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.

Current concepts in the pathology and epigenetics of endometrial carcinoma.

In the Western world, endometrial carcinoma is the most common malignant tumour of the female genital tract and is the fourth most common cancer in women. Two different clinicopathological subtypes are recognised: the oestrogen-related (type I, endometrioid) and the non-oestrogen related (type II, non-endometrioid). This article reviews the epidemiology, risk factors, genetic alterations during endometrial carcinogenesis, features of tumours and precursors and early detection of the disease. Insights into the epigenetic alterations, with emphasis on DNA methylation during endometrial carcinogenesis, and their diagnostic value are also provided.

DNA methylation and cancer diagnosis: new methods and applications.

Methylation of cytosines in cytosine-guanine (CpG) dinucleotides is one of the most important epigenetic alterations in animals. The presence of methylcytosine in the promoter of specific genes has profound consequences on local chromatin structure and on the regulation of gene expression. Changes in DNA methylation play a central role in carcinogenesis. Hypermethylation and consecutive transcriptional silencing of tumor-suppressor genes has been documented in numerous cancers. The identification of target genes silenced by this modification has a great impact on diagnosis, classification, definition of risk groups and prognosis of cancer patients. Here we outline genome-wide techniques aiming at the identification of relevant methylated promoters. Methods and applications allowing clinicians to monitor the methylation of target genes will be also reviewed.

Sequence-optimised E2 constructs from BVDV-1b and BVDV-2 for DNA immunisation in cattle.

We report DNA immunisation experiments in cattle using plasmid constructs that encoded glycoprotein E2 from bovine viral diarrhoea virus (BVDV)-1 (E2.1) and BVDV-2 (E2.2). The coding sequences were optimised for efficient expression in mammalian cells. A modified leader peptide sequence from protein gD of BoHV1 was inserted upstream of the E2 coding sequences for efficient membrane export of the proteins. Recombinant E2 were efficiently expressed in COS7 cells and they presented the native viral epitopes as judged by differential recognition by antisera from cattle infected with BVDV-1 or BVDV-2. Inoculation of pooled plasmid DNA in young cattle elicited antibodies capable of neutralising viral strains representing the major circulating BVDV genotypes.