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      Whole-Genome Saliva and Blood DNA Methylation Profiling in Individuals with a Respiratory Allergy

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          Abstract

          The etiology of respiratory allergies (RA) can be partly explained by DNA methylation changes caused by adverse environmental and lifestyle factors experienced early in life. Longitudinal, prospective studies can aid in the unravelment of the epigenetic mechanisms involved in the disease development. High compliance rates can be expected in these studies when data is collected using non-invasive and convenient procedures. Saliva is an attractive biofluid to analyze changes in DNA methylation patterns. We investigated in a pilot study the differential methylation in saliva of RA (n = 5) compared to healthy controls (n = 5) using the Illumina Methylation 450K BeadChip platform. We evaluated the results against the results obtained in mononuclear blood cells from the same individuals. Differences in methylation patterns from saliva and mononuclear blood cells were clearly distinguishable (P Adj<0.001 and |Δβ|>0.2), though the methylation status of about 96% of the cg-sites was comparable between peripheral blood mononuclear cells and saliva. When comparing RA cases with healthy controls, the number of differentially methylated sites (DMS) in saliva and blood were 485 and 437 (P<0.05 and |Δβ|>0.1), respectively, of which 216 were in common. The methylation levels of these sites were significantly correlated between blood and saliva. The absolute levels of methylation in blood and saliva were confirmed for 3 selected DMS in the PM20D1, STK32C, and FGFR2 genes using pyrosequencing analysis. The differential methylation could only be confirmed for DMS in PM20D1 and STK32C genes in saliva. We show that saliva can be used for genome-wide methylation analysis and that it is possible to identify DMS when comparing RA cases and healthy controls. The results were replicated in blood cells of the same individuals and confirmed by pyrosequencing analysis. This study provides proof-of-concept for the applicability of saliva-based whole-genome methylation analysis in the field of respiratory allergy.

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          International Study of Asthma and Allergies in Childhood (ISAAC): rationale and methods.

          Richard Beasley, Melissa L. Anderson, Linda Asher … (1995)
          The aetiology of asthma and allergic disease remains poorly understood, despite considerable research. The International Study of Asthma and Allergies in Childhood (ISAAC), was founded to maximize the value of epidemiological research into asthma and allergic disease, by establishing a standardized methodology and facilitating international collaboration. Its specific aims are: 1) to describe the prevalence and severity of asthma, rhinitis and eczema in children living in different centres, and to make comparisons within and between countries; 2) to obtain baseline measures for assessment of future trends in the prevalence and severity of these diseases; and 3) to provide a framework for further aetiological research into genetic, lifestyle, environmental, and medical care factors affecting these diseases. The ISAAC design comprises three phases. Phase 1 uses core questionnaires designed to assess the prevalence and severity of asthma and allergic disease in defined populations. Phase 2 will investigate possible aetiological factors, particularly those suggested by the findings of Phase 1. Phase 3 will be a repetition of Phase 1 to assess trends in prevalence.
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            DNA extracted from saliva for methylation studies of psychiatric traits: evidence tissue specificity and relatedness to brain.

            Alicia K. Smith, Varun Kilaru, Torsten Klengel … (2015)
            DNA methylation has become increasingly recognized in the etiology of psychiatric disorders. Because brain tissue is not accessible in living humans, epigenetic studies are most often conducted in blood. Saliva is often collected for genotyping studies but is rarely used to examine DNA methylation because the proportion of epithelial cells and leukocytes varies extensively between individuals. The goal of this study was to evaluate whether saliva DNA is informative for studies of psychiatric disorders. DNA methylation (HumanMethylation450 BeadChip) was assessed in saliva and blood samples from 64 adult African Americans. Analyses were conducted using linear regression adjusted for appropriate covariates, including estimated cellular proportions. DNA methylation from brain tissues (cerebellum, frontal cortex, entorhinal cortex, and superior temporal gyrus) was obtained from a publically available dataset. Saliva and blood methylation was clearly distinguishable though there was positive correlation overall. There was little correlation in CpG sites within relevant candidate genes. Correlated CpG sites were more likely to occur in areas of low CpG density (i.e., CpG shores and open seas). There was more variability in CpG sites from saliva than blood, which may reflect its heterogeneity. Finally, DNA methylation in saliva appeared more similar to patterns from each of the brain regions examined overall than methylation in blood. Thus, this study provides a framework for using DNA methylation from saliva and suggests that DNA methylation of saliva may offer distinct opportunities for epidemiological and longitudinal studies of psychiatric traits.
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              Blood-based profiles of DNA methylation predict the underlying distribution of cell types: a validation analysis.

