Plasticity of Functional MAOA Gene Methylation in Acrophobia

Plasticity of Functional MAOA Gene Methylation in Acrophobia Epigenetic mechanisms have been proposed to mediate fear extinction in animal models. Here, MAOA methylation was analyzed via direct sequencing of sodium bisulfite-treated DNA extracted from blood cells before and after a 2-week exposure therapy in a sample of n = 28 female patients with acrophobia as well as in n = 28 matched healthy female controls. Clinical response was measured using the Acrophobia Questionnaire and the Attitude Towards Heights Questionnaire. The functional relevance of altered MAOA methylation was investigated by luciferase-based reporter gene assays. MAOA methylation was found to be significantly decreased in patients with acrophobia compared with healthy controls. Furthermore, MAOA methylation levels were shown to significantly increase after treatment and correlate with treatment response as reflected by decreasing Acrophobia Questionnaire/Attitude Towards Heights Questionnaire scores. Functional analyses revealed decreased reporter gene activity in presence of methylated compared with unmethylated pCpGfree_MAOA reporter gene vector constructs. The present proof-of-concept psychotherapy-epigenetic study for the first time suggests functional MAOA methylation changes as a potential epigenetic correlate of treatment response in acrophobia and fosters further investigation into the notion of epigenetic mechanisms underlying fear extinction. Keywords: monoamine oxidase A, anxiety, extinction, epigenetics, DNA methylation Received: January 30, 2018; Revised: May 9, 2018; Accepted: May 30, 2018 © The Author(s) 2018. Published by Oxford University Press on behalf of CINP. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, 822 provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Downloaded from https://academic.oup.com/ijnp/article-abstract/21/9/822/5026103 by Ed 'DeepDyve' Gillespie user on 04 September 2018 Schiele et al. | 823 prefrontal cortex in a lying position (10 Hz, verum or sham; Introduction CBT[-rTMS] and CBT[+rTMS]; see Herrmann et al., 2016). An “epigenetic switch is the key to persistent extinction” Psychometric assessment of fear of heights was evaluated (Stafford et  al., 2011), a notion supported by experimental evi- before (T0) and after (T1) the therapeutic intervention using the dence suggesting that pharmacological modulation of epi- Acrophobia Questionnaire (AQ, Anxiety Subscale; Cohen, 1977) genetic mechanisms such as DNA methylation or histone and the Attitude Towards Heights Questionnaire (ATHQ; Abelson acetylation can produce lasting suppression of memories (for and Curtis, 1989). Patients were instructed not to change any review, see Lattal et al., 2013). This seems to be particularly rele- lifestyle patterns including smoking behavior from T0 to T1. vant for the psychotherapeutic treatment of anxiety disorders essentially entailing fear extinction (Whittle et al., 2014). MAOA Methylation Analysis In female patients with panic disorder, we recently iden- tified hypomethylation of a promoter/exon I/intron I  region In the patient sample, DNA was isolated from EDTA blood of the monoamine oxidase A  (MAOA) gene on chromosome taken at T0 and T1 using the FlexiGene DNA Kit (Qiagen). After Xp11.4-p11.3 as a disease risk factor (Domschke et  al., 2012), sodium bisulfite conversion (EpiTect 96 Bisulfite Kit, Qiagen), an which was found to be reversible with a successful cognitive amplicon comprising MAOA exon I as well as the adjacent pro- behavioral psychotherapeutic intervention (Ziegler et al., 2016). moter and intron I regions (chromosome X, GRCh38.p2 Primary The MAOA gene is a prime candidate gene also for specific pho- Assembly, NCBI Reference Sequence: NC_000023.11, 43656260– bias based on evidence from animal studies (Dubrovina et  al., 43656613) was PCR-amplified following a published proto- 2006) and genetic association studies in patients with specific col (Domschke et  al., 2012, 2015; Ziegler et  al., 2016, 2017) and phobias (Samochowiec et al., 2004). sequenced (ABI 3730XL, LGC Genomics). The obtained sequences Thus, in the present proof-of-concept “psychotherapy-epi- were quantitatively analyzed using the Epigenetic Sequencing genetic” study, we for the first time investigated DNA methy- Methylation software (ESME;  Lewin et  al., 2004; cf. Domschke lation in the above-mentioned promoter/exon I/intron I region et al., 2012, 2013, 2014, 2015; Tadić et al., 2014; Ziegler et al., 2015, of the MAOA gene in female patients with acrophobia during 2016, 2018). Electropherograms were robustly readable for 13 a standardized 2-week cognitive behavioral psychotherapeutic CpG sites (CpG1  =  43 656 316; CpG2  =  43 656 327; CpG3  =  43 656 intervention including exposure exercises using virtual reality 362; CpG4  =  43 656 368; CpG5  =  43 656 370; CpG6  =  43 656 383; technology, partly enhanced by repetitive transcranial magnetic CpG7  =  43 656 386; CpG8  =  43 656 392; CpG9  =  43 656 398; stimulation (rTMS). In analogy to a previous study in panic dis- CpG10  =  43 656 427; CpG11  =  43 656 432; CpG12  =  43 656 514; order we predicted MAOA methylation to increase and thus to CpG13  =  43 656 553). Healthy controls had been assessed for “normalize” along with treatment response. Additionally, the MAOA methylation using the same methods as applied to the functional relevance of altered MAOA promoter/exon I/intron present patient sample (see Domschke et al., 2012). Quality con- I  methylation was investigated by means of luciferase-based trol was performed as described previously (cf. Domschke et al., reporter gene assays. 