Exposing the DNA methylome iceberg

DNA methylation was the first epigenetic modification discovered. Until recently, comprehensive coverage of the composition and distribution of methylated cytosines across the genome was lacking. Technological advances, however, are providing methylation maps that can reveal the genomic distribution of DNA methylation in different cell states or phenotypes. The emerging picture includes extensive gene body methylation that is highly conserved in eukaryotes, the presence of DNA methylation in previously unappreciated sequence contexts, and the discovery of another modified DNA base, 5-hydroxymethylcytosine. These new data point to the role of DNA methylation both in gene silencing and gene activation; reconciliation of these seemingly contradictory roles will be essential to fully unravel the biological function of DNA methylation in eukaryotes. Here we review how these recently exposed features of the DNA methylome are challenging previously held dogmas in the field.     

Genome-wide hypomethylation of LINE-1 and Alu retroelements in cell-free DNA of blood is an epigenetic biomarker of human aging

Aging associated DNA hypomethylation of LINE-1 and Alu retroelements may be a crucial determinant of loss of genomic integrity, deterioration and cancer. In peripheral blood LINE-1 hypomethylation has been reported to increase during aging, but other studies did not observe significant changes. We hypothesized that these apparently inconsistent reports might relate to differences between cellular and cell-free DNA. Using the technique of idiolocal normalization of real-time methylation-specific PCR (IDLN-MSP) for genetic imbalanced DNA specimens we obtained evidence that LINE-1 hypomethylation in cell-free DNA, but not cellular DNA from peripheral blood is an epigenetic biomarker for human aging. Furthermore, hypomethylation of cell-free DNA is more extensive in smokers, suggesting that it might be used as a surrogate marker for monitoring the improvement of smoking-induced adverse effects after cancelling smoking.     

Drugging the methylome: DNA methylation and memory

Over the past decade, since epigenetic mechanisms were first implicated in memory formation and synaptic plasticity, dynamic DNA methylation reactions have been identified as integral to long-term memory formation, maintenance, and recall. This review incorporates various new findings that DNA methylation mechanisms are important regulators of non-Hebbian plasticity mechanisms, suggesting that these epigenetic mechanisms are a fundamental link between synaptic plasticity and metaplasticity. Because the field of neuroepigenetics is so young and the biochemical tools necessary to probe gene-specific questions are just now being developed and used, this review also speculates about the direction and potential of therapeutics that target epigenetic mechanisms in the central nervous system and the unique pharmacokinetic and pharmacodynamic properties that epigenetic therapies may possess. Mapping the dynamics of the epigenome in response to experiential learning, even a single epigenetic mark in isolation, remains a significant technical and bioinformatic hurdle facing the field, but will be necessary to identify changes to the methylome that govern memory-associated gene expression and effectively drug the epigenome.     

Uncovering the DNA methylome in chronic lymphocytic leukemia

Over the past two decades, aberrant DNA methylation has emerged as a key player in the pathogenesis of chronic lymphocytic leukemia (CLL), and knowledge regarding its biological and clinical consequences in this disease has evolved rapidly. Since the initial studies relating DNA hypomethylation to genomic instability in CLL, a plethora of reports have followed showing the impact of DNA hypermethylation in silencing vital single gene promoters and the reversible nature of DNA methylation through inhibitor drugs. With the recognition that DNA hypermethylation events could potentially act as novel prognostic and treatment targets in CLL, the search for aberrantly methylated genes, gene families and pathways has ensued. Subsequently, the advent of microarray and next-generation sequencing technologies has supported the hunt for such targets, allowing exploration of the methylation landscape in CLL at an unprecedented scale. In light of these analyses, we now understand that different CLL prognostic subgroups are characterized by differential methylation profiles; we recognize DNA methylation of a number of signaling pathways genes to be altered in CLL, and acknowledge the role of DNA methylation outside of traditional CpG island promoters as fundamental players in the regulation of gene expression. Today, the significance and timing of altered DNA methylation within the complex epigenetic network of concomitant epigenetic messengers such as histones and miRNAs is an intensive area of research. In CLL, it appears that DNA methylation is a rather stable epigenetic mark occurring rather early in the disease pathogenesis. However, other consequences, such as how and why aberrant methylation marks occur, are less explored. In this review, we will not only provide a comprehensive summary of the current literature within the epigenetics field of CLL, but also highlight some of the novel findings relating to when, where, why and how altered DNA methylation materializes in CLL.     

