The duodenal cell population-specific methylome of celiac disease
Abstract
Different factors are involved in the development of celiac disease (CD) and the importance of the HLA region is known since 1972. Fernandez-Jimenez and collaborators studied the contribute of the methylome on the CD pathology. They found a characteristic methylation pattern in duodenal cell populations in CD patients, especially for the epithelial fraction, which is differentially methylated compared to controls.
Celiac disease develops in genetically susceptible individuals who generate an immune response consequently to the ingestion of gluten, but are also involved other factors, for example environmental factors and the well-known genetic factors linked to the HLA-region. The immune response within the duodenal epithelium is caused by the activity of the transglutaminase which deaminates gliadine, the main component of gluten, allowing its interaction with HLA-DQ2 and HLA-DQ8 surface molecules of APC. This process triggers also an intraepithelial lymphocytosis. [1,2] The article by Nora Fernandez-Jimenez and collaborators [3], published on scientific reports last February, aims at defining the duodenal cell population-specific methylome of celiac disease, understanding its interaction with the CD-associated genetic variations, studying possible changes at transcriptional level, and finally, verifying whether some of the genetic associations in CD could be related to allele-specific DNA methylation.
The authors focused their attention on two principal cell populations of the duodenal mucosa: enterocytes and immune cells, characterized by epithelial cell adhesion molecules EpCAM and CD45 antigen, respectively. Both have been separated by magnetic cell separation technology and then RNA and DNA have been extracted for sequencing. Epithelial fractions and immune fractions of patients and controls were treated with bisulfite and sequenced by the Illumina Infinium HumanMethylation450 microarray to find out the methylated positions on the genome. In this way they could detect, differentially methylated positions (DMPs), differentially methylated regions (DMRs), and differentially variable and methylated CpG sites (DVMCs). Comparison between CD patients and controls identified 43 DMPs (11 hypo and 32 hypermethylated) for the epithelial fraction, and 310 DMPs (40 hypo and 270 hypermethylated) for the immune fractions. They seemed to be more localized in TSS proximal regions and promoters causing a slight decrease of CpG islands, but these results are not significant in every position. They examine the positions of the CGI subject to greater variability and greater difference in methylation associated with celiac disease. The results for the epithelial fraction of CD patients showed a significant different methylation compared to controls and 4 genes were characterized by a high level of variability in CD samples. Not significant DVMCs were found for the immune fractions.
Significant DMPs were taken under consideration to study DMRs on single genes. The direct bisulfite sequencing has been used to confirm the differential methylation of single genes, in this specific case TAP1 and HLA-B, of an independent cohort of complete biopsies from 7 gluten free diet treated patients and 7 controls.
They found the most important HLA-DMRs: one made of two small regions surrounding the gene promoter of TAP1 and another consisted of an hypomethylated CpG shore in HLA-B promoter region. The DMR mapped to TAP1 showed a stronger methylation trend in the epithelial compartment than in the immune cells. DMR on HLA-B was significant only for the epithelial fraction, instead. These results were confirmed by the validation of the differential methylation of the TAP1 and HLA-B promoters, performing RNA sequencing. As expected, TAP1 was overexpressed in both immune and epithelial fractions, while HLA-B was upregulated only in the epithelial fraction. This epigenetic association to gene expression appears tissue-specific. The degree of DNA methylation at a CpG dinucleotide can correlate with the SNP genotype, creating a methylation quantitative trait locus. The mQTLs may affect the methylation across the genome and underline direct SNP associations or gene-environment interactions. [4] The samples were previously genotyped to individuate the SNPs, then the Immunochip SNP genotypes were analysed together with the methylation results to detect if methylation changes overlap sequence variants and to study the phenotypic effect due to the interplay of these two factors. Methylation levels of the CpGs regulated by the mQTLs did not differ between CD patients and controls, this could indicate that the celiac methylation signature is independent from the mQTL-SNP genotypes. Several studies have suggested that there may be additional alleles in the HLA region that confer increased disease risk over and above HLA-DQ [5], moreover it is known that DQ2 and DQ8 aplotipes are necessary but not sufficient for celiac disease development. For this reason, authors focalized their research on the whole HLA-region conducing the first methylome study in duodenal mucosa samples from CD patients. The limited number of samples taken in account could represent a limit of the study, and so the use of much larger numbers of patients and controls is necessary to have more significant results. From our point of view, it would be advantageous having DQ2 or DQ8 positive symptomatic patients allowing a research on genetic homogeneous samples.
This study opens the way for new researches about duodenal specific methylome and its interaction with the micro-environmental factors linked to celiac disease.
References
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- Lebwohl B, Sanders DS, Green PHR. Coeliac disease. 391, 70–81 (2018).
- Fernandez-Jimenez N et al. The methylome of the celiac intestinal epithelium harbours genotype independent alterations in the HLA region. Scientific Reports. 9: 1298 (2019).
- Smith AK et al. Methylation quantitative trait loci (meQTLs) are consistently detected across ancestry, developmental stage, and tissue type. BMC Genomics. 15, 145 (2014)
- Van Heel DA, Hunt K, Greco L, Wijmenga C. Genetics in coeliac disease. Best Practice & Reserch: Clinical Gastroenterology. 19, 323-339 (2005).