Serotonin Transporter Gene Promoter Polymorphism and Somatoform Symptoms
Introduction: Symptoms of somatoform and affective disorders are thought to be connected to serotonergic neurotransmission because serotonin is known to regulate the functions relevant in these disorders, such as pain and mood. Previous studies have reported associations of these disorders with a functional polymorphism in the promoter region of the serotonin transporter gene, a limiting factor of the serotonergic neuronal system, as its alleles have been associated with differences in levels of synthesized transporter and therefore differences in reuptake efficiency.
Method: Ninety-one patients with at least 2 unexplained physical symptoms were clinically evaluated and genotyped for the triallelic genotypes of the serotonin transporter gene polymorphism; patients were recruited from 2001 until 2004. DSM-IV diagnoses were assessed using the International Checklists for ICD-10 and DSM-IV. Somatic complaints were quantified with an interview version of the Screening for Somatoform Symptoms, persistent symptoms in the last 2 years (SOMS-2) and the SOMS-7 (current symptoms in the last 7 days). Depressive symptoms were quantified with the Beck Depression Inventory (BDI).
Results: Subjects with higher-expressing allele variants of the serotonin transporter gene (L‘ ²L‘ ² and L‘ ²S‘ ²) had significantly more somatic symptoms in the last 2 years (trait) than those with lower-expressing variants (S‘ ²S‘ ²) (P < .01). No differences could be found in regard to short-term somatic symptoms (ie, in the last 7 days). Neither depressive symptoms nor a comorbid diagnosis of major depression was associated with allelic variants.
Conclusion: Somatoform symptoms may be associated with a functional polymorphism in the promoter region of the serotonin transporter gene.
J Clin Psychiatry 2009;70(11):1536-1539
© Copyright 2009 Physicians Postgraduate Press, Inc.
Submitted: August 14, 2008; accepted October 9, 2008.
Online ahead of print: June 16, 2009 (doi:10.4088/JCP.08m04613).
Corresponding author and reprints: Anika Hennings, Philipps University of Marburg, Division of Clinical Psychology and Psychotherapy, Gutenbergstrasse 18, 35032, Marburg, Germany ([email protected]).
The term somatoform disorders refers to a group of psychiatric disorders with somatic symptoms not caused by a well-known medical condition. These bodily symptoms tend to persist and tend to occur together with symptoms of other organ systems ("multisomatoform disorders").1 The pathophysiology of these syndromes is not fully understood, but neurotransmitters are considered to be relevant for symptom development and maintenance.2
Serotonin (5-hydroxytryptamine [5-HT]) is a prominent neurobiologic marker and is known to regulate functions such as pain, sleep, and mood,3-5 which are related to somatoform disorders as well as affective disorders. In depression, possible actions of 5-HT might be primarily central, modulating cognition and affect regulation,6,7 whereas pathways of contribution for serotonin to somatoform symptoms could be 2-fold: through modulation of pain perception via direct cerebral action, or through peripheral pathways, eg, via contribution to muscle metabolism or action on different receptor subtypes in pain.5,8
One limiting factor for the 5-HT neuronal system is the 5-HT transporter gene (SLC6A4). The 5-HT transporter regulates the action of 5-HT by transporting it from synaptic spaces into presynaptic neurons, thus terminating its action. SLC6A4 encodes this transporter. A polymorphism within the promoter region (5-HT transporter gene-linked polymorphic region, 5-HTTLPR) has been reported, and its allelic variants are associated with differences in the regulations of gene expression and in the efficiency of 5-HT reuptake.3,9 The 5-HTTLPR was thought to be biallelic, with a less efficient S ("short") and an L ("long") variant, but recent findings suggest that the L allele can be subtyped into LA ("long-A") and LG ("long-G"), the latter of which is thought to be similar to the S allele in terms of reuptake efficiency.10-13
Studies on "somatoform-associated disorders"2 such as chronic fatigue syndrome or temporomandibular disorder have found higher rates of high-expressing genotypes compared to control groups.3,4 In affective disorders, there may be associations between the high-expressing allele (LA or "L" in a biallelic model) and higher response rates to antidepressant treatment.11,14 Other studies did not find an association between 5-HTTLPR and depression.9 These inconsistencies could result from differences in study design.
