scholarly journals Identification of novel differentially methylated sites with potential as clinical predictors of impaired respiratory function and COPD

2018 ◽  
Author(s):  
Mairead L Bermingham ◽  
Rosie M Walker ◽  
Riccardo E. Marioni ◽  
Stewart M Morris ◽  
Konrad Rawlik ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Axon Guidance ◽  
Peripheral Blood ◽  
Respiratory Function ◽  
Population Sample ◽  
Smoking History ◽  
Blood Samples ◽  
Cpg Sites ◽  
Genome Wide ◽  
Epic Array

AbstractBackgroundThe causes of poor respiratory function and COPD are incompletely understood, but it is clear that genes and the environment play a role. As DNA methylation is under both genetic and environmental control, we hypothesised that investigation of differential methylation associated with these phenotypes would permit mechanistic insights, and improve prediction of COPD. We investigated genome-wide differential DNA methylation patterns using the recently released 850K Illumina EPIC array in the largest single population sample to date.MethodsEpigenome-wide association studies (EWASs) of respiratory function and COPD were performed in peripheral blood samples from the Generation Scotland: Scottish Family Health Study (GS:SFHS) cohort (N=3,791; 274 COPD cases and 2,928 controls). In independent COPD incidence data (N=150), significantly differentially methylated sites (DMSs; p<3.6×10−8) were evaluated for their added predictive power when added to a model including clinical variables, age, sex, height and smoking history using receiver operating characteristic analysis. The Lothian Birth Cohort 1936 (LBC1936) was used to replicate association (N=895) and prediction (N=178) results.FindingsWe identified 29 respiratory function and/or COPD associated DMSs, which mapped to genes involved in alternative splicing, JAK-STAT signalling, and axon guidance. In prediction analyses, we observed significant improvement in discrimination between COPD cases and controls (p<0.05) in independent GS:SFHS (p=0.014) and LBC1936 (p=0.018) datasets by adding DMSs to a clinical model.InterpretationIdentification of novel DMSs has provided insight into the molecular mechanisms regulating respiratory function and aided prediction of COPD risk.FundingWellcome Trust Strategic Award 10436/Z/14/Z.Research in contextEvidence before this studyWe searched for articles in PubMed published in English up to July 25, 2018, with the search terms “DNA methylation” and “respiratory function”, or “COPD”. We found some evidence for association between differential DNA methylation and both respiratory function and COPD. Of the twelve previous studies identified, eight used peripheral blood samples (sample size [N] range = 100-1,085) and four used lung tissue samples (N range = 24-160). The number of CpG loci analysed range from 27,578 to 485,512. These studies have not identified consistent changes in methylation, most likely due to a combination of factors including small sample sizes, technical issues, phenotypic definitions, and study design. In addition, no previous study has: analysed a sample from a large single cohort; used the recently released Illumina EPIC array (which assesses ~850,000 CpG loci); adjusted methylation data and phenotype for smoking history, or used both prevalent and incident COPD electronic health record data.Added value of this studyTo our knowledge, this is the largest single cohort epigenome-wide association study (EWAS) of respiratory function and COPD to date (N=3,791). After applying stringent genome-wide significance criteria (P <3.6×10−8), we found that DNA methylation levels at 29 CpG sites in peripheral blood were associated with respiratory function or COPD. Of these 29, seven were testable in an independent population sample: all seven showed consistent direction of effect between the two samples and three showed replication (p<0.007 [0.05/7 CpG sites tested]). Our results suggest that adjustment of both the phenotypic and the DNA methylation probe data for smoking history, which has not been carried out in previous studies, reduces the confounding effects of smoking, identifies larger numbers of associations, and reduces the heterogeneity of effects across smoking strata. We used gene set enrichment and pathway analyses, together with an approach that combines DNA methylation results with gene expression data to provide evidence for enrichment of differentially methylated sites in genes linked to alternative splicing, and JAK-STAT signalling and axon guidance. Finally, we demonstrated that the inclusion of DNA methylation data improves COPD risk prediction over established clinical variables alone in two independent datasets.Implications of all the available evidenceThere is now accumulating evidence that DNA methylation in peripheral blood is associated with respiratory function and COPD.Our study has shown that DNA methylation levels at 29 CpG sites are robustly associated with respiratory function and COPD, provide mechanistic insights, and can improve prediction of COPD risk. Further studies are warranted to improve understanding of the aetiology of COPD and to assess the utility of DNA methylation profiling in the clinical management of this condition.

