Analysis Of Methylation Status Research Articles

Methylation patterns at the adjacent CpG sites within enhancers are a part of cell identity

BackgroundIt is generally accepted that methylation status of CpG sites spaced up to 50 bp apart is correlated, and accumulation of locally disordered methylation at adjacent CpG sites is involved in neoplastic transformation, acting in similar way as stochastic accumulation of mutations.ResultsWe used EPIC microarray data from 596 samples, representing 12 healthy tissue and cell types, as well as 572 blood cancer specimens to analyze methylation status of adjacent CpG sites across human genome, and subsequently validated our findings with NGS and Sanger sequencing. Our analysis showed that there is a subset of the adjacent CpG sites in human genome, with cytosine at one CpG site methylated and the other devoid of methyl group. These loci map to enhancers that are targeted by families of transcription factors involved in cell differentiation. Moreover, our results suggest that the methylation at these loci differ between alleles within a cell, what allows for remarkable level of heterogeneity of methylation patterns. However, different types of specialized cells acquire only one specific and stable pattern of methylation at each of these loci and that pattern is to a large extent lost during neoplastic transformation.ConclusionsWe identified a substantial number of adjacent CpG loci in human genome that display remarkably stable and cell type specific methylation pattern. The methylation pattern at these loci appears to reflect different methylation of alleles in cells. Furthermore, we showed that changes of methylation status at those loci are likely to be involved in regulation of the activity of enhancers and contribute to neoplastic transformation.

Multiplex methylation detection assays using a blocking FRET probe with machine learning-assisted quantitative melting curve method targeting early-stage breast cancer

Breast cancer (BC) is the second most prevalent form of cancer, and poses a significant threat to public health. DNA methylation is an ideal marker for the early detection of BC. Fluorescence quantitative polymerase chain reaction (PCR)-based DNA methylation detection is simpler and faster but is constrained by its multiplexing capability and specificity. To address this, we developed a multiplex quantitative methylation PCR assay for the simultaneous analysis of methylation status at multiple sites specific to BC (cg11754974, cg13828440, cg18637238, and cg16652347). The machine learning model was trained using 1200 cases of multipeak data to enhance the melting curve resolution. Performance testing demonstrated the method’s ability to selectively amplify methylated genes at a DNA concentration of 1 × 105 copies μL−1, with high replicability (coefficient of variation <5 %) and mutation detection capabilities as low as 10 %. When applied to 80 clinical BC samples, the assay effectively distinguished patients with early-stage BC from normal controls, achieving an area under the curve of 0.8938, sensitivity of 83.33 %, and specificity of 84.62 %. Our essay exhibits superior clinical performance when compared to the quantitative methylation-specific PCR assays for noninvasive detection of early-stage BC, which is poised to become a favorable clinical diagnostic method for early-stage BC owing to its simplicity, speed, and capacity to improve diagnostic accuracy.

Droplet digital PCR analysis of CDH13 methylation status in Slovak women with invasive ductal breast cancer

Identifying novel epigenetic biomarkers is a promising way to improve the clinical management of patients with breast cancer. Our study aimed to determine the methylation pattern of 25 tumor suppressor genes (TSG) and select the best methylation biomarker associated with clinicopathological features in the cohort of Slovak patients diagnosed with invasive ductal carcinoma (IDC). Overall, 166 formalin-fixed, paraffin-embedded (FFPE) tissues obtained from patients with IDC were included in the study. The methylation status of the promoter regions of 25 TSG was analyzed using semiquantitative methylation-specific MLPA (MS-MLPA). We identified CDH13 as the most frequently methylated gene in our cohort of patients. Further analysis by ddPCR confirmed an increased level of methylation in the promoter region of CDH13. A significant difference in CDH13 methylation levels was observed between IDC molecular subtypes LUM A versus HER2 (P = 0.0116) and HER2 versus TNBC (P = 0.0234). In addition, significantly higher methylation was detected in HER2+ versus HER2- tumors (P = 0.0004) and PR− versus PR+ tumors (P = 0.0421). Our results provide evidence that alteration in CDH13 methylation is associated with clinicopathological features in the cohort of Slovak patients with IDC. In addition, using ddPCR as a methylation-sensitive method represents a promising approach characterized by higher precision and technical simplicity to measure the methylation of target CpGs in CDH13 compared to other conventional methods such as MS-MLPA.

