Session Title: Genetics and Genomics of Rheumatic Disease II
Session Type: Abstract Submissions (ACR)
Background/Purpose: Multiple reports now describe changes to the DNA methylome in a variety of autoimmune disorders, such as rheumatoid arthritis. In many cases, these studies have analysed methylation in mixed cell populations from whole blood. However, the cellular heterogeneity inherent in these approaches may preclude the identification of cell type-specific methylation differences, which may in turn bias identification of disease-specific changes in methylation. To address this possibility, we used genome-wide DNA methylation profiling to identify differences within matched pairs of T-lymphocytes and B-lymphocytes isolated from the blood of healthy individuals.
Methods: Peripheral blood samples were collected from 10 healthy female donors (all Caucasian non-smokers). T- and B-lymphocyte populations were isolated from each individual using magnetic beads and genomic DNA was extracted. Sodium bisulphite converted DNA was hybridised to HumanMethylation450 BeadChips to establish quantitative genome-wide DNA methylation at more than 450,000 CpG sites. Methylation status at individual CpGs was reported as a β-value on a continuous scale ranging from 0 (unmethylated) to 1 (completely methylated). Array processing and identification of differential methylation was performed using NIMBL software. Pyrosequencing was used to validate array data.
Results: Genome-wide methylation was initially determined by analysis of LINE-1 sequences and was significantly higher in B-lymphocytes than matched T-lymphocytes (69.8 vs. 65.2%, p ≤ 0.01). Pairwise analysis identified 679 CpGs, representing 250 genes, which were differentially methylated between T-lymphocytes and B-lymphocytes. Approximately half of the sites (326; 48%) displayed β-value differences of at least 0.5, and a similar number were associated with a CpG island. The majority of the sites (76.6%) were hypermethylated in B-lymphocytes compared with T-lymphocytes. Pyrosequencing analysis of selected candidate CpGs/genes confirmed the array data in all cases. Hierarchical clustering revealed perfect segregation of the samples into two distinct clusters based on cell type. Differentially methylated genes showed enrichment for biological functions/pathways associated with leukocytes and T-lymphocytes.
Conclusion: Matched pairs of T- and B-lymphocytes from healthy individuals possess intrinsic differences in DNA methylation within a restricted set of functionally-related genes. These data provide a foundation for investigating DNA methylation in diseases in which these cell types play important and distinct roles.
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ACR Meeting Abstracts - https://acrabstracts.org/abstract/genome-wide-profiling-identifies-significant-differences-between-the-t-lymphocyte-and-b-lymphocyte-methylomes-in-healthy-individuals/