Comparing DNA methylation profiles in saliva and intestinal mucosa

- Comparing DNA methylation profiles in saliva and intestinal mucosa.
- DNA methylation studies in these diseases have utilised intestinal mucosal tissue or blood which can be difficult to collect, particularly for large-scale research studies.
- Saliva is an attractive alternative for epigenetic studies as it is easy to collect and provides high quality methylation profiles.
- The aim of the study was to determine the utility of saliva as an alternative for DNA methylation studies of intestinal disorders..
- Results: DNA methylation in saliva and intestinal mucosa samples were compared in individuals ( n = 10) undergoing endoscopies using the Illumina Infinium Methylation 450 K Beadchip array.
- Of the 48,541 probes (approximately 11% of CpG sites) that were differentially methylated between saliva and intestinal mucosa (adjusted p <.
- Conclusions: This study suggests that saliva has the potential to be used as an alternate DNA source to invasive intestinal mucosa for DNA methylation research into intestinal conditions..
- The involvement of both genetic and environmental fac- tors suggests that epigenetic mechanisms have a role in the pathogenesis of these intestinal inflammatory dis- eases [3].
- In the mammalian genome approxi- mately 15% of the CpG dinucleotides are found in CpG islands, which are defined as stretches of DNA >.
- CpG islands are often found in the promoter regions of both constitutively expressed genes and tissue specific genes [8].
- DNA methylation has been implicated in the pathogen- esis of intestinal inflammatory diseases, with studies in Crohn’s disease, inflammatory bowel disease, and ulcera- tive colitis demonstrating that individuals with these conditions have altered DNA methylation profiles in their intestinal, colon, or rectal tissue, when compared to healthy controls .
- Full list of author information is available at the end of the article.
- Many studies have investigated the utility of DNA methylation biomarkers for disease screening, diagnosis, and management [12, 13].
- Saliva is an attractive alternative for epigen- etic studies as it is easy to collect and provides high quality methylation profiles [16].
- In the clinical setting, saliva is also an attractive alternative compared to blood or intestinal mucosa, as individuals can collect their own saliva using commercially available kits which stabilises the sample at room temperature, enabling individuals to mail the sample back to the clinic.
- DNA methylation studies comparing profiles between tissue types within individuals have shown that regions of tissue-specific differential methylation mainly map to CpG poor regions [19].
- including brain [20], lung/bronchial epithelium [21], and peripheral blood mononuclear cells [16], for DNA methylation studies has been previously demonstrated, with methylation profiles correlating positively between saliva and the tis- sue in question.
- water, and also contains white blood cells and epithelial cells, which represent the cell types of the oral mucosa [22].
- environmental exposures can drive epigenetic change, saliva and the oral mucosa is an ideal alternative for the intestinal mucosa given the similarities in cellular com- position and environmental exposures between the two tissue sites..
- To date, no studies have compared DNA methylation profiles between saliva and the intestinal mucosa.
- The aim of the current study was to determine the utility of saliva for large scale DNA methylation research studies in intestinal conditions by investigating tissue-specific methylation differences..
- Intestinal mucosa and saliva samples were obtained from 10 individuals.
- Demographic and clinical characteristics of the participants are summarised in Table 1..
- DNA methylation was measured using Illumina Infi- nium Methylation 450 K Beadchip array for all 20 sam- ples (10 saliva and 10 matched intestinal samples)..
- Following quality control filtering and removal of probes on sex chromosomes and probes on non-cpg sites, 456,148 probes were included in the analysis..
- Hierarchical clustering based on average linkage and correlation-based distances showed that the saliva and intestinal tissue samples clustered into two distinct groups, and within the saliva cluster, individuals with coeliac disease also appear to cluster together (Fig.
- Multidimensional scaling (MDS) of the top 1000 most variable sites showed intestinal.
- Overall, global DNA methylation of saliva and intes- tinal mucosa was positively correlated within an indi- vidual (Pearson’s correlation co-efficient r p <.
- For 68.9% of CpG sites positive Pearson correlations between methylation levels of saliva and tissue samples were observed, and of these 14.8% were statistically significant (Fig.
- A scatter plot of average DNA methylation values for 4000 ran- dom CpG sites indicates methylation is in the same direction in saliva versus intestinal mucosal tissue samples, r = 0.94 (Fig.
- The distribution of differ- ence in DNA methylation (|Δβ|) between saliva and tissue samples is shown in Fig.