scholarly journals TANC1 methylation as a novel biomarker for the diagnosis of patients with anti-tuberculosis drug-induced liver injury

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Dongxue Wu ◽  
Yuhong Li ◽  
Qi Ren ◽  
Shengfei Pei ◽  
Lin Wang ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Peripheral Blood ◽  
Target Genes ◽  
Characteristic Analysis ◽  
Drug Induced ◽  
Blood Samples ◽  
Cpg Sites ◽  
Genome Wide ◽  
Methylation Patterns ◽  
Genomic Methylation

AbstractWe aimed to elucidate the differences in genomic methylation patterns between ADLI and non-ADLI patients to identify DNA methylation-based biomarkers. Genome-wide DNA methylation patterns were obtained using Infinium MethylationEPIC (EPIC) BeadChip array to analyze 14 peripheral blood samples (7 ADLI cases, 7 non-ADLI controls). Changes in the mRNA and DNA methylation in the target genes of another 120 peripheral blood samples (60 ADLI cases, 60 non-ADLI controls) were analyzed by real-time polymerase chain reaction and pyrosequencing, respectively. A total of 308 hypermethylated CpG sites and 498 hypomethylated CpG sites were identified. Significantly, hypermethylated CpG sites cg06961147 and cg24666046 in TANC1 associated with ADLI was identified by genome-wide DNA methylation profiling. The mRNA expression of TANC1 was lower in the cases compared to the controls. Pyrosequencing validated these two differentially methylated loci, which was consistent with the results from the EPIC BeadChip array. Receiver operating characteristic analysis indicated that the area under the curve of TANC1 (cg06961147, cg24666046, and their combinations) was 0.812, 0.842, and 0.857, respectively. These results indicate that patients with ADLI have different genomic methylation patterns than patients without ADLI. The hypermethylated differentially methylated site cg06961147 combined with cg24666046 in TANC1 provides evidence for the diagnosis of ADLI.

scholarly journals A validation of Illumina EPIC array system with bisulfite-based amplicon sequencing

PeerJ ◽  
2021 ◽  
Vol 9 ◽  
pp. e10762
Author(s):  
Alexandra J. Noble ◽  
John F. Pearson ◽  
Joseph M. Boden ◽  
L. John Horwood ◽  
Neil J. Gemmell ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Large Scale ◽  
Amplicon Sequencing ◽  
Pilot Studies ◽  
Array Data ◽  
Cpg Sites ◽  
Genome Wide ◽  
The Individual ◽  
Epic Array ◽  
Large Scale Screening

The Illumina Infinium® MethylationEPIC BeadChip system (hereafter EPIC array) is considered to be the current gold standard detection method for assessing DNA methylation at the genome-wide level. EPIC arrays are often used for hypothesis generation or pilot studies, the natural conclusion to which is to validate methylation candidates and expand these in a larger cohort, in a targeted manner. As such, an accurate smaller-scale, targeted technique, that generates data at the individual CpG level that is equivalent to the EPIC array, is needed. Here, we tested an alternative DNA methylation detection technique, known as bisulfite-based amplicon sequencing (BSAS), to determine its ability to validate CpG sites detected in EPIC array studies. BSAS was able to detect differential DNA methylation at CpG sites to a degree which correlates highly with the EPIC array system at some loci. However, BSAS correlated less well with EPIC array data in some instances, and most notably, when the magnitude of change via EPIC array was greater than 5%. Therefore, our data suggests that BSAS can be used to validate EPIC array data, but each locus must be compared on an individual basis, before being taken forward into large scale screening. Further, BSAS does offer advantages compared to the probe-based EPIC array; BSAS amplifies a region of the genome (∼500 bp) around a CpG of interest, allowing analyses of other CpGs in the region that may not be present on the EPIC array, aiding discovery of novel CpG sites and differentially methylated regions of interest. We conclude that BSAS offers a valid investigative tool for specific regions of the genome that are currently not contained on the array system.

