Methylation-sensitive Restriction Enzyme Research Articles

Optimising Transformation Efficiency in Borrelia: Unravelling the Role of the Restriction-Modification System of Borrelia afzelii and Borrelia garinii.

Borrelia spp. are transmitted to humans by the bite of an infected tick. In Europe, Borrelia afzelii and Borrelia garinii are the main causative agents of Lyme borreliosis, one of the most prevalent tick-borne diseases in the northern hemisphere. In bacteria such as Borrelia spp., a restriction-modification system (RMS) protects against the harmful introduction of foreign DNA. The RMS comprises two activities: methyltransferase and endonuclease. This study is aimed to characterize the RMS of B. afzelii and B. garinii. First, we identified potential RMS genes. The predicted genes were cloned into a methylase-deficient Escherichia coli strain and digested with methylation-sensitive restriction enzymes to verify methyltransferase activity. Additionally, the RMS proteins were purified to evaluate endonuclease activity. Subsequently, methylated and unmethylated plasmids were used to investigate the effect of methylation on endonuclease activity and transformation efficiency. We identified four possible RMS genes in B. afzelii and four RMS genes in B. garinii. We analyzed the presence of these genes in patient isolates and observed a high degree of heterogeneity. The restriction pattern of DNA methylated by each of the four recombinantly expressed genes provided strong evidence that all encode adenine-specific methyltransferases. After 24 h of incubation with purified RMS proteins, we observed complete digestion of unmethylated plasmid DNA, demonstrating endonuclease activity. Finally, we proved that methylation protects against endonuclease activity and increases transformation efficiency.

Glyphosate and a glyphosate-based herbicide dysregulate the epigenetic landscape of Homeobox A10 (Hoxa10) gene during the endometrial receptivity in Wistar rats.

We observed that gestational plus lactational exposure to glyphosate (Gly), as active ingredient, or a glyphosate-based herbicide (GBH) lead to preimplantation losses in F1 female Wistar rats. Here, we investigated whether GBH and/or Gly exposure could impair Hoxa10 gene transcription by inducing epigenetic changes during the receptive stage in rats, as a possible herbicide mechanism implicated in implantation failures. F0 dams were treated with Gly or a GBH through a food dose of 2mg Gly/kg bw/day from gestational day (GD) 9 up to lactational day 21. F1 female rats were bred, and uterine tissues were analyzed on GD5 (preimplantation period). Transcripts levels of Hoxa10, DNA methyltransferases (Dnmt1, Dnmt3a and Dnmt3b), histone deacetylases (Hdac-1 and Hdac-3) and histone methyltransferase (EZH2) were assessed by quantitative polymerase chain reaction (qPCR). Four CpG islands containing sites targeted by BstUI methylation-sensitive restriction enzyme and predicted transcription factors (TFs) were identified in Hoxa10 gene. qPCR-based methods were used to evaluate DNA methylation and histone post-translational modifications (hPTMs) in four regulatory regions (RRs) along the gene by performing methylation-sensitive restriction enzymes and chromatin immunoprecipitation assays, respectively. GBH and Gly downregulated Hoxa10 mRNA. GBH and Gly increased DNA methylation levels and Gly also induced higher levels than GBH in all the RRs analyzed. Both GBH and Gly enriched histone H3 and H4 acetylation in most of the RRs. While GBH caused higher H3 acetylation, Gly caused higher H4 acetylation in all RRs. Finally, GBH and Gly enhanced histone H3 lysine 27 trimethylation (H3K27me3) marker at 3 out of 4 RRs studied which was correlated with increased EZH2 levels. In conclusion, exposure to GBH and Gly during both gestational plus lactational phases induces epigenetic modifications in regulatory regions of uterine Hoxa10 gene. We show for the first time that Gly and a GBH cause comparable gene expression and epigenetic changes. Our results might contribute to delineate the mechanisms involved in the implantation failures previously reported. Finally, we propose that epigenetic information might be a valuable tool for risk assessment in the near future, although more research is needed to establish a cause-effect relationship.