Abstract LB197: Methylation patterns at adjacent CpG sites within enhancers are part of cell identity and are disrupted during malignant transformation

Abstract Background: Following the principles of physiology of DNA methylation, it is generally accepted that methylation status of CpG sites spaced up to 50 bp is correlated and accumulation of locally disordered methylation at CpG sites is a part of neoplastic transformation, acting in similar way as stochastic accumulation of mutations during malignant transformation. Methods: We developed a new algorithm allowing to analyze methylation status at the adjacent CpG sites. We then, used EPIC microarray data to analyze genome wide methylation patterns at the adjacent CpG sites in almost 600 samples, representing 12 healthy tissues and cell types as well as 572 cancer specimens. We also used bisulfite next generation sequencing data and Sanger sequencing to validate our findings. Results: Our analysis, identified in human genome a subset of loci with adjacent CpG sites spaced less than 50bp (average 28bp), harboring one cytosine methylated and the other devoid of methyl group. These loci are enriched in enhancers that are targeted by families of transcription factors involved in cell differentiation. The methylation patterns at these loci can differ between alleles within a cell, what allows for remarkable level of heterogeneity of methylation patterns. However, our analysis also showed that, different types of specialized cells, acquire during differentiation only one specific and remarkably stable pattern of methylation at each of these loci and that pattern is to a large extent lost during neoplastic transformation. Conclusions: Contrary to generally accepted principle of identical methylation status of the adjacent CpG sites being essential for regulatory function of DNA methylation. We have shown that methylation at a subset of adjacent CpG sites in human genome located within important functional genomic elements differs in healthy cells. These CpG sites acquire remarkably stable and tissue specific patterns of methylation during cell differentiation. Moreover, the methylation patterns at these CpG sites, are lost during malignant transformation suggesting the involvement of identified phenomenon in cancer pathology. Funding: This work was funded by grant OPUS22 from Polish National Science Centre (grant number: 2021/43/B/NZ2/02979) and Polish National Agency for Academic Exchange (grant number: PPN/PPO/2018/1/00088/U). Citation Format: Olga Taryma-Leśniak, Jan Bińkowski, Patrycja K. Przybylowicz, Katarzyna E. Sokolowska, Konrad Borowski, Tomasz K. Wojdacz. Methylation patterns at adjacent CpG sites within enhancers are part of cell identity and are disrupted during malignant transformation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB197.

Methylation‑sensitive restriction enzyme‑droplet digital PCR assay for the one‑step highly sensitive analysis of DNA methylation hotspots.

DNA methylation is an epigenetic modification that plays a key role in several cellular processes mediating the fine regulation of gene expression. Aberrant DNA methylation is observed in a wide range of pathologies, including cancer. Since these DNA modifications are transferred to the cell progenies and are stable over the time, the analysis of DNA methylation status has been proposed for diagnostic and prognostic purposes in cancer. Currently, DNA bisulfite conversion is the gold standard method for the high‑throughput analysis of DNA methylation alterations. However, bisulfite treatment induces DNA fragmentation affecting its quality for the downstream analyses. In this field, it is mandatory to identify novel methods to overcome the limits of conventional approaches. In the present study, the Methylation‑Sensitive Restriction Enzyme‑droplet digital PCR (MSRE‑ddPCR) assay was developed as a novel sensitive method for the analysis of DNA methylation of short genomic regions, combining the MSRE assay with the high‑sensitivity ddPCR and using an exogenous methylation sequence as control. Setup and validation experiments were performed analyzing a methylation hotspot of the Solute Carrier Family 22 Member 17 in DNA samples derived from melanoma cell lines as well as from tissues and serum samples obtained from patients with melanoma and healthy controls. Compared with the standard MSRE approaches, the MSRE‑ddPCR assay is more appropriate for the analysis of DNA methylation (methDNA) in samples with low amounts of DNA (up to 0.651 ng) showing a greater sensitivity. These findings suggested the potential clinical application of MSRE‑ddPCR paving the way to the analysis of other methDNA hotspots in different tumors.

Glycerophosphoinositol modulates FGA and NOTCH3 in exercise-induced muscle adaptation and colon cancer progression.

Fibroleukin (FGA) and NOTCH3 are vital in both exercise-induced muscle adaptation and colon adenocarcinoma (COAD) progression. This study aims to elucidate the roles of FGA and NOTCH3 in phenotypic variations of striated muscle induced by exercise and in COAD development. Additionally, it seeks to evaluate the prognostic significance of these proteins. Gene Set Variation Analysis (GSVA) and protein-protein interaction (PPI) network analysis were employed to identify differentially expressed genes (DEGs). Molecular docking studies were conducted to assess the binding affinities of 39 compounds to the NOTCH3 protein. In vitro assays, including mobileular viability, gene expression, and apoptosis assays, were performed to evaluate the effects of glycerophosphoinositol on FGA and NOTCH3 expression. Additionally, copy number variation (CNV), methylation status, and survival analyses were conducted across multiple cancers types. The NOTCH signaling pathway was consistently upregulated in exercise-induced muscle samples. High NOTCH3 expression was associated with poor prognosis in COAD, extracellular matrix organization, immune infiltration, and activation of the PI3K-Akt pathway. Molecular docking identified gamma-Glu-Trp, gamma-Glutamyltyrosine, and 17-Deoxycortisol as strong binders to NOTCH3. Glycerophosphoinositol treatment modulated FGA and NOTCH3 expression, influencing cell proliferation and apoptosis. CNV and methylation analyses revealed specific changes in FGA and NOTCH3 across 20 cancers types. Survival analyses showed strong associations between FGA/NOTCH3 expression and survival metrics, with negative correlations for FGA and positive correlations for NOTCH3. FGA and NOTCH3 play significant roles in exercise-induced muscle adaptation and colon cancer progression. The expression profiles and interactions of these proteins provide promising prognostic markers and therapeutic targets. These findings offer valuable insights into the post-translational modifications (PTMs) in human cancer, highlighting novel pharmacological and therapeutic opportunities.