scholarly journals Comparison of methylation capture sequencing and Infinium MethylationEPIC array in peripheral blood mononuclear cells

2020 ◽  
Vol 13 (1) ◽  
Author(s):  
Chang Shu ◽  
Xinyu Zhang ◽  
Bradley E. Aouizerat ◽  
Ke Xu
Keyword(s):  
Dna Methylation ◽  
Peripheral Blood ◽  
Cpg Island ◽  
Association Studies ◽  
Pearson Correlation ◽  
Peripheral Blood Mononuclear ◽  
Cpg Sites ◽  
Cpg Site ◽  
Highly Correlated ◽  
Epic Array

Abstract Background Epigenome-wide association studies (EWAS) have been widely applied to identify methylation CpG sites associated with human disease. To date, the Infinium MethylationEPIC array (EPIC) is commonly used for high-throughput DNA methylation profiling. However, the EPIC array covers only 30% of the human methylome. Methylation Capture bisulfite sequencing (MC-seq) captures target regions of methylome and has advantages of extensive coverage in the methylome at an affordable price. Methods Epigenome-wide DNA methylation in four peripheral blood mononuclear cell samples was profiled by using SureSelectXT Methyl-Seq for MC-seq and EPIC platforms separately. CpG site-based reproducibility of MC-seq was assessed with DNA sample inputs ranging in quantity of high (> 1000 ng), medium (300–1000 ng), and low (150 ng–300 ng). To compare the performance of MC-seq and the EPIC arrays, we conducted a Pearson correlation and methylation value difference at each CpG site that was detected by both MC-seq and EPIC. We compared the percentage and counts in each CpG island and gene annotation between MC-seq and the EPIC array. Results After quality control, an average of 3,708,550 CpG sites per sample were detected by MC-seq with DNA quantity > 1000 ng. Reproducibility of DNA methylation in MC-seq-detected CpG sites was high among samples with high, medium, and low DNA inputs (r > 0.96). The EPIC array captured an average of 846,464 CpG sites per sample. Compared with the EPIC array, MC-seq detected more CpGs in coding regions and CpG islands. Among the 472,540 CpG sites captured by both platforms, methylation of a majority of CpG sites was highly correlated in the same sample (r: 0.98–0.99). However, methylation for a small proportion of CpGs (N = 235) differed significantly between the two platforms, with differences in beta values of greater than 0.5. Conclusions Our results show that MC-seq is an efficient and reliable platform for methylome profiling with a broader coverage of the methylome than the array-based platform. Although methylation measurements in majority of CpGs are highly correlated, a number of CpG sites show large discrepancy between the two platforms, which warrants further investigation and needs cautious interpretation.

scholarly journals Deoxyribonucleic acid methylation in the enhancer region of the CDKN2A/2B and CDKN2B-AS1 genes in blood vessels and cells in patients with carotid atherosclerosis

2020 ◽  
Vol 25 (10) ◽  
pp. 4060
Author(s):  
Yu. A. Koroleva ◽  
A. V. Markov ◽  
I. A. Goncharova ◽  
A. A. Sleptsov ◽  
N. P. Babushkina ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Peripheral Blood ◽  
Methylation Level ◽  
Deoxyribonucleic Acid ◽  
Healthy Individuals ◽  
Peripheral Blood Leukocytes ◽  
Blood Leukocytes ◽  
Blood Samples ◽  
Cpg Sites ◽  
Atherosclerotic Lesions

