversion kits: A guide for optimal sensitivity and specificity of circulating cell-free DNA methylation-based biomarkers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3804. doi:10.1158/1538-7445.AM2017-3804

A modified bisulfite conversion method for the detection of DNA methylation.

Our purpose is to improve the conventional procedures for bisulfite conversion used to detect 5-methylcytosine in DNA. Impacts of different bisulfite salts, bisulfite conversion temperature, antioxidants and denaturants on DNA conversion and degradation were assessed by methylation-sensitive melt curve analysis. The modified method was tested on different genes and the conversion efficiency was analyzed by bisulfite sequencing. We developed a modified bisulfite conversion method that completes this process within 2 h. We demonstrate that high temperature denaturation is the major cause for DNA degradation, and the addition of ethylene glycol dimethyl ether is an effective way to accelerate the bisulfite conversion. The conversion efficiency is comparable to many other commercial kits. Our modified bisulfite conversion method is simple, cost efficient and less time consuming and is compatible with different genes and samples, thus has a great potential for the future research and clinical applications.

FAT4 hypermethylation and grade dependent downregulation in gastric adenocarcinoma.

Gastric cancer is one of the major causes of death due to cancer in the world. It is a multi-factorial disease with epigenetic factors being also involved in its development. FAT4 is a tumor suppressor gene exerting an important role in cell adhesion. This study aimed at analyzing FAT4 expression and promoter methylation in gastric cancer. FAT4 expression was studied in 30 tumoral tissues and their non-tumoral counterparts using Taqman real time PCR method. Promoter methylation was assessed using bisulfite conversion method followed by sequencing. Tumor tissues showed reduced FAT4 expression (P=0.04). FAT4 downregulation was associated with tumor grade, with higher repression at advanced grades. Significant increase of promoter methylation was observed in tumoral tissues. Reduced expression of FAT4 and increased methylation of its promoter may be one of the effective processes in turning a healthy stomach tissue into a tumor tissue.

Pyrosequencing Evaluation of Widely Available Bisulfite Conversion Methods: Considerations for Application

Introduction: Bisulfite treatment of DNA introduces methylation-dependent sequence changes through selective chemical conversion of nonmethylated cytosine to uracil and serves as pretreatment step for the majority of DNA methylation analysis methods. Methods: We have evaluated the conversion performance of five of the most commonly used bisulfite treatment kits [MethylDetector (Active Motif), Epitect+ (Qiagen), Zymo Methylation, Zymo Gold and Zymo Lightning (all from Zymo Research)] by pyrosequencing four different regions with variable methylation levels, including: a repetitive element (ALUSX), a gene with low levels of methylation (IL6ST), an imprinted gene expected to be approximately 50% methylated (IGF2), and a fully methylated gene (ST3GAL2). In addition, we have studied the influence of duration (3 vs. 16 h) and type (fixed temperature vs. cycling program) of incubation protocol on the conversion efficiency of each evaluated kit. Results: All kits produced similar conversion rates of ALUSX,IGF2 and ST3GAL2, while the conversion of the low methylated IL6ST gene was variable between kits. The Zymo kits were highly consistent in their performance even when different protocols of incubation were applied, generating full conversion at the low methylated gene IL6; this was not true for the MethylDetector and Epitect+ kits. However, long-cycling incubation could produce similar conversion rates for the same locus in combination with Active Motif and Qiagen kits. Conclusions: The selection of a long-cycling protocol during conversion permits standardization of protocols, improving the reproducibility of methylation estimates across laboratories for gene-specific, genome-wide and bisulfite-based sequencing analyses.

Abstract 182: Direct bisulfite conversion from archived tumor samples for methylation detection

Abstract DNA methylation is an important epigenetic mechanism of transcriptional control. DNA methylation plays an essential role in maintaining cellular function, and changes in methylation patterns may contribute to the development of cancer. Aberrant methylation of DNA is frequently found in tumor cells. Formalin-fixed and paraffin-embedded (FFPE) tissue sections are used for routine histopathological diagnostics, but they have increasingly become material for molecular studies of genome using molecular biological techniques such as PCR, qPCR, sequencing, genotyping etc. A major limitation of FFPE samples is the significant degree of degradation of the nucleic acids recovered from fixed tissues especially for the aged, long term archived samples. Since FFPE samples provide such huge collections of clinical tissue bank, more and more efforts have been spent in recovering the high quality of DNA from these specimens. Although the number of epigenetic cancer studies continues to grow, the wealth of FFPE samples available remains largely untapped. The value of FFPE samples and the potential they have to contribute to studies of the epigenome and the role of DNA methylation in numerous biological processes and disease, including cancer has been recognized more and more. Couple methods have been wildly used for methylation detection nowadays. With all the methods available, The bisulfite conversion method remains to be the most commonly used technique today as it can provide single CpG site methylation detection. Currently available methods for genome study or epigenetic study require isolation of gDNA from FFPE samples. With that, all the protocols require deparaffinization of the specimen prior to DNA extraction. Deparaffinization is usually accomplished with 2 to 3 incubations in xylene and can be a nuisance, particularly if there are more than just a few specimens to deal with. We, hereby, develop a new method to not only eliminate the deparaffinization but also eliminate the genomic DNA purification procedure. In our procedure, FFPE samples are directly threw in the denaturation/lysis reagent followed by bisulfite conversion and desulfonation procedure. The whole process can be completed within 2 hours. Even for long term archived samples, fair length of DNA fragments can be captured after the conversion. High quality of converted gDNA fragmented are produced which can be used for methylation detection at single CpG site level such as methylation profiling, deep sequencing etc. Clinical archived breast cancer and adjacent normal tissues as well as paired lung cancer samples were directly bisulfite converted and both breast cancer and lung cancer related promoter regions such as APC, BCL2, PTEN, and SCGB3A1 were compared for the methylation pattern changes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 182.