Construction of lncRNA/Pseudogene-miRNA Network Based on In Silico Approaches for Glycolysis Pathway to Identify Prostate Adenocarcinoma-Related Potential Biomarkers.

LncRNAs, pseudogenes, and miRNAs participate a fundamental function in tumorigenesis, metabolism, and invasion of cancer cells, although their regulation of tumor glycolysis in prostate adenocarcinoma (PRAD) is thoroughly not well studied. In this study, we applied transcriptomic, proteomic, and medical information to identify glycolysis-related key genes and modules associated with PRAD. Then, the glycolysis-related lncRNA/lncRNAs/pseudogenes-miRNA-mRNA network was constructed. Analysis of DNA methylation status and expression data determined a DNA methylation-dysregulated three-DE-mRNAs signature for predicting diagnosis, ANGPTL4, GNE, and HSPA in PRAD patients and healthy control. Several lncRNAs/pseudogenes, significantly correlated with the overall survival PVT1, CA5BP1, MIRLET7BHG, SNHG12, and ZNF37BP and disease-free survival status, MALAT1, GUSBP11, MIRLET7BHG, and SNHG1, of patients with PRAD were determined. The methylation profile of DE-lncRNA/pseudogenes was significantly proper for predicting PRAD prognostic model. The transcription level of 6 DE-mRNA ANGPTL4, QSOX1, BIK, CLDN3, DDIT4, and TFF3 was correlated with cancer-related fibroblast infiltration in PRAD. The mutated form of 7 mRNAs, COL5A1, IDH1, HK2, DDIT4, GNE, and QSOX1, was associated with PRAD. In addition to the glycolysis pathway, DE-RNAs play regulatory roles on several pathways, including DNA damage, RTK, cell cycle, RAS/MAPK, TSC/mTOR and PI3K/AKT, AR hormone, and EMT. Overall, our study improves our knowledge of the relation between lncRNAs/pseudogenes and miRNA related to glycolysis and PRAD pathogenesis.

Methylation status of various gene loci in localized prostate cancer: Novel biomarkers for diagnostics and biochemical recurrence.

Oncologic outcomes for patients with localized prostate cancer (PCa) undergoing radical prostatectomy (RP) can vary widely. Hypermethylation of tumor-associated genes has potential as a novel diagnostic tool and predictive biomarker in PCa. We investigated the methylation status of tumor-associated genes in patients who underwent RP. Patients who underwent RP during 2004 to 2008 were matched retrospectively based on post-operative D'Amico risk stratification. Quantitative pyrosequencing was used to analyze methylation status of 10 gene loci in cancerous and adjacent benign tissue from histological specimen. Follow-up was performed according to EAU guideline recommendations. Statistical analyses were performed to correlate methylation levels in cancerous and benign tissue with risk profiles and biochemical recurrence (BCR). The cohort included 71 patients: 22 low-risk, 22 intermediate-risk, and 27 high-risk. Mean follow-up time was 74 months. Methylation status differed significantly between cancerous and adjacent benign tissue for the 5 gene loci GSTP1, APC, RASSF1, TNFRFS10c, and RUNX3 (each P < 0.001). Also, the methylation level was significantly higher in high-risk than in low-risk patients for Endoglin2 and APC (P = 0.026; P = 0.032). Using ROC analysis, hypermethylation of APC in PCa tissue was associated with higher risk of BCR (P = 0.005). Methylation status of various gene loci holds diagnostic and predictive potential in PCa. Hypermethylation of APC, RASSF1, TNFRFS10c and RUNX3 were identified as novel PCa-specific biomarkers. Furthermore, increased methylation levels of APC and Endoglin2 were associated with high-risk PCa. Additionally, hypermethylation of APC was associated with increased risk of BCR after RP.

Combined SEPT9 and BMP3 methylation in plasma for colorectal cancer early detection and screening in a Brazilian population.