Epigenetic alteration of uterine Leukemia Inhibitory Factor gene after glyphosate or a glyphosate-based herbicide exposure in rats

Glyphosate-based herbicides (GBHs) or its active ingredient, glyphosate (Gly), induce implantation failure in rats. We aimed to elucidate a mechanism of action of these compounds assessing the transcriptional and epigenetic status of the receptivity marker, leukemia inhibitory factor (Lif) gene. F0 rats were orally exposed to GBH or Gly at 3.8 or 3.9 mg Gly/kg/day, respectively, from gestational day (GD) 9 until weaning. F1 females were mated and uterine samples collected at GD5. Methylation-sensitive restriction enzymes (MSRE) sites and transcription factors were in silico predicted in regulatory regions of Lif gene. DNA methylation status and histone modifications (histone 3 and 4 acetylation (H3Ac and H4Ac) and H3 lysine-27-trimethylation (H3K27me3)) were assessed. GBH and Gly decreased Lif mRNA levels and caused DNA hypermethylation. GBH increased H3Ac levels, whereas Gly reduced them; both compounds enhanced H3K27me3 levels. Finally, both GBH and Gly induced similar epigenetic alterations in the regulatory regions of Lif.

Universal penalized regression (Elastic-net) model with differentially methylated promoters for oral cancer prediction

BackgroundDNA methylation showed notable potential to act as a diagnostic marker in many cancers. Many studies proposed DNA methylation biomarker in OSCC detection, while most of these studies are limited to specific cohorts or geographical location. However, the generalizability of DNA methylation as a diagnostic marker in oral cancer across different geographical locations is yet to be investigated.MethodsWe used genome-wide methylation data from 384 oral cavity cancer and normal tissues from TCGA HNSCC and eastern India. The common differentially methylated CpGs in these two cohorts were used to develop an Elastic-net model that can be used for the diagnosis of OSCC. The model was validated using 812 HNSCC and normal samples from different anatomical sites of oral cavity from seven countries. Droplet Digital PCR of methyl-sensitive restriction enzyme digested DNA (ddMSRE) was used for quantification of methylation and validation of the model with 22 OSCC and 22 contralateral normal samples. Additionally, pyrosequencing was used to validate the model using 46 OSCC and 25 adjacent normal and 21 contralateral normal tissue samples.ResultsWith ddMSRE, our model showed 91% sensitivity, 100% specificity, and 95% accuracy in classifying OSCC from the contralateral normal tissues. Validation of the model with pyrosequencing also showed 96% sensitivity, 91% specificity, and 93% accuracy for classifying the OSCC from contralateral normal samples, while in case of adjacent normal samples we found similar sensitivity but with 20% specificity, suggesting the presence of early disease methylation signature at the adjacent normal samples. Methylation array data of HNSCC and normal tissues from different geographical locations and different anatomical sites showed comparable sensitivity, specificity, and accuracy in detecting oral cavity cancer with across. Similar results were also observed for different stages of oral cavity cancer.ConclusionsOur model identified crucial genomic regions affected by DNA methylation in OSCC and showed similar accuracy in detecting oral cancer across different geographical locations. The high specificity of this model in classifying contralateral normal samples from the oral cancer compared to the adjacent normal samples suggested applicability of the model in early detection.

A novel index combining fecal immunochemical test, DNA test, and age improves detection of advanced colorectal adenoma.

Although the fecal immunochemical test for hemoglobin (FIT) is a widely used screening test for colorectal cancer, it is not sensitive enough to detect advanced colorectal adenoma. To address this issue, we performed this study to investigate whether combining the FIT and fecal DNA testing of methylated somatostatin (SST) could improve diagnostic performance for advanced colorectal adenoma. We collected feces from 79 healthy subjects with negative results on colonoscopy, 43 patients with non-advanced colorectal adenoma, 117 patients with advanced colorectal adenoma, and 126 patients with colorectal cancer. After fecal DNA was incubated with methylation-sensitive restriction enzymes, SST methylation levels were measured by droplet digital PCR. Using logistic multivariate analysis, we established a prediction formula for detecting colorectal neoplasia and named it the FAMS (FIT, age, methylated SST) index. The diagnostic performance of a single use of FIT for advanced colorectal adenoma showed a sensitivity of 29.1% (34/117) and specificity of 89.3% (109/122). In contrast, the FAMS index showed a sensitivity of 56.4% (66/117) at a similar specificity point of 91.0% (111/122). Furthermore, even at the higher specificity point of 94.3% (115/122), the sensitivity was still higher than that of FIT, reaching 42.7% (50/117). As the FAMS index showed better diagnostic performance for advanced colorectal adenoma than a single use of FIT, the FAMS index could be a promising tool for detecting advanced colorectal adenoma.