2012; Ziegler et al., 2016). No participant had to be excluded from the reported analyses when applying quality control (data miss- ing for CpG1 in 3 patients). Methods Additionally, all patients and controls were genotyped for the MAOA VNTR according to published protocols (Reif et al., 2012) Sample and stratified into low (33/34/3a4/35) and high expression (44/45) Twenty-eight female Caucasian patients (44.86  ±  13.67  years) genotype groups (cf. Reif et al., 2012; Domschke et al., 2015). with acrophobia (SCID-I) were recruited at the Department of Psychiatry, University of Würzburg, Germany. Exclusion criteria Functional Analysis were any psychiatric comorbidities or previous treatment of acro- phobia within the last 6 months, metal parts in the head, med- Functional analysis was accomplished using the pCpGfree- ical implants, increased intracranial pressure, pregnancy, current promoter-Lucia vector (InvivoGen) expressing a Lucia luciferase involvement in psycho- or pharmacotherapy, cardiovascular under a human elongation factor-1 (hEF1) promoter. The insert or neurological diseases, a history of tinnitus, or family history comprising CpGs 1–13 was PCR-amplified and ligated into the of epilepsy (for details see Herrmann et  al., 2016). Additionally, linearized vector using T4 DNA Ligase (Invitrogen). The vectors 28 healthy female controls drawn from a larger, well-character - were methylated using the CpG methyltransferase M.SssI (NEB) ized pool of healthy probands (see Domschke et  al., 2012) were and transfected to HEK293 cells (ECACC) using the TransFast matched to the sample of patients with acrophobia by age reagent (Promega). Luciferase assays were performed using the (39.61 ± 6.68  years, t = -1.8, n.s.). This study was approved by the Stop & Glo® Reagent (Promega) on a GloMax+ Luminometer ethics committees of the Universities of Würzburg and Münster, (Promega), and Lucia luciferase activity was normalized using a Germany and was conducted according to the ethical princi- co-transfected pGL4.74 Renilla luciferase control vector. All anal- ples of the Helsinki Declaration. Written informed consent was yses were conducted in technical triplicates. obtained from all participants. Statistical Analysis Treatment Differences in MAOA methylation levels between patients and After receiving psycho-educative information in written form, controls as well as between genotype groups were tested by patients performed 2 exposure sessions within 2 weeks in a means of independent samples t tests. The association between 3- x 4-m, 5-sided Cave Automatic Virtual Environment oper - baseline methylation and treatment response as defined as the ated by the Department of Psychology, University of Würzburg intra-individual relative change (in percent) in clinical symptom (PsyCave; see Mühlberger et al., 2015). Prior to the exposure ses- scores (AQ Anxiety) was assessed by means of linear regression sion, 20 minutes of excitatory rTMS was applied over the medial analysis and controlled for genotype and TMS status. Changes Downloaded from https://academic.oup.com/ijnp/article-abstract/21/9/822/5026103 by Ed 'DeepDyve' Gillespie user on 04 September 2018 824 | International Journal of Neuropsychopharmacology, 2018 in MAOA methylation levels following therapy were tested using A positive change in AQ Anxiety scores indicating treat- repeated-measures ANOVA and controlled for grouped genotype ment response was seen in 20 patients (71.4%). While no signifi- and TMS randomization. Symptom changes were evaluated by cant association between baseline average MAOA methylation means of paired sample t tests. A  partial correlation was run and treatment response could be discerned, differences were to determine the relationship between individual changes in observed regarding CpG1 (β  =  .567, P  =  .003) and, approaching methylation (T1-T0) and symptom improvement (T1-T0) whilst trend levels of significance, CpG5 (β  =  .340, P  =  .086) and CpG6 controlling for baseline methylation (T0), genotype, and TMS (β  =  .331, P  =  .091), with lower baseline methylation predicting status. Luciferase assay data were normalized to transfection impaired treatment response. efficiency by Renilla luciferase control and z-transformed to Following therapy, MAOA methylation increased significantly eliminate day and measurement specific fluctuations. All tests in the patient group for average methylation (T1: 0.441 ± 0.029, were carried out 2-sided, and an alpha level of <0.05 was consid- F  =  4.703, P  =  .040) and specifically at CpG4 (T1: 0.420 ± 0.040, ered significant. Given the present proof-of-concept approach F = 4.708, P = .040), CpG6 (T1: 0.375 ± 0.051, F = 4.954, P = .036), CpG8 and high correlation between the 13 individual CpG sites (data (T1: 0.334 ± 0.053, F = 10.204, P = .004) and CpG9 (T1: 0.484 ± 0.035, not shown), no correction for multiple testing was applied when F = 10.249, P = .004) (Figure 1A). Methylation at all other CpG sites analyzing single CpG sites. did not change significantly from T0 to T1 (all P > .05). In the main analyses regarding methylation change depend- ent on treatment response as measured by changes in AQ Results Anxiety or ATHQ Total/Danger scores, respectively, negative cor - Mean scores on both questionnaires decreased significantly relations indicated clinical symptom improvement to go along with an increase in average MAOA methylation (AQ Anxiety: after the therapeutic intervention (AQ Anxiety T0: 57.05 ± 18.11, T1: 43.47 ± 18.91, t  =  4.61, P < .001; ATHQ Total T0: 37.86± 16.56, r = -0.465, P = .019, Figure 1B; ATHQ Danger: r = -0.496, P = .012). This pattern held true for several individual CpG sites (AQ T1: 26.89 ± 9.53, t  =  4.59, P < .001; ATHQ Danger T0: 18.32 ± 8.19, T1: 12.61 ± 5.34, t  =  5.19, P < .001). For this female patient sub- Anxiety: CpG2: r = -0.455, P = .022; CpG3: r = -0.398, P = .049; CpG7: r  =  -0.459, P = .021; CpG9: r  =  -0.399, P = .048; CpG10: r  =  -0.527, sample, no difference was discerned between the CBT(-rTMS) and CBT(+rTMS) groups regarding treatment response (data not P  =  .007; ATHQ Total: CpG9: r  =  -0.402, P  =  .047; ATHQ Danger: CpG2: r = -.412, P = .041; CpG3: r = -0.401, P = .047; CpG7: r = -0.473, shown), allowing for a joint epigenetic analysis of the 