Aim. Comparative analysis of the deoxyribonucleic acid (DNA) methylation level in the enhancer region of the CDKN2A/2B and CDKN2B-AS1 genes (9p21.3 locus) in vessels with/without atherosclerotic lesions, as well as in leukocytes of patients with clinically relevant carotid artery (CA) atherosclerosis and healthy individuals.Material and methods. The group of patients with clinically relevant atherosclerosis included 22 individuals with severe stenosis (>80%) of CA. Samples of atherosclerotic plaques, presenting CA regions, and great saphenous veins, as well as peripheral blood samples (leukocytes) were obtained from patients. The control group consisted of 14 individuals with the mild CA stenosis (£24%) and without hemodynamically relevant changes; peripheral blood samples were obtained from each of them. DNA methylation level was assessed by targeted bisulfite sequencing of amplicons.Results. The tissue-specific methylation of 31 CpG-site in the CDKN2A/2B and CDKN2B-AS1 gene enhancer was established: the vascular tissues significantly differed from the peripheral blood leukocytes. At the same time, there was an increase in the methylation level of both certain CpG sites and whole analyzed CA region affected by atherosclerosis (48,6 [34,8; 62,0]%), compared with intact vessels, both arteries (25,2 [23,1; 41,60]%, p=0,0001) and veins (35,0 [31,6; 40,0]%, p=0,0039). Patients had lower methylation levels in all CpG sites in blood leukocytes compared to blood vessel samples (8,7 [6,1; 9,7]%; p<0,05). At the same time, the level of DNA methylation in the blood leukocytes of atherosclerotic patients does not differ from that in healthy individuals (9,3 [8,3; 13,6]%; p>0,8).Conclusion. In the present study, the relationship between an increase in the DNA methylation in the enhancer of the CDKN2A/2B and CDKN2B-AS1 genes in CA and their atherosclerotic lesions was revealed, as well as the tissue-specific DNA methylation between vessels and peripheral blood leukocytes.

scholarly journals A validation of Illumina EPIC array system with bisulfite-based amplicon sequencing

2020 ◽  
Author(s):  
Alexandra Noble ◽  
John Pearson ◽  
Joseph Boden ◽  
John Horwood ◽  
Neil Gemmell ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Detection Method ◽  
Pcr Amplification ◽  
Amplicon Sequencing ◽  
Pilot Studies ◽  
Cpg Sites ◽  
Genome Wide ◽  
Lower Specificity ◽  
The Individual ◽  
Epic Array

AbstractThe Illumina Infinium® MethylationEPIC BeadChip system (hereafter EPIC array) is considered to be the current gold standard detection method for assessing DNA methylation at the genome-wide level. EPIC arrays are used for hypothesis generation or pilot studies, the natural conclusion is to validate methylation candidates and expand these in a larger cohort, in a targeted manner. As such, an accurate smaller-scale, targeted technique, that generates data at the individual CpG level that is equivalent to the EPIC array, is needed. Here, we tested an alternative DNA methylation detection technique, known as bisulfite-based amplicon sequencing (BSAS), to determine its ability to validate CpG sites detected in EPIC array studies. BSAS was able to detect differential DNA methylation at CpG sites to a degree which correlates highly with the EPIC array system. However, BSAS correlated less well with EPIC array data when the magnitude of change via EPIC array was greater than 5%, suggesting that this lower specificity at larger differential methylation values is a consequence of PCR amplification that BSAS requires. However, our data suggests that BSAS does offer advantages that the EPIC array: BSAS amplifies a region of the genome (~500bp) around a CpG of interest, allowing analyses of other CpGs in the region that may not be present on the EPIC array, aiding discovery of novel CpG sites and differentially methylated regions of interest. We conclude that BSAS offers a valid investigative tool for specific regions of the genome that are currently not contained on the array system.

scholarly journals Comparison of Methylation Capture Sequencing and Infinium EPIC Methylation Array in Peripheral Blood Mononuclear Cells

2020 ◽  
Author(s):  
Chang Shu ◽  
Xinyu Zhang ◽  
Bradley E. Aouizerat ◽  
Ke Xu
Keyword(s):  
Dna Methylation ◽  
Peripheral Blood ◽  
Cpg Island ◽  
Association Studies ◽  
Pearson Correlation ◽  
Peripheral Blood Mononuclear ◽  
Cpg Sites ◽  
Cpg Site ◽  
Highly Correlated ◽  
Epic Array

