Clinical Rheumatology

, Volume 23, Issue 4, pp 338–344 | Cite as

Mutational analysis of serotonin receptor genes: HTR3A and HTR3B in fibromyalgia patients

  • Bernd Frank
  • Beate Niesler
  • Brigitta Bondy
  • Michael Späth
  • Dieter E. Pongratz
  • Manfred Ackenheil
  • Christine Fischer
  • Gudrun Rappold
Original Article

Abstract

The neurotransmitter serotonin (5-hydroxytryptamine, 5-HT) has been implicated in numerous human disorders. Dysfunction of serotonergic neurotransmission is thought to play a major role in the pathophysiology of the fibromyalgia syndrome (FMS) which is characterised by non-restorative sleep and severe pain. In our study, both serotonin receptor subunit genes, HTR3A and HTR3B, have been investigated for sequence variations in FMS patients in order to reveal a possible involvement in the aetiology of FMS. We examined DNA samples from 48 patients with FMS representing sporadic cases by single-strand conformation polymorphism (SSCP) and denaturing high-performance liquid chromatography (dHPLC) analysis, sequenced samples with conspicuous patterns and performed statistical calculations. HTR3A mutational analysis revealed one novel as well as five known sequence variations. Investigating HTR3B, we detected seven formerly described mutations and one novel sequence variant. Statistical computation rated all variants as probably non-disease-related polymorphisms. Nevertheless, one might speculate about an effect of the respective sequence variants on the severity of the disease. Sequence variants of the serotonin receptor subunit genes HTR3A and HTR3B indicate no obvious significance in the aetiology of fibromyalgia, yet they represent the basis for future studies on their pharmacogenetic relevance.

Keywords

Fibromyalgia HTR3A HTR3B Mutational analysis Serotonin receptor genes 

Introduction

In the nervous system serotonin (5-hydroxytryptamine, 5-HT) represents a key neurotransmitter. The effect of serotonin is mediated by different 5-HT receptor subtypes: 5-HT1R to 5-HT7R [1]. They are encoded by a multigene family of receptors coupled to G-protein binding proteins except for the 5-HT3R, representing a ligand-gated ion channel. 5-HT3R is formed by an oligomeric complex of five subunits. The 5-HT3 receptor subunit genes HTR3A and HTR3B are predominantly expressed in brain regions such as amygdala, caudate nucleus and hippocampus [2, 3]. Interestingly, serotonergic dysfunction has been suggested to be involved in neurogenetic diseases such as bipolar affective disorder, schizophrenia and Tourette syndrome [4, 5, 6]. According to animal studies, serotonin is also proposed to play a major role in the pathophysiology of the fibromyalgia syndrome (FMS) [7] which affects 1% of the world’s population and is characterised by chronic, widespread and persistent pain associated with symptoms such as stiffness, fatigue and psychological distress [8, 9]. Clinical features of fibromyalgia include depression, anxiety and sleep disturbances. FM runs within families which show an increased loading with depressive disorders leading to the suggestion that FMS might be a “depressive spectrum disorder” [10]. Positive therapeutic response to antidepressant drugs as well as a decreased pain perception threshold during depression were considered as being attributable to dysfunction in several neurotransmitter systems [11, 12]. The aetiology of fibromyalgia is yet unknown, but on the basis of multiple biological findings, 5-HT is strongly suggested to contribute to the aetiology of FMS. These findings include disturbances in the serotonin pathway such as low levels of serotonin and tryptophan in serum and 5-hydroxyindole acetic acid (5-HIAA) in cerebrospinal fluid (CSF) of idiopathic pain patients [13, 14, 15, 16]. Despite familial aggregation, a genetic involvement in FMS development has not yet been confirmed [17]. This basically arises from limited studies on the role of genetics in FMS. Analysing the polymorphisms of the promoter region of the serotonin transporter gene in patients e.g. pointed to a contribution to the clinical severity of FMS [18]. Bondy et al. e.g. defined the silent T102C mutation of the 5-HT2A receptor gene as a fibromyalgia-associated variant [19]. Recent studies have revealed 5-HT3 receptor antagonists such as ondansetron, granisetron and tropisetron as potential drugs in the therapy of fibromyalgia patients [20].

Therefore, we studied 48 DNA samples of male and female patients suffering from FMS in order to detect nucleotide variants in the serotonin receptor genes HTR3A and HTR3B. For that purpose, mutational analysis was carried out using single-strand conformational polymorphism (SSCP) and denaturing high-performance liquid chromatography (dHPLC) analysis. Sequence variations in the respective genes were investigated to reveal a putative involvement in fibromyalgia aetiology.