              Scott M Langevin, Margaret R. Karagas, Brock C. Christensen … (2013)
              The potential influence of underlying differences in relative leukocyte distributions in studies involving blood-based profiling of DNA methylation is well recognized and has prompted development of a set of statistical methods for inferring changes in the distribution of white blood cells using DNA methylation signatures. However, the extent to which this methodology can accurately predict cell-type proportions based on blood-derived DNA methylation data in a large-scale epigenome-wide association study (EWAS) has yet to be examined. We used publicly available data deposited in the Gene Expression Omnibus (GEO) database (accession number GSE37008), which consisted of both blood-derived epigenome-wide DNA methylation data assayed using the Illumina Infinium HumanMethylation27 BeadArray and complete blood cell (CBC) counts among a community cohort of 94 non-diseased individuals. Constrained projection (CP) was used to obtain predictions of the proportions of lymphocytes, monocytes and granulocytes for each of the study samples based on their DNA methylation signatures. Our findings demonstrated high consistency between the average CBC-derived and predicted percentage of monocytes and lymphocytes (17.9% and 17.6% for monocytes and 82.1% and 81.4% for lymphocytes), with root mean squared error (rMSE) of 5% and 6%, for monocytes and lymphocytes, respectively. Similarly, there was moderate-high correlation between the CP-predicted and CBC-derived percentages of monocytes and lymphocytes (0.60 and 0.61, respectively), and these results were robust to the number of leukocyte differentially methylated regions (L-DMRs) used for CP prediction. These results serve as further validation of the CP approach and highlight the promise of this technique for EWAS where DNA methylation is profiled using whole-blood genomic DNA.
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                Author and article information

                Contributors
                Esteban Ballestar: Role: Editor
                Journal
                Journal ID (nlm-ta): PLoS One
                Journal ID (iso-abbrev): PLoS ONE
                Journal ID (publisher-id): plos
                Journal ID (pmc): plosone
                Title: PLoS ONE
                Publisher: Public Library of Science (San Francisco, CA USA )
                ISSN (Electronic): 1932-6203
                Publication date (Electronic): 21 March 2016
                Publication date Collection: 2016
                Volume: 11
                Issue: 3
                Electronic Location Identifier: e0151109
                Affiliations
                [1 ]Environmental Risk and Health unit, Flemish Institute for Technological Research (VITO), Mol, Belgium
                [2 ]Laboratory of Protein Chemistry, Proteomics and Epigenetic Signaling (PPES), University of Antwerp, Wilrijk, Belgium
                [3 ]Department of Biomedical Sciences, University of Antwerp, Wilrijk, Belgium
                [4 ]Laboratory of Cancer Research and Clinical Oncology, Center for Medical Genetics, University of Antwerp, Edegem, Belgium
                [5 ]University of Southern Denmark, Institute of Public Health, Department of Environmental Medicine, Odense, Denmark
                [6 ]Centre for Environmental Sciences, Hasselt University, Diepenbeek, Belgium
                Bellvitge Biomedical Research Institute (IDIBELL), SPAIN
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Conceived and designed the experiments: SL KSVS PDB. Performed the experiments: SL ST KSVS. Analyzed the data: KD KSVS SL PDB. Contributed reagents/materials/analysis tools: SL GVC WVB PDB. Wrote the paper: SL KSVS PDB KD WVB. Evaluated and interpreted results: SL KSVS PDB KD WVB. Evaluated manuscript text: SL KSVS KD ST GS GK GVC WVB PDB.