Abstract Background: Epigenome-wide association studies (EWAS) have been widely applied to identify methylation CpG sites associated with human disease. To date, the Infinium Methylation EPIC array (EPIC) is commonly used for high-throughput DNA methylation profiling. However, the EPIC array covers only 30% of the human methylome. Methylation Capture bisulfite sequencing (MC-seq) captures target regions of methylome and has advantages of extensive coverage in the methylome at an affordable price. Methods: Epienome-wide DNA methylation in four peripheral blood mononuclear cell samples was profiled by using SureSelectXT Methyl-Seq for MC-seq and EPIC platforms separately. CpG site-based reproducibility of MC-seq was assessed with DNA sample inputs ranging in quantity of high (> 1000ng), medium (300-1000ng), and low (150ng-300ng). To compare the performance of MC-seq and the EPIC arrays, we conducted a Pearson correlation and methylation value difference at each CpG site that was detected by both MC-seq and EPIC. We compared the percentage and counts in each CpG island and gene annotation between MC-seq and the EPIC array.Results: After quality control, an average of 3,708,550 CpG sites per sample was detected by MC-seq with DNA quantity >1000ng. Reproducibility of MC-seq detected CpG sites was high with strong correlation estimates for CpG methylation among samples with high, medium, and low DNA inputs (r > 0.96). The EPIC array captured an average of 846,464 CpG sites per sample. Compared with the EPIC array, MC-seq detected more CpGs in coding regions and CpG islands. Among the 472,540 CpG sites captured by both platforms, methylation of a majority of CpG sites was highly correlated in the same sample (r: 0.98~0.99). However, methylation for a small proportion of CpGs (N=235) differed significantly between the two platforms, with differences in beta values of greater than 0.5.Conclusions: Our results show that MC-seq is an efficient and reliable platform for methylome profiling with a broader coverage of the methylome than the array-based platform. Although methylation measurements in majority of CpGs are highly correlated, a number of CpG sites show large discrepancy between the two platforms, which warrants further investigation and needs cautious interpretation.

scholarly journals Comparison of Methylation Capture Sequencing and Infinium EPIC Methylation Array in Peripheral Blood Mononuclear Cells

2020 ◽  
Author(s):  
Chang Shu ◽  
Xinyu Zhang ◽  
Bradley E. Aouizerat ◽  
Ke Xu
Keyword(s):  
Dna Methylation ◽  
Peripheral Blood ◽  
Cpg Island ◽  
Association Studies ◽  
Pearson Correlation ◽  
Peripheral Blood Mononuclear ◽  
Cpg Sites ◽  
Cpg Site ◽  
Highly Correlated ◽  
Epic Array

Abstract Background: Epigenome-wide association studies (EWAS) have been widely applied to identify methylation CpG sites associated with human disease. To date, the Infinium Methylation EPIC array (EPIC) is commonly used for high-throughput DNA methylation profiling. However, the EPIC array covers only 30% of the human methylome. Methylation Capture bisulfite sequencing (MC-seq) captures target regions of methylome and has advantages of extensive coverage in the methylome at an affordable price. Methods: Epigenome-wide DNA methylation in four peripheral blood mononuclear cell samples was profiled by using SureSelectXT Methyl-Seq for MC-seq and EPIC platforms separately. CpG site-based reproducibility of MC-seq was assessed with DNA sample inputs ranging in quantity of high (> 1000ng), medium (300-1000ng), and low (150ng-300ng). To compare the performance of MC-seq and the EPIC arrays, we conducted a Pearson correlation and methylation value difference at each CpG site that was detected by both MC-seq and EPIC. We compared the percentage and counts in each CpG island and gene annotation between MC-seq and the EPIC array. Results: After quality control, an average of 3,708,550 CpG sites per sample was detected by MC-seq with DNA quantity >1000ng. Reproducibility of MC-seq detected CpG sites was high with strong correlation estimates for CpG methylation among samples with high, medium, and low DNA inputs (r > 0.96). The EPIC array captured an average of 846,464 CpG sites per sample. Compared with the EPIC array, MC-seq detected more CpGs in coding regions and CpG islands. Among the 472,540 CpG sites captured by both platforms, methylation of a majority of CpG sites was highly correlated in the same sample (r: 0.98~0.99). However, methylation for a small proportion of CpGs (N=235) differed significantly between the two platforms, with differences in beta values of greater than 0.5. Conclusions: Our results show that MC-seq is an efficient and reliable platform for methylome profiling with a broader coverage of the methylome than the array-based platform. Although methylation measurements in majority of CpGs are highly correlated, a number of CpG sites show large discrepancy between the two platforms, which warrants further investigation and needs cautious interpretation.