IMPRESS: Improved methylation profiling using restriction enzymes and smMIP sequencing, combined with a new biomarker panel, creating a multi-cancer detection assay

BackgroundDespite the worldwide progress in cancer diagnostics, more sensitive diagnostic biomarkers are needed. The methylome has been extensively investigated in the last decades, but a low-cost, bisulfite-free detection method for multiplex analysis is still lacking.MethodsWe developed a methylation detection technique called IMPRESS, which combines methylation-sensitive restriction enzymes and single-molecule Molecular Inversion Probes. We used this technique for the development of a multi-cancer detection assay for eight of the most lethal cancer types worldwide. We selected 1791 CpG sites that can distinguish tumor from normal tissue based on DNA methylation. These sites were analysed with IMPRESS in 35 blood, 111 tumor and 114 normal samples. Finally, a classifier model was built.ResultsWe present the successful development of IMPRESS and validated it with ddPCR. The final classifier model discriminating tumor from normal samples was built with 358 CpG target sites and reached a sensitivity of 0.95 and a specificity of 0.91. Moreover, we provide data that highlight IMPRESS’s potential for liquid biopsies.ConclusionsWe successfully created an innovative DNA methylation detection technique. By combining this method with a new multi-cancer biomarker panel, we developed a sensitive and specific multi-cancer assay, with potential use in liquid biopsies.

Single Methylation Sensitive Restriction Endonuclease-Based Cascade Exponential Amplification Assay for Visual Detection of DNA Methylation at Single-Molecule Level.

Function as a potential cancer biomarker, DNA methylation shows great significance in cancer diagnosis, prognosis, and treatment monitoring. While the lack of an ultrasensitive, specific, and accurate method at the single-molecule level hinders the analysis of the exceedingly low levels of DNA methylation. Herein, based on the outstanding recognition and digestion ability of methylation-sensitive restriction endonuclease (MSRE), we established a single MSRE-based cascade exponential amplification method, which requires only two ingeniously designed primers and only one recognition site of MSRE for the detection of DNA methylation. Differentiated by MSRE digestion, the cleaved unmethylated DNA is too short to induce any amplification reactions, while methylated DNA remains intact to trigger cascade exponential amplification and the subsequent CRISPR/Cas12a system. By integrating the two exponential amplification reactions, as low as 1 aM methylated DNA can be accurately detected, which corresponds to 6 molecules in a 10 μL system, indicating that our method is more sensitive than single amplification-based methods with the ability to detect DNA methylation at the single-molecule level. In addition, 0.1% methylated DNA can be effectively distinguished from large amounts of unmethylated DNA. Our method is further introduced to exploit the expression difference of DNA methylation among normal cells and cancer cells. Moreover, the visual detection of DNA methylation is also realized by the full hybridization between amplification products and the crRNA of CRISPR/Cas12a. Therefore, the proposed method has great potential to be a promising and robust bisulfite-free method for the detection of DNA methylation at the single-molecule level, which is of great importance for early diagnosis of cancer.

Determination of global DNA methylation level by methylation-sensitive comet assay in patients with urinary bladder cancer.

DNA methylation is an important mechanism in the regulation of gene expression and maintenance of genomic integrity. Aberrant DNA methylation is an early event in carcinogenesis. DNA methyltransferase inhibitors are used to restore aberrant DNA methylation and inhibit tumor growth. Evaluation of DNA methylation level is important for an effective anti-cancer therapy. In the present study, the determination of global DNA methylation levels in patients with urinary bladder cancer was proposed. The methylation-sensitive comet assay determined the global DNA methylation level at the level of single cells. McrBC enzyme, a methylation-sensitive restriction endonuclease was used for enzymatic digestion to generate additional breaks at methylated sites. % DNA methylation level was significantly higher in patients with bladder cancer compared to the control group. The clinical performance of % DNA methylation analysis by methylation-sensitive comet assay was evaluated by ROC curve. Using the cut-off value of 6,5% DNA methylation, 92% sensitivity, and 42% specificity were obtained. In conclusion, global DNA methylation measured by methylation-sensitive comet assay may be a promising non-invasive biomarker that reduces interventional tests required in the diagnosis and follow-up of urinary bladder cancer.

Implementation of a MSRE ddPCR method for the detection of methylated WIF1 and NPY genes in colorectal cancer patients.

