Chol-Hee Jung

The use of DNA from archival dried blood spots with the Infinium HumanMethylation450 array

BackgroundDried blood (Guthrie card) spots provide an efficient way to collect and store blood specimens. DNA from this source has been utilised for a number of molecular analyses including genome-wide association studies, but only few studies have tested the feasibility of using it for epigenetic applications, particularly at a genome-wide level.ResultsIn this study, we demonstrate the successful use of DNA isolated from archived dried blood spots for the Infinium HumanMethylation450 Beadchip, along with DNA from matched frozen buffy coats. We obtained high quality and reproducible genome-wide DNA methylation profiles using both sample types. We also report high correlations (r > 0.9907) between DNA obtained from matched dried blood spots and frozen buffy coats, sufficient to distinguish between unrelated individuals.ConclusionsWe, thus, demonstrate that DNA from archived dried blood spots is suitable for genome-wide DNA methylation profiling.

DNA methylation changes measured in pre-diagnostic peripheral blood samples are associated with smoking and lung cancer risk.

DNA methylation changes are associated with cigarette smoking. We used the Illumina Infinium HumanMethylation450 array to determine whether methylation in DNA from pre‐diagnostic, peripheral blood samples is associated with lung cancer risk. We used a case‐control study nested within the EPIC‐Italy cohort and a study within the MCCS cohort as discovery sets (a total of 552 case‐control pairs). We validated the top signals in 429 case‐control pairs from another 3 studies. We identified six CpGs for which hypomethylation was associated with lung cancer risk: cg05575921 in the AHRR gene (p‐valuepooled = 4 × 10−17), cg03636183 in the F2RL3 gene (p‐valuepooled = 2 × 10 − 13), cg21566642 and cg05951221 in 2q37.1 (p‐valuepooled = 7 × 10−16 and 1 × 10−11 respectively), cg06126421 in 6p21.33 (p‐valuepooled = 2 × 10−15) and cg23387569 in 12q14.1 (p‐valuepooled = 5 × 10−7). For cg05951221 and cg23387569 the strength of association was virtually identical in never and current smokers. For all these CpGs except for cg23387569, the methylation levels were different across smoking categories in controls (p‐valuesheterogeneity ≤ 1.8 x10 − 7), were lowest for current smokers and increased with time since quitting for former smokers. We observed a gain in discrimination between cases and controls measured by the area under the ROC curve of at least 8% (p‐values ≥ 0.003) in former smokers by adding methylation at the 6 CpGs into risk prediction models including smoking status and number of pack‐years. Our findings provide convincing evidence that smoking and possibly other factors lead to DNA methylation changes measurable in peripheral blood that may improve prediction of lung cancer risk.

Reliability of DNA methylation measures from dried blood spots and mononuclear cells using the HumanMethylation450k BeadArray

The reliability of methylation measures from the widely used HumanMethylation450 (HM450K) microarray has not been assessed for DNA from dried blood spots (DBS) or peripheral blood mononuclear cells (PBMC), nor for combined data from different studies. Repeated HM450K methylation measures in DNA from DBS and PBMC samples were available from participants in six case-control studies nested within the Melbourne Collaborative Cohort Study. Reliability was assessed for individual CpGs by calculating the intraclass correlation coefficient (ICC) based on technical replicates (samples repeated in a single study; 126 PBMC, 136 DBS) and study duplicates (samples repeated across studies; 280 PBMC, 769 DBS) using mixed-effects models. Reliability based on technical replicates was moderate for PBMC (median ICC = 0.42), but lower for DBS (median ICC = 0.20). Study duplicates gave lower ICCs than technical replicates. CpGs that were either highly methylated or unmethylated generally had lower ICCs, which appeared to be mostly related to their lower variability. The ICCs for global methylation measures were high, typically greater than 0.70. The reliability of methylation measures determined by the HM450K microarray is wide-ranging and depends primarily on the variability in methylation at individual CpG sites. The power of association studies is low for a substantial proportion of CpGs in the HM450K assay.

