Environmental DNA Methylation Signatures of Adolescent Victimization: Analysis of a Longitudinal Monozygotic Twin Sample, 2015-2020

Accumulating evidence suggests that individuals exposed to victimization at key developmental stages may have different epigenetic fingerprints compared to those exposed to no/minimal stressful events, however results are inconclusive. This study aimed to strengthen causal inference regarding the impact of adolescent victimization on the epigenome by controlling for genetic variation, age, gender, and shared environmental exposures. We conducted longitudinal epigenome-wide association analyses (EWAS) on DNA methylation (DNAm) profiles of 118 monozygotic (MZ) twin pairs from the Environmental Risk study with and without severe adolescent victimization generated using buccal DNA collected at ages 5, 10 and 18, and the Illumina EPIC array. Additionally, we performed cross-sectional EWAS on age-18 blood and buccal DNA from the same individuals to elucidate tissue-specific signatures of severe adolescent victimization. Our analyses identified 20 suggestive differentially methylated positions (DMPs) (P < 5e-05), with altered DNAm trajectories between ages 10–18 associated with severe adolescent victimization (∆Beta range = −5.5%−5.3%). Age-18 cross-sectional analyses revealed 72 blood (∆Beta range = −2.2%−3.4%) and 42 buccal (∆Beta range = −3.6%−4.6%) suggestive severe adolescent victimization-associated DMPs, with some evidence of convergent signals between these two tissue types. Downstream regional analysis identified significant differentially methylated regions (DMRs) in LGR6 and ANK3 (Šidák P = 5e-09 and 4.07e-06), and one upstream of CCL27 (Šidák P = 2.80e-06) in age-18 blood and buccal EWAS, respectively. Our study represents the first longitudinal MZ twin analysis of DNAm and severe adolescent victimization, providing initial evidence for altered DNA methylomic signatures in individuals exposed to adolescent victimization.<p>Stress is a normal, adaptive response to stressors (e.g. events that make a person feel threatened or upset) in our environment. However extensive research on the biology of stress now shows that healthy development can be derailed by excessive or prolonged activation of stress response systems in the body especially during important developmental periods in life such as adolescence. Exposure to severe stress during early life unfortunately is not uncommon. A World Health Organization survey reported that nearly 40% of adults experienced some form of severe stress during childhood and adolescence. Exposure to severe stress may have immediate or long lasting damaging effects on learning, behavior, and health. Therefore, it is imperative that we develop a better understanding of how exposure to psychosocial stress during adolescence gets under the skin to leave lasting biological imprints.

There is now increasing evidence to show that one of the ways in which severe psychosocial stress exposure can lead to physical and emotional problems is by getting underneath the skin and influencing the degree to which genes are turned on and off. This regulation of gene expression is known as epigenetics and often occurs through changes in DNA methylation. Initial studies have shown that individuals exposed to severe psychosocial stress have different patterns of DNA methylation (epigenetic 'signatures') compared to individuals exposed to no/minimal stressful life events. However limited conclusions can be drawn from these studies as they often do not fully account for factors that are important in one's reactivity to stress including age, sex and genetic difference between individuals.

Taking advantage of an established longitudinal twin study, our team of biological and social science experts will investigate epigenetic differences within 100 genetically identical twin pairs that exposed to different level of severe psychosocial stress during adolescence. In particular, all of these carefully selected twin pairs were not exposed to any major psychosocial stressors during childhood but in adolescence one twin is exposed to severe psychosocial stress while the co-twin is not. Severe psychosocial stress includes being physically attacked, beaten by parent, frequently bullied, sexually assaulted, persistently harassed on the internet or via a mobile phone, witnessing domestic violence, having a serious illness and being involved in an accident (e.g., a car accident). The use of genetically identical twins who essentially have the same genes and family experiences as well as age and sex will allow us to ascertain the 'purer' impact of psychosocial stress on the epigenetic signatures. Utilising repeated assessments over time (i.e. before and after exposure to stress) is a powerful design as it enables exploration of changes that occur within the same child rather than simply comparing one child to another. We will also investigate whether these stress-associated epigenetic signatures are similar or different in DNA extracted from cheek swabs and blood as this will help us to know whether these 'signatures' can be detected using minimally invasive procedures. The findings from this study will shed light on how biological systems operate under environmental challenge with the potential to ultimately advance understanding of how to sustain lifelong health and wellbeing and prevent health inequalities through early detection of the biological impact of psychosocial stress exposure which will aid prevention efforts. In addition, our multidisciplinary team is highly committed to increase awareness about epigenetics and how the environment may influence the way our genes are expressed and ultimately impact upon cognition and behaviour. This will be achieved through our team's ongoing public engagement with schools and science open days, as well as hosting workshops for university students across London.</p>