- region analysis identi- fied 292 tissue-specific regions (tDMR hypomethylated and hypermethylated re- gions between saliva and intestinal mucosa, which mapped to 278 annotated genes (Additional file 4:.
- Functional annotation analysis of the 278 annotated genes identified cell adhesion (GO:0098609, GO:0007156, GO:0098742, GO:0007155), biological adhesion (GO:00 22610) and calcium ion binding (GO:0005509) as the top most significant terms (Table 3).
- 1 Data clustering and comparison between saliva and tissue DNA methylation profiles.
- a) Hierarchical clustering based on correlation distances of methylation profiles in 20 samples – intestinal mucosa and saliva samples from 10 individuals.
- b) Pearson correlation coefficients for methylation levels of saliva vs tissue for each of the CpG sites (positive correlations in boxed area with statistically significant correlations (adjusted P <.
- The present study compared DNA methylation profiles between saliva and intestinal mucosa within an individ- ual, to determine the feasibility of saliva as an alternative for intestinal mucosal tissue for DNA methylation re- search studies in intestinal conditions.
- We found that methylation profiles were highly correlated between sal- iva and intestinal mucosa (r within an indi- vidual.
- This indicates that saliva has the potential to be a viable alternative for intestinal tissue for future DNA methylation studies..
- Saliva is an attractive alternative for DNA methylation profiling as collection is simple, non-invasive, and high quality methylation profiles can be generated [16, 18]..
- Methylation profiles comparing buccal epi- thelial swabs and bronchial epithelium obtained from in- dividuals with adenocarcinoma lung cancers who smoke tobacco, to assess the utility of saliva for screening and diagnosing lung cancer found the methylation profiles of two genes involved in early lung carcinogenesis, p16 and FHIT, correlated strongly between saliva and bronchial samples [21].
- It was suggested that since the entire air- way from oral cavity to lungs is exposed to the carcino- gens from tobacco, the carcinogens would be able to induce similar epigenetic alterations in the oral mucosa as well as in the distant lung tissue.
- This study was the first to suggest that an environmental agent could in- duce the same epigenetic changes in the oral mucosa as it does in a distant tissue, prompting the suggestion that oral mucosa could be used as a surrogate tissue in early screening interventions for preventing lung cancer [21]..
- Further comparisons between saliva and blood in a population of African Americans recruited at urban public hospitals found 88.6% of CpG sites were comparable.
- This study also found that DNA methylation profiles between saliva and brain tissues were more similar than methylation profiles between saliva and blood [20]..
- Enzymes found in saliva are also essential in the breakdown of dietary starches and fats..
- The similar composition and function of mucosa be- tween the oral mucosa and intestinal mucosa alongside our findings of comparable methylation profiles between saliva and intestinal mucosa strengthens the idea that saliva has the potential to be used as an alternative for more invasive tissues..
- Table 2 Correlation of average DNA methylation across all CpG sites between saliva and intestinal mucosa within an individual Participant r-value p -value Confidence Interval.
- 2 Scatter plot of average DNA methylation beta values for 4000 random CpG sites between intestinal and saliva samples.
- Functional analysis of the tDMR found the genes that were hypomethylated in certain tissues were frequently associated with tissue-specific functions, while none of the.
- In the present study, 292 tDMR were identified be- tween intestinal mucosa and saliva which corresponded to 278 unique genes.
- Oxytocin has been suggested to con- tribute to the control of the gastrointestinal motility, as it is expressed in nerve fibres along the entire human gastrointestinal tract, [28].
- Table 3 GO terms for differentially methylated genes between intestinal mucosa and saliva samples.
- Table 4 Pathway analysis for differentially methylated genes between intestinal mucosa and saliva samples.
- Comparisons of methylation profiles were made within individuals which likely minimised any ef- fect of the different gastric issues on the methylation profiles.
- Similarly, in coeliac disease where the typical symptoms are observed in the abdominal region, the disease may manifest in the oral cavity as oral ulcerations, changes in the salivary and parotid glands, and changes in the tongue [31]..
- The gut microbiome can effect DNA methylation pro- files via the production of metabolites, which alters the pool of compounds used in epigenetics modifications, or through inhibiting enzymes involved in epigenetic path- ways [32].
- The differences in methylation profiles ob- served may be due to differences in the microbiota of these two tissues.
- Further investigations would be needed to assess if there are differences in the micro- biome profiles between the oral and intestinal micro- biota and if these are causing the differences observed in the methylation profiles..