Colorectal cancer is a worldwide leading cause of death accounting for high-rate mortality. Mutations in the epidermal growth factor receptor and RAS/MAPK pathways, as well as altered methylation genes profiles, have been described as molecular mechanisms promoting and sustaining tumour development and progression. Aberrant methylation is a well-known epigenetic mechanism involved in gene regulation; particularly several genes were reported as hypermethylated in CRC. Recently, it was shown that epigenetic alterations in genes such as neuropeptide y, proenkephalin and Wnt inhibitory factor 1 can be used as promising disease biomarkers. Almost all methods developed for the DNA methylation analysis combined next generation sequencing, conventional qRT-PCR or ddPCR with the prior DNA modification with sodium bisulfite. We implemented a ddPCR method to assess the methylation status of Wnt inhibitory factor 1 and neuropeptide y using the methylation sensitive restriction enzyme approach that does not impact on DNA quality and guarantees the discrimination of DNA methylation independent of bisulfite conversion. We showed that this method is robust and sensitive also allowing the monitoring of CRC disease progression when applied to circulating free DNA samples from liquid biopsies, proving to be a fast and easy to implement assay to be used for the monitoring of the methylation pattern of clinically relevant target genes.

Association of XRCC2 with breast cancer, a multi-omics analysis at genomic, transcriptomic, and epigenomic level.

One of the main causes of cancer-related mortality for women worldwide is breast cancer (BC). The XRCC2 gene, essential for DNA repair, has been implicated in cancer susceptibility. This study aims to evaluate the association between XRCC2 and BC risk. The study was conducted at Zheen International Hospital in Erbil, Iraq, between 2021 and 2024 with a total of 88 samples, including 44 paired normal and cancer tissue samples. Mutation analysis was performed using Next-Generation Sequencing, coupled with in silico tools for variant impact prediction. Expression levels were assessed through RT-PCR, and methylation status was determined using methylation-sensitive restriction enzyme digestion PCR. The study identified seven inherited germline variants in the XRCC2 gene, with five of these mutations being Uncertain Significance, one being Likely Pathogenic, and one being Likely benign. RNA purity was found high with mean A260/280 ratios of 1.986 ± 0.097 in normal (N) and 1.963 ± 0.092 in tumor (T) samples. Tumor samples exhibited a higher RNA concentration (78.56 ± 40.87 ng/µL) than normal samples (71.44 ± 40.79 ng/µL). XRCC2 gene expression was significantly upregulated in tumor tissue, with marked increases in patients aged 40-55 and >56 years and in higher cancer grades (II and III) and invasive ductal carcinoma (p-values ranging from <0.0001 to 0.0392). DNA methylation rates in tumor tissues were low (7%), suggesting limited regulation by methylation. The study suggests that XRCC2 can be classified as an oncogene and that its structural investigation by targeted NGS and expression evaluation can be used as a potential biomarker in BC.

Genotyping-by-sequencing targets genic regions and improves resolution of genome-wide association studies in autotetraploid potato

Key messageDe novo genotyping in potato using methylation-sensitive GBS discovers SNPs largely confined to genic or gene-associated regions and displays enhanced effectiveness in estimating LD decay rates, population structure and detecting GWAS associations over ‘fixed’ SNP genotyping platform. Study also reports the genetic architectures including robust sequence-tagged marker–trait associations for sixteen important potato traits potentially carrying higher transferability across a wider range of germplasm.This study deploys recent advancements in polyploid analytical approaches to perform complex trait analyses in cultivated tetraploid potato. The study employs a ‘fixed’ SNP Infinium array platform and a ‘flexible and open’ genome complexity reduction-based sequencing method (GBS, genotyping-by-sequencing) to perform genome-wide association studies (GWAS) for several key potato traits including the assessment of population structure and linkage disequilibrium (LD) in the studied population. GBS SNPs discovered here were largely confined (~ 90%) to genic or gene-associated regions of the genome demonstrating the utility of using a methylation-sensitive restriction enzyme (PstI) for library construction. As compared to Infinium array SNPs, GBS SNPs displayed enhanced effectiveness in estimating LD decay rates and discriminating population subgroups. GWAS using a combined set of 30,363 SNPs identified 189 unique QTL marker–trait associations (QTL-MTAs) covering all studied traits. The majority of the QTL-MTAs were from GBS SNPs potentially illustrating the effectiveness of marker-dense de novo genotyping platforms in overcoming ascertainment bias and providing a more accurate correction for different levels of relatedness in GWAS models. GWAS also detected QTL ‘hotspots’ for several traits at previously known as well as newly identified genomic locations. Due to the current study exploiting genome-wide genotyping and de novo SNP discovery simultaneously on a large tetraploid panel representing a greater diversity of the cultivated potato gene pool, the reported sequence-tagged MTAs are likely to have higher transferability across a wider range of potato germplasm and increased utility for expediting genomics-assisted breeding for the several complex traits studied.

Pterostilbene Reverses Epigenetic Silencing of Nrf2 and Enhances Antioxidant Response in Endothelial Cells in Hyperglycemic Microenvironment.