We hypothesized a positive association between 5-HTTLPR and the number of unexplained somatic symptoms because the serotonergic system plays a major role in pain perception and pain thresholds. We examined patients with at least 2 unexplained symptoms and measured the frequency of the triallelic form of 5-HTTLPR from blood samples. We also controlled for the possible influence of depression.
METHOD
Sample and Psychometric Instruments
Ninety-one subjects (mean [SD] age = 50.89 [12.65] years; 39.6% men, 60.4% women) with a history of at least 2 unexplained physical symptoms were included in this study (see Table 1); patients were recruited from 2001 until 2004. Participants were a subsample of 2 prior studies and were recruited from primary care practices. The procedures were fully explained to the participants. Additionally, they were asked whether they agreed to let their blood be drawn and tested for genetic variants. All participants gave their informed consent to the procedures. Study protocol was approved by the Ethics committee of the German Society of Psychology (DGPs). Detailed inclusion criteria are described elsewhere.15,16 Of 289 subjects, 104 agreed to have their blood tested for 5-HTTLPR, and 13 were excluded because the correct alleles could not be determined. Eighty- six (94.51%) of the participants were diagnosed with at least 1 DSM-IV somatoform disorder—ie, somatization disorder, undifferentiated somatization disorder, pain disorder, conversion disorder, or hypochondriasis (mean [SD] number of disorders = 1 [0.58]). Participants were mainly of German (ie, Caucasian) origin (n = 89, 97.8%), with 2 (2.2%) reporting other origins.
Subjects were evaluated by laboratory analyses, medical history, interviews, and self-rating scales. We used the International Diagnostic Checklists for ICD-10 and DSM-IV17 to assess psychiatric morbidity, and the interview version of the Screening for Somatoform Symptoms, persistent symptoms in the last 2 years (SOMS-2)18 was used to assess somatoform complaints during the last 2 years. This interview is equivalent to the symptom list used in the DSM-IV criteria for somatization disorder. The questionnaires assessed current somatic symptoms using the SOMS-7 (persistent symptoms in the last 7 days, reflecting the "state" version of the SOMS-2),18 and depressive symptoms were quantified with the Beck Depression Inventory (BDI).19
Genotyping
Genomic DNA was isolated from whole blood according to standard procedures. The 5-HTTLPR polymorphism was determined in its triallelic form, in which the A/G SNP rs25531 leads to a further differentiation of the L allele into LA and LG.10 The alleles were genotyped by polymerase chain reaction (PCR) and subsequent restriction fragment length polymorphisms (RFLP) analysis using the following primers: forward primer: 5-CTC CCT GTA CCC CTC CTA GG-3‘ ²; reverse primer: 5‘ ²-TGC AAG GAG AAT GCT GGA G-3‘ ². PCR was performed with 50 ng DNA in a total volume of 15 µL containing 1.5 µL PCR buffer, 0.6 µM each primer, 3 µL Q-solution (Qiagen, Hilden, Germany), and 0.15 µL (0.75 units) Taq polymerase (Qiagen, Hilden, Germany). After denaturation at 95°C for 15 minutes, 35 cycles of PCR were performed with the following conditions: 94°C for 30 seconds, 60°C for 30 seconds, 72°C for 30 seconds, and a final extension at 72°C for 7 minutes. 15 µL PCR product was digested with 1 µL MspI and 2 µL Puffer Y-Tango (Fermentas, St. Leon-Rot, Germany) in a total volume of 20 µL at 37°C overnight. The samples were run on a 3% agarose gel and visualized by ethidium bromide staining. For the LA-allele bands of 245 bp and 38 kb and for the LG-allele bands of 162 kb, 83 kb, and 38 kb were obtained, whereas the S allele was visible by an uncut band of 211 bp.