Genome-wide detection of additional fetal chromosomal abnormalities by cell-free DNA testing of 15,626 consecutive pregnant women

Cell-free DNA(cfDNA) testing for common fetal trisomies(T21, T18, T13) is highly effective. However, the usefulness of cfDNA testing in detecting other chromosomal abnormalities is unclear. We evaluated the performance of cfDNA testing for genome-wide abnormalities, and analyzed the incremental yield by reporting extra abnormalities. We performed genome-wide cfDNA testing in 15,626 consecutive pregnancies prospectively enrolled in this study. cfDNA testing results were reported and counseling was given depending on the presence of extra chromosomal abnormalities. cfDNA testing identified 190 cases(1.2%)of chromosomal abnormalities including 100 common trisomies and 90 additional abnormalities. By expanding the cfDNA reporting range to genome-wide abnormalities, the false positive rate increased to 0.39%(P0.001) and positive predictive value(PPV) was reduced to 65.58%(P=0.42). However, the detection yield increased from 0.44% to 0.65%(P=0.014), and cfDNA testing detected 38.61%(39/101) additional abnormalities with no ultrasound and biochemical screening findings. cfDNA testing outperformed biochemical screening by showing 60 times higher true positive rate and fewer false negative results.Genome-wide cfDNA testing significantly increased the diagnostic yield by detecting extra abnormalities, especially those without diagnostic indications. Genome-wide cfDNA testing has fewer false positive and false negative results compared with biochemical screening.     

Alterations of DNA methylome in human bladder cancer

Bladder cancer is the fourth most common cancer in men in the United States, and its recurrence rate is highest among all malignancies. The unmet need for improved strategies for early detection, treatment, and monitoring of the progression of this disease continues to translate into high mortality and morbidity. The quest for advanced diagnostic, therapeutic, and prognostic approaches for bladder cancer is a high priority, which can be achieved by understanding the molecular mechanisms of the initiation and progression of this malignancy. Aberrant DNA methylation in single or multiple cancer-related genes/loci has been found in human bladder tumors and cancer cell lines, and urine sediments, and correlated with many clinicopathological features of this disease, including tumor relapse, muscle-invasiveness, and survival. The present review summarizes the published research on aberrant DNA methylation in connection with human bladder cancer. Representative studies are highlighted to set forth the current state of knowledge, gaps in the knowledgebase, and future directions in this prime epigenetic field of research. Identifying the potentially reversible and ‘drugable’ aberrant DNA methylation events that initiate and promote bladder cancer development can highlight biological markers for early diagnosis, effective therapy and accurate prognosis of this malignancy.     

The human blood DNA methylome displays a highly distinctive profile compared with other somatic tissues

In mammals, DNA methylation profiles vary substantially between tissues. Recent genome-scale studies report that blood displays a highly distinctive methylomic profile from other somatic tissues. In this study, we sought to understand why blood DNA methylation state is so different to the one found in other tissues. We found that whole blood contains approximately twice as many tissue-specific differentially methylated positions (tDMPs) than any other somatic tissue examined. Furthermore, a large subset of blood tDMPs showed much lower levels of methylation than tDMPs for other tissues. Surprisingly, these regions of low methylation in blood show no difference regarding genomic location, genomic content, evolutionary rates, or histone marks when compared to other tDMPs. Our results reveal why blood displays a distinctive methylation profile relative to other somatic tissues. In the future, it will be important to study how these blood specific tDMPs are mechanistically involved in blood-specific functions.     