Methods

Patients and controls

After approval by the Ethics Committee of the University of Munich, Germany, a total of 48 unrelated Caucasian FMS patients (all German, 38 females and 10 males, age range: 29–66 years, mean±SD: 51.12±9.1 years) were recruited by the Friedrich-Baur-Institute, University of Munich, after admission because of acute pain symptoms. Diagnoses were made according to the American College of Rheumatology (ACR) criteria, which require the presence of widespread pain for at least 3 months [8]. All patients were sporadic cases and underwent a clinical examination at baseline inception, and a tender point count (TPC) was used for collecting self-reported information on pain with 245-point pain severity items. The TPC developed by Lautenschläger et al. [21] consists of a body image illustrating 24 regions on the back and front which are commonly indicated as painful by FMS patients. The patient has to rate each region with a scale from 0–5 (no pain to extreme pain). A total score is calculated by adding the rates of all regions with 120 being the highest possible score. All patients gave informed consent to participate in the study. As controls we used the DNA of 62 healthy, unrelated subjects from Centre d’etude du polymorphisme humaine (CEPH) families [22] originating from Europe and the United States. The control sample consisted of 31 male and 31 female unrelated adult individuals. The extended control sample of 129–156 persons used for HTR3A exon 1, 2 and 9 analysis (Table 1 and Table 2) comprised both the CEPH families and individuals originating from Germany. Both HTR3A (accession number: D49394) and HTR3B (accession number: AF169255) variants were termed as recommended by den Dunnen and Antonarakis [23].
Table 1

Distribution of HTR3A variants in alleles of fibromyalgia patients and control individuals

Exon

Sequence variation

n

p value

HTR3A exon 1

-42C>T

30C>T (Leu10Leu)

  Σ FMS patients

16 (16.7%)

2 (2.1%)

96

0.3424

  Controls

35 (11.2%)

5 (1.6%)

312

HTR3A exon 2

97G>A (Ala33Thr)

  Σ FMS patients

2 (2.1%)

96

0.180

  Controls

1 (0.4%)

258

HTR3A exon 3

IVS3+7A>C

  Σ FMS patients

8 (8.3%)

96

0.3888

  Controls

5 (5.2%)

96

HTR3A exon 6

576G>A (Leu192Leu)

  Σ FMS patients

4 (4.2%)

96

0.6826

  Controls

2 (2.1%)

94

HTR3A exon 9

1377A>G (Leu459Leu)

  Σ FMS patients

21 (21.9%)

96

0.6865

  Controls

74 (23.9%)

310

Table 2

Distribution of HTR3B variants in alleles of fibromyalgia patients and control individuals

Exon

Sequence variation

n

p value

HTR3B exon 1

-102_-100delAGA

  Σ FMS patients

17 (17.7%)

96

0.4158

  Controls

17 (13.7%)

124

HTR3B exon 4

IVS4+12G>A

IVS4+11C>T

  Σ FMS patients

31 (32.3%)

5 (5.2%)

96

0.9257

  Controls

40 (32.3%)

8 (6.5%)

124

HTR3B exon 5

386A>C (Tyr129Ser)

[386A>C;466A>C] ([Tyr129Ser;Ser156Arg])

462G>A (Ala154Ala)

  Σ FMS patients

26 (27.1%)

0 (0%)

0 (0%)

96

0.4105

  Controls

25 (20.2%)

1 (0.8%)

1 (0.8%)

124

HTR3B exon 6

IVS6+72A>G

547G>A (Val183Ile)

  Σ FMS patients

22 (22.9%)

1 (1.0%)

96

0.1275

  Controls

17 (13.7%)

1 (0.8%)

124

Determination of HTR3B exon-intron boundaries

Searching the human database (http://www.ncbi.nlm.nih.gov/BLAST/) with the HTR3B cDNA sequence (accession number: AF169255), we detected the BAC clone RP11–799K20 (accession number: AC020742) containing all HTR3B exons. The BAC was derived from the Resource Center, Oakland, Calif., USA (RPCI-11, Roswell Park Cancer Institute Human BAC Library). Exon-intron organisation of HTR3B was determined based on the available sequence data of RP11–799K20 using the SIM4 algorithm (http://pbil.univ-lyon1.fr/sim4.php). This enabled us to establish exon-flanking HTR3B primers for polymerase chain reaction (PCR) analysis (Table 3). RP11–799K20 was used as positive control.
Table 3

Primer sequences for HTR3B analysis using dHPLC

Exon

Primer

Sequence (5’->3’)

Size

TA (°C)

1

HTR3B-5’-UTR/E1FOR

ATC AAT TCC AAA ACA TTT GC

299 bp

54

HTR3BIN1REV

AAG AGT TAA TCC TTA ATG CCT AGT

2

HTR3BIN2FOR

AAC CAA GCT CCT CTT ACT TTT

259 bp

53

HTR3BIN2REV

TGT TTC AAA AGA CGT TAA CAA G

3

HTR3BIN3FOR

TCT TCT GAA TTT CCT TAC TGC TGA

195 bp

56

HTR3BIN3REV

AAC TAT ATT TTG CAG CAG ATC CA

4

HTR3BIN4FOR

CAG TCT AAA CCC CAC AGA AA

197 bp

62/58/54 (step-down)