Colorectal cancer (CRC) screening can help to reduce its incidence and mortality. Noninvasive strategies, such as plasma analysis of epigenetic alterations, can constitute important biomarkers of CRC detection. This study aimed to evaluate the plasma methylation status of SEPT9 and BMP3 promoters as biomarkers for detection of CRC and its precursor lesions in a Brazilian population. Plasma samples from 262 participants of the CRC screening program of Barretos Cancer Hospital who had a positive fecal occult blood test and underwent colonoscopy and cancer patients were analyzed. Participants were grouped according to the worst lesion detected in the colonoscopy. Cell-free circulating DNA (cfDNA) was bisulfite treated followed by the analysis of SEPT9 and BMP3 methylation status using a droplet digital PCR system (ddPCR). The best methylation cutoff value for group discrimination was calculated by receiver operating characteristic (ROC) curve analysis. Among the 262 participants, 38 were diagnosed with CRC, 46 with advanced adenomas 119 with nonadvanced adenomas, three with sessile serrated lesions, and 13 with hyperplastic polyps. In 43 participants, no lesion was detected in the colonoscopy and were used as controls. The CRC group showed the highest cfDNA concentration (10.4 ng/mL). For the SEPT9 gene, a cutoff of 2.5% (AUC = 0.681) that discriminates between CRC and the control group resulted in CRC sensitivity and specificity of 50% and 90%, respectively. Concerning the BMP3 gene, a cutoff of 2.3% (AUC = 0.576) showed 40% and 90% of sensitivity and specificity for CRC detection, respectively. Combining SEPT9, BMP3 status, and age over 60 years resulted in a better performance for detecting CRC (AUC = 0.845) than the individual gene models, yielding 80% and 81% of sensitivity and specificity, respectively. The present study suggests that a combination of SEPT9 and BMP3 plasma methylation, along with age over 60 years, showed the highest performance in detecting CRC in a Brazilian population. These noninvasive biomarkers can potentially serve as useful tools for CRC screening programs.

Comprehensive Profiling of EBV Gene Expression and Promoter Methylation Reveals Latency II Viral Infection and Sporadic Abortive Lytic Activation in Peripheral T-Cell Lymphomas.

Epstein-Barr virus (EBV) latency patterns are well defined in EBV-associated epithelial, NK/T-cell, and B-cell malignancies, with links between latency stage and tumorigenesis deciphered in various studies. In vitro studies suggest that the oncogenic activity of EBV in T-cells might be somewhat different from that in EBV-tropic B lymphoid cells, prompting us to study this much less investigated viral gene expression pattern and its regulation in nine EBV+ peripheral T-cell lymphoma (PTCL) biopsies. Using frozen specimens, RT-PCR showed 6/7 cases with a latency II pattern of EBV gene expression. Analyses of EBNA1 promoter usage and CpG methylation status in these six cases showed that only Qp was used, while Cp, Wp, and Fp were all silent. However, the remaining case showed an exceptionally unique latency III type with lytic activation, as evidenced by EBV lytic clonality and confirmed by the full usage of Cp and Qp as well as weakly lytic Fp and Wp, fully unmethylated Cp and marginally unmethylated Wp. Further immunostaining of the eight cases revealed a few focally clustered LMP1+ cells in 7/8 cases, with rare isolated LMP1+ cells detected in another case. Double immunostaining confirmed that the LMP1+ cells were of the T-cell phenotype (CD3+). In 6/8 cases, sporadically scattered Zta+ cells were detected. Double staining of EBER-ISH with T-cell (CD45RO/UCHL1) or B-cell (CD20) markers confirmed that the vast majority of EBER+ cells were of the T-cell phenotype. Predominant type-A EBV variant and LMP1 30-bp deletion variant were present, with both F and f variants detected. In summary, the EBV gene expression pattern in PTCL was found to be mainly of latency II (BART+EBNA1(Qp)+LMP1+LMP2A+BZLF1+), similar to that previously reported in EBV-infected nasopharyngeal epithelial, NK/T-cell, and Hodgkin malignancies; however, fully lytic infection could also be detected in occasional cases. Rare cells with sporadic immediate-early gene expression were commonly detected in PTCL. These findings have implications for the future development of EBV-targeting therapeutics for this cancer.