Whole Methylome Analysis by Ultra-Deep Sequencing Using Two-Base Encoding

Methylation, the addition of methyl groups to cytosine (C), plays an important role in the regulation of gene expression in both normal and dysfunctional cells. During bisulfite conversion and subsequent PCR amplification, unmethylated Cs are converted into thymine (T), while methylated Cs will not be converted. Sequencing of this bisulfite-treated DNA permits the detection of methylation at specific sites. Through the introduction of next-generation sequencing technologies (NGS) simultaneous analysis of methylation motifs in multiple regions provides the opportunity for hypothesis-free study of the entire methylome. Here we present a whole methylome sequencing study that compares two different bisulfite conversion methods (in solution versus in gel), utilizing the high throughput of the SOLiD™ System. Advantages and disadvantages of the two different bisulfite conversion methods for constructing sequencing libraries are discussed. Furthermore, the application of the SOLiD™ bisulfite sequencing to larger and more complex genomes is shown with preliminary in silico created bisulfite converted reads.

Developmental and Differentiation-Specific Patterns of γ- and β-Globin Promoter DNA Methylation in Primary Human Erythroid Cells.

Despite years of investigation in a variety of experimental systems, the mechanisms underlying human β-globin locus developmental gene switching remain elusive. Several lines of evidence implicate DNA methylation in this process. As an initial step in studying the role of epigenetic modifications in the human switching process and in determining the mechanisms by which DNA methyltransferase inhibitors reverse the switch, we have characterized the DNA methylation patterns of the individual CpGs in the γ- and β-globin promoters in fetal liver (FL) and adult bone marrow (BM) primary erythroid cells and during in vitro differentiation of adult erythroid cells. Using the bisulfite conversion method we evaluated all CpGs in the ~500 bp regions centered on the γ- and β-globin promoter start sites. Fetal liver (FL) and adult bone marrow (BM) samples were obtained using IRB approved protocols and informed consent procedures. Erythroid cells were purified using anti-glycophorin A (glyA) magnetic beads. Purity was confirmed to be greater than 95%. Samples from five independent BM and FL samples were analyzed and 8–20 bisulfite converted sequences were determined for each promoter in each sample. Our results show that all 8 CpGs between −249 and +210 of the Gγ and Aγ-globin promoters are less than 20% methylated in FL and greater than 80% methylated in BM except for the −158 CpG which is only 40% methylated in BM(p<0.002). The 6 CpGs between −415 and +110 of the β-globin promoter show an inverse pattern with lower levels of DNA methylation in BM. Histone H3 acetylation of the γ-globin promoter, as determined by ChIP analysis, showed a complimentary pattern with higher levels in FL than BM. We next evaluated γ-globin promoter methylation patterns during in vitro erythroid differentiation from CD34+ BM cells. In this experiment, cells were grown with SCF, Flt3 ligand and IL-3 for 7 days and then in EPO for 14 days producing erythroid cells which express 99%HbA and 1% HbF. The initial day 0 CD34+ cells showed 90–100% methylation of all γ promoter sites. By day 3 in culture, before the initiation of erythroid differentiation, methylation at all sites upstream of the promoter had decreased to less than 60% and the CpG at −53 (the site of Stage Selector Protein complex binding) had decreased to less than 20%. The three CpG sites down-stream of the promoter (+6, +17 and +50) remained highly methylated. The pattern was unchanged at day 10, early in erythroid differentiation, when γ-globin mRNA expression was beginning. By day 14, when β-globin expression was peaking, methylation of the upstream promoter had increased back to the 70–100% level at all CpGs. These experiments provide a comprehensive picture of γ- and β-globin promoter methylation during the fetal and adult stages of erythroid development and of the γ-globin promoter during adult erythroid differentiation. The finding of transient γ-promoter hypomethylation during differentiation offers a potential mechanism to explain the transient γ-globin gene expression seen during normal adult erythropoiesis. Our results also raise the possibility that, just as domains of altered histone modification exist in β-globin gene loci, there may also be developmentally-specific domains of DNA methylation.