The epigenetic regulation of nuclear factor erythroid 2-related factor 2 (Nrf2), a pivotal redox transcription factor, plays a crucial role in maintaining cellular homeostasis. Recent research has underscored the significance of epigenetic modifications of Nrf2 in the pathogenesis of diabetic foot ulcers (DFUs). This study investigates the epigenetic reversal of Nrf2 by pterostilbene (PTS) in human endothelial cells in a hyperglycemic microenvironment (HGM). The activation potential of PTS on Nrf2 was evaluated through ARE-Luciferase reporter assays and nuclear translocation studies. Following 72 h of exposure to an HGM, mRNA expression and protein levels of Nrf2 and its downstream targets NAD(P)H quinone oxidoreductase 1 (NQO1), heme-oxygenase 1(HO-1), superoxide dismutase (SOD), and catalase (CAT) exhibited a decrease, which was mitigated in PTS-pretreated endothelial cells. Epigenetic markers, including histone deacetylases (HDACs class I-IV) and DNA methyltransferases (DNMTs 1/3A and 3B), were found to be downregulated under diabetic conditions. Specifically, Nrf2-associated HDACs, including HDAC1, HDAC2, HDAC3, and HDAC4, were upregulated in HGM-induced endothelial cells. This upregulation was reversed in PTS-pretreated cells, except for HDAC2, which exhibited elevated expression in endothelial cells treated with PTS in a hyperglycemic microenvironment. Additionally, PTS was observed to reverse the activity of the methyltransferase enzyme DNMT. Furthermore, CpG islands in the Nrf2 promoter were hypermethylated in cells exposed to an HGM, a phenomenon potentially counteracted by PTS pretreatment, as shown by methyl-sensitive restriction enzyme PCR (MSRE-qPCR) analysis. Collectively, our findings highlight the ability of PTS to epigenetically regulate Nrf2 expression under hyperglycemic conditions, suggesting its therapeutic potential in managing diabetic complications.

Identification of skewed X chromosome inactivation using exome and transcriptome sequencing in patients with suspected rare genetic disease.

X-chromosome inactivation (XCI) is an epigenetic process that occurs during early development in mammalian females by randomly silencing one of two copies of the X chromosome in each cell. The preferential inactivation of either the maternal or paternal copy of the X chromosome in a majority of cells results in a skewed or non-random pattern of X inactivation and is observed in over 25% of adult females. Identifying skewed X inactivation is of clinical significance in patients with suspected rare genetic diseases due to the possibility of biased expression of disease-causing genes present on the active X chromosome. The current clinical test for the detection of skewed XCI relies on the methylation status of the methylation-sensitive restriction enzyme (Hpall) binding site present in proximity of short tandem polymorphic repeats on the androgen receptor (AR) gene. This approach using one locus results in uninformative or inconclusive data for 10-20% of tests. Further, recent studies have shown inconsistency between methylation of the AR locus and the state of inactivation of the X chromosome. Herein, we develop a method for estimating X inactivation status, using exome and transcriptome sequencing data derived from blood in 227 female samples. We built a reference model for evaluation of XCI in 135 females from the GTEx consortium. We tested and validated the model on 11 female individuals with different types of undiagnosed rare genetic disorders who were clinically tested for X-skew using the AR gene assay and compared results to our outlier-based analysis technique. In comparison to the AR clinical test for identification of X inactivation, our method was concordant with the AR method in 9 samples, discordant in 1, and provided a measure of X inactivation in 1 sample with uninformative clinical results. We applied this method on an additional 81 females presenting to the clinic with phenotypes consistent with different hereditary disorders without a known genetic diagnosis. This study presents the use of transcriptome and exome sequencing data to provide an accurate and complete estimation of X-inactivation and skew status in a cohort of female patients with different types of suspected rare genetic disease.

Abstract 7009: Quantification of DNA methylation using methylation-sensitive restriction enzymes and digital PCR