                Article
                Publisher ID: PONE-D-15-43771
                DOI: 10.1371/journal.pone.0151109
                PMC ID: 4801358
                PubMed ID: 26999364
                SO-VID: f3f3fa80-a84e-4d78-aac7-14a2c5f7845c
                Copyright © © 2016 Langie et al
                License:

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                Date received : 5 October 2015
                Date accepted : 22 February 2016
                Page count
                Figures: 5, Tables: 2, Pages: 17
                Funding
                SL is the beneficiary of a post-doctoral grant from the AXA Research Fund and the Cefic-LRI Innovative Science Award 2013.
                Categories
                Subject: Research Article
                Subject: Biology and life sciences
                Subject: Cell biology
                Subject: Chromosome biology
                Subject: Chromatin
                Subject: Chromatin modification
                Subject: DNA methylation
                Subject: Biology and life sciences
                Subject: Genetics
                Subject: Epigenetics
                Subject: Chromatin
                Subject: Chromatin modification
                Subject: DNA methylation
                Subject: Biology and life sciences
                Subject: Genetics
                Subject: Gene expression
                Subject: Chromatin
                Subject: Chromatin modification
                Subject: DNA methylation
                Subject: Biology and life sciences
                Subject: Genetics
                Subject: DNA
                Subject: DNA modification
                Subject: DNA methylation
                Subject: Biology and life sciences
                Subject: Biochemistry
                Subject: Nucleic acids
                Subject: DNA
                Subject: DNA modification
                Subject: DNA methylation
                Subject: Biology and life sciences
                Subject: Genetics
                Subject: Epigenetics
                Subject: DNA modification
                Subject: DNA methylation
                Subject: Biology and life sciences
                Subject: Genetics
                Subject: Gene expression
                Subject: DNA modification
                Subject: DNA methylation
                Subject: Biology and Life Sciences
                Subject: Anatomy
                Subject: Body Fluids
                Subject: Saliva
                Subject: Medicine and Health Sciences
                Subject: Anatomy
                Subject: Body Fluids
                Subject: Saliva
                Subject: Biology and Life Sciences
                Subject: Physiology
                Subject: Body Fluids
                Subject: Saliva
                Subject: Medicine and Health Sciences
                Subject: Physiology
                Subject: Body Fluids
                Subject: Saliva
                Subject: Biology and Life Sciences
                Subject: Anatomy
                Subject: Body Fluids
                Subject: Blood
                Subject: Medicine and Health Sciences
                Subject: Anatomy
                Subject: Body Fluids
                Subject: Blood
                Subject: Biology and Life Sciences
                Subject: Physiology
                Subject: Body Fluids
                Subject: Blood
                Subject: Medicine and Health Sciences
                Subject: Physiology
                Subject: Body Fluids
                Subject: Blood
                Subject: Medicine and Health Sciences
                Subject: Hematology
                Subject: Blood
                Subject: Medicine and Health Sciences
                Subject: Clinical Medicine
                Subject: Clinical Immunology
                Subject: Allergies
                Subject: Biology and Life Sciences
                Subject: Immunology
                Subject: Clinical Immunology
                Subject: Allergies
                Subject: Medicine and Health Sciences
                Subject: Immunology
                Subject: Clinical Immunology
                Subject: Allergies
                Subject: Biology and Life Sciences
                Subject: Cell Biology
                Subject: Cellular Types
                Subject: Animal Cells
                Subject: Blood Cells
                Subject: Medicine and Health Sciences
                Subject: Pulmonology
                Subject: Asthma
                Subject: Biology and Life Sciences
                Subject: Genetics
                Subject: Epigenetics
                Subject: Biology and Life Sciences
                Subject: Molecular Biology
                Subject: Molecular Biology Techniques
                Subject: Artificial Gene Amplification and Extension
                Subject: Polymerase Chain Reaction
                Subject: Research and Analysis Methods
                Subject: Molecular Biology Techniques
                Subject: Artificial Gene Amplification and Extension
                Subject: Polymerase Chain Reaction
                Custom metadata
                Data Availability The data discussed in this publication have been deposited in NCBI's Gene Expression Omnibus (Edgar et al., 2002) and are accessible through GEO Series accession number GSE73745 ( https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE73745).

                ScienceOpen disciplines: Uncategorized
                Data availability:
                ScienceOpen disciplines: Uncategorized

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