Epigenetic profiling of cardiovascular ageing

Cardiovascular ageing is a complex phenomenon affected by a range of intrinsic and extrinsic factors. This doctoral dissertation studied the role epigenetics in cardiovascular ageing of volunteers from the longitudinal Asklepios Study. This study showed that telomere length in the first round of the Asklepios Study was correlated with sensitive, clinically relevant parameters of diastolic function. This suggests that telomere length may be involved early on in certain aspects of cardiovascular ageing. Further, blood samples were profiled with respect to DNA methylation in a genome-wide fashion. DNA methylation regulates gene expression and plays a role in establishing and maintaining cellular identity. Subtle, independent methylation changes were observed which correlated either with atherosclerosis progression or ageing between study rounds. More research is needed to determine whether these observations extend to the general populace

Systemic suppression of the shoot metabolism upon rice root nematode infection

Hirschmanniella oryzae is the most common plant-parasitic nematode in flooded rice cultivation systems. These migratory animals penetrate the plant roots and feed on the root cells, creating large cavities, extensive root necrosis and rotting. The objective of this study was to investigate the systemic response of the rice plant upon root infection by this nematode. RNA sequencing was applied on the above-ground parts of the rice plants at 3 and 7 days post inoculation. The data revealed significant modifications in the primary metabolism of the plant shoot, with a general suppression of for instance chlorophyll biosynthesis, the brassinosteroid pathway, and amino acid production. In the secondary metabolism, we detected a repression of the isoprenoid and shikimate pathways. These molecular changes can have dramatic consequences for the growth and yield of the rice plants, and could potentially change their susceptibility to above-ground pathogens and pests

RAB25 expression is epigenetically downregulated in oral and oropharyngeal squamous cell carcinoma with lymph node metastasis

Oral and oropharyngeal squamous cell carcinoma (OOSCC) have a low survival rate, mainly due to metastasis to the regional lymph nodes. For optimal treatment of these metastases, a neck dissection is required; however, inaccurate detection methods results in under- and over-treatment. New DNA prognostic methylation biomarkers might improve lymph node metastases detection. To identify epigenetically regulated genes associated with lymph node metastases, genome-wide methylation analysis was performed on 6 OOSCC with (pN+) and 6 OOSCC without (pN0) lymph node metastases and combined with a gene expression signature predictive for pN+ status in OOSCC. Selected genes were validated using an independent OOSCC cohort by immunohistochemistry and pyrosequencing, and on data retrieved from The Cancer Genome Atlas. A two-step statistical selection of differentially methylated sequences revealed 14 genes with increased methylation status and mRNA downregulation in pN+ OOSCC. RAB25, a known tumor suppressor gene, was the highest-ranking gene in the discovery set. In the validation sets, both RAB25 mRNA (P = 0.015) and protein levels (P = 0.012) were lower in pN+ OOSCC. RAB25 mRNA levels were negatively correlated with RAB25 methylation levels (P < 0.001) but RAB25 protein expression was not. Our data revealed that promoter methylation is a mechanism resulting in downregulation of RAB25 expression in pN+ OOSCC and decreased expression is associated with lymph node metastasis. Detection of RAB25 methylation might contribute to lymph node metastasis diagnosis and serve as a potential new therapeutic target in OOSCC

Staphylococcal enterotoxin B influences the DNA methylation pattern in nasal polyp tissue : a preliminary study

Staphylococcal enterotoxins may influence the pro-inflammatory pattern of chronic sinus diseases via epigenetic events. This work intended to investigate the potential of staphylococcal enterotoxin B (SEB) to induce changes in the DNA methylation pattern. Nasal polyp tissue explants were cultured in the presence and absence of SEB; genomic DNA was then isolated and used for whole genome methylation analysis. Results showed that SEB stimulation altered the methylation pattern of gene regions when compared with non stimulated tissue. Data enrichment analysis highlighted two genes: the IKBKB and STAT-5B, both playing a crucial role in T- cell maturation/activation and immune response

Discovery of new methylation markers to improve screening for cervical intraepithelial neoplasia grade 2/3

Background: Assessment of DNA promoter methylation markers in cervical scrapings for the detection of cervical intraepithelial neoplasia (CIN) and cervical cancer is feasible, but finding methylation markers with both high sensitivity as well as high specificity remains a challenge. In this study, we aimed to identify new methylation markers for the detection of high-grade CIN (CIN2/3 or worse, CIN2+) by using innovative genome-wide methylation analysis (MethylCap-seq). We focused on diagnostic performance of methylation markers with high sensitivity and high specificity considering any methylation level as positive. Results: MethylCap-seq of normal cervices and CIN2/3 revealed 176 differentially methylated regions (DMRs) comprising 164 genes. After verification and validation of the 15 best discriminating genes with methylation-specific PCR (MSP), 9 genes showed significant differential methylation in an independent cohort of normal cervices versus CIN2/3 lesions (p < 0.05). For further diagnostic evaluation, these 9 markers were tested with quantitative MSP (QMSP) in cervical scrapings from 2 cohorts: (1) cervical carcinoma versus healthy controls and (2) patients referred from population-based screening with an abnormal Pap smear in whom also HPV status was determined. Methylation levels of 8/9 genes were significantly higher in carcinoma compared to normal scrapings. For all 8 genes, methylation levels increased with the severity of the underlying histological lesion in scrapings from patients referred with an abnormal Pap smear. In addition, the diagnostic performance was investigated, using these 8 new genes and 4 genes (previously identified by our group: C13ORF18, JAM3, EPB41L3, and TERT). In a triage setting (after a positive Pap smear), sensitivity for CIN2+ of the best combination of genes (C13ORF18/JAM3/ANKRD18CP) (74 %) was comparable to hrHPV testing (79 %), while specificity was significantly higher (76 % versus 42 %, p <= 0.05). In addition, in hrHPV-positive scrapings, sensitivity and specificity for CIN2+ of this best-performing combination was comparable to the population referred with abnormal Pap smear. Conclusions: We identified new CIN2/3-specific methylation markers using genome-wide DNA methylation analysis. The diagnostic performance of our new methylation panel shows higher specificity, which should result in prevention of unnecessary colposcopies for women referred with abnormal cytology. In addition, these newly found markers might be applied as a triage test in hrHPV-positive women from population-based screening. The next step before implementation in primary screening programs will be validation in population-based cohorts