越来越多的证据表明，在关键发育阶段遭受受害经历的个体，其表观遗传指纹（epigenetic fingerprints）可能与未经历或仅经历极少应激事件的个体存在差异，但相关结果尚未明确。本研究旨在通过控制遗传变异、年龄、性别及共同环境暴露因素，强化青少年受害经历对表观基因组（epigenome）影响的因果推断。我们对环境风险研究（Environmental Risk study）中118对同卵（MZ）双胞胎的DNA甲基化（DNAm）谱进行了纵向表观基因组关联分析（EWAS），这些双胞胎在青少年时期存在或不存在严重受害经历；DNA样本为5岁、10岁及18岁时采集的颊黏膜DNA，采用Illumina EPIC芯片检测。此外，我们对同一批个体18岁时血液及颊黏膜DNA进行了横断面表观基因组关联分析（EWAS），以阐明青少年严重受害经历相关的组织特异性特征。

我们的分析鉴定出20个提示性差异甲基化位点（DMPs）（P < 5e-05），这些位点在10至18岁期间的DNA甲基化（DNAm）轨迹因青少年严重受害经历而改变（ΔBeta值范围：-5.5%至5.3%）。18岁时的横断面分析显示，血液中存在72个、颊黏膜中存在42个与青少年严重受害经历相关的提示性差异甲基化位点（DMPs），其ΔBeta值范围分别为-2.2%至3.4%和-3.6%至4.6%；两种组织类型间存在部分收敛信号的证据。下游区域分析在18岁血液和颊黏膜的EWAS中分别鉴定出显著的差异甲基化区域（DMRs）：血液中LGR6和ANK3基因区域（Šidák校正P值分别为5e-09和4.07e-06），以及颊黏膜中CCL27基因上游区域（Šidák校正P值为2.80e-06）。本研究是首个针对DNA甲基化与青少年严重受害经历的纵向同卵双胞胎分析，为遭受青少年受害经历个体的DNA甲基化特征改变提供了初步证据。

应激是机体对环境中应激源（如使人感到威胁或不安的事件）的正常适应性反应。然而，当前针对应激生物学的大量研究表明，过度或长期激活体内应激反应系统可能干扰健康发育，尤其在青春期等生命关键发育阶段。遗憾的是，生命早期暴露于严重应激并不少见：世界卫生组织（World Health Organization）的一项调查显示，近40%的成年人在童年和青少年时期经历过某种形式的严重应激。暴露于严重应激可能对学习、行为及健康产生即时或长期的损害。因此，深入理解青少年时期心理社会应激暴露如何对身体产生深远影响并留下持久生物印记至关重要。

目前越来越多的证据表明，严重心理社会应激暴露导致生理和情绪问题的途径之一，是通过深入影响基因的表达调控——这种基因表达调控被称为表观遗传学（epigenetics），且常通过DNA甲基化的改变实现。初步研究显示，经历严重心理社会应激的个体，其DNA甲基化模式（即表观遗传“特征”）与未经历或仅经历极少应激事件的个体存在差异。然而，这些研究的结论有限，因其往往未能充分考虑应激反应性的重要影响因素，如年龄、性别及个体间的遗传差异。

借助已建立的纵向双胞胎研究，我们的生物与社会科学专家团队将探究100对基因完全相同的双胞胎在青少年时期暴露于不同程度严重心理社会应激后的表观遗传差异。具体而言，这些精心筛选的双胞胎在童年时期均未暴露于任何重大心理社会应激源，但青春期时其中一方经历了严重心理社会应激，而另一方未经历。严重心理社会应激包括身体攻击、父母殴打、频繁欺凌、性侵犯、持续的网络或手机骚扰、目睹家庭暴力、患严重疾病及遭遇事故（如车祸）。利用基因、家庭经历、年龄及性别基本一致的同卵双胞胎，可帮助我们确定心理社会应激对表观遗传特征的“更纯粹”影响。通过随时间重复评估（即应激暴露前后）的设计极具优势，因其能探索同一儿童体内发生的变化，而非简单比较不同儿童。我们还将探究这些应激相关表观遗传特征在颊拭子和血液提取的DNA中是否相似或不同，这将有助于了解是否可通过微创方法检测这些“特征”。

本研究的发现将阐明生物系统在环境挑战下的运作机制，有望最终增进对如何维持终身健康与福祉、如何通过早期检测心理社会应激暴露的生物影响来预防健康不平等的理解，从而为预防工作提供助力。此外，我们的多学科团队致力于提高公众对表观遗传学及环境如何影响基因表达进而最终作用于认知与行为的认知。这将通过团队持续的公众参与（如与学校合作、举办科学开放日）以及为伦敦各高校学生举办研讨会来实现。