The effect of different sample collection methods on rabbit blood parameters

Nowadays great deal of research is physiological field is conducted on experimental animals and there is a lot of criticism from the wide public on methods used. Therefore, recently there is a lot of effort focused on the welfare of the animals. Main aim of this study is to determine the effect of experimental sample collection method on the selected parameters of stress. In the experiment two sample collections of rabbit blood from marginal ear vein were realized – first using standard method with one person fixing the animal and other collecting the blood using gently fixating the animal. In the second groups experimental method of inserting the experimental animal into a sack and further collection in dark was realized. During the experiment the levels of cortisol – main stress indicator in organism and other health parameters of animals including mineral profile and haematological parameters were observed. Our results show no significant changes in levels of cortisol but also a decreasing tendency in the sample from the second (dark) collection. Haematological parameters were generally in the reference values and any significant changes except levels of lymphocytes and percent of lymphocytes which shown significant increase in the second collection period were found. Also the levels of mean corpuscular haemoglobin and percent of neutrophils unveiled a significant decrease in values. Values of mineral profile parameters have indicated no significant changes except the levels of phosphorus. Based on the result we can state that the experimental sample collection has no effect on blood parameters of the animals but we spectated a statistically insignificant decrease in the levels of cortisol which can suggest that the dark collection is possibly less stressful to the animals.     

Genome-wide association study of stem rust resistance in a world collection of cultivated barley

Key message

QTL conferring a 14–40% reduction in adult plant stem rust severity to multiple races of Pgt were found on chromosome 5H and will be useful in barley breeding.

Abstract

Stem rust, caused by Puccinia graminis f. sp. tritici (Pgt) is an important disease of barley. The resistance gene Rpg1 has protected the crop against stem rust losses for over 70 years in North America, but is not effective against the African Pgt race TTKSK (and its variants) nor the domestic race QCCJB. To identify resistance to these Rpg1-virulent races, the Barley iCore Collection, held by the United States Department of Agriculture-Agricultural Research Service National Small Grains Collection was evaluated for adult plant resistance (APR) and seedling resistance to race TTKSK and APR to race QCCJB and the Pgt TTKSK composite of races TTKSK, TTKST, TTKTK, and TTKTT. Using a genome-wide association study approach based on 6224 single nucleotide polymorphic markers, seven significant loci for stem rust resistance were identified on chromosomes 1H, 2H, 3H, and 5H. The most significant markers detected were 11_11355 and SCRI_RS_177017 at 71–75 cM on chromosome 5H, conferring APR to QCCJB and TTKSK composite. Significant markers were also detected for TTKSK seedling resistance on chromosome 5H. All markers detected on 5H were independent of the rpg4/Rpg5 complex at 152–168 cM. This study verified the importance of the 11_11355 locus in conferring APR to races QCCJB and TTKSK and suggests that it may be effective against other races in the Ug99 lineage.

     

Decoding the transcriptome and DNA methylome of human primordial germ cells

<正>Primordial germ cells(PGCs)are the embryonic founder cells of the gametes—the oocytes and sperms that are vital for transmitting genetic information faithfully and efficiently from one generation to the next and for maintaining the continuation of a species[1].It is therefore critical to understand the crucial epigenetic processes during the de-     

Genome-wide association study of the level of blood components in Pekin ducks

Blood components are considered to reflect nutrient metabolism and immune activity in both humans and animals. In this study, we measured 12 blood components in Pekin ducks and performed genome-wide association analysis to identify the QTLs (quantitative trait locus) using a genotyping-by-sequencing strategy. A total of 54 QTLs were identified for blood components. One genome-wide significant QTL for alkaline phosphatase was identified within the intron-region of the OTOG gene (P = 1.31E-07). Moreover, 21 genome-wide significant SNPs for the level of serum cholinesterase were identified on six different scaffolds. In addition, for serum calcium, one genome-wide significant QTL was identified in the upstream region of gene RAB11B. These results provide new markers for functional studies in Pekin ducks, and several candidate genes were identified, which may provide additional insights into specific mechanisms for blood metabolism in ducks and their potential application for duck breeding programs.     