2 groups in the present context. P = .017; CpG9: r = -0.541, P = .005; CpG11: r = -0.424, P = .035). Applying in vitro luciferase assays, nonmethylated pCpGfree- At T0, average MAOA methylation (0.432± 0.021) did not cor - relate with age or disease severity according to AQ Anxiety or promoter Lucia_MAOA vectors showed a significant increase in normalized Lucia luciferase activity compared with pCpGfree- ATHQ Total/Danger scores (all P > .05). No influence of grouped genotype (low expression [33/34/3a4/35]: n  =  17 patients and promoter Lucia_MAOA vectors methylated with M.SssI prior to transfection (t  =  4.157,p < .001; Figure  2). PCpGfree-promoter n  =  19 controls; high expression [44/45]: n  =  11 patients and n = 9 controls) was discerned on average MAOA methylation or Lucia vectors without insert showed no significant difference between the methylated and the nonmethylated state (t = 0.394, questionnaire scores at T0 (all P > .05). Furthermore, patients and controls did not differ in genotype distribution ( Χ =  .311, p = .70; Figure 2). P = .577). Average MAOA methylation as well as methylation at CpGs Discussion 1–8 and 10–13 differed significantly between patients with acro- phobia at baseline (T0) and healthy controls. Patients were The present finding of (I) decreased MAOA methylation in characterized by significantly lower average methylation levels, patients with acrophobia compared with healthy controls, (II) specifically driven by single CpGs 1–8, 10, and 11 (for details see decreased methylation at a single MAOA CpG site to predict Table 1). treatment response, and (III) increasing MAOA methylation along Table 1. MAOA Methylation Levels in Patients with Acrophobia and Healthy Controls  Statistics Patients (n = 28) Controls (n = 28) Mean (SE) Mean (SE) t value P value Average methylation 0.432 (0.004) 0.4712 (0.008) 4.488 <.001 CpG1 0.359 (0.009) 0.4594 (0.011) 7.040 <.001 CpG2 0.358 (0.010) 0.3927 (0.011) 2.366 .022 CpG3 0.370 (0.008) 0.4097 (0.011) 2.873 .006 CpG4 0.403 (0.008) 0.5895 (0.020) 8.756 <.001 CpG5 0.286 (0.009) 0.3375 (0.016) 2.800 .008 CpG6 0.361 (0.009) 0.4246 (0.011) 4.403 <.001 CpG7 0.441 (0.007) 0.4964 (0.009) 4.900 <.001 CpG8 0.318 (0.009) 0.3751 (0.010) 4.284 <.001 CpG9 0.467 (0.006) 0.4836 (0.009) 1.575 .121 CpG10 0.482 (0.005) 0.5049 (0.007) 2.758 .008 CpG11 0.282 (0.009) 0.3138 (0.008) 2.571 .013 CpG12 0.913 (0.007) 0.8484 (0.020) -3.082 .003 CpG13 0.569 (0.011) 0.4896 (0.018) -3.670 .001 P-values from independent samples t test. Downloaded from https://academic.oup.com/ijnp/article-abstract/21/9/822/5026103 by Ed 'DeepDyve' Gillespie user on 04 September 2018 Schiele et al. | 825 Figure 2. Functional analysis of MAOA promoter/exon I/intron I DNA methyla- tion using luciferase-based reporter gene assays. Left: Normalized reporter gene activity was significantly decreased in the presence of pCpGfree-promoter Lucia_ MAOA vectors containing the methylated insert spanning CpGs 1–13 compared with those carrying a nonmethylated insert; ***P < .001. Right: No significant dif- ference in normalized reporter gene activity was discerned between methylated or non-methylated pCpGfree-promoter Lucia control vectors lacking the insert of the sequence spanning CpGs 1–13. In anxiety disorders, besides the above-mentioned study (Ziegler et al., 2016) only 2 other studies have investigated methylation dynamics with respect to treatment effects. Here, methylation changes in the serotonin transporter (5-HTT) gene as well as in the FK506-binding protein 5 (FKBP5) gene were found to be asso- ciated with response to psychotherapy in children with mixed primary anxiety disorder diagnoses and obsessive-compulsive disorder (Roberts et al., 2014, 2015). A major limitation of the present study entailing the risk of Figure  1. MAOA methylation and treatment response in acrophobia. (A) false positive results is the relatively small sample size owed to Significant changes in MAOA methylation from baseline (T0; black bars) to post the proof-of-concept design, strict inclusion/exclusion criteria, therapy (T1; grey bars) in n = 28 female patients with acrophobia at single CpG and inclusion of female patients only due to the X-chromosomal sites 4, 6, 8, and 9 and for average methylation (CpGs 1–13); mean ± SE. *P < location of the MAOA gene and previous observations of female- .05, **P < .01. (B) Correlation between reduction in anxiety severity (Acrophobia specific effects of genetic as well as epigenetic MAOA variation Questionnaire [AQ] Anxiety score from T0 to T1) and MAOA average methylation in anxiety disorders (Domschke et  al., 2012; Reif et  al., 2012; change from T0 to T1 (r = -0.449, P = .019). Ziegler et  al., 2016). Thus, despite comparability to previous with treatment response further strengthens and extends the treatment-epigenetic studies regarding size (e.g., Ziegler et  al., emerging body of evidence for a potential role of MAOA methyl- 2016), replication in larger samples as well as in mixed samples ation in the pathogenesis of fear-related disorders. Additionally, including also male patients is warranted. Given that no control in line with the hypothesis of epigenetically driven neuroplasti- group was available paralleling the time course of treatment in city underlying response to extinction-related psychotherapeu- the patient sample, the observed changes in MAOA methylation tic interventions (Stafford et al., 2011 M ), AOA methylation might in patients cannot conclusively be attributed to the effects of the be worthwhile to be further investigated as a possible correlate intervention but could rather be an inconsequential side effect of successful extinction treatment (cf. in panic disorder: Ziegler arising from a significant environmental stimulus. Also, despite et al., 2016). Furthermore, using luciferase-based reporter gene no statistically significant influence having been detected, psy- assays, we demonstrated increased methylation of the therapy- chotherapeutic