Global DNA methylation of peripheral blood leukocytes from dogs bearing multicentric non-Hodgkin lymphomas and healthy dogs: A comparative study

Abstract

Non-Hodgkin lymphomas are among the most common types of tumors in dogs, and they are currently accepted as comparative models of the disease in humans. Aberrant patterns of DNA methylation seem to play a key role in the development of hematopoietic neoplasms in humans, constitute a special mechanism of transcriptional control, and may be influenced by genetic and environmental factors. Blood leukocyte DNA global methylation has been poorly investigated in dogs. The aim of this study is to examine whether peripheral blood global DNA methylation is associated with canine multicentric lymphomas. Peripheral venous blood samples from ten healthy dogs and nine dogs bearing multicentric lymphomas were collected, and the buffy coat was separated. Global DNA methylation was analyzed by High Performance Liquid Chromatography (HPLC) and immunocytochemistry (ICC). In both analyses, leukocytes from dogs with lymphoma presented lower global DNA methylation than in healthy dogs (HPLC: p = 0.027/ 5MeCyt immunoreactivity scores: p = 0.015). Moderate correlation was observed between the results obtained by HPLC and ICC (correlation coefficient = 0.50). For the identification of differently methylated genes between both groups, the Infinium Human Methylation (HM) EPIC BeadChip (850K) was used. Of the 853,307 CpGs investigated in the microarray, there were 34,574 probes hybridized in the canine samples. From this total, significant difference was observed in the methylation level of 8433 regions, and through the homologous and orthologous similarities 525 differently methylated genes were identified between the two groups. This study is pioneer in suggesting that dogs bearing non-Hodgkin lymphoma presented DNA global hypomethylation of circulating leukocytes compared with healthy dogs. Although canine samples were used in an assay developed specifically for human DNA, it was possible to identify differently methylated genes and our results reiterate the importance of the use of peripheral blood leukocytes in cancer research and possible new biomarkers targets.

Cross-species examination of X-chromosome inactivation highlights domains of escape from silencing

“…Having male DNAme as a control and an upper threshold for calling genes as subject to XCI should reduce the chance of calling a gene as subject to XCI if it is instead silenced on both copies of the X in a tissue-specific manner. For the primate and dog samples which used the human 450k DNAme array, only probes which mapped consistently between the species were kept by the source publications [ 50 , 51 ], and so these species may be enriched for genes with a conserved XCI status. Utilizing datasets from different studies confounds the species differences with other experimental differences including sample size as well as inclusion of male samples.…”

Cross-species examination of X-chromosome inactivation highlights domains of escape from silencing

“…Past studies of XCI status calls using DNAme in human did not see many differences in DNAme-based XCI status across tissues [23], so different tissues analyzed may not cause many discordancies. For the primate and dog samples which used the human 450k methylation array, only probes which mapped consistently between the species were kept by the source publications [50,51], and so these species may be enriched for genes with a conserved XCI status. Utilizing datasets from different studies confounds the species differences with other experimental differences including sample size as well as inclusion of male samples.…”

Epigenetic Mechanisms in Canine Cancer

“…A recent study has shown that genome-wide hypomethylation was frequently found in grade III canine mast cell tumor, which is the most common skin tumor in dogs, thus correlating DNA hypomethylation with the aggressiveness of this type of cancer (53). In addition, dogs bearing non-Hodgkin lymphoma (NHL) exhibit higher DNA global hypomethylation of circulating leukocytes in comparison with healthy dogs (54). DNA hypomethylation was also observed in canine lung cancer samples and in metastatic osteosarcoma from the primary lung cancer (55).…”