Genome-wide DNA methylation profiling in rheumatoid arthritis identifies disease-associated methylation changes that are distinct to individual T- and B-lymphocyte populations

Figures & data

Figure 1. LINE-1 DNA methylation in T-lymphocytes and B-lymphocytes from patients with rheumatoid arthritis. Methylation at three adjacent CpG sites within LINE-1 repetitive sequences was quantified in purified T- and B-lymphocytes from 12 RA patients by sodium bisulfite pyrosequencing. Unfilled circles and crosses represent T-lymphocytes and B-lymphocytes, respectively. The mean levels of methylation for each of the CpG sites are shown by the short black horizontal bars.* P ≤ 0.01 (Wilcoxon Signed-Rank Test).

Figure 2. DNA methylation heatmap for the 679 CpG methylation signature in RA derived T- and B-lymphocytes. CpGs presented in the heatmap were described in our previous work (22) and represent those sites which define a unique methylation signature distinguishing healthy T-lymphocytes from B-lymphocytes. Each row represents an individual CpG, ordered according to the clustering output in healthy individuals (22), and each column a different sample (listed beneath the heatmap). Color gradation from yellow to blue represents low to high DNA methylation respectively, with β-values ranging from 0 (no methylation; yellow) to 1 (complete methylation; blue). Intrinsic methylation differences previously observed between the cell types in healthy individuals are accurately preserved in RA patients.

Figure 3. Filtering criteria for identification of CpGs differentially methylated between RA patients and healthy individuals. For T-lymphocytes and B-lymphocytes separately, the starting number of CpGs (477,312 and 479,639 for T- and B-lymphocytes respectively) was derived through the removal of unreliable sites (CpGs with detection p-values > 0.05) and those with missing β-values, as described in the Methods. Numbers indicate the number of CpGs remaining at each step.† Refers to CpGs for which all RA samples showing a change from the healthy mean ± two-times the SD in the preceding step were either hypermethylated or hypomethylated in RA.

Table 1. Summary characteristics for the differentially methylated CpGs identified in RA derived T-lymphocytes and B-lymphocytes.*

	T-lymphocyte CpG candidates	B-lymphocyte CpG candidates	All differentially methylated CpGs
CpGs differentially methylated†	32	20	52
In CpG islands	6 (18.8)	1 (5.0)	7 (13.5)
In CpG island shores	10 (31.3)	2 (10.0)	12 (23.1)
In CpG island shelves	3 (9.4)	5 (25.0)	8 (15.4)
Non-island associated CpGs	13 (40.6)	12 (60.0)	25 (48.1)
Hyper/Hypomethylated CpGs	17/15	0/20	17/35
CpGs associated with a gene†	18 (56.3)	10 (50.0)	28 (50.0)
In TSS200/TSS1500	7 (38.9)	2 (20.0)	9 (32.1)
In 5′ UTR/1st Exon	1 (5.6)	0 (0.0)	1 (3.6)
In Gene body	8 (44.4)	7 (70.0)	15 (53.6)
In 3′ UTR	2 (11.1)	1 (10.0)	3 (10.7)
CpGs associated with a promoter	12 (37.5)	2 (10.0)	14 (26.9)
Unique genes represented	15	10	23‡

Unless otherwise indicated, all figures are the number (%); * Differentially methylated CpGs were identified according to the criteria described in the Results section and Figure 3;† Annotation describing CpG island, shore and shelf status, and genetic location relative to defined gene regions, is derived from the University of California at Santa Cruz (UCSC) database;‡ Two genes were common to both the T-lymphocyte and B-lymphocyte candidate sites; Abbreviations: TSS200, 200-bp block upstream of the transcription start site; TSS1500, 1500-bp block upstream of the transcription start site; 5′ UTR, 5′ untranslated region; 3′ UTR, 3′ untranslated region.

Figure 4. Validation by bisulfite pyrosequencing of differentially methylated CpG candidates in RA derived T- and B-lymphocytes. For each cell type, four candidate CpGs that were differentially methylated between RA patients and healthy individuals were selected. This included two sites each that were hypermethylated (ARSB and DUSP22) and hypomethylated (GALNT9 and MGMT) in RA derived T-lymphocytes (A), and included four hypomethylated sites in B-lymphocytes (B). In each plot, unfilled triangles indicate healthy individuals and where the mean methylation and two-times the SD of the mean are indicated for the array data by short blue horizontal bars with vertical stops and with arrowheads, respectively. RA patients are indicated by circles, where filled circles in the array data indicate the individual samples that were identified as differentially methylated relative to healthy individuals (as defined by the criteria in Figure 3). These same samples are also indicated by filled circles in the pyrosequencing data. Abbreviations: Pyro., bisulfite pyrosequencing.

Figure 5. Identification of additional differentially methylated CpGs for two array-identified candidates in RA derived T-lymphocytes. Shown are additional CpGs adjacent to the array-identified candidate site for DUSP22 (two additional sites) (A) and GALNT9 (four additional sites) (B) that were also found to be differentially methylated in RA patients relative to healthy individuals, as determined by pyrosequencing. Negative base pair numbers on the x-axis indicate bases upstream of the array CpG site. In each plot, unfilled triangles indicate healthy individuals and where the mean methylation is indicated by short blue horizontal bars with vertical stops. RA patients are indicated by circles, where filled circles for the array site indicate the individual samples that were identified as differentially methylated relative to healthy individuals (as defined by the criteria in Figure 3). These same samples are also indicated by filled circles in each of the adjacent CpGs presented. Abbreviations: bp, base pairs.