scholarly journals DNA methylation mediates the association between breastfeeding and early-life growth trajectories

2021 ◽  
Vol 13 (1) ◽  
Author(s):  
Laurent Briollais ◽  
Denis Rustand ◽  
Catherine Allard ◽  
Yanyan Wu ◽  
Jingxiong Xu ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Signaling Pathway ◽  
Exclusive Breastfeeding ◽  
Child Growth ◽  
Postnatal Period ◽  
Overweight And Obesity ◽  
Breastfeeding Duration ◽  
Ampk Signaling ◽  
Cpg Sites ◽  
Genome Wide

Abstract Background The role of breastfeeding in modulating epigenetic factors has been suggested as a possible mechanism conferring its benefits on child development but it lacks evidence. Using extensive DNA methylation data from the ALSPAC child cohort, we characterized the genome-wide landscape of DNA methylation variations associated with the duration of exclusive breastfeeding and assessed whether these variations mediate the association between exclusive breastfeeding and BMI over different epochs of child growth. Results Exclusive breastfeeding elicits more substantial DNA methylation variations during infancy than at other periods of child growth. At the genome-wide level, 13 CpG sites in girls (miR-21, SNAPC3, ATP6V0A1, DHX15/PPARGC1A, LINC00398/ALOX5AP, FAM238C, NATP/NAT2, CUX1, TRAPPC9, OSBPL1A, ZNF185, FAM84A, PDPK1) and 2 CpG sites in boys (IL16 and NREP), mediate the association between exclusive breastfeeding and longitudinal BMI. We found enrichment of CpG sites located within miRNAs and key pathways (AMPK signaling pathway, insulin signaling pathway, endocytosis). Overall DNA methylation variation corresponding to 3 to 5 months of exclusive breastfeeding was associated with slower BMI growth the first 6 years of life compared to no breastfeeding and in a dose–response manner with exclusive breastfeeding duration. Conclusions Our study confirmed the early postnatal period as a critical developmental period associated with substantial DNA methylation variations, which in turn could mitigate the development of overweight and obesity from infancy to early childhood. Since an accelerated growth during these developmental periods has been linked to the development of sustained obesity later in life, exclusive breastfeeding could have a major role in preventing the risks of overweight/obesity and children and adults through DNA methylation mechanisms occurring early in life.

scholarly journals Butyrate and related epigenetic changes link Parkinson's disease to inflammatory bowel disease and depressive symptoms

2021 ◽  
Author(s):  
Aoji Xie ◽  
Elizabeth Ensink ◽  
Peipei Li ◽  
Juozas Gordevicius ◽  
Lee L. Marshall ◽  
...  
Keyword(s):  
Parkinson’S Disease ◽  
Dna Methylation ◽  
Parkinson's Disease ◽  
Depressive Symptoms ◽  
Gut Microbiome ◽  
Whole Blood ◽  
Blood Samples ◽  
Targeted Bisulfite Sequencing ◽  
Genome Wide ◽  
Whole Blood Samples

Background The gut microbiome and its metabolites can impact brain health and are altered in Parkinson's disease (PD) patients. It has been recently demonstrated that PD patients have reduced fecal levels of the potent epigenetic modulator butyrate and its bacterial producers. Here, we investigate whether the changes in the gut microbiome and associated metabolites are linked to PD symptoms and epigenetic markers in leucocytes and neurons. Methods Stool, whole blood samples, and clinical data were collected from 55 PD patients and 55 controls. We performed DNA methylation analysis on whole blood samples and analyzed the results in relation to fecal short-chain fatty acid concentrations and microbiota composition. In another cohort, prefrontal cortex neurons were isolated from control and PD brains. We identified the genome-wide DNA methylation by targeted bisulfite sequencing. Results We show that lower fecal butyrate and reduced Roseburia, Romboutsia, and Prevotella counts are linked to depressive symptoms in PD patients. Genes containing butyrate-associated methylation sites include PD risk genes and significantly overlap with sites epigenetically altered in PD blood leucocytes, predominantly neutrophils, and in brain neurons, relative to controls. Moreover, butyrate-associated methylated-DNA (mDNA) regions in PD overlap with those altered in gastrointestinal, autoimmune, and psychiatric diseases.

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