and rTMS effects on DNA methylation changes modulated region to drive decreased gene expression, which is along with clinical symptom improvement cannot be differenti- line with a previous functional in  vitro assay (Checknita et al., ated. In general, DNA methylation was measured in peripheral 2015) and blood MAOA methylation correlating inversely with blood samples entailing cell composition effects driven by a vast brain MAO-A levels in  vivo using [(11)C]clorgyline positron amount of factors such as inflammation, diet, exercise, stress, or emission tomography (Shumay et  al., 2012). These functional hormonal status as a possible confounder. Also, while patients results also support the notion that not only methylation of were instructed not to change smoking patterns from T0 to the promoter but also of exon I with its adjacent promoter and T1, smoking patterns could still have confounded the present intron I regions seems to be linked to transcriptional repression results. Furthermore, data from in vitro luciferase assays span- (Brenet et al., 2011). In sum, we hypothesize that in acrophobia, ning 13 CpG sites do not allow for evaluating MAOA gene regu- decreased availability of monoamines conferred by decreased lation in toto or at single CpG sites and thus are to be considered MAOA methylation might be reversible by remethylation during suggestive, but not conclusive, particularly since different CpG the course of a successful therapeutic intervention. sites were found to predict or correlate with treatment response, The present study thereby adds to the only recently burgeon- respectively. Data on MAOA mRNA expression in apt tissue ing evidence for epigenetic mechanisms possibly constituting dependent on individual MAOA methylation status would aid in dynamic biological correlates of therapeutic interventions in further defining the in vivo physiological consequences of MAOA mental disorders (for review, see Schiele and Domschke, 2018). promoter hypomethylation (cf. Hotamisligil and Breakefield, Downloaded from https://academic.oup.com/ijnp/article-abstract/21/9/822/5026103 by Ed 'DeepDyve' Gillespie user on 04 September 2018 826 | International Journal of Neuropsychopharmacology, 2018 1991). Along these lines, despite some evidence for their poten- (GAD1) DNA hypomethylation? Prog Neuropsychopharmacol tial as viable markers of central processes (e.g., Shumay et  al., Biol Psychiatry 46:189–196. 2012), peripheral methylation patterns do not necessarily allow Domschke K, Tidow N, Schwarte K, Deckert J, Lesch KP, Arolt for deductions regarding MAOA methylation status in brain tis- V, Zwanzger P, Baune BT (2014) Serotonin transporter gene sue. Finally, localized changes such as the presently identified hypomethylation predicts impaired antidepressant treat- MAOA methylation patterns might reflect global changes in gen- ment response. Int J Neuropsychopharmacol 17:1167–1176. omic DNA methylation either by CpG hyper- and hypomethyla- Domschke K, Tidow N, Schwarte K, Ziegler C, Lesch KP, Deckert J, tion necessitating investigation by means of epigenome-wide Arolt V, Zwanzger P, Baune BT (2015) Pharmacoepigenetics of association studies in larger, sufficiently powered samples. depression: no major influence of MAO-A DNA methylation In sum, while warranting replication and considering all on treatment response. J Neural Transm 122:99–108. caveats mentioned above, the present psychotherapy-epige- Dubrovina NI, Popova NK, Gilinskii MA, Tomilenko RA, Seif I netic study for the first time suggests functionally relevant (2006) Acquisition and extinction of a conditioned passive MAOA methylation changes as a potential epigenetic correlate avoidance reflex in mice with genetic knockout of monoam- of response to cognitive behavioral psychotherapy in acropho- ine oxidase A. Neurosci Behav Physiol 36:335–339. bia and corroborates a previous independent finding in panic Herrmann MJ, Katzorke A, Busch Y, Gromer D, Polak T, Pauli P, disorder (Ziegler et  al., 2016). The emerging notion of epigen- Deckert J (2016) Medial prefrontal cortex stimulation accel- etic signatures as a core mechanism of action of fear extinc- erates therapy response of exposure therapy in acrophobia. tion might aid in probing pharmacological treatment enhancers Brain Stimul 10:291–297. such as MAO-inhibitors or, in a more general sense, drugs induc- Hotamisligil GS, Breakefield XO (1991) Human monoamine oxi- ing epigenetic changes as augmentation strategies for lasting dase A gene determines levels of enzyme activity. Am J Hum extinction effects and is thereby hoped to contribute to a more Genet. 49:383–392. effective treatment based on individual epigenetic information. Lattal KM, Wood MA (2013) Epigenetics and persistent memory: implications for reconsolidation and silent extinction beyond the zero. Nat Neurosci 16:124–129. Funding Lewin J, Schmitt AO, Adorján P, Hildmann T, Piepenbrock C (2004) Quantitative DNA methylation analysis based on four- This work was supported by the German Research Foundation, SFB-TRR-58, projects C02 (to K.D.), C06 (to M.J.H.) and Z02 (to dye trace data from direct sequencing of PCR amplificates. Bioinformatics 20:3005–3012. K.D., P.P., A.R., and J.D.), and the German Ministry of Research and Education (01EE1402F, PROTECT-AD, P5 to K.D. and J.D.). Mühlberger A, Kinateder M, Brütting J, Eder S, Müller M, Gromer D, Pauli P (2015) Influence of information and instructions on human behavior in tunnel accidents: a virtual reality study. Acknowledgments JVRB 12:1–13. 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Abstract