Statistical Analyses
SPSS software (version 15.0 for Windows; SPSS Inc., Chicago, Illinois) was used for statistical analyses. Missing values for the self-report scales were imputed using an expectation-maximization algorithm. The triallelic genotypes were reclassified into a biallelic model by their levels of expression. LG and S, which are associated with lower levels of expression than LA, were reclassified as S‘ ², and LA was reclassified as L‘ ².13 Group differences were described using Kruskal-Wallis rank analysis (or Mann-Whitney test/χ2 test where appropriate). Associations were assessed using the Cramer V; since P values calculated in this statistical procedure are actually approximations of P values, designations of these values are shown within quotation marks ("P").
RESULTS
Genotype distribution was in Hardy-Weinberg equilibrium in the triallelic (χ2 = 6.25, df = 4, P > .05) and the biallelic model (χ2 = 5.89, df = 2, P > .05). 5-HTTLPR biallelic genotype groups did not differ in terms of age, sex, and comorbid major depressive disorder (Table 1). For the biallelic model, there was a significant group difference for somatic complaints during the 2 years prior (SOMS-2) (H = 12.11, df = 2, P < .01). Participants with the L‘ ²L‘ ² (mean [SD] number of symptoms = 9.13 [4.44]) and the L‘ ²S‘ ² (mean [SD] number of symptoms = 7.41 [3.7]) variants reported significantly more persistent somatic complaints than those with the S‘ ²S‘ ² variant (mean [SD] number of symptoms = 5.4 [3.24]) (U = 282.5, P < .05, and U = 193.5, P < .01, respectively). There was no significant group difference for current depressive symptoms (BDI) (H = 3.71, df = 2, P > .10) or somatic complaints during the previous 7 days (SOMS-7) (H = 0.53, df = 2, P > .10). Figure 1 shows the "trait" somatic complaints for the 2 allelic models; for the means and standard deviations (SOMS-2, SOMS-7, BDI) for the biallelic model, see Table 1. For the triallelic model, the groups were too small to test for differences, but the Cramer V revealed a significant association between genotype and "trait" somatic complaints (Cramer’s V = 0.28, "P" < .05); 40.6% of the participants with the LALA genotype reported a high number of somatic complaints in the last 2 years (compared to 10% of those with the SS genotype). There were no significant associations for "state" somatic complaints or current depressive symptoms (Cramer’s V = 0.16 and 0.21, "P" > .05).
Further analysis on a possible association between depression and 5-HTTLPR also failed to show significant results (binary logistic regression with diagnosis of major depression as dependent variable and SOMS-2 scores and biallelic genotype as covariates; data not shown).
Click figure to enlarge
Click figure to enlarge
DISCUSSION
We expected the allelic variation in 5-HTTLPR to be positively associated with the number of unexplained physical symptoms in patients with somatoform disorders. Accordingly, we found that persistent somatic symptoms are related to higher-expressing alleles of SLC6A4, which are thought to facilitate higher reuptake rates of serotonin. Similar findings have been shown for chronic fatigue syndrome and temporomandibular disorders.3,4 It is possible that higher reuptake rates cause lowered 5-HT concentrations in the extracellular space, thus creating a relative hypofunction of the serotonergic system that in turn might cause or amplify somatic symptoms.3,4 However, the causality of the connection between 5-HTTLPR allelic variants and somatoform symptoms remains to be clarified as there are also findings contrary to our results. For other somatoform-associated disorders such as fibromyalgia, links to lower-expressing allele (S) were reported.20 Our results suggest that serotonergic pathways are associated with a lasting tendency to experience physical symptoms ("trait") but not with the perception of acute symptoms ("state"). Considering the reduced tryptophan concentration found in patients with somatization disorders8 and the efficacy of serotonergic medication on pain symptoms in somatoform disorders,21 there is evidence for the influence of serotonergic pathways in the development and maintenance of somatoform disorders.