Abstract DNA methylation is a common epigenetic modification, characterized by the presence of the signature 5-methylcytosine without altering the sequence of DNA. The study of DNA methylation in mammals has gained significant attention due to its broad impact on numerous biological processes and its critical role in the onset and progression of many diseases, such as cancer and aging. Consequently, there is a pressing need for a rapid and precise method to assess methylation status. Currently, most approaches for quantifying DNA methylation rely on sodium bisulfite treatment. However, such approaches do not align with the uracil DNA glycosylase PCR system. In this study, we demonstrate the application of methylation-sensitive restriction enzymes (MSRE) and digital PCR to determine the methylation status of the Ras association domain family 1 isoform A (RASSF1A) in cancer cell lines. Digital PCR enables the precise detection and absolute quantification without the reliance on reference standards. We developed a multiplex digital PCR assay that includes the target gene RASSF1A, along with two reference genes for the purpose of monitoring digestion completion and correcting DNA input. Each sample of interest was measured both before and after a MSRE digestion. To ensure the quantification accuracy, we employed the commercially available CpGenome human methylated DNA standard with a known concentration from MilliporeSigma, treating it with and without a MSRE, and subsequently performing digital PCR experiments using Roche Digital LightCycler® IVD system with a Universal nanowell plate featuring 28,000 partitions. The expected concentrations lay within the 95% confidence interval, affirming the accurate quantification achieved through digital PCR. Next, we measured the fraction of methylated alleles in various lung cancer and breast cancer cell lines, as well as in healthy human gDNA. The methylation of RASSF1A promoter in healthy human gDNA is ~0.7%, while a broad range of methylation levels was observed in cancer cell lines, ranging from ~0.7% to ~100.2%. Notably, the influence of GC bias was identified in this study. To overcome this critical challenge, we optimized the usage of several high GC enhancers. The results demonstrated that the concentration of 5-7.5% DMSO, 7.5% glycerol, and commercially available OneTaq or Q5 GC enhancers from New England Biolabs were effectively incorporated into the Roche digital PCR system, thereby enhancing the accuracy of quantification from 30% to 100%. Together, we demonstrated a remarkable approach for DNA methylation analysis using Roche digital PCR system in combination with MSREs and suitable high GC enhancers. This study suggests a promising rapid, accurate and cost-effective tool to advance research in the field. *Data on file at Roche Diagnostics, Wilmington, MA, USA Citation Format: Yu Zhao, Lindsey Cambria. Quantification of DNA methylation using methylation-sensitive restriction enzymes and digital PCR [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 7009.

Abstract 1722: Subtype classification of small cell lung cancer (SCLC) tissues using the EpiCheck methylation sensitive restriction-based PCR platform

Abstract Small cell lung cancer (SCLC) is an aggressive neuroendocrine malignancy with poor survival rates. Despite molecular and clinical heterogeneity, SCLC is currently treated as a single entity without guidance of predictive biomarkers leading to expectedly poor outcomes. A recent study suggested classifying SCLC into four subtypes (“A”, “N”, “P” and “I”), each with unique molecular features and therapeutic vulnerabilities. While initially this classification was based on gene expression (RNA-seq) data, subsequent data suggest it can be recapitulated using reduced-representation bisulfite sequencing (RRBS) methylation profile. While the classification system accurately predicts responses to therapies, including immunotherapies, in retrospective analysis, both tissue-based RNAseq and RRBS are cumbersome, time-consuming and impractical for prospective treatment assignment in an aggressive malignancy. In this pilot study, we developed a methylation-based PCR assay for classification of SCLC subtypes based on the EpiCheck platform, which combines methylation-sensitive restriction endonuclease (MSRE) digestion with qPCR amplification to detect differential methylation at the DNA level. We developed a 13-marker assay and successfully classified, in a blinded study, 96.55% of FFPE tissue samples originating from SCLC patient of A, N and P subtypes. First, a bioinformatic analysis identified 41 candidate biomarkers that are differentially methylated between pairs of SCLC subtypes (A-N, A-P, and N-P) using RRBS data from 56 RNA-seq classified SCLC tumor samples (34 “A”, 19 “N”, 3 “P”). Development of primers and probes for these markers was followed by a biochemical selection process, identifying 13 markers that displayed optimal performance. A PCR EpiCheck assay, employing these 13 markers, was applied to a blinded set of 29 DNA samples from SCLC FFPE tumor tissues (21 “A”, 6 “N”, 2 “P”). Each sample received two ranks based on methylation levels of 5 A-N and 9 P-AN markers and was classified using determined thresholds for each rank. Finally, when comparing PCR-based and RNA-seq-based classifications, the 13-marker PCR assay classified 28/29 (96.55%) of the samples concordantly, misclassifying one "P" sample as "A". This pilot demonstrates the potential of a simple PCR EpiCheck-based assay to accurately differentiate between SCLC subtypes. For complete subtype classification, “I” markers need to be added to the panel, and the assay should be further validated using a larger cohort, particularly for the “P” subtype. Early detection and classification are critical for timely personalized therapy, and such an assay, performed on cfDNA samples, could potentially be used to shorten the time between a patient diagnosis and tailored treatment initiation or clinical study inclusion from a month in best case scenarios to a few days. Citation Format: Orna Savin, Dvir Netanely, Simon Heeke, Carl Michael Gay, Marcos Roberto Estecio, Shacade Danan, Sarah Zaouch, Aharona Shuali, Mathias Ehrich, Lauren Averett Byers, John Victor Heymach, Adam Wasserstrom, Danny Frumkin. Subtype classification of small cell lung cancer (SCLC) tissues using the EpiCheck methylation sensitive restriction-based PCR platform [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 1722.