Epigenetic mechanisms have been proposed to mediate fear extinction in animal models. Here, MAOA methylation was analyzed via direct sequencing of sodium bisulfite-treated DNA extracted from blood cells before and after a 2-week exposure therapy in a sample of n = 28 female patients with acrophobia as well as in n = 28 matched healthy female controls. Clinical response was measured using the Acrophobia Questionnaire and the Attitude Towards Heights Questionnaire. The functional relevance of altered MAOA methylation was investigated by luciferase-based reporter gene assays. MAOA methylation was found to be significantly decreased in patients with acrophobia compared with healthy controls. Furthermore, MAOA methylation levels were shown to significantly increase after treatment and correlate with treatment response as reflected by decreasing Acrophobia Questionnaire/Attitude Towards Heights Questionnaire scores. Functional analyses revealed decreased reporter gene activity in presence of methylated compared with unmethylated pCpGfree_MAOA reporter gene vector constructs. The present proof-of-concept psychotherapy-epigenetic study for the first time suggests functional MAOA methylation changes as a potential epigenetic correlate of treatment response in acrophobia and fosters further investigation into the notion of epigenetic mechanisms underlying fear extinction. Keywords: monoamine oxidase A, anxiety, extinction, epigenetics, DNA methylation Received: January 30, 2018; Revised: May 9, 2018; Accepted: May 30, 2018 © The Author(s) 2018. Published by Oxford University Press on behalf of CINP. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, 822 provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Downloaded from https://academic.oup.com/ijnp/article-abstract/21/9/822/5026103 by Ed 'DeepDyve' Gillespie user on 04 September 2018 Schiele et al. | 823 prefrontal cortex in a lying position (10 Hz, verum or sham; Introduction CBT[-rTMS] and CBT[+rTMS]; see Herrmann et al., 2016). An “epigenetic switch is the key to persistent extinction” Psychometric assessment of fear of heights was evaluated (Stafford et  al., 2011), a notion supported by experimental evi- before (T0) and after (T1) the therapeutic intervention using the dence suggesting that pharmacological modulation of epi- Acrophobia Questionnaire (AQ, Anxiety Subscale; Cohen, 1977) genetic mechanisms such as DNA methylation or histone and the Attitude Towards Heights Questionnaire (ATHQ; Abelson acetylation can produce lasting suppression of memories (for and Curtis, 1989). Patients were instructed not to change any review, see Lattal et al., 2013). This seems to be particularly rele- lifestyle patterns including smoking behavior from T0 to T1. vant for the psychotherapeutic treatment of anxiety disorders essentially entailing fear extinction (Whittle et al., 2014). MAOA Methylation Analysis In female patients with panic disorder, we recently iden- tified hypomethylation of a promoter/exon I/intron I  region In the patient sample, DNA was isolated from EDTA blood of the monoamine oxidase A  (MAOA) gene on chromosome taken at T0 and T1 using the FlexiGene DNA Kit (Qiagen). After Xp11.4-p11.3 as a disease risk factor (Domschke et  al., 2012), sodium bisulfite conversion (EpiTect 96 Bisulfite Kit, Qiagen), an which was found to be reversible with a successful cognitive amplicon comprising MAOA exon I as well as the adjacent pro- behavioral psychotherapeutic intervention (Ziegler et al., 2016). moter and intron I regions (chromosome X, GRCh38.p2 Primary The MAOA gene is a prime candidate gene also for specific pho- Assembly, NCBI Reference Sequence: NC_000023.11, 43656260– bias based on evidence from animal studies (Dubrovina et  al., 43656613) was PCR-amplified following a published proto- 2006) and genetic association studies in patients with specific col (Domschke et  al., 2012, 2015; Ziegler et  al., 2016, 2017) and phobias (Samochowiec et al., 2004). sequenced (ABI 3730XL, LGC Genomics). The obtained sequences Thus, in the present proof-of-concept “psychotherapy-epi- were quantitatively analyzed using the Epigenetic Sequencing genetic” study, we for the first time investigated DNA methy- Methylation software (ESME;  Lewin et  al., 2004; cf. Domschke lation in the above-mentioned promoter/exon I/intron I region et al., 2012, 2013, 2014, 2015; Tadić et al., 2014; Ziegler et al., 2015, of the MAOA gene in female patients with acrophobia during 2016, 2018). Electropherograms were robustly readable for 13 a standardized 2-week cognitive behavioral psychotherapeutic CpG sites (CpG1  =  43 656 316; CpG2  =  43 656 327; CpG3  =  43 656 intervention including exposure exercises using virtual reality 362; CpG4  =  43 656 368; CpG5  =  43 656 370; CpG6  =  43 656 383; technology, partly enhanced by repetitive transcranial magnetic CpG7  =  43 656 386; CpG8  =  43 656 392; CpG9  =  43 656 398; stimulation (rTMS). In analogy to a previous study in panic dis- CpG10  =  43 656 427; CpG11  =  43 656 432; CpG12  =  43 656 514; order we predicted MAOA methylation to increase and thus to CpG13  =  43 656 553). Healthy controls had been assessed for “normalize” along with treatment response. Additionally, the MAOA methylation using the same methods as applied to the functional relevance of altered MAOA promoter/exon I/intron present patient sample (see Domschke et al., 2012). Quality con- I  methylation was investigated by means of luciferase-based trol was performed as described previously (cf. Domschke et al., reporter gene assays. 