SHORT COMMUNICATION
DNA methylation in the 5-HTT regulatory region is associated with CO2-induced fear in panic disorder patients

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Abstract

A polymorphism in the gene encoding the serotonin (5-HT) transporter (5-HTT) has been shown to moderate the response to CO2 inhalation, an experimental model for panic attacks (PAs). Recurrent, unpredictable PAs represent, together with anticipatory anxiety of recurring attacks, the core feature of panic disorder (PD) and significantly interfere with patients’ daily life. In addition to genetic components, accumulating evidence suggests that epigenetic mechanisms, which regulate gene expression by modifying chromatin structure, also play a fundamental role in the etiology of mental disorders. However, in PD, epigenetic mechanisms have barely been examined to date. In the present study, we investigated the relationship between methylation at the regulatory region of the gene encoding the 5-HTT and the reactivity to a 35% CO2 inhalation in PD patients. We focused on four specific CpG sites and found a significant association between the methylation level of one of these CpG sites and the fear response. This suggests that the emotional response to CO2 inhalation might be moderated by an epigenetic mechanism, and underlines the implication of the 5-HT system in PAs. Future studies are needed to further investigate epigenetic alterations in PD and their functional consequences. These insights can increase our understanding of the underlying pathophysiology and support the development of new treatment strategies.

Introduction

Panic attacks (PAs) are characterized by intense fear and physical symptoms, and strongly impair patients’ quality of life (Davidoff et al., 2012). Unexpected PAs represent the core symptom of panic disorder (PD) and are now listed as a severity specifier applicable to all mental disorders in the Diagnostic and Statistical Manual of Mental Disorders (DSM)−5 (American Psychiatric Association, 2013).

PAs can be reliably provoked in PD patients using a brief 35% CO2-inhalation, and interestingly also in healthy individuals when the CO2 dosage is increased (for review see Leibold et al., 2015). This suggests the existence of a CO2-sensitivity continuum in which PD patients are hypersensitive to CO2. By now, CO2-hypersensitivity is a well-validated endophenotype of PD. A series of studies linked this endophenotype to the serotonergic (5-HT) system. Briefly, tryptophan depletion to reduce 5-HT levels increased the panic response to CO2-inhalation (Schruers et al., 2000). In contrast, acute administration of the 5-HT precursor to increase 5-HT levels reduced the response to CO2 (Schruers et al., 2002). Next, natural variation in 5-HT levels was studied by focusing on functional polymorphisms that alter gene expression. Participants homozygous for the long (L)-allele of the serotonin transporter (5-HTT) gene-linked polymorphic region (5-HTTLPR) reported the highest fear to CO2 (Schruers et al., 2011). The l-allele has been linked to a higher 5-HTT expression compared to the short (S)-allele (Lesch et al., 1996). In the brain, the 5-HTT is located on the presynaptic neuron and a higher expression leads to a relatively more efficient reuptake of 5-HT into the neuron, thereby affecting neurotransmission.

Research in the last few years has shown that not only static genetic variants contribute to how an individual's brain network is activated and works, but that gene expression is also influenced by environmental cues, mediated through dynamic epigenetic modifications (Guo et al., 2011). Genetically identical individuals can have a distinct risk to develop a disorder, as they have different gene expression and thus differently functioning networks caused by these epigenetic modifications. One of the most studied types of epigenetic regulation is DNA methylation occurring at cytosine residues. Although DNA methylation has gained attention in recent years, few studies have examined epigenetic modifications in PD. Only two genome-wide association studies (Iurato et al., 2017; Shimada-Sugimoto et al., 2017) and, based on evidence that the 5-HT system dysfunction is implicated in mental disorders, some candidate gene-based studies, focusing on crucial factors of the 5-HT system such as the monoamine oxidase A (MAO-A) gene, attempted to determine the involvement of DNA methylation in the etiology of PD (Domschke et al., 2012; Ziegler et al., 2016). Interestingly, although variation in the gene encoding the 5-HTT (SLC6A4) has been associated with PD and CO2-sensitivity, its methylation status has not yet been specifically examined in this context.

The aim of this study was to address this gap and to explore the relationship between DNA methylation in the regulatory region of the SLC6A4 gene and CO2-responsivity in PD patients, taking 5-HTT genotype into account. Previously, we showed that 5-HTTLPR l-allele carriers, presumably having a relatively higher expression of 5-HTT, displayed a stronger response to CO2 (Schruers et al., 2011). Based on this, we hypothesized that relatively lower DNA methylation levels, generally considered to be concomitant with a higher gene transcription and thus 5-HTT expression, were associated with higher CO2-reactivity.

Section snippets

Experimental procedures

All experimental procedures were approved by the Medical Ethics Committee of Maastricht University and Maastricht University Hospital, the Netherlands, and were conducted in accordance with the Declaration of Helsinki. Participants gave written informed consent.

Results

Overall, methylation levels of the four investigated CpG sites were low. Individual percentages ranged from 0% to 8.74%. Mean methylation (± SD) was as follows: CpG1 2.30±1.63, CpG2 3.64±1.94, CpG3 1.46±1.46, and CpG4 3.05±1.89. Linear regression revealed a significant negative association between CpG3 methylation level and changes in fear scores in PD patients (p=0.004, B=−3.729, Beta=−0.233, t=−2.922) (Table 1). CpG1, CpG2 and CpG4 were not significantly associated with fear scores (p

Discussion

This study showed a significant negative association between CO2-induced fear and the degree of CpG3 methylation (chr17:30,236,084) in the regulatory region of the SLC6A4 gene in PD patients, in line with our hypothesis. In our previous genetic study, we observed a higher fear response to CO2 in 5-HTTLPR LL-allele carriers (Schruers et al., 2011) who presumably have a higher 5-HTT expression. The 5-HTT is located on presynaptic 5-HT neurons and is responsible for reuptake of extracellular 5-HT.

CRediT authorship contribution statement

N.K. Leibold: Conceptualization, Formal analysis, Investigation, Methodology, Visualization, Writing - original draft. M.T. Weidner: Conceptualization, Formal analysis, Investigation, Methodology, Writing - review & editing. C. Ziegler: Methodology, Writing - review & editing. G. Ortega: Investigation, Methodology, Writing - review & editing. K. Domschke: Methodology, Writing - review & editing. K.P. Lesch: Conceptualization, Methodology, Project administration, Supervision, Writing - review &

Role of funding source

Klaus-Peter Lesch (KPL) and his team are supported by the Deutsche Forschungsgemeinschaft (DFG: CRU 125, CRC TRR 58 A1/A5, No. 44,541,416), the European Union's Horizon 2020 Research and Innovation Programme under Grant No. 728,018 (Eat2beNICE), ERA-Net NEURON/RESPOND,No. 01EW1602B, and 5–100 Russian Academic Excellence Project. Katharina Domschke (KD) received funding by the German Research Foundation (DFG) – project no. 44,541,416 – TRR 58, subprojects C02 and Z02. The funders had no role in

Contributors

KRS designed the study and wrote the protocol. NKL did the experiments. NKL, MTW, CZ, OG, designed and did the epigenetic analysis. NKL and MTW undertook the statistical analysis, and NKL wrote the first draft of the manuscript. DvdH, KD, and KPL contributed to overall discussion. All authors contributed to and have approved the final manuscript.

Conflict of interest

KPL served as a speaker for Eli Lilly and received research support from Medice, and travel support from Shire, all outside the submitted work. Other authors declare no conflict of interest.

Acknowledgement

None.

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