Array-based sequencing of filaggrin gene for comprehensive detection of disease-associated variants

The filaggrin gene (FLG) is essential for skin differentiation and epidermal barrier formation. FLG loss-of-function (LoF) variants are associated with ichthyosis vulgaris and the major genetic risk factor for developing atopic dermatitis (AD).1, 2, 3 Genetic stratification of patients with AD according to FLG LoF risk is a common practice for both research and clinical studies; however, few studies comprehensively sequence the entire FLG coding region. Most studies that include FLG genotyping have screened for common predominant LoF variants to report allele frequencies after full Sanger sequencing of a smaller batch of test patient samples or previously published data. This strategy potentially results in underreporting of the genetic contribution especially in ethnicities where FLG LoF variants are highly diverse.4 Distinct LoF variants have been reported for most ethnicities studied to date. For example, 2 predominant sequence variants (p.R501X and c.2282del4) make up approximately 80% of the mutation burden in northern Europeans,5 whereas in East Asian ethnicities, a larger FLG LoF mutation spectrum is found with fewer predominating variants.6, 7 However, routinely Sanger sequencing the entire FLG coding region for large cohorts is not always feasible, although desirable as it is essential to correctly stratify patients. To address this, we developed a robust and cost-effective high-throughput PCR-based method for analyzing the entire coding region of FLG using Fluidigm microfluidics technology and next-generation sequencing (NGS). We have applied this method to fully resequence cohorts of Chinese, Malay, and Indian patients with AD from the Singaporean population.ASTAR (Agency for Sci., Tech. and Research, S’pore)Published versio

Quality evaluation of methyl binding domain based kits for enrichment DNA-methylation sequencing

DNA-methylation is an important epigenetic feature in health and disease. Methylated sequence capturing by Methyl Binding Domain (MBD) based enrichment followed by second-generation sequencing provides the best combination of sensitivity and cost-efficiency for genome-wide DNA-methylation profiling. However, existing implementations are numerous, and quality control and optimization require expensive external validation. Therefore, this study has two aims: 1) to identify a best performing kit for MBD-based enrichment using independent validation data, and 2) to evaluate whether quality evaluation can also be performed solely based on the characteristics of the generated sequences. Five commercially available kits for MBD enrichment were combined with Illumina GAIIx sequencing for three cell lines (HCT15, DU145, PC3). Reduced representation bisulfite sequencing data (all three cell lines) and publicly available Illumina Infinium BeadChip data (DU145 and PC3) were used for benchmarking. Consistent large-scale differences in yield, sensitivity and specificity between the different kits could be identified, with Diagenode's MethylCap kit as overall best performing kit under the tested conditions. This kit could also be identified with the Fragment CpG-plot, which summarizes the CpG content of the captured fragments, implying that the latter can be used as a tool to monitor data quality. In conclusion, there are major quality differences between kits for MBD-based capturing of methylated DNA, with the MethylCap kit performing best under the used settings. The Fragment CpG-plot is able to monitor data quality based on inherent sequence data characteristics, and is therefore a cost-efficient tool for experimental optimization, but also to monitor quality throughout routine applications

Charting the methylome

Epigenetics, with DNA-methylation as its most stable feature, translates the genetic background into a particular phenotype. Massively parallel sequencing technologies opened up new possibilities for genome-wide profiling of DNA-methylation. Particularly Methyl Binding Domain capturing based Sequencing (MethylCap-Seq) is a low-cost, high-resolution technology to uncover DNA-methylation in a truly genome-wide manner and is becoming increasingly popular. Methods : To chart the map of the methylome, we used raw MethylCap-Seq data of 80 different samples, including different healthy tissues, cell lines and tumor samples. Since no normalization procedures are applied, artefacts are avoided. A Poisson background model is used to identify significantly methylated regions. A conservative set of rules was derived that identifies adjacent methylation prone regions in a single region. Results : Based on this methodology, we provide a reference map of ~1.5 million methylation cores. Together they make up about 10.4% of the human genome and 40% of the approximately 28 million human CpGs dinucleotides. Validation by a different MBD kit and targeted bisulfite sequencing data indicates that the Map of the Human Methylome is approximately 95% complete. Conclusions : We found that although CpG-islands (CGIs) and exon regions are higly enriched in methylation cores with high methylation levels , they show less variability between samples compared to promotor, intergenic and intronic regions. The accuracy of the methylome map will increase with more samples from different tissues and diseases. Comparing the map of the methylome with expression and other data such as histone marks will enable functional annotation of the methylation prone regions, providing a better understanding of the mechanisms involved in epigenetic regulation. This approach is a flexible methodology that can be ported to other genome wide high- throughput methods such as third generation sequencing technologies