Modifying Effect of the Interleukin-18 Level on the Association between BDNF Methylation and Long-Term Cardiovascular Outcomes in Patients with Acute Coronary Syndrome

This study investigated the potential modifying effects of the level of the serum interleukin-18 (IL-18) on the association between BDNF methylation status and long-term cardiovascular outcomes in patients with acute coronary syndrome (ACS). Hospitalized ACS patients were recruited sequentially from 2006 to 2012. At baseline, the IL-18 level and BDNF methylation status were evaluated in 969 patients who were followed for major adverse cardiac events (MACEs) for 5-12 years, until 2017 or death. The time to first composite or individual MACE was compared between individuals with lower and higher average BDNF methylation levels (in the low- and high-IL-18 groups, respectively) using a Cox proportional hazards model. After adjusting for potential covariates, the modifying effects of IL-18 and average BDNF methylation levels on the initial composite and individual MACEs were examined. In the high-IL-18 group, but not in the low-IL-18 group, a higher average BDNF methylation level was associated with increases in composite MACEs (HR (95% CI) = 2.15 (1.42-3.26)), all-cause mortality (HR (95% CI) = 1.89 (1.11-3.22)), myocardial infarction (HR (95% CI) = 1.98 (1.07-3.67)), and percutaneous coronary intervention (HR (95% CI) = 1.81 (1.01-3.23)), independent of confounding variables. The interaction effect between the IL-18 and average BDNF methylation levels on composite MACEs (p = 0.019) and myocardial infarction (p = 0.027) was significant after adjusting for covariates. Analysis of BDNF methylation status and IL-18 levels may help identify ACS patients who are most likely to have adverse clinical outcomes.

Biomarkers of Clinical Resistance to Decitabine and Venetoclax Therapy Inferred By Transcriptome and Methylation Analyses

The combination of the selective BCL-2 inhibitor venetoclax (VEN) with the hypomethylating agent (HMA) decitabine (DEC) is highly effective in treating older ineligible for intensive chemotherapy AML patients. However, approximately 15-34% of newly diagnosed (ND) and 60% of relapsed/refractory (R/R) patients fail to achieve complete remission (CR) (DiNardo et al. New Engl J Med. 2020, DiNardo et al. Lancet Haematol. 2020). Factors that reduce efficacy remain to be elucidated. To dissect underlying mechanisms of HMA-VEN resistance, we performed RNA-seq utilizing primary samples from patients treated on a prospective clinical trial of 10-day DEC with VEN (NCT03404193). Previously, we reported that alterations of transcriptome signatures of energy-metabolism and cell migration/adhesion pathways were upregulated in primary resistant ND group, and immune-system-associated genes were downregulated in R/R group (Yamatani et al. ASH 2021). This study investigated transcriptomic and DNA methylation signatures associated with DEC10-VEN response or resistance in ND and R/R AML. Because DEC was reported to strongly induce interferon-stimulated genes (ISGs) through gene methylation modifications, we focused on 116 ISGs. An integrated analysis of DNA methylation status around the transcription start site (TSS) was performed using the Infinium methylated EPIC array (Illumina). Forty-four pre-treatment samples from 32 patients with AML from DEC-VEN clinical trial, ND patient group (CR/CRi 14; non-responder, NR 11) and R/R patient group (CR/CRi 6; NR 13), were analyzed. First, we performed the comparative analysis of NR vs CRs. In the ND group, NR cases showed significantly higher gene expression and lower TSS methylation of an urea cycle enzyme, ASS1 (Argininosuccinate Synthase 1) with RNA-seq Log2 fold-change (FC) 3.6 and methylation array Δβ (differential methylation β-value) -0.11, p < 0.05, and of an actin-based molecular motor, MYO1C (Myosin 1C), Log2FC 2.5, Δβ = -0.18, p< 0.05, than in CR cases. In the R/R group, gene expression levels of seven ISGs, including CCL3 (C-C motif chemokine 3), were significantly lower in NR than in CRs, with no difference in methylation status. Next, changes in gene expression levels and TSS methylation status after DEC-VEN treatment were investigated in non-responders in ND group (pre 19, post 12) and R/R group (pre 16, post 4). After DEC-VEN treatment, PPARG (Peroxisome Proliferator-Activated Receptor γ), a transcription factor that activates fatty acid metabolism and negatively regulates subsets of IFN-γ target genes, was significantly upregulated along with its TSS demethylation both in ND (Log2FC 4.9, Δβ = -0.14, p < 0.05), and R/R groups (Log2FC 3.7, Δβ = -0.06, p < 0.05). CDC42EP4, an effector protein of CDC42 involved in the organization of the actin cytoskeleton, was upregulated after the treatment in the R/R group (Log2FC 2.7, Δβ = -0.14, p < 0.05). Based on these findings, we validated transcriptional and methylation changes of ASS1, PPARG, MYO1C and CDC42EP4 by RT-qPCR and bisulfite pyrosequencing using HL60 and MV4-11 AML cell lines in which DEC (IC50, HL60, 249 nM; MV4-11, 2090 nM) and VEN (IC50, HL60, 18 nM; MV4-11, 64 nM) produced additive or synergistic combination effect. Cells were treated by DEC and/or VEN under BM-derived mesenchymal stem cells (MSCs) co-culture conditions to mimic the BM microenvironment. DEC upregulated gene expression levels with TSS demethylation of PPARG, MYO1C and CDC42EP4 both in HL60 and MV4-11, and of ASS1 only in HL-60. VEN treatment alone or in combination with DEC did not induce significant changes in the expression of these tested genes. Of note, PPARG expression was further upregulated under MSCs co-culture condition without TSS methylation status change in AML cells. DEC treatment induced no significant changes of PPARG in MSC. These findings suggest that not only TSS demethylation but also interaction with BM stromal cells may be involved in PPARG upregulation associated with energy production in AML cells. Taken together, DEC-VEN treatment stimulates two key factors, energy metabolism and interaction with the BM microenvironment, which may reduce therapeutic efficacy by increasing gene expression of PPARG,MYO1C,ASS1, and CDC42EP4 through TSS demethylation and/or interaction with BM stromal cells. Experimentally, the consequences of overexpression / knockdown of these genes will be further validated.