2012; Ziegler et al., 2016). No participant had to be excluded from the reported analyses when applying quality control (data miss- ing for CpG1 in 3 patients). Methods Additionally, all patients and controls were genotyped for the MAOA VNTR according to published protocols (Reif et al., 2012) Sample and stratified into low (33/34/3a4/35) and high expression (44/45) Twenty-eight female Caucasian patients (44.86  ±  13.67  years) genotype groups (cf. Reif et al., 2012; Domschke et al., 2015). with acrophobia (SCID-I) were recruited at the Department of Psychiatry, University of Würzburg, Germany. Exclusion criteria Functional Analysis were any psychiatric comorbidities or previous treatment of acro- phobia within the last 6 months, metal parts in the head, med- Functional analysis was accomplished using the pCpGfree- ical implants, increased intracranial pressure, pregnancy, current promoter-Lucia vector (InvivoGen) expressing a Lucia luciferase involvement in psycho- or pharmacotherapy, cardiovascular under a human elongation factor-1 (hEF1) promoter. The insert or neurological diseases, a history of tinnitus, or family history comprising CpGs 1–13 was PCR-amplified and ligated into the of epilepsy (for details see Herrmann et  al., 2016). Additionally, linearized vector using T4 DNA Ligase (Invitrogen). The vectors 28 healthy female controls drawn from a larger, well-character - were methylated using the CpG methyltransferase M.SssI (NEB) ized pool of healthy probands (see Domschke et  al., 2012) were and transfected to HEK293 cells (ECACC) using the TransFast matched to the sample of patients with acrophobia by age reagent (Promega). Luciferase assays were performed using the (39.61 ± 6.68  years, t = -1.8, n.s.). This study was approved by the Stop & Glo® Reagent (Promega) on a GloMax+ Luminometer ethics committees of the Universities of Würzburg and Münster, (Promega), and Lucia luciferase activity was normalized using a Germany and was conducted according to the ethical princi- co-transfected pGL4.74 Renilla luciferase control vector. All anal- ples of the Helsinki Declaration. Written informed consent was yses were conducted in technical triplicates. obtained from all participants. Statistical Analysis Treatment Differences in MAOA methylation levels between patients and After receiving psycho-educative information in written form, controls as well as between genotype groups were tested by patients performed 2 exposure sessions within 2 weeks in a means of independent samples t tests. The association between 3- x 4-m, 5-sided Cave Automatic Virtual Environment oper - baseline methylation and treatment response as defined as the ated by the Department of Psychology, University of Würzburg intra-individual relative change (in percent) in clinical symptom (PsyCave; see Mühlberger et al., 2015). Prior to the exposure ses- scores (AQ Anxiety) was assessed by means of linear regression sion, 20 minutes of excitatory rTMS was applied over the medial analysis and controlled for genotype and TMS status. Changes Downloaded from https://academic.oup.com/ijnp/article-abstract/21/9/822/5026103 by Ed 'DeepDyve' Gillespie user on 04 September 2018 824 | International Journal of Neuropsychopharmacology, 2018 in MAOA methylation levels following therapy were tested using A positive change in AQ Anxiety scores indicating treat- repeated-measures ANOVA and controlled for grouped genotype ment response was seen in 20 patients (71.4%). While no signifi- and TMS randomization. Symptom changes were evaluated by cant association between baseline average MAOA methylation means of paired sample t tests. A  partial correlation was run and treatment response could be discerned, differences were to determine the relationship between individual changes in observed regarding CpG1 (β  =  .567, P  =  .003) and, approaching methylation (T1-T0) and symptom improvement (T1-T0) whilst trend levels of significance, CpG5 (β  =  .340, P  =  .086) and CpG6 controlling for baseline methylation (T0), genotype, and TMS (β  =  .331, P  =  .091), with lower baseline methylation predicting status. Luciferase assay data were normalized to transfection impaired treatment response. efficiency by Renilla luciferase control and z-transformed to Following therapy, MAOA methylation increased significantly eliminate day and measurement specific fluctuations. All tests in the patient group for average methylation (T1: 0.441 ± 0.029, were carried out 2-sided, and an alpha level of <0.05 was consid- F  =  4.703, P  =  .040) and specifically at CpG4 (T1: 0.420 ± 0.040, ered significant. Given the present proof-of-concept approach F = 4.708, P = .040), CpG6 (T1: 0.375 ± 0.051, F = 4.954, P = .036), CpG8 and high correlation between the 13 individual CpG sites (data (T1: 0.334 ± 0.053, F = 10.204, P = .004) and CpG9 (T1: 0.484 ± 0.035, not shown), no correction for multiple testing was applied when F = 10.249, P = .004) (Figure 1A). Methylation at all other CpG sites analyzing single CpG sites. did not change significantly from T0 to T1 (all P > .05). In the main analyses regarding methylation change depend- ent on treatment response as measured by changes in AQ Results Anxiety or ATHQ Total/Danger scores, respectively, negative cor - Mean scores on both questionnaires decreased significantly relations indicated clinical symptom improvement to go along with an increase in average MAOA methylation (AQ Anxiety: after the therapeutic intervention (AQ Anxiety T0: 57.05 ± 18.11, T1: 43.47 ± 18.91, t  =  4.61, P < .001; ATHQ Total T0: 37.86± 16.56, r = -0.465, P = .019, Figure 1B; ATHQ Danger: r = -0.496, P = .012). This pattern held true for several individual CpG sites (AQ T1: 26.89 ± 9.53, t  =  4.59, P < .001; ATHQ Danger T0: 18.32 ± 8.19, T1: 12.61 ± 5.34, t  =  5.19, P < .001). For this female patient sub- Anxiety: CpG2: r = -0.455, P = .022; CpG3: r = -0.398, P = .049; CpG7: r  =  -0.459, P = .021; CpG9: r  =  -0.399, P = .048; CpG10: r  =  -0.527, sample, no difference was discerned between the CBT(-rTMS) and CBT(+rTMS) groups regarding treatment response (data not P  =  .007; ATHQ Total: CpG9: r  =  -0.402, P  =  .047; ATHQ Danger: CpG2: r = -.412, P = .041; CpG3: r = -0.401, P = .047; CpG7: r = -0.473, shown), allowing for a joint epigenetic analysis of the 2 groups in the present context. P = .017; CpG9: r = -0.541, P = .005; CpG11: r = -0.424, P = .035). Applying in vitro luciferase assays, nonmethylated pCpGfree- At T0, average MAOA methylation (0.432± 0.021) did not cor - relate with age or disease severity according to AQ Anxiety or promoter Lucia_MAOA vectors showed a significant increase in normalized Lucia luciferase activity compared with pCpGfree- ATHQ Total/Danger scores (all P > .05). No influence of grouped genotype (low expression [33/34/3a4/35]: n  =  17 patients and promoter Lucia_MAOA vectors methylated with M.SssI prior to transfection (t  =  4.157,p < .001; Figure  2). PCpGfree-promoter n  =  19 controls; high expression [44/45]: n  =  11 patients and n = 9 controls) was discerned on average MAOA methylation or Lucia vectors without insert showed no significant difference between the methylated and the nonmethylated state (t = 0.394, questionnaire scores at T0 (all P > .05). Furthermore, patients and controls did not differ in genotype distribution ( Χ =  .311, p = .70; Figure 2). P = .577). Average MAOA methylation as well as methylation at CpGs Discussion 1–8 and 10–13 differed significantly between patients with acro- phobia at baseline (T0) and healthy controls. Patients were The present finding of (I) decreased MAOA methylation in characterized by significantly lower average methylation levels, patients with acrophobia compared with healthy controls, (II) specifically driven by single CpGs 1–8, 10, and 11 (for details see decreased methylation at a single MAOA CpG site to predict Table 1). treatment response, and (III) increasing MAOA methylation along Table 1. MAOA Methylation Levels in Patients with Acrophobia and Healthy Controls  Statistics Patients (n = 28) Controls (n = 28) Mean (SE) Mean (SE) t value P value Average methylation 0.432 (0.004) 0.4712 (0.008) 4.488 <.001 CpG1 0.359 (0.009) 0.4594 (0.011) 7.040 <.001 CpG2 0.358 (0.010) 0.3927 (0.011) 2.366 .022 CpG3 0.370 (0.008) 0.4097 (0.011) 2.873 .006 CpG4 0.403 (0.008) 0.5895 (0.020) 8.756 <.001 CpG5 0.286 (0.009) 0.3375 (0.016) 2.800 .008 CpG6 0.361 (0.009) 0.4246 (0.011) 4.403 <.001 CpG7 0.441 (0.007) 0.4964 (0.009) 4.900 <.001 CpG8 0.318 (0.009) 0.3751 (0.010) 4.284 <.001 CpG9 0.467 (0.006) 0.4836 (0.009) 1.575 .121 CpG10 0.482 (0.005) 0.5049 (0.007) 2.758 .008 CpG11 0.282 (0.009) 0.3138 (0.008) 2.571 .013 CpG12 0.913 (0.007) 0.8484 (0.020) -3.082 .003 CpG13 0.569 (0.011) 0.4896 (0.018) -3.670 .001 P-values from independent samples t test. Downloaded from https://academic.oup.com/ijnp/article-abstract/21/9/822/5026103 by Ed 'DeepDyve' Gillespie user on 04 September 2018 Schiele et al. | 825 Figure 2. Functional analysis of MAOA promoter/exon I/intron I DNA methyla- tion using luciferase-based reporter gene assays. Left: Normalized reporter gene activity was significantly decreased in the presence of pCpGfree-promoter Lucia_ MAOA vectors containing the methylated insert spanning CpGs 1–13 compared with those carrying a nonmethylated insert; ***P < .001. Right: No significant dif- ference in normalized reporter gene activity was discerned between methylated or non-methylated pCpGfree-promoter Lucia control vectors lacking the insert of the sequence spanning CpGs 1–13. In anxiety disorders, besides the above-mentioned study (Ziegler et al., 2016) only 2 other studies have investigated methylation dynamics with respect to treatment effects. Here, methylation changes in the serotonin transporter (5-HTT) gene as well as in the FK506-binding protein 5 (FKBP5) gene were found to be asso- ciated with response to psychotherapy in children with mixed primary anxiety disorder diagnoses and obsessive-compulsive disorder (Roberts et al., 2014, 2015). A major limitation of the present study entailing the risk of Figure  1. MAOA methylation and treatment response in acrophobia. (A) false positive results is the relatively small sample size owed to Significant changes in MAOA methylation from baseline (T0; black bars) to post the proof-of-concept design, strict inclusion/exclusion criteria, therapy (T1; grey bars) in n = 28 female patients with acrophobia at single CpG and inclusion of female patients only due to the X-chromosomal sites 4, 6, 8, and 9 and for average methylation (CpGs 1–13); mean ± SE. *P < location of the MAOA gene and previous observations of female- .05, **P < .01. (B) Correlation between reduction in anxiety severity (Acrophobia specific effects of genetic as well as epigenetic MAOA variation Questionnaire [AQ] Anxiety score from T0 to T1) and MAOA average methylation in anxiety disorders (Domschke et  al., 2012; Reif et  al., 2012; change from T0 to T1 (r = -0.449, P = .019). Ziegler et  al., 2016). Thus, despite comparability to previous with treatment response further strengthens and extends the treatment-epigenetic studies regarding size (e.g., Ziegler et  al., emerging body of evidence for a potential role of MAOA methyl- 2016), replication in larger samples as well as in mixed samples ation in the pathogenesis of fear-related disorders. Additionally, including also male patients is warranted. Given that no control in line with the hypothesis of epigenetically driven neuroplasti- group was available paralleling the time course of treatment in city underlying response to extinction-related psychotherapeu- the patient sample, the observed changes in MAOA methylation tic interventions (Stafford et al., 2011 M ), AOA methylation might in patients cannot conclusively be attributed to the effects of the be worthwhile to be further investigated as a possible correlate intervention but could rather be an inconsequential side effect of successful extinction treatment (cf. in panic disorder: Ziegler arising from a significant environmental stimulus. Also, despite et al., 2016). Furthermore, using luciferase-based reporter gene no statistically significant influence having been detected, psy- assays, we demonstrated increased methylation of the therapy- chotherapeutic and rTMS effects on DNA methylation changes modulated region to drive decreased gene expression, which is along with clinical symptom improvement cannot be differenti- line with a previous functional in  vitro assay (Checknita et al., ated. In general, DNA methylation was measured in peripheral 2015) and blood MAOA methylation correlating inversely with blood samples entailing cell composition effects driven by a vast brain MAO-A levels in  vivo using [(11)C]clorgyline positron amount of factors such as