Introduction
Coeliac disease (CD) is an autoimmune disorder characterised by chronic inflammation of the small intestine in response to dietary gluten. Genetic predisposition, including the presence of human leukocyte antigen (HLA) haplotypes, HLA-DQ2 or HLA-DQ8, is widely recognised in CD pathogenesis.1,2 However, these haplotypes are common and non-specific, and perhaps half of the disease heritability remains unexplained despite GWAS studies. Epigenetics, as an interface between genome and environment, may also partly explain disease susceptibility, as well as identify novel biological processes, therapeutic targets and biomarkers.3
Aims & Methods
Previously, altered DNA methylation profiles have been found in the intestinal tissue and saliva of patients with CD.4 Our objective is to compare the DNA methylation profiles of whole blood between children with CD and healthy controls (HC) to identify unique DNA methylation profiles, an approach that has proven to be fruitful in inflammatory bowel diseases (IBD). Most studies have focused on epigenetic alterations in adult CD populations despite it being more commonly diagnosed in children and young adults. Moreover, we aim to determine whether CD-associated age acceleration exists, as observed in adult IBD patients.5
Paediatric patients with CD (n=36) and HCs (n=35) well-matched for age and sex were recruited at the John Radcliffe Hospital, Oxford, UK. DNA methylation was assayed at 850,000 CpGs with HumanMethylationEPIC arrays (Illumina). After pre-processing in minfi, epigenome-wide methylation changes were analysed with limma, using age, sex, Marsh scores, and cell counts as covariates, and with Benjamini-Hochberg correction for multiple testing. Epigenetic age was determined using general (Horvath6) and paediatric-specific (Wu7) clock models. Genotyping was assayed using Global Screening Arrays (Illumina), with data prepared in GenomeStudio and PLINK. Cis-meQTLs within 300kbp were evaluated using linear regression.
Results
Fifty-three CpGs were significant genome-wide (FDR <0.05) between CD and HCs. Among these, 23 CpGs exhibited hypermethylation while 30 were hypomethylated, with top loci including protein tyrosine phosphatase PTPN6 (p=4.79×10-9); CDC42 (1.28×10-9), involved in cell migration, structure, and phagocytosis; and collagen gene COL4A1 (9.95×10-8). The significant probes were found to be associated with ontology terms concerning cytidine metabolism (p=6.68×10-5), collagen metabolism (1.0×10-3) and filopodium assembly (8.6×10-4). No significant CpGs were found in the HLA region in this study. Differentially methylated regions containing clusters of significant CpGs were identified within the genes APOBEC3B (3 CpGs), ASGR1 (2), and CCNG1 (2). The significant CpG with the strongest evidence of genetic regulation in a meQTL was NOP10 (p=4.48×106). There was some evidence of genetic regulation of methylation at other significant CpGs including CDC42 and PTPN6 but these did not survive correction for multiple testing due to low power in this small cohort. No age acceleration was observed in CD patients with either epigenetic clock.
Conclusion
Paediatric patients with coeliac disease showed altered blood DNA methylation compared to controls, identifying novel targets for further investigation, although HLA region methylation was not altered in this cohort in contrast to some reports in adult CD. Age acceleration was not observed in paediatric CD patients, perhaps reflecting a short disease duration and mild systemic inflammation.
References
1. Karell, K. et al. HLA types in celiac disease patients not carrying the DQA1 *05-DQB1 *02 (DQ2) heterodimer: Results from the European genetics cluster on celiac disease. Hum Immunol 64, 469–477 (2003).
2. Sollid, L. M. et al. Evidence for a primary association of celiac disease to a particular HLA-DQ alpha/beta heterodimer. J Exp Med 169, 345–350 (1989).
3. Anderson, R. P. et al. A novel serogenetic approach determines the community prevalence of celiac disease and informs improved diagnostic pathways. BMC Med 11, 1–13 (2013).
4. Hearn, N. L., Chiu, C. L. & Lind, J. M. Comparison of DNA methylation profiles from saliva in Coeliac disease and non-coeliac disease individuals. BMC Med Genomics 13, 1–11 (2020).
5. Kalla, R. et al. Analysis of Systemic Epigenetic Alterations in Inflammatory Bowel Disease: Defining Geographical, Genetic and Immune-Inflammatory influences on the Circulating Methylome. J Crohns Colitis 17, 170–184 (2023).
6. Horvath, S. DNA methylation age of human tissues and cell types. Genome Biol 14, (2013).
7. Wu, X. et al. DNA methylation profile is a quantitative measure of biological ageing in children. Ageing 11, 10031–10051 (2019).