Global measures of peripheral blood-derived DNA methylation as a risk factor in the development of mature B-cell neoplasms

AIM To examine whether peripheral blood methylation is associated with risk of developing mature B-cell neoplasms (MBCNs). MATERIALS & METHODS We conducted a case-control study nested within a large prospective cohort. Peripheral blood was collected from healthy participants. Cases of MBCN were identified by linkage to cancer registries. Methylation was measured using the Infinium(®) HumanMethylation450. RESULTS During a median of 10.6-year follow-up, 438 MBCN cases were evaluated. Global hypomethylation was associated with increased risk of MBCN (odds ratio: 2.27, [95% CI: 1.59-3.25]). Within high CpG promoter regions, hypermethylation was associated with increased risk (odds ratio: 1.76 [95% CI: 1.25-2.48]). Promoter hypermethylation was observed in HOXA9 and CDH1 genes. CONCLUSION Aberrant global DNA methylation is detectable in peripheral blood collected years before diagnosis and is associated with increased risk of MBCN, suggesting changes to DNA methylation are an early event in MBCN development.

Genetic and Environmental Causes of Variation in the Difference Between Biological Age Based on DNA Methylation and Chronological Age for Middle-Aged Women

The disease- and mortality-related difference between biological age based on DNA methylation and chronological age (Δage) has been found to have approximately 40% heritability by assuming that the familial correlation is only explained by additive genetic factors. We calculated two different Δage measures for 132 middle-aged female twin pairs (66 monozygotic and 66 dizygotic twin pairs) and their 215 sisters using DNA methylation data measured by the Infinium HumanMethylation450 BeadChip arrays. For each Δage measure, and their combined measure, we estimated the familial correlation for MZ, DZ and sibling pairs using the multivariate normal model for pedigree analysis. We also pooled our estimates with those from a former study to estimate weighted average correlations. For both Δage measures, there was familial correlation that varied across different types of relatives. No evidence of a difference was found between the MZ and DZ pair correlations, or between the DZ and sibling pair correlations. The only difference was between the MZ and sibling pair correlations (p < .01), and there was marginal evidence that the MZ pair correlation was greater than twice the sibling pair correlation (p < .08). For weighted average correlation, there was evidence that the MZ pair correlation was greater than the DZ pair correlation (p < .03), and marginally greater than twice the sibling pair correlation (p < .08). The varied familial correlation of Δage is not explained by additive genetic factors alone, implying the existence of shared non-genetic factors explaining variation in Δage for middle-aged women.

Genome-wide measures of DNA methylation in peripheral blood and the risk of urothelial cell carcinoma: a prospective nested case-control study

Background:Global DNA methylation has been reported to be associated with urothelial cell carcinoma (UCC) by studies using blood samples collected at diagnosis. Using the Illumina HumanMethylation450 assay, we derived genome-wide measures of blood DNA methylation and assessed them for their prospective association with UCC risk.Methods:We used 439 case–control pairs from the Melbourne Collaborative Cohort Study matched on age, sex, country of birth, DNA sample type, and collection period. Conditional logistic regression was used to compute odds ratios (OR) of UCC risk per s.d. of each genome-wide measure of DNA methylation and 95% confidence intervals (CIs), adjusted for potential confounders. We also investigated associations by disease subtype, sex, smoking, and time since blood collection.Results:The risk of superficial UCC was decreased for individuals with higher levels of our genome-wide DNA methylation measure (OR=0.71, 95% CI: 0.54–0.94; P=0.02). This association was particularly strong for current smokers at sample collection (OR=0.47, 95% CI: 0.27–0.83). Intermediate levels of our genome-wide measure were associated with decreased risk of invasive UCC. Some variation was observed between UCC subtypes and the location and regulatory function of the CpGs included in the genome-wide measures of methylation.Conclusions:Higher levels of our genome-wide DNA methylation measure were associated with decreased risk of superficial UCC and intermediate levels were associated with reduced risk of invasive disease. These findings require replication by other prospective studies.

