Genome-wide DNA methylation profiles according to Chlamydophila psittaci infection and the response to doxycycline treatment in ocular adnexal lymphoma

Abstract

Purpose: To compare genome-wide DNA methylation profiles according to Chlamydophila psittaci (Cp) infection status and the response to doxycycline treatment in Korean patients with ocular adnexal extranodal marginal zone B-cell lymphoma (EMZL).

Methods: Twelve ocular adnexal EMZL cases were classified into two groups (six Cp-positive cases and six Cp-negative cases). Among the 12 cases, eight were treated with doxycycline as first-line therapy, and they were divided into two groups according to their response to the treatment (four doxy-responders and four doxy-nonresponders). The differences in the DNA methylation states of 27,578 methylation sites in 14,000 genes were evaluated using Illumina bead assay technology. We also validated the top-ranking differentially methylated genes (DMGs) with bisulfite direct sequencing or pyrosequencing.

Results: The Infinium methylation chip assay revealed 180 DMGs in the Cp-positive group (74 hypermethylated genes and 106 hypomethylated genes) compared to the Cp-negative group. Among the 180 DMGs, DUSP22, which had two significantly hypomethylated loci, was validated, and the correlation was significant for one CpG site (Spearman coefficient=0.6478, p=0.0262). Regarding the response to doxycycline treatment, a total of 778 DMGs were revealed (389 hypermethylated genes and 336 hypomethylated genes in the doxy-responder group). In a subsequent replication study for representative hypomethylated (IRAK1) and hypermethylated (CXCL6) genes, the correlation between the bead chip analysis and pyrosequencing was significant (Spearman coefficient=0.8961 and 0.7619, respectively, p<0.05).

Conclusions: Ocular adnexal EMZL showed distinct methylation patterns according to Cp infection and the response to doxycycline treatment in this genome-wide methylation study. Among the candidate genes, DUSP22 has a methylation status that was likely attributable to Cp infection. Our data also suggest that the methylation statuses of IRAK1 and CXCL6 may reflect the response to doxycycline treatment.

Figure 1

Amplification of Chlamydophila psittaci (Cp) DNA using PCR in ocular adnexal extranodal marginal zone B-cell lymphoma (EMZL). Six cases of 12 ocular adnexal EMZL showed positive bands and defined as Cp-positive samples, and the others were grouped as Cp-negative group. P, positive control for C. psittaci; N, negative control; L, size marker 485 (100-bp DNA ladder).

Figure 2

Hierarchical clustering analysis based on the DNA methylation data obtained from six Chlamydophila psittaci–positive and six Chlamydophila psittaci–negative ocular adnexal extranodal marginal zone B-cell lymphoma cases (Chlamydophila psittaci [Cp]-positive: P1–P6, Cp-negative: N1–N6). The 184 significant methylated CpG sites were selected with the criteria |delta mean| >0.06 and p<0.05. The color scale of the heat map represents densely methylated loci (red) to sparsely methylated loci (green). All cases were clearly clustered into two groups.

Figure 3

Results of direct bisulfite sequencing for two differentially methylated CpG sites of DUSP22 in ocular adnexal extranodal marginal zone B-cell lymphoma. A: The first CpG site, target ID cg15383120, promoter, CpG island. B: The second CpG site, target ID cg11235426, nonpromoter, CpG island. Each row represents a bacterial clone with a circle symbolizing a CpG site. Methylated and unmethylated CpG sites are indicated by black and white circles, respectively. Mutated sites are indicated by gray circles. Correlation analysis between the Infinium methylation chip assay and bisulfite sequencing revealed that only the second CpG site showed significant correlation between the two methods.

Figure 4

Cluster analysis and heatmap of methylation level between ocular adnexal extranodal marginal zone B-cell lymphoma doxy-responders (n=4) and doxy-nonresponders (n=4). The methylation levels at the 778 CpG sites were used for hierarchical clustering. All cases were clearly clustered into two groups in this dendrogram.

Figure 5

Validation of Infinium methylation chip assay by pyrosequencing. Pyrogram data of A: CXCL6 and B: IRAK1 in ocular adnexa extranodal marginal zone B-cell lymphoma. C: Correlation analysis between Infinium Methylation Chip Assay and pyrosequencing of IRAK1 and CXCL6.