Lin et al. Biology of Sex Differences (2019) 10:2 https://doi.org/10.1186/s13293-018-0213-7 RESEARCH Open Access Sex-related DNA methylation differences in B cell chronic lymphocytic leukemia Shuchun Lin1, Yun Liu2, Lynn R. Goldin3, Chen Lyu4, Xiangyin Kong1, Yan Zhang5, Neil E. Caporaso3, Song Xiang1 and Ying Gao1* Abstract Background: Men are at higher risk of developing chronic lymphocytic leukemia (CLL) than women. DNA methylation has been shown to play important roles in a number of cancers. There are differences in the DNA methylation pattern between men and women. In this study, we investigated whether this contributes to the sex-related difference of B cell CLL risk. Methods: Using the HumanMethylation450 BeadChip, we profiled the genome-wide DNA methylation pattern of CD19+ B cells from 48 CLL patients (29 female patients and 19 male patients) and 28 healthy people (19 women and 9 men). Results: We identified 1043 sex-related differentially methylated positions (DMPs) related to CLL, 56 of which are located on autosomes and 987 on the X chromosome. Using published B cell RNA-sequencing data, we found 18 genes covered by the DMPs also have different expression levels in male and female CLL patients. Among them, TRIB1, an autosome gene, has been shown to promote tumor growth by suppressing apoptosis. Conclusions: Our study represents the first epigenome-wide association study (EWAS) that investigates the sex-related differences in cancer, and indicated that DNA methylation differences might contribute to the sex-related difference in CLL risk. Keywords: DNA methylation, Chronic lymphocytic leukemia, B cell, Sex, EWAS Background DNA methylation plays important roles in regulating Chronic lymphocytic leukemia (CLL) is characterized by gene expression. There are considerable differences in proliferation and accumulation of malignant B lympho- the DNA methylation pattern between men and women. cytes in the peripheral lymphoid tissues and bone For instance, recent studies on human blood DNA re- marrow. It is one of the most common leukemias among vealed significant sex-related differences in its methyla- adults in the western world [1]. Its occurrence in men tion pattern [6–8]. DNA methylation changes are linked and women is drastically different [2]. For instance, the to many diseases [9]. In CLL patients, a strong change in Surveillance, Epidemiology, and End Results (SEER) DNA methylation pattern is reported [10]. This suggests database indicated that in 1975–2001, the US CLL that DNA methylation could play a role in the sex- incidence per 100,000 per year was 5.0 for men and 2.5 related differences in CLL. However, to date, solid evidence for women [3, 4]. In addition, female CLL patients have is lacking. better 10-year survival rates and show better response to We report here an epigenome-wide association study treatment [5]. Understanding the mechanism behind (EWAS) of CLL. Our study revealed 1043 sex-related these sex-related differences will provide valuable insights differentially methylated positions (DMPs) in CLL. Using into CLL. available RNA-sequencing data, we found 18 sex-related differentially expressed genes (DEGs) that overlapped with these DMPs. A number of these genes have been * Correspondence: [email protected] 1Shanghai Institutes for Biological Sciences, University of Chinese Academy reported to be associated with aggressive CLL progres- of Sciences, Chinese Academy of Sciences, Shanghai, China sion. To our knowledge, this study is the first EWAS Full list of author information is available at the end of the article © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Lin et al. Biology of Sex Differences (2019) 10:2 Page 2 of 11 that investigates the sex-related differences in cancer. Age was adjusted in the model. P values were corrected The differently methylated/expressed genes we identified for multiple testing by Benjamini-Hochberg FDR (q could be potential markers for CLL risk assessment and value). Probes with FDR under a threshold of 0.05 (q drug targets for CLL treatment. value < 0.05) were considered significant. For the auto- somal probes, 71 were significant between male and fe- Methods male CLL patients, and 101 were significant between Sample preparation healthy men and women. These 2 groups shared 15 In this study, 48 CLL subjects and 28 unrelated healthy common probes, which had the same methylation dif- controls were recruited from the NCI CLL Registry ference direction, but showed no difference