Genetic and environmental determinants of

Background: Epigenetic inactivation of O‐methylguanine DNA‐methyltransferase (MGMT) is associated with increased sensitivity to alkylating chemotherapeutic agents in glioblastoma patients. The genetic background underlying MGMT gene methylation may explain individual differences in treatment response and provide a clue to a personalized treatment strategy. Making use of the longitudinal twin design, we aimed, for the first time, to estimate the genetic contributions to MGMT methylation in a Danish twin cohort. Methods: DNA‐methylation from whole blood (18 monozygotic (MZ) and 25 dizygotic (DZ) twin pairs) repeated 10 years apart from the Longitudinal Study of Aging Danish Twins (LSADT) were used to search for genetic and envi‐ ronmental contributions to DNA‐methylation at 170 CpG sites of across the MGMT gene. Both univariate and bivariate twin models were applied. The intraclass correlations, performed on cross‐sectional data (246 MZ twin pairs) from an independent study population, the Middle‐Aged Danish Twins (MADT), were used to assess the genetic influence at each CpG site of MGMT for replication. Results: Univariate twin model revealed twelve CpG sites showing significantly high heritability at intake (wave 1, h > 0.43), and seven CpG sites with significant heritability estimates at end of follow‐up (wave 2, h > 0.5). There were six significant CpG sites, located at the gene body region, that overlapped among the two waves (h > 0.5), of which five remained significant in the bivariate twin model, which was applied to both waves. Within MZ pair correlation in these six CpGs from MADT demarks top level of genetic influence. There were 11 CpGs constantly have substantial common environmental component over the 10 years. Conclusions: We have identified 6 CpG sites linked to the MGMT gene with strong and persistent genetic control based on their DNA methylation levels. The genetic basis of MGMT gene methylation could help to explain individual differences in glioblastoma treatment response and most importantly, provide references for mapping the methyla‐ tion Quantitative Trait Loci (meQTL) underlying the genetic regulation. © The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/publi cdoma in/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Open Access *Correspondence: qtan@health.sdu.dk 1 Epidemiology, Biostatistics and Biodemography, Department of Public Health, University of Southern Denmark, J.B. Winsløws Vej 9 B., 5000 Odense C, Denmark Full list of author information is available at the end of the article Page 2 of 14 Wang et al. Clin Epigenet (2021) 13:35 Background Glioblastoma (GBM) is the most common malignant brain tumor which is highly fatal as its five-year relative survival is only 6.8% [1, 2]. The current standard therapeutic management for newly diagnosed GBM is maximal safe surgical resection, followed by systematic radiotherapy combined with concomitant and adjuvant temozolomide (TMZ). The addition of TMZ to radiotherapy has successively improved the long-time survival for GBM patients [3]. However, many of the GBM patients are insensitive to alkylating chemotherapeutic agents (e.g., TMZ) and thus cannot get benefit from the standard treatment [4]. One major cause is the silencing of the O6-methylguanine DNA-methyltransferase (MGMT) gene [5–7]. The MGMT gene resides in chromosome 10q26 and encodes a DNA-repair enzyme [8, 9]. Aalkylating agents-induced cytotoxicity is triggered by adding its methyl group to specific sites, especially O6 positions of guanine. The O6-MeG adduct causes cell killing by inaccurate pairing of methylated guanine with thymine during DNA replication. The MGMT protein restores alkylation-induced DNA lesion by transferring the methyl group from the O6-MeG adduct to a cysteine residue in its active site irreversibly, thus blunts the therapeutic effect of alkylating agents [10, 11]. The silencing of the MGMT gene expression can be affected by both genetic and epigenetic factors [12, 13]. It is widely accepted that the MGMT promoter methylation is the leading regulation mechanisms which reduce gene expression. The study of the relationship between gene expression and the methylation patterns of the overall and specific CpG sites [14] in the promoter of MGMT has been a topic of wide interest [15]. Everhard et al. [16] found six CpG sites, which were located in the promoter region of MGMT, highly correlated with expression in GBMs. Bady et al. [17] identified two distinct regions in the CpG island of the promoter with high importance for MGMT silencing in GBM. Though numerous studies have demonstrated that the MGMT promoter methylation status may determine the efficiency of TMZ treatment for the GBM patients [15–17], this biomarker has not yet been used in routine clinical practice to guide therapy for glioblastoma [18]. Methylation could also occur in the MGMT gene body. Gene body hypermethylation was positively correlated with MGMT expression in some GBM patients [19], which could partially explain the inconsistencies between the MGMT promoter methylation, gene expression level and different patient prognosis. There are 176 CpG sites annotated for MGMT by HumanMethylation450 (450 k) beadchips. The total amount of discrete CpG methylation patterns, and intratumoral methylation homogeneity of MGMT is variable in GBM [16, 20–22], and also other tumors [23]. In addition, Markus et al. [24] reported that there is considerable variation of MGMT activity in normal tissues. These findings indicate that a degree of inter-individuals methylation heterogeneity and intra-individual variability exists. DNA methylation is dynamic and changes throughout the life course, while its levels are affected by environmental factors, as well as genetic variation. Cisor trans-acting genetic factors, known as methylation Quantitative Trait Loci (meQTL) can introduce or disrupt CpG sites and have a significant effect on the methylation status of the specific gene. To our knowledge, genetic contribution or heritability in MGMT methylation is not well established and therefore, need more attention. Heritability is estimated by the correlation between genetic sharing and phenotypic sharing. Twin studies are regarded as the some of the best ways for assessing human heritability. Comparison of phenotype correlation in monozygotic (MZ) twin pairs who share their genetic makeups and dizygotic (DZ) twin pairs who share on average half of their genetic materials allows for better interpretation and quantification of genetic factors. Longitudinal twin studies on long term conservation of individual molecular phenotypes contribute to the exploration of genetic and environmental bases for maintaining molecular homeostasis [25–27]. This study introduces, for the first time, the twin design for disease studies to assess the genetic contribution to the molecular phenotype of MGMT methylation to provide (1) reference for mapping meQTL of the MGMT gene; and (2) explanation to the observed individual differences in treatment response.