HTR3BIN4REV

AGA TAT TGC CCC ATT TTG AT

5

HTR3BIN5FOR

TCA CCT ATG TGC ACC AAC C

299 bp

48

HTR3BIN5REV

ACC CTC TCC CTA TTC AGC CTA T

6

HTR3BIN6FOR

TGA GAA CAT GCA ATA GAG CAG

303 bp

62/58/54 (step-down)

HTR3BIN6REV

TTG GCA TCT CTT TCT CTC TG

7

HTR3BIN7FOR

AAA TCT GAG TCT GTT GGC CT

305 bp

58

HTR3BIN7REV

TGT AAG ATG AGT GTC CAG TG

8

HTR3BIN8FOR

AGA ATC ACT TCA GCC CAA TAC G

485 bp

64/62/58 (step-down)

HTR3BIN8REV

AAA TGC CAC ACC CAG GCT

9

HTR3BIN9FOR

CCT TGA AGG ATG AGG CCA T

521 bp

64/62/58 (step-down)

HTR3BIN9REV

TGT GGT CTC ACT ATG TTG CTC TG

PCR

HTR3A mutational analysis

PCR for SSCP analysis was carried out as described [24].

HTR3B mutational analysis

Standard PCR amplifications were performed using 50 ng of genomic DNA (20 ng of BAC DNA as positive control, respectively), 12.5 pmole of each primer, 200 µM dNTPs (MBI Fermentas, St. Leon-Rot, Germany), 1.5 mM MgCl2, PCR buffer pH 8.7 (1×), Q-Solution (1×) and 1 U HotStarTaq DNA Polymerase (Qiagen, Hilden, Germany) in a total volume of 25 µl. Thermal cycling was carried out using the Thermocycler PTC-200 (MJ Research, Waterdown, Mass., USA) with the following conditions: Initial denaturation for 15 min at 96°C was followed by 35 cycles consisting of 30 s at 96°C, 30 s at annealing temperature (TA), 30 s to 1 min at 72°C and a final extension step for 5 min at 72°C. The formation of heteroduplexes (re-annealing for the dHPLC analysis) was achieved by heating the amplified products for 5 min at 95°C and cooling them gradually down to 4°C within 45 cycles (−2.0°C/cycle). For the HTR3B analysis exon-flanking primers were designed amplifying exon-intron junctions as well. Names of primers, primer sequences, sizes of the respective products, TA and dHPLC conditions are given in Table 3. Exon 4 was analysed by SSCP analysis.

SSCP analysis

SSCP analysis was carried out as described elsewhere [24, 25].

dHPLC analysis

dHPLC analysis was performed using a WAVE DNA fragment analysis system (Transgenomics, Santa Clara, Calif., USA). PCR products were automatically loaded on a DNASep column. Optimal column temperatures were selected based on recommendations of the WAVEMAKER software (Transgenomics, Santa Clara, Calif., USA) and confirmed by test runs of the respective amplicon. Using a 7.5-min linear acetonitrile gradient, the amplified products were eluted from the column at a constant flow rate of 0.9 ml/min. The gradient was created by combining 0.1 M triethylammonium acetate buffers (TEAA, Transgenomics, Santa Clara, Calif., USA) pH 7.65 with both 0.025% (v/v) acetonitrile (buffer A, Sigma-Aldrich, Taufkirchen, Germany) and 25% (v/v) acetonitrile (buffer B). The elution of the PCR products was monitored by a UV detector and analysed with the D-7000 HSM program version 3.0-2.1 (Transgenomics, Santa Clara, Calif., USA).

Sequencing

PCR products were sequenced either with unlabelled gene-specific primers using a MEGABACE Sequencer (Amersham Biosciences, Uppsala, Sweden) or—after subcloning into the pCRII Topo vector (pTOPO kit, Invitrogen, Breda, The Netherlands)—with Cy5-labelled vector primers on an ALFExpress automated sequencer (Amersham Biosciences, Uppsala, Sweden). Selection of clones to be sequenced was based on allele identification on SSCP level as described [24]. Sequencing reactions were performed according to the manufacturer’s protocol (Cycle Reader Kit, MBI Fermentas/DYEnamic ET Terminator Cycle Sequencing Kit, Amersham Biosciences, Uppsala, Sweden).