- In addition to the clustering of saliva and intestinal tissue into two distinct groups, saliva samples also clustered into two distinct groups.
- Diet has been shown to modify microbiota populations as well as methylation profiles [33–35].
- Further investigations comparing DNA methylation profiles from saliva samples between coeliac and non-coeliac individuals in a larger dataset would be of interest.
- This suggests that saliva is a po- tentially more informative tissue compared to intestinal mucosa with regard to DNA methylation due to the ability to detect differences between disease groups..
- Half of our participants were former smokers, while smoking can have long-lasting effects on DNA methyla- tion patterns, it has been shown that individuals who quit smoking have DNA methylation profiles similar to those of non-smokers [36].
- It has been suggested how- ever that environmental agents can induce the same epi- genetic changes in the oral mucosa as it does in distant.
- Therefore, any methylation changes caused by past smoking in the oral mucosa could occur in the gut as well.
- Comparisons were conducted within an indi- vidual to account for the effects of past smoking on DNA methylation profiles..
- The results from this study provide a framework for the use of saliva in DNA methylation research studies in in- testinal conditions.
- Overall, positive correla- tions in global DNA methylation were observed between saliva and intestinal mucosa tissues.
- Further studies investigating the influence of a gluten-free diet on saliv- ary DNA methylation profiles would be of interest..
- Biopsies were excised from the duodenal region of the gastrointestinal tract and stored at − 80 °C..
- Methylation measurements of a subset of probes on the microarray can be confounded by underlying SNPs and cross-hybridisation to other areas of the genome.
- 5% within 5 bp of the single base exten- sion site were removed from all analyses based on the SNP annotation data provided by Illumina, the Bioconductor package minfi [39], and Chen and colleagues [41].
- While DNA methylation studies can report effect sizes as small as 5% [15], a more stringent definition of ab- solute beta difference of 20% was used as we would expect larger differences between tissue types.
- Furthermore, these cut-offs are in line with previous studies comparing DNA methylation between tissues .
- Correla- tions between methylation in saliva and intestinal mucosa were measured using the cor.test function, at each probe site between paired samples, using Pearson’s product mo- ment correlation coefficient.
- Following this, the topGO or topKEGG function of the limma package was used to identify the most significant GO terms and KEGG pathways..
- Significantly differentially methylated probe sites between intestinal mucosa and saliva.
- Significantly differentially methylated regions between intestinal mucosa and saliva.
- Manhattan plot of each CpG site plotted according to genomic position on the x-axis and the strength of the association.
- The dataset(s) supporting the conclusions of this article is(are) available in the NCBI ’ s Gene Expression Ombnibus repository [48], and are accessible through GEO Series accession number GSE118260 (https://www.ncbi.nlm.nih..
- Identification of disease-associated DNA methylation in intestinal tissues from patients with inflammatory bowel disease.
- DNA methylation dynamics during the mammalian life cycle.
- CpG-rich islands and the function of DNA methylation.
- Tissue specific DNA methylation of CpG islands in normal human adult.
- DNA methylation of colon mucosa in ulcerative colitis patients: correlation with inflammatory status.
- Prognostic DNA methylation biomarkers in ovarian cancer.
- DNA methylation analysis in the intestinal epithelium-effect of cell separation on gene expression and methylation profile.
- DNA methylation profiling in inflammatory bowel disease provides new insights into disease pathogenesis.
- Whole-genome saliva and blood DNA methylation profiling in individuals with a respiratory allergy..
- Salivary DNA methylation profiling: aspects to consider for biomarker identification.
- Oral epithelium as a surrogate tissue for assessing smoking-induced molecular alterations in the lungs.
- comparison of the response rate and quality of genomic DNA.
- Comparison of whole-genome DNA methylation patterns in whole blood, saliva, and lymphoblastoid cell lines.
- The role of apelin in the modulation of gastric and pancreatic enzymes activity in adult rats.
- Tobacco smoking leads to extensive genome-wide changes in DNA methylation.
- Minfi: a flexible and comprehensive Bioconductor package for the analysis of Infinium DNA methylation microarrays.
- Discovery of cross-reactive probes and polymorphic CpGs in the Illumina Infinium HumanMethylation450 microarray.
- An evaluation of methods correcting for cell-type heterogeneity in DNA methylation studies.
- De novo identification of differentially methylated regions in the human genome

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