Journal of Psychiatric Research 132 (2021) 38–43 Contents lists available at ScienceDirect Journal of Psychiatric Research journal homepage: www.elsevier.com/locate/jpsychires Short communication Childhood adversity increases methylation in the GRIN2B gene Elin Engdahl a,*,1, Ali Alavian-Ghavanini b,2, Yvonne Forsell c, Catharina Lavebratt d,e, Jo¨elle Rüegg f a Karolinska Institutet, Institute of Environmental Medicine (IMM), Unit of Integrative Toxicology, Stockholm, Sweden b Swetox, Karolinska Institutet, Unit of Toxicology Sciences, Sodert¨ alje,¨ Sweden c Karolinska Institutet, Department of Global Public Health, Stockholm, Sweden d Karolinska Institutet, Department of Molecular Medicine and Surgery, Stockholm, Sweden e Center for Molecular Medicine (CMM), Karolinska University Hospital, Stockholm, Sweden f Uppsala University, Department of Organismal Biology, Uppsala, Sweden ARTICLE INFO ABSTRACT Keywords: Childhood adversity is an early life stressor associated with increased risk of several psychiatric disorders such as GRIN2B depression. Epigenetic changes, primarily DNA methylation, can be affected by early life stress, which in turn DNA methylation might contribute to altered disease susceptibility later in life. One plausible biomarker of early life stress is Childhood adversity methylation of the ionotropic glutamate receptor NMDA type subunit 2B (GRIN2B) gene, which has been pre­ Early life stress viously shown to be epigenetically affected by prenatal environmental stressors. Here, we set out to investigate if Depression Epigenetics stress-inducing adversity during childhood is associated with changes in methylation of GRIN2B in adulthood. We studied 186 individuals from a Swedish naturalistic population-based cohort who had provided saliva samples (DNA) as well as information regarding both childhood adversity (CA) and depressive symptoms (dep) (nCA,dep = 41, nCA,no-dep = 56, nno-CA,dep = 40, Nno-CA,no-dep = 49). Methylation at four CpG sites in a regulatory region of GRIN2B was analysed using bisulfitepyrosequencing. Associations for methylation status to childhood adversity and to depression status were investigated using linear regression models. Our study shows that childhood adversity is associated with increased methylation levels of GRIN2B in adulthood, for three of the measured CpGs (p = 0.007, 0.006 and 5 × 10 14). This indicates that GRIN2B methylation is susceptible to early life stress, and that methylation at this gene is persistent over time. No association was found between GRIN2B methylation and depression status. Yet, this does not rule out a role for alterations in GRIN2B methylation for other neuropsychological outcomes not studied here. 1. Introduction Childhood adversity (CA), such as parental death, neglect, sexual, physical or emotional abuse, and friction within the family, is an early On top of the genetic information, environmental factors, especially life stressor that has been associated with an increased risk of psychiatric early in life, shape an organism’s phenotype and disease susceptibility disorders like depression (Collishaw et al., 2007; Widom et al., 2007; later in life (Gluckman and Hanson, 2004; Suzuki, 2018). During Liu, 2017), psychosis (Varese et al., 2012) and schizophrenia (Wells development, epigenetic processes play a crucial role in organising the et al., 2020), but also with cognitive impairments (Pechtel and Pizza­ functional genome (Champagne, 2013). Changes in epigenetic patterns, galli, 2011; Martins et al., 2019; Wells et al., 2020). On a molecular such as DNA methylation, induced during development can be stable level, CA can induce long lasting changes in DNA methylation patterns throughout life and are thought to be one link between early life expo­ (Provencal and Binder, 2015; Burns et al., 2018; Park et al., 2019). sures and health outcomes later in life (Marsit, 2015; Provencal and Primarily, methylation of genes involved in glucocorticoid signalling (e. Binder, 2015; Tran and Miyake, 2017; Burns et al., 2018; Goyal et al., g. NR3C1, FKBP5 (Tyrka et al., 2012; Klengel et al., 2013; Turecki and 2019; Park et al., 2019). Meaney, 2016)) and cortisol stress reactivity (e.g. KITLG (Houtepen * Corresponding author. E-mail address: [email protected] (E. Engdahl). 1 Present address: Uppsala University, Department of Organismal Biology, Uppsala, Sweden. 