A patient with mosaic USP9X gene variant

The finding of USP9X variants in females has been associated with female-restricted X-linked mental retardation (MRXS99F), a rare syndrome featured by developmental delay and distinct congenital anomalies. Here, we report a female fetus with MRXS99F due to a novel frameshift variant, c.6679_6685delAAATTATinsTCCTG (p.Lys2227SerfsTer2) in USP9X, which was present in a mosaic state in the amniocytes and in the peripheral blood after birth (14% and 30%, respectively). The X methylation status analysis displayed a partially skewed X-inactivation with 80% of inactive paternal X. The first signs of the MRXS99F were prenatally detected at 20 weeks, with an enlarged posterior cranial fossa, an upward rotation of the cerebellar vermis and partial corpus callosum agenesis, together with a cardiac septal defect and a single umbilical artery. After birth and during postnatal follow-up the anal anteriorization and the presence of a bilateral membranous choanal atresia were noted, whereas the MRI revealed the hypo/aplasia of the olfactory bulbs, a widening of the subarachnoid spaces and a delay in the myelinisation. During the 18-months follow-up a severe growth and global developmental delay, together with a bilateral moderate deafness with a threshold at 40 dB, dental enamel erosions and an initial scoliosis were observed. We report the prenatal and postnatal features of a classical MRXS99F phenotype and a mosaic USP9X frameshift variant with a partially skewed X inactivation and discuss genotype/phenotype correlations in view of the published studies so far.

208 Proposed Revision to Meningioma Classification System to Utilize Methylation Status of EGFR

INTRODUCTION: Meningiomas are among the most common brain tumors, making up about 13-26% of all intracranial tumors. They arise from the arachnoid cap and most commonly appear along the lining of the skull base and the venous cavities. The current classification system for meningiomas was established by the World Health Organization (WHO) and designates tumors as WHO grades I-III depending on histological characteristics. While WHO grade does strongly correlate with tumor recurrence, it has serious shortcomings. In particular, some WHO grade I tumors, which are supposedly benign, end up becoming highly aggressive and recurrent. A more accurate method of classifying tumors to provide better insight into prognosis is needed. Epidermal growth factor receptor (EGFR) is a highly expressed protein in meningiomas that has been linked to angiogenesis and other tumor-promoting properties. METHODS: A retrospective chart analysis was first performed to gather clinical and demographic information for 291 patients diagnosed with meningioma and treated with surgical intervention. Primary tissue samples were available for 25 patients and an analysis of EGFR promoter methylation status was performed. RESULTS: A significant association was found between EGFR promoter methylation and tumor recurrence (p = 0.02176*). Of note, there was a 100% positivity rate for EGFR hypermethylation in patients with tumor progression within 10 months post-surgery (n =9). Every tumor sample classified as WHO grade II-III had EGFR hypermethylation (n = 10), as well as 7 out of the 15 WHO grade I tumors. In total, over half of the meningioma patients with EGFR hypermethylation had tumor progression within 10 months of surgery. CONCLUSION: These findings suggest that assessing methylation status of EGFR could be a valuable tool for identifying highly aggressive meningiomas and determining patient prognosis early in the game. This would allow for improved postoperative treatment regimens to be developed, reducing the chances of tumor progression.