Analysis of X chromosome inactivation and prenatal diagnosis for a Chinese pedigree with loss of heterozygosity at Xq22.1q22.3

To explore the correlation between skewed X chromosome inactivation (XCI) and clinical phenotype of a Chinese pedigree with loss of heterozygosity at Xq22.1q22.3. A pedigree diagnosed at Taizhou Hospital on November 10, 2021 was selected as the study subject. G-banded chromosomal karyotyping and copy number variation sequencing (CNV-seq) were carried out to analyze the amniotic fluid and peripheral blood samples from the couple. XCI was detected by PCR amplification of CAG repeats in exon 1 of androgen receptor gene before and after the digestion with methylation-sensitive restriction enzyme Hpa II. Correlation between the genotype and clinical phenotype was analyzed. The karyotypes of the pregnant woman and the fetus were both determined as 46,X,del(X)(q22), and the result of CNV-seq was seq[hg19]del(X)(q22.1q22.3) chrX: g.10046000_105740000del, suggesting that both had harbored a 5.28 Mb deletion on the X chromosome. No obvious abnormality was found in the husband. XCI analysis showed that the activity ratio of the two X chromosomes of the pregnant woman and her fetus was 0 : 100. The X chromosome harboring the q22.1q22.3 deletion was completely inactivated, and the inactivated X chromosome of the fetus was derived from its mother. The fetus has harbored a maternally derived inactivated X chromosome del(X)(q22) , and its phenotype is closely associated with the activity of the abnormal X chromosome. Pedigree XCI analysis combined with the clinical phenotype has facilitated recognition of the maternal phenotype and prognosis of female fetus with loss of heterozygosity at Xq22.1q22.3.

A female patient carrying a novel DMD mutation with non-random X-chromosome inactivation from a DMD family

ObjectiveTo analyze the clinical phenotype and genetic characteristics of a female proband carrying a novel mutation in the DMD gene with non-random X-chromosome inactivation in a large pedigree with pseudohypertrophic muscular dystrophy.MethodsClinical information of the female proband, her monozygotic twin sister, and other family members were collected. Potential pathogenic variants were detected with Multiplex Ligation-dependent Probe Amplification (MLPA) and whole-exome sequencing (WES). Methylation-sensitive restriction enzyme (HhaI) was employed for X-chromosome inactivation analysis.ResultsThe proband was a female over 5 years old, displayed clinical manifestations such as elevated creatine kinase (CK) levels and mild calf muscle hypertrophy. Her monozygotic twin sister exhibited normal CK levels and motor ability. Her uncle and cousin had a history of DMD. WES revealed that the proband carried a novel variant in the DMD (OMIM: 300,377) gene: NM_004006.3: c.3051_3053dup; NP_003997.2: p.Tyr1018*. In this pedigree, five out of six female members were carriers of this variant, while the cousin and uncle were hemizygous for this variant. X-chromosome inactivation analysis suggested non-random inactivation in the proband.ConclusionThe c.3051_3053dup (p.Tyr1018*) variant in the DMD gene is considered to be the pathogenic variant significantly associated with the clinical phenotype of the proband, her cousin, and her uncle within this family. Integrating genetic testing with clinical phenotype assessment can be a valuable tool for physicians in the diagnosis of progressive muscular dystrophies, such as Becker muscular dystrophy (BMD) and Duchenne muscular dystrophy (DMD).

Investigating FGFR2 gene as a blood-based epigenetic biomarker in gastric cancer.