The results of previous studies on mood disorders have been inconsistent, sometimes revealing associations with 5-HTTLPR alleles that were not confirmed in other studies.9 We found no significant associations between allelic genotypes and depressive symptoms or comorbid major depression, possibly because affective disorders were not the predominant diagnoses for participants.
There are some limitations to the interpretation of our findings. External validity may be limited, as we did not include a symptom-free control group in our design. In addition, the participants of this study were mainly of German origin. Previous studies have shown considerable variations in the distribution of 5-HTTLPR alleles in different cultures22; therefore, our assumptions might be limited to Caucasian populations. Furthermore, we examined only a single candidate gene. In regard to the heterogeneity of symptoms in both somatoform and affective disorders, interactions of multiple genes as biologic bases for the psychiatric phenotypes are thought to be more likely.9 Future research, therefore, should control for gene-gene interactions.
It might also be interesting to compare differences in allelic genotypes for patients with subsyndromes to further investigate the effects of serotonin in somatoform symptomatology. Due to the small sample size, it was not suitable to compare subgroups in our study, and the results might only be considered preliminary. However, our results confirm the association of somatoform symptoms with the serotonergic system, and to our knowledge this is one of the first reports on somatization and gene expression.
Author affiliations: Division of Clinical Psychology and Psychotherapy, Philipps University of Marburg (Ms Hennings and Prof Dr Rief); and Psychiatric and Psychotherapeutic Hospital, Ludwig-Maximilian University, Munich (Dr Zill), Germany.
Financial disclosure: The authors declare no conflicts of interest that could have influenced this study report.
Funding/support: This study was funded by 2 grants to Prof Dr Rief, both from the German Ministry of Education and Research (BMBF). One of the 2 grants was part of the German Competence Network on Depression and Suicidality.
Previous presentation: The results were presented previously in a poster at the "26. Symposium der Fachgruppe Klinische Psychologie und Psychotherapie der Deutschen Gesellschaft für Psychologie," May 1-3, 2008, Potsdam, Germany.
Acknowledgment: We wish to thank Dr Elisabeth Rauh, Thomas Zech, and Andrea Bender for supporting this study. We are also grateful to the "Steering Committee Genetics" of the Competence networks of medicine: depression and suicidality, namely Prof Dr Wolfgang Maier, Prof Dr Marcella Rietschel, and Dr Dan Rujescu. None of the individuals acknowledged here report any financial affiliation or other relationship relevant to the subject of this article.
REFERENCES
1. Kroenke K, Mangelsdorff D. Common symptoms in ambulatory care: incidence, evaluation, therapy, and outcome. Am J Med. 1989;86:262-266. PubMed doi:10.1016/0002-9343(89)90293-3
2. Rief W, Barsky AJ. Psychobiological perspectives on somatoform disorders. Psychoneuroendocrinology. 2005;30:996-1002. PubMed doi:10.1016/j.psyneuen.2005.03.018
3. Narita M, Nishigami N, Narita N, et al. Association between serotonin transporter gene polymorphism and chronic fatigue syndrom. Biochem Biophys Res Commun. 2003;311:264-266. PubMed doi:10.1016/j.bbrc.2003.09.207
4. Ojima K, Watanabe N, Narita N, et al. Temporomandibular disorder is associated with a serotonin transporter gene polymorphism in the Japanese population. Biopsychosoc Med. 2007;1:3. PubMed doi:10.1186/1751-0759-1-3