Defining the purity of exosomes required for diagnostic profiling of small RNA suitable for biomarker discovery

ABSTRACT Small non-coding RNAs (ncRNA), including microRNAs (miRNA), enclosed in exosomes are being utilised for biomarker discovery in disease. Two common exosome isolation methods involve differential ultracentrifugation or differential ultracentrifugation coupled with Optiprep gradient fractionation. Generally, the incorporation of an Optiprep gradient provides better separation and increased purity of exosomes. The question of whether increased purity of exosomes is required for small ncRNA profiling, particularly in diagnostic and biomarker purposes, has not been addressed and highly debated. Utilizing an established neuronal cell system, we used next-generation sequencing to comprehensively profile ncRNA in cells and exosomes isolated by these 2 isolation methods. By comparing ncRNA content in exosomes from these two methods, we found that exosomes from both isolation methods were enriched with miRNAs and contained a diverse range of rRNA, small nuclear RNA, small nucleolar RNA and piwi-interacting RNA as compared with their cellular counterparts. Additionally, tRNA fragments (30–55 nucleotides in length) were identified in exosomes and may act as potential modulators for repressing protein translation. Overall, the outcome of this study confirms that ultracentrifugation-based method as a feasible approach to identify ncRNA biomarkers in exosomes.

iSRAP - a one-touch research tool for rapid profiling of small RNA-seq data.

Small non-coding RNAs have been significantly recognized as the key modulators in many biological processes, and are emerging as promising biomarkers for several diseases. These RNA species are transcribed in cells and can be packaged in extracellular vesicles, which are small vesicles released from many biotypes, and are involved in intercellular communication. Currently, the advent of next-generation sequencing (NGS) technology for high-throughput profiling has further advanced the biological insights of non-coding RNA on a genome-wide scale and has become the preferred approach for the discovery and quantification of non-coding RNA species. Despite the routine practice of NGS, the processing of large data sets poses difficulty for analysis before conducting downstream experiments. Often, the current analysis tools are designed for specific RNA species, such as microRNA, and are limited in flexibility for modifying parameters for optimization. An analysis tool that allows for maximum control of different software is essential for drawing concrete conclusions for differentially expressed transcripts. Here, we developed a one-touch integrated small RNA analysis pipeline (iSRAP) research tool that is composed of widely used tools for rapid profiling of small RNAs. The performance test of iSRAP using publicly and in-house available data sets shows its ability of comprehensive profiling of small RNAs of various classes, and analysis of differentially expressed small RNAs. iSRAP offers comprehensive analysis of small RNA sequencing data that leverage informed decisions on the downstream analyses of small RNA studies, including extracellular vesicles such as exosomes.

Genome-Wide Measures of Peripheral Blood Dna Methylation and Prostate Cancer Risk in a Prospective Nested Case-Control Study

Global measures of peripheral blood DNA methylation have been associated with risk of some malignancies, including breast, bladder, and gastric cancer. Here, we examined genome‐wide measures of peripheral blood DNA methylation in prostate cancer and its non‐aggressive and aggressive disease forms.

Variant effect prediction tools assessed using independent, functional assay-based datasets: implications for discovery and diagnostics

BackgroundGenetic variant effect prediction algorithms are used extensively in clinical genomics and research to determine the likely consequences of amino acid substitutions on protein function. It is vital that we better understand their accuracies and limitations because published performance metrics are confounded by serious problems of circularity and error propagation. Here, we derive three independent, functionally determined human mutation datasets, UniFun, BRCA1-DMS and TP53-TA, and employ them, alongside previously described datasets, to assess the pre-eminent variant effect prediction tools.ResultsApparent accuracies of variant effect prediction tools were influenced significantly by the benchmarking dataset. Benchmarking with the assay-determined datasets UniFun and BRCA1-DMS yielded areas under the receiver operating characteristic curves in the modest ranges of 0.52 to 0.63 and 0.54 to 0.75, respectively, considerably lower than observed for other, potentially more conflicted datasets.ConclusionsThese results raise concerns about how such algorithms should be employed, particularly in a clinical setting. Contemporary variant effect prediction tools are unlikely to be as accurate at the general prediction of functional impacts on proteins as reported prior. Use of functional assay-based datasets that avoid prior dependencies promises to be valuable for the ongoing development and accurate benchmarking of such tools.