Gastric adenocarcinoma is a prevalent form of cancer that often remains undetected in its early stages due to the lack of specific symptoms. This delayed diagnosis leads to poor clinical outcomes, underscoring the need for an effective and non-invasive method for early detection. Recent advances in cancer epigenetics have led to the identification of biomarkers that have the potential to revolutionize the early detection and monitoring of this disease. One such promising biomarker is the methylation of the FGFR2 promoter. This study aims to measure the methylation levels of a specific CpG site in the FGFR2 promoter gene in DNA extracted from blood leukocytes from patients with intestinal metaplasia, gastric cancer, and healthy control. The CpG site of the FGFR2 gene promoter was identified in its control region. Methylation alteration of the selected FGFR2 CpG site was determined through the (methylation-sensitive restriction enzyme) MSRE-qPCR. Genomic DNA was extracted from one hundred twenty-five participants. The normal group had mean methylation levels of 93.23 ± 4.929%, while the IM group had a level of 69.85 ± 27.15%. In GC patients, the levels varied, with 25.96 ± 18.98% in the intestinal type and 28.30 ± 16.07% in the diffuse type. The methylation levels in the IM and GC patients were significantly lower than those in the normal control group. However, no significant difference was observed between the methylation status of the intestinal type of GC and the diffuse type. The Receiver operating characteristic (ROC) curve analysis showed that FGFR2 CpG methylation levels in GC patients compared to normal controls had a high sensitivity of 100% and specificity of 100%, with a cut-off of < 74.25%; when GC patients were compared to IM patients, the sensitivity was 85%, and the specificity was 80%, with a cut-off < 44.45%. The potential of the FGFR2 methylation status as a non-invasive biomarker lies in its ability to be detected in blood leukocytes, which makes it a promising tool for the early detection of intestinal metaplasia and gastric cancer. This could significantly improve the detection and management of these gastric conditions.

A noninvasive method for the detection of foetal DNA in early pregnancy based on differential methylation pattern of Ras association domain family member 1A

Background The detection of foetal DNA and extravillus trophoblasts (EVTs) in early pregnancy in cervical and uterine samples offers a potential pathway for non-invasive prenatal diagnostics. However, the challenge lies in effectively quantifying these samples. This study introduces a novel approach using the Ras association domain family 1 A (RASSF1A), which exhibits hypermethylation in foetal cells and hypomethylation in maternal cells. The differential methylation pattern of RASSF1A provides a unique biomarker for quantifying foetal cells in cervical and intrauterine samples. Methods This study was conducted between September 2022 and May 2023. In total, 23 samples (12 cervical cell samples and 11 intrauterine samples) were collected from women in the Sichuan Jinxin Women & Children Hospital, Jingxiu District, Chengdu, China. The cervical cell samples were collected via lavage and brush techniques, and the intrauterine cell samples were obtained via uterine lavage. These samples were collected as part of a broader effort to advance our understanding of foetal cell dynamics during early pregnancy. The sampling methods were chosen for their minimally invasive nature and their potential in capturing a representative cell population from the respective sites. After digestion of the cell samples using a methylation-sensitive restriction enzyme cocktail, a critical step to differentiate between maternal and foetal DNA, the quantitative polymerase chain reaction (qPCR) of RASSF1A and β-actin (ACTB) were employed to measure foetal DNA and cell concentrations. Immunofluorescence techniques targeting histocompatibility complex, class I G (HLA-G) and GATA binding protein 3 (GATA-3) were employed to detect EVTs in the cell samples and in decidual tissue, with the latter providing an additional layer of confirmation for the presence of foetal cells. Results The results showed no hypermethylated RASSF1A was detected in any of the cervical samples, irrespective of whether the samples were obtained by brush or lavage. However, an average of 17,236 ± 7490 foetal cells per sample were detected in the uterine lavage samples. Foetal cells accounted for approximately 0.14% ± 0.10% of the total cell population in these samples. The presence of EVTs in these samples was confirmed by their expression of both HLA-G and GATA-3. Conclusion The detection of foetal cells in uterine cavity samples based on hypermethylation of RASSF1A and quantification of foetal cells can be used to prenatal screening. GATA-3 can be used to label EVTs.

MEnrich-seq: methylation-guided enrichment sequencing of bacterial taxa of interest from microbiome.

Metagenomics has enabled the comprehensive study of microbiomes. However, many applications would benefit from a method that sequences specific bacterial taxa of interest, but not most background taxa. We developed mEnrich-seq (in which 'm' stands for methylation and seq for sequencing) for enriching taxa of interest from metagenomic DNA before sequencing. The core idea is to exploit the self versus nonself differentiation by natural bacterial DNA methylation and rationally choose methylation-sensitive restriction enzymes, individually or in combination, to deplete host and background taxa while enriching targeted taxa. This idea is integrated with library preparation procedures and applied in several applications to enrich (up to 117-fold) pathogenic or beneficial bacteria from human urine and fecal samples, including species that are hard to culture or of low abundance. We assessed 4,601 bacterial strains with mapped methylomes so far and showed broad applicability of mEnrich-seq. mEnrich-seq provides microbiome researchers with a versatile and cost-effective approach for selective sequencing of diverse taxa of interest.