Comparative study of chemical pathology sample collection tubes at the largest hospital in South Africa

BackgroundBarricor^TM Lithium heparin plasma tubes are new blood tubes that have been introduced to overcome the effects of gel in serum separator tubes (SST) and the shortcomings of standard Lithium heparin plasma. We aimed to evaluate Barricor^TM tubes as an alternative to serum separator tubes and compare the stability between the tubes.MethodsForty-four paired samples were collected using both Barricor^TM and SST. We compared five analytes at baseline (<6 h) and after every 24 h using the PassingBablok and Bland-Altman plots. Aspartate aminotransferase (AST), potassium (K), phosphate (PO4) , lactate dehydrogenase (LDH), and creatinine were analysed in both tubes. We calculated the percentage difference for each analyte between the baseline and time intervals to assess analyte stability. The percentage difference was compared to the desirable specification for bias and reference change value (RCV).ResultsAll analytes were comparable at baseline. Statistical differences (p<0.001) became evident after 24 h. PO4, K, and creatinine had a mean difference that exceeded the desirable specification for bias (-9.59%, - 9.35%, and -4.59%, respectively). Potassium was stable up to 24 h in both tubes. LDH showed better stability in SST (144 h vs 96 h). PO4 concentrations were more stable in both tubes with the SST (96 h vs 72 h). Creatinine and AST had the longest stability in both tubes compared to other analytes (144 h).ConclusionsData demonstrated variability and similarities in analyte concentrations and stability, respectively, in both tubes.     

Genotyping cell-free tumor DNA in the blood to detect residual disease and drug resistance

DNA fragments released from cancer cells into the blood can be used to generate molecular profiles of tumors. Non-invasive ''liquid biopsies'' can be used to monitor minimal residual disease and detect the emergence of drug resistance.     

Methyl-DNA immunoprecipitation (MeDIP): hunting down the DNA methylome

One of the most challenging projects in the field of epigenetics is the generation of detailed functional maps of DNA methylation in different cell and tissue types in normal and disease-associated conditions. This information will help us not only understand the role of DNA methylation but also identify targets for therapeutic treatment. The completion of the various epigenome projects depends on the design of novel strategies to survey and generate detailed cartograms of the DNA methylome. Methyl-DNA immunoprecipitation (MeDIP) assays, in combination with hybridization on high-resolution microarrays or high-throughput sequencing (HTS) techniques, are excellent methods for identifying methylated CpG-rich sequences. We provide a critical overview of different genome-wide techniques for DNA methylation analysis and propose that MeDIP assays may constitute a key method for elucidating the hypermethylome of cancer cells.     

Evaluation of blood collection tubes using selected reaction monitoring MS: Implications for proteomic biomarker studies

An emphasis of current proteomic research is the validation of plasma protein biomarkers. The process of blood collection itself is critical to the accuracy and reproducibility of quantitative biomarker assays. We have developed selected reaction monitoring (SRM) assays to analyse thirteen abundant plasma proteins and evaluated the impact of three different blood collection tubes on the levels of these proteins. We also assessed the implications of the time taken to analyse plasma samples by evaluating the recovery of these proteins. We showed that SRM detects minor differences in the levels of some proteins which can be attributed to collection tube type. The average recovery for 12 of 18 assays was higher for proteins that were collected in tubes containing protease inhibitors compared to conventional collection tubes. For five of the assays, the differential recovery was statistically significant. Delaying MS analysis of a freeze‐thawed sample for 1 hour showed greatly reduced recovery of these analytes; however differences attributed to tube type were only evident at the baseline timepoint. Finally, we assessed the natural variation of circulating levels of these proteins in a cohort of seven healthy individuals. This study provides useful information for researchers contemplating blood collection for undertaking protein biomarker studies.