inflammation, diet, exercise, stress, or emission tomography (Shumay et  al., 2012). These functional hormonal status as a possible confounder. Also, while patients results also support the notion that not only methylation of were instructed not to change smoking patterns from T0 to the promoter but also of exon I with its adjacent promoter and T1, smoking patterns could still have confounded the present intron I regions seems to be linked to transcriptional repression results. Furthermore, data from in vitro luciferase assays span- (Brenet et al., 2011). In sum, we hypothesize that in acrophobia, ning 13 CpG sites do not allow for evaluating MAOA gene regu- decreased availability of monoamines conferred by decreased lation in toto or at single CpG sites and thus are to be considered MAOA methylation might be reversible by remethylation during suggestive, but not conclusive, particularly since different CpG the course of a successful therapeutic intervention. sites were found to predict or correlate with treatment response, The present study thereby adds to the only recently burgeon- respectively. Data on MAOA mRNA expression in apt tissue ing evidence for epigenetic mechanisms possibly constituting dependent on individual MAOA methylation status would aid in dynamic biological correlates of therapeutic interventions in further defining the in vivo physiological consequences of MAOA mental disorders (for review, see Schiele and Domschke, 2018). promoter hypomethylation (cf. Hotamisligil and Breakefield, Downloaded from https://academic.oup.com/ijnp/article-abstract/21/9/822/5026103 by Ed 'DeepDyve' Gillespie user on 04 September 2018 826 | International Journal of Neuropsychopharmacology, 2018 1991). Along these lines, despite some evidence for their poten- (GAD1) DNA hypomethylation? Prog Neuropsychopharmacol tial as viable markers of central processes (e.g., Shumay et  al., Biol Psychiatry 46:189–196. 2012), peripheral methylation patterns do not necessarily allow Domschke K, Tidow N, Schwarte K, Deckert J, Lesch KP, Arolt for deductions regarding MAOA methylation status in brain tis- V, Zwanzger P, Baune BT (2014) Serotonin transporter gene sue. Finally, localized changes such as the presently identified hypomethylation predicts impaired antidepressant treat- MAOA methylation patterns might reflect global changes in gen- ment response. Int J Neuropsychopharmacol 17:1167–1176. omic DNA methylation either by CpG hyper- and hypomethyla- Domschke K, Tidow N, Schwarte K, Ziegler C, Lesch KP, Deckert J, tion necessitating investigation by means of epigenome-wide Arolt V, Zwanzger P, Baune BT (2015) Pharmacoepigenetics of association studies in larger, sufficiently powered samples. depression: no major influence of MAO-A DNA methylation In sum, while warranting replication and considering all on treatment response. J Neural Transm 122:99–108. caveats mentioned above, the present psychotherapy-epige- Dubrovina NI, Popova NK, Gilinskii MA, Tomilenko RA, Seif I netic study for the first time suggests functionally relevant (2006) Acquisition and extinction of a conditioned passive MAOA methylation changes as a potential epigenetic correlate avoidance reflex in mice with genetic knockout of monoam- of response to cognitive behavioral psychotherapy in acropho- ine oxidase A. Neurosci Behav Physiol 36:335–339. bia and corroborates a previous independent finding in panic Herrmann MJ, Katzorke A, Busch Y, Gromer D, Polak T, Pauli P, disorder (Ziegler et  al., 2016). The emerging notion of epigen- Deckert J (2016) Medial prefrontal cortex stimulation accel- etic signatures as a core mechanism of action of fear extinc- erates therapy response of exposure therapy in acrophobia. tion might aid in probing pharmacological treatment enhancers Brain Stimul 10:291–297. such as MAO-inhibitors or, in a more general sense, drugs induc- Hotamisligil GS, Breakefield XO (1991) Human monoamine oxi- ing epigenetic changes as augmentation strategies for lasting dase A gene determines levels of enzyme activity. Am J Hum extinction effects and is thereby hoped to contribute to a more Genet. 49:383–392. effective treatment based on individual epigenetic information. Lattal KM, Wood MA (2013) Epigenetics and persistent memory: implications for reconsolidation and silent extinction beyond the zero. Nat Neurosci 16:124–129. Funding Lewin J, Schmitt AO, Adorján P, Hildmann T, Piepenbrock C (2004) Quantitative DNA methylation analysis based on four- This work was supported by the German Research Foundation, SFB-TRR-58, projects C02 (to K.D.), C06 (to M.J.H.) and Z02 (to dye trace data from direct sequencing of PCR amplificates. Bioinformatics 20:3005–3012. K.D., P.P., A.R., and J.D.), and the German Ministry of Research and Education (01EE1402F, PROTECT-AD, P5 to K.D. and J.D.). Mühlberger A, Kinateder M, Brütting J, Eder S, Müller M, Gromer D, Pauli P (2015) Influence of information and instructions on human behavior in tunnel accidents: a virtual reality study. Acknowledgments JVRB 12:1–13. Reif A, Weber H, Domschke K, Klauke B, Baumann C, Jacob CP, We gratefully acknowledge the skillful technical support by Ströhle A, Gerlach AL, Alpers GW, Pauli P, Hamm A, Kircher Carola Gagel. T, Arolt V, Wittchen HU, Binder EB, Erhardt A, Deckert J (2012) Meta-analysis argues for a female-specific role of MAOA- Statement of Interest uVNTR in panic disorder in four European populations. Am J Med Genet B Neuropsychiatr Genet 159B:786–793. None. Roberts S, Lester KJ, Hudson JL, Rapee RM, Creswell C, Cooper PJ, Thirlwall KJ, Coleman JRI, Breen G, Wong CCY, Eley TC (2014) References Serotonin transporter [corrected] methylation and response Abelson JL, Curtis GC (1989) Cardiac and neuroendo- to cognitive behaviour therapy in children with anxiety dis- crine responses to exposure therapy in height phobics: orders. Transl Psychiatry 4:444. Desynchrony within the “physiological response system.” Roberts S et  al. 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