5. Suzuki R, Rygh LJ, Dickenson AF. Bad news from the brain: descending 5-HT pathways that control spinal pain processing. Trends Pharmacol Sci. 2004;25:613-617. PubMed doi:10.1016/j.tips.2004.10.002
6. Lucki I. The spectrum of behaviors influenced by serotonin. Biol Psychiatry. 1998;44:151-162. PubMed doi:10.1016/S0006-3223(98)00139-5
7. Ressler KJ, Nemeroff CB. Role of serotonergic and noradrenergic systems in the pathophysiology of depression and anxiety disorders. Depress Anxiety. 2000;12(suppl.2):2-19. PubMed doi:10.1002/1520-6394(2000)12:1+<2::AID-DA2>3.0.CO;2-4
8. Rief W, Pilger F, Ihle D, et al. Psychobiological aspects of somatoform disorders: contribution of monoaminergic transmitter systems. Neuropsychobiology. 2004;49:24-29. PubMed doi:10.1159/000075335
9. Glatt CE, Freimer NB. Association analysis of candidate genes for neuropsychiatric disease: the perpetual campaign. Trends Genet. 2002;18:307-312. PubMed doi:10.1016/S0168-9525(02)02670-7
10. Hu X, Oroszi G, Chun J, et al. An expanded evaluation of the relationship of four alleles to the level of response to alcohol and the alcoholism risk. Alcohol Clin Exp Res. 2005;29:8-16. PubMed doi:10.1097/01.ALC.0000150008.68473.62
11. Smeraldi E, Serretti A, Artioli P, et al. Serotonin transporter gene-linked polymorphic region: implications of rare variants. Psychiatr Genet. 2006;16:153-158. PubMed doi:10.1097/01.ypg.0000218611.53064.a0
12. Stein MB, Seedat S, Gelernter J. Serotonin transporter gene promoter polymorphism predicts SSRI response in generalized social anxiety disorder. Psychopharmacology (Berl). 2006;187(1):68-72. PubMed doi:10.1007/s00213-006-0349-8
13. Parsey RV, Hastings RS, Oquendo MA, et al. Effect of a triallelic functional polymorphism of the serotonin-transporter-linked promoter region on expression of serotonin transporter in the human brain. Am J Psychiatry. 2006;163:48-51. PubMed doi:10.1176/appi.ajp.163.1.48
14. Smits KM, Smits LJ, Schouten JS, et al. Influence of SERTPR and sTin2 in the serotonin transporter gene on the effect of selective serotonin reuptake inhibitors in depression: a systematic review. Mol Psychiatry. 2004;9:433-441. PubMed doi:10.1038/sj.mp.4001488
15. Rief W, Martin A, Rauh E, et al. Evaluation of general practitioners’ training: how to manage patients with unexplained physical symptoms. Psychosomatics. 2006;47:304-311. PubMed doi:10.1176/appi.psy.47.4.304
16. Martin A, Rauh E, Fichter M, et al. A one-session treatment for patients suffering from medically unexplained symptoms in primary care: a randomised clinical trial. Psychosomatics. 2007;48:294-303. PubMed doi:10.1176/appi.psy.48.4.294
17. Hiller W, Zaudig M, Mombour W. IDCL: Internationale Diagnosen Checklisten für ICD-10 und DSM-IV. Bern, Switzerland: Verlag Hans Huber; 1995.
18. Rief W, Hiller W, Heuser J. SOMS: Screening for Somatoform Symptoms. Bern, Switzerland: Verlag Hans Huber; 1997.
19. Hautzinger M, Bailer M, Worall H, et al. Beck-Depressions-Inventar, 2. Aufl. Bern, Switzerland: Verlag Hans Huber; 1995.
20. Offenbaecher M, Bondy B, de Jonge S, et al. Possible association of fibromyalgia with a polymorphism in the serotonin transporter gene regulatory region. Arthritis Rheum. 1999;42:2482-2488. PubMed doi:10.1002/1529-0131(199911)42:11<2482::AID-ANR27>3.0.CO;2-B
21. Fishbain DA, Cutler RB, Rosomoff HL, et al. Do antidepressants have an analgesic effect in psychogenic pain and somatoform pain disorder? a meta-analysis. Psychosom Med. 1998;60:503-509. PubMed
22. Hu XZ, Lipsky RH, Zhu G, et al. Serotonin transporter promoter gain-of-function genotypes are linked to obsessive-compulsive disorder. Am J Hum Genet. 2006;78(5):815-826. PubMed doi:10.1086/503850