Fine structure and transcription dynamics of bread wheat ribosomal DNA loci deciphered by a multi‐omics approach

Three out of four RNA components of ribosomes are encoded by 45S ribosomal DNA (rDNA) loci, which are organized as long head-to-tail tandem arrays of nearly identical units, spanning several megabases of sequence. Due to this structure, the rDNA loci are the major sources of gaps in genome assemblies, and gene copy number, sequence composition, and expression status of particular arrays remain elusive, especially in complex genomes harboring multiple loci. Here we conducted a multi-omics study to decipher the 45S rDNA loci in hexaploid bread wheat. Coupling chromosomal genomics with optical mapping, we reconstructed individual rDNA arrays, enabling locus-specific analyses of transcription activity and methylation status from RNA- and bisulfite-sequencing data. We estimated a total of 6,650 rDNA units in the bread wheat genome, with approximately 2,321, 3,910, 253, and 50 gene copies located in short arms of chromosomes 1B, 6B, 5D, and 1A, respectively. Only 1B and 6B loci contributed substantially to rRNA transcription at a roughly 2:1 ratio. The ratio varied among five tissues analyzed (embryo, coleoptile, root tip, primary leaf, mature leaf), being the highest (2.64:1) in mature leaf and lowest (1.72:1) in coleoptile. Cytosine methylation was considerably higher in CHG context in the silenced 5D locus as compared with the active 1B and 6B loci. In conclusion, a fine genomic organization and tissue-specific expression of rDNA loci were deciphered, for the first time, in a complex polyploid species. The results are discussed in the context of wheat evolution and transcription regulation.

Identification and validation of plasma biomarkers for diagnosis of breast cancer in South Asian women

Breast cancer is the most common malignancy among women globally. Development of a reliable plasma biomarker panel might serve as a non-invasive and cost-effective means for population-based screening of the disease. Transcriptomic profiling of breast tumour, paired normal and apparently normal tissues, followed by validation of the shortlisted genes using TaqMan® Low density arrays and Quantitative real-time PCR was performed in South Asian women. Fifteen candidate protein markers and 3 candidate epigenetic markers were validated first in primary breast tumours and then in plasma samples of cases [N = 202 invasive, 16 DCIS] and controls [N = 203 healthy, 37 benign] using antibody array and methylation specific PCR. Diagnostic efficiency of single and combined markers was assessed. Combination of 6 protein markers (Adipsin, Leptin, Syndecan-1, Basic fibroblast growth factor, Interleukin 17B and Dickopff-3) resulted in 65% sensitivity and 80% specificity in detecting breast cancer. Multivariate diagnostic analysis of methylation status of SOSTDC1, DACT2, WIF1 showed 100% sensitivity and up to 91% specificity in discriminating BC from benign and controls. Hence, combination of SOSTDC1, DACT2 and WIF1 was effective in differentiating breast cancer [non-invasive and invasive] from benign diseases of the breast and healthy individuals and could help as a complementary diagnostic tool for breast cancer.

Identification of prostate cancer specific methylation biomarkers from a multi-cancer analysis

BackgroundDetecting prostate cancer at a non-aggressive stage is the main goal of prostate cancer screening. DNA methylation has been widely used as biomarkers for cancer diagnosis and prognosis, however, with low clinical translation rate. By taking advantage of multi-cancer data from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO), we aimed to identify prostate cancer specific biomarkers which can separate between non-aggressive and aggressive prostate cancer based on DNA methylation patterns.ResultsWe performed a comparison analysis of DNA methylation status between normal prostate tissues and prostate adenocarcinoma (PRAD) samples at different Gleason stages. The candidate biomarkers were selected by excluding the biomarkers existing in multiple cancers (pan-cancer) and requiring significant difference between PRAD and other urinary samples. By least absolute shrinkage and selection operator (LASSO) selection, 8 biomarkers (cg04633600, cg05219445, cg05796128, cg10834205, cg16736826, cg23523811, cg23881697, cg24755931) were identified and in-silico validated by model constructions. First, all 8 biomarkers could separate PRAD at different stages (Gleason 6 vs. Gleason 3 + 4: AUC = 0.63; Gleason 6 vs. Gleason 4 + 3 and 8–10: AUC = 0.87). Second, 5 biomarkers (cg04633600, cg05796128, cg23523811, cg23881697, cg24755931) effectively detected PRAD from normal prostate tissues (AUC ranged from 0.88 to 0.92). Last, 6 biomarkers (cg04633600, cg05219445, cg05796128, cg23523811, cg23881697, cg24755931) completely distinguished PRAD with other urinary samples (AUC = 1).ConclusionsOur study identified and in-silico validated a panel of prostate cancer specific DNA methylation biomarkers with diagnosis value.

Clinical Significance of ADAMTS19, BMP7, SIM1, and SFRP1 Promoter Methylation in Renal Clear Cell Carcinoma.