2 Present address: Departments of Physiology, University of Toronto, Canada. https://doi.org/10.1016/j.jpsychires.2020.09.022 Received 31 January 2020; Received in revised form 14 September 2020; Accepted 25 September 2020 Available online 3 October 2020 0022-3956/© 2020 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). E. Engdahl et al. Journal of Psychiatric Research 132 (2021) 38–43 et al., 2016)) has been reported to be affected by CA. Yet, methylation of study participants (controls) did not show any pathological symptoms genes with other cellular functions such as cellular/neuronal plasticity of depression, anxiety, obsessive compulsive disorder, agoraphobia, has also been reported to be affected by CA (Labonte et al., 2012; Yang eating disorder or social disability due to a psychological problem. In et al., 2013; Checknita et al., 2018), indicating that several molecular addition, controls had never received health care for a psychiatric dis­ mechanisms may underlie associations between CA and mental and order or nervous discomfort. cognitive health outcomes later in life. Alcohol consumption was assessed using the Alcohol Use Disorders One gene important for mental and cognitive development is Identification Test (AUDIT) consisting of 10 questions on alcohol use GRIN2B, encoding the NR2B subunit of the N-methyl-D-aspartate re­ (Saunders et al., 1993), and the average score between wave 1 and 2 was ceptor (NMDAR). NMDARs are receptors for the excitatory neurotrans­ used. Smoking (never or stopped/sometimes/regularly), education mitter glutamate and are involved in synaptic plasticity (reviewed in (9y/12y/>12y), occupation (no/yes), and financial stability (ability to (Liu and Zhao, 2013; Baez et al., 2018)). Mutations in GRIN2B have been pay 14 000 Swedish Crowns within a week in case of an unexpected associated with neurodevelopmental and neuropsychiatric disorders situation: No/No, probably not/Yes, probably/Yes, certainly) such as intellectual disability, developmental dyslexia, autism spectrum (Hallstr¨ om¨ et al., 2003) were self-reported in wave 2. disorders and schizophrenia (Platzer and Lemke, 1993; Mascheretti In total, 3018 DNA samples were collected. Of these, all individuals et al., 2015; Guo et al., 2016; Hu et al., 2016; Myers et al., 2019). In with CA among the depression cases and the non-depressed controls addition, specific genetic variants in GRIN2B have also been associated were selected (ndep = 46, nnon-dep = 57). Additionally, participants with major depressive disorder (MDD), specifically treatment resistant without CA were included, trying to match for depression status, sex and depression (Zhang et al., 2014), and suicide attempts (Sokolowski et al., smoking (ndep = 46, nnon-dep = 56). Demographic characteristics for all 2013). In post mortem brain samples from females, but not males, higher selected participants is presented in Table S1. GRIN2B gene expression has been observed in MDD patients compared to controls, where GRIN2B expression was even higher in MDD-suicide 2.2. DNA methylation of GRIN2B patients (Gray et al., 2015). These reports indicate that GRIN2B may play a role in depression, possibly in a sex-dependent way. Methylation status of 4 CpG sites in the GRIN2B regulatory region We have previously shown that methylation in a GRIN2B regulatory (figure S1) were assessed by targeted bisulfite pyrosequencing. First, region is affected by chemical exposure during development (Alavian-­ genomic DNA obtained from saliva samples was bisulfite converted Ghavanini et al., 2018), and others have shown that GRIN2B methyl­ using the EZ-96 DNA Methylation-Gold MagPrep kit (Zymo Research ation is altered in schizophrenia patients (Fachim et al., 2019). In the Corporation). After that, a 119 base pair (bp) fragment was amplified present study we hypothesised that not only chemical exposure, but also using the PyroMark PCR kit (Qiagen) together with 0.2 μM forward 0 0 the psychological stressor CA affects GRIN2B methylation in humans. In primer 5 -TGATTTAGGGGGGAGGAGAAATT-3 and biotinylated reverse 0 0 addition, we set out to investigate if GRIN2B methylation is associated primer 5 -AAACTACCTCCCCCAAAATCTTAACA-3 and with the addi­ with depressive disorders, which are commonly stress-induced and tion of 1 μl MgCl2 (25 mM) per 25 μl reaction, according to manufac­ associated with impaired cognitive function (Airaksinen et al., 2004). turer’s protocol. Pyrosequencing was performed on a PyroMark Q96 ID instrument (Qiagen) using PyroMark Gold Q96 reagents (Qiagen) and 0 0 2. Material and method 0.4 μM sequencing primer 5 -GGAAGATATTGTTTTTGTTTTTAG-3 ac­ cording to the manufacturer’s protocol. Methylation level at the four 2.1. PART cohort sequenced CpG sites were obtained using the PyroMark Q96 software. The first CpG site in this fragment (hereafter referred to as CpG1) is The samples included in this work were derived from the PART study identical with the Illumina 450K probe ID cg10091102. The assay was (Lundberg et al., 2005; Liu et al., 2015), a longitudinal population based validated