in the [11]. A total of 92 blood samples were collected, with interaction term. Autosomal probes that had significant multiple samples collected from 8 subjects. Then, B methylation differences between male and female CLL lymphocytes were selected from cryopreserved periph- patients, but not between healthy men and women, were eral blood lymphocytes using a CD19 antibody. Cell defined as sex-related autosomal DMPs (N =56,Fig. 1a). purity was evaluated with flow cytometry using propi- For the X chromosomal probes, 7042 were significant dium iodide and CD45/CD19 antigens. Samples with between male CLL and female CLL patients, and 6772 greater than 90% purity were processed for DNA ex- were significant between healthy men and women. These traction and methylation analysis. 2 groups shared 6094 common probes, 39 of which were significant in the interaction term. X chromosomal probes Datasets for DMP replication that had significant methylation differences between male To replicate the DMPs we detected, we requested two and female CLL patients, but not between healthy men DNA methylation datasets accessed by 450K from The and women (N = 948), as well as those significant in the European Genome-phenome Archive (EGA), EGAD000 interaction term (N =39),weredefinedasXchromosomal 10000254 [12] and EGAD00010000871 [13]. Both con- DMPs (Fig. 2a). The same method was applied to the tain B cell samples from CLL patients and healthy people datasets for DMP replication. Details of the interaction (Additional file 1: Table S1). term were in Additional file 2. Datasets for DEG analysis DEG analysis We requested two RNA-sequencing datasets for B cell DEGs were identified by the limma package [19] using from CLL patients: EGAD00001000258 [14]fromEGA an interaction linear model adjusted for the study batch. and GSE66117 [15] from Gene Expression Omnibus P values corrected by FDR under the cutoff of 0.05 (q (GEO). Sex information of GSE66117 was obtained from value < 0.05) were considered significant. Sex-related the author. Data for healthy immortalized B cells (GSE DEGs were defined as genes with significantly different 16921 [16]) was used as a control. Since the mRNA expression levels between male and female CLL patients, expression of immortalized B cells might differ from but not between healthy men and women. normal B cells, two additional control datasets were re- quested. One contains two collections of CD19+ Bcells Other analyses from healthy women (GSM1523501 and GSM1523502 Methods for analysis of 450K BeadChip data, differen- from GSE62246 [17]); the other contains five collec- tially methylated region (DMR), functional epigenetic tions of CD19+ B cells from healthy men (GSM182 module (FEM), and Gene Ontology (GO), are shown in 0115, GSM1820116, GSM1820117, GSM1820118, and Additional file 2. GSM1820119 from GSE70830). Sex information was obtained from the author. Results Autosomal DMPs DMP analysis The characteristics of subjects are summarized in Table 1. After passing quality control of 450K BeadChip, our We identified 56 DMPs among 450k autosomal CpG sites, dataset contains 361,732 autosomal probes and 9482 X which had significant methylation differences between chromosomal probes from 89 samples. Samples obtained male and female CLL patients, but not between healthy after the first diagnosis of CLL (N = 76) were next used men and women (Fig. 1a). Among them, 22 were hyper- to identify DMP. Autosomal DMPs and X chromosomal methylated (hyper-DMPs) and 34 were hypomethylated DMPs were detected separately. We used linear regres- (hypo-DMPs) in male CLL patients, compared to female sion following the R package limma [18] to detect patients (Fig. 1b). Both hypo-DMPs and hyper-DMPs were probes with significant DNA methylation differences be- mainly enriched in CpG islands and promoter regions tween male and female CLL patients and between (Fig. 1c, both p values < 0.01 in Fisher’s test). These 56 healthy men and women, along with an interaction term. autosomalDMPsshowedlittledifferenceinDNA Lin et al. Biology of Sex Differences (2019) 10:2 Page 3 of 11 ab c Autosomes DMP(N=56) 1.00 MvsFinCLL MvsFinCon 0.75 56 15 85 Group CLLFemale 0 0.50 CLLMale 0 1 conFemale MeanBeta conMale 0 0.25 Diff 361575 0.00 HyperCLLM HypoCLLM 22 34 State d e 5 4 3 State HyperCLLM HypoCLLM 2 DMP counts 1 0 12345678101112131617181920 Chromosome f Autosomes DMP Top 25 Group conFemale conMale CLLFemale CLLMale 1.6e−01 3.7e−01 1.0e+00 1.0e+00 1.0e+00 1.0e+00 5.9e−01 1.0e+00 1.0e+00