BackgroundClear cell renal cell carcinoma (ccRCC) is the most common subtype of kidney tumors, accounting for the majority of deaths from genitourinary cancers. The currently used nomograms for predicting patient outcomes are based on clinical-pathological characteristics only; however, a significant number of ccRCC survivors with similar radiological and histological features still demonstrate a different clinical course of the disease. This study aimed at the identification of novel DNA methylation biomarkers for the monitoring of patients with ccRCC.MethodsGene expression profiling by SurePrint G3 Human GE 8×60K Microarrays was performed in 4 ccRCC tissues and adjacent non-cancerous renal tissue (NRT) samples. Four down-regulated genes were selected for further DNA methylation status analysis in 123 ccRCC and 45 NRT samples using methylation-specific PCR (MSP).ResultsDNA methylation changes of ADAMTS19, BMP7, SIM1, and SFRP1 were cancer-specific with significantly (P<0.050) higher methylation frequency (37%, 20%, 18%, and 42%, respectively) in tumor tissues. The methylated status of at least one gene was significantly related to various clinical-pathological parameters, including tumor size, Fuhrman and WHO/ISUP grades, intravascular invasion, and necrosis. Moreover, the methylated status of multimarker panel ADAMTS19, BMP7 & SFRP1 was predictive for poorer overall survival (HR, 4.11; 95% CI, 1.22–13.86).ConclusionIn conclusion, DNA methylation of the three-gene panel consisting of ADAMTS19, BMP7 & SFRP1 supposedly predicts the outcome of patients diagnosed with ccRCC and possibly might be used to enrich the current prognostic tools.

Immunohistochemical expression of cyclin-dependent kinase inhibitors p16 and p57 in rhabdomyosarcoma

Rhabdomyosarcoma (RMS) is a highly malignant cancer and is the most common soft tissue sarcoma in children and adolescents, but it is rare in adults (<1% of all adult malignancies). Altered expression and molecular abnormalities of cell-cycle-regulatory proteins are one of the most prominent features in RMS. Therefore, we evaluated the expression of cyclin-dependent kinase inhibitors p57 and p16, as well as p16 methylation status, along with clinicopathological characteristics and overall survival (OS) in RMS patients. This analysis was conducted on 23 pediatric and 44 adult patients. There was a male predominance in both groups and extremities were the most frequent tumor site. In adults, alveolar and pleomorphic types were almost equally represented. The majority of pediatric tumors were low grade, whereas, in adults, only one patient had a low-grade tumor. Seven pediatric (30.43%) and eight adult (18.18%) patients had a low p16 expression. The analysis of methylation status of the p16 promoter showed the presence of methylated allele only in one sample with pleomorphic histology. Six (26.1%) pediatric and 15 (34.1%) adult patients had low p57 expression, while in 17 (73.9%) pediatric and 29 (65.9%) adult patients it was assessed as high. Ninetyone percent of the pediatric patients and 32.6% of adults were alive at the end of the observational period. In adults, significant associations were found between OS and age (P = 0.020), gender (P = 0.027), tumor size (P < 0.001), lymph node status (P < 0.001), presence of metastases (P = 0.015), and p57 expression (P = 0.039). Stratification by histological type showed the correlation of low p57 expression (P = 0.030) and worse OS of patients with alveolar RMS. Univariate analysis identified age > 50 yrs. (HR 2.447), tumors > 5 cm (HR 21.31), involvement of regional lymph nodes (HR 3.96), the presence of metastases (HR 2.53), and low p57 expression (HR 2.11) as predictors of lower OS. Tumor size, regional lymph nodes involvement, and metastases were the independent predictors after multivariate analysis, while p57 did not predict OS in an independent way. In summary, although p57 was not confirmed to be an independent predictor of OS, our results indicate that its low expression may be the marker of aggressive phenotype and poor prognosis in adult RMS patients. Also, our findings suggest that epigenetic inactivation of p16 is not important in the pathogenesis of rhabdomyosarcoma.

Key Macrophage Responses to Infection With Mycobacterium tuberculosis Are Co-Regulated by microRNAs and DNA Methylation.

Tuberculosis (TB) is the leading cause of death from infection with a single bacterial pathogen. Host macrophages are the primary cell type infected with Mycobacterium tuberculosis (Mtb), the organism that causes TB. Macrophage response pathways are regulated by various factors, including microRNAs (miRNAs) and epigenetic changes that can shape the outcome of infection. Although dysregulation of both miRNAs and DNA methylation have been studied in the context of Mtb infection, studies have not yet investigated how these two processes may jointly co-regulate critical anti-TB pathways in primary human macrophages. In the current study, we integrated genome-wide analyses of miRNA abundance and DNA methylation status with mRNA transcriptomics in Mtb-infected primary human macrophages to decipher which macrophage functions may be subject to control by these two types of regulation. Using in vitro macrophage infection models and next generation sequencing, we found that miRNAs and methylation changes co-regulate important macrophage response processes, including immune cell activation, macrophage metabolism, and AMPK pathway signaling.