Statistical analysis

Comparison of allele frequencies was performed using two-sided χ2 tests or, in case of cell counts smaller than five, Fisher’s exact test (SAS 8.02 on PC, Cavey, N.C., USA). Significance level was 0.05, not adjusted for multiple comparisons. Differences of allele frequencies and 95% confidence intervals were estimated as described [26] using a tool accessible via the Internet (http://ihg.gsf.de/cgi-bin/hw/hwa1.pl).

Results

Mutational analysis of HTR3A

We analysed a total of 110 DNA samples including 48 fibromyalgia patients and 62 control subjects. Exon-specific primers were used for all PCRs except for exon 1 and exon 3 [24]. The sizes of the analysed fragments ranged from 104 to 295 bp. We used the SSCP technique [25] in order to detect point mutations, small deletions, insertions and other small rearrangements in HTR3A. Sequencing of PCR products showing mobility shifts led to the identification of six sequence variants. Five of them had already been described [24, 27]: a nucleotide exchange in the 5’ untranslated region (UTR) of the gene (-42C>T, homo- and heterozygous) encoding a putative amino acid exchange in an upstream open reading frame (uORF2: Pro16Ser), three variants leading to silent mutations residing in exon 1 [30C>T (Leu10Leu) heterozygous], exon 6 [576G>A (Leu192Leu), heterozygous] and exon 9 [1377A>G (Leu459Leu), homo- and heterozygous] and a heterozygous point mutation in intron 3 [IVS3+7A>C] (Table 1 and Fig. 1). The 5’ UTR variant, the three silent mutations as well as the point mutation in intron 3 were detected with comparable frequencies in fibromyalgia patients and control individuals (Table 1), p values not being below the critical value of p=0.05.
Fig. 1

Genomic structure of HTR3A and HTR3B including detected sequence variants (indicated by black arrows). Exons are drawn as boxes (with sizes below) and introns as interrupted lines (not drawn to scale). Conserved regions (transmembrane domains and cysteine loop) are characterised as TM1-TM4 and C-C. 3’ untranslated region (UTR) and 5’ UTR [containing uORF (upstream open reading frame) 1 and uORF2] are marked as grey boxes. Novel variants are indicated in light grey, known variants in black. Tyr129Ser and Ser156Arg exclusively occur in combination affecting one single allele (compound allelic variant) in a control bearing Ala154Ala on the second allele. Detailed information about the detected variants is given as follows: 42C>T (GGCCTCG [C/T] CCTGAGC) (dbSNP: rs1062613); Leu10Leu (CGCTGCT [C/T] GCCTTGC); Ala33Thr (CAGGCCC [G/A] CTCTGCT); IVS3+7A>C (GGTGAGC [A/C] GACCCGC); Leu192Leu (GGCGCTT [G/A] CCAGAAA); Leu459Leu (ACCTGCT [A/G] GCGGTGC) (dbSNP: rs1176713); -102–100delAAG (AACGGAG -/AAG GAGGAGG); IVS4+11C>T (AGTGTGC [C/T] GGTGTGT); IVS4±12G>A (GTGTGCC [G/A] GTGTGTA) (dbSNP: rs1176746); Tyr129Ser (GAAAGAT [A/C] CCCTGAC) (dbSNP: rs1176744); Ala154Ala (TCTCTGC [G/A] TGCAGTT) (dbSNP: rs2276305); Ser156Arg (TGCGTGC [A/C] GTTTAGA); Val183Ile (GGAAGAC [G/A] TAGACCT) and IVS6±72A>G (CCAAGGA [A/G] TTTCTGC) (dbSNP: rs2276307)

Additionally, we identified a novel missense mutation in exon 2 (97G>A, heterozygous) leading to an amino acid exchange from alanine to threonine (Ala33Thr) in two patients. For this reason, the number of control subjects was extended from 62 to 129. Within this extended control group one individual showed the respective sequence variant in a heterozygous manner (Table 1) suggesting that this variant represents a polymorphism. According to the statistical calculation, that particular variant failed to reach significance with regard to FMS (p=0.18). For further description, estimated differences of allele frequencies and 95% confidence intervals are shown in Table 4.
Table 4

Allele frequency differences between FMS patients and controls. Patients-controls are given in % with 95% confidence intervals

HTR3A exon 1

-42C>T

Patients-controls

5.4 (-2.4;15.2)

HTR3A exon 1

30C>T (Leu10Leu)

Patients-controls

0.48 (-2.0;6.1)

HTR3A exon 2

97G>A (Ala33Thr)

Patients-controls

1.7 (-0.7;7.8)

HTR3A exon 3

IVS3+7A>C

Patients-controls

3.1 (-5;11.1)

HTR3A exon 6

576G>A (Leu192Leu)

Patients-controls

2.0 (-1;8.7)

HTR3A exon 9

1377G>A (Leu459Leu)

Patients-controls

-2.0 (-4.0;8.7)

HTR3B exon 1

-102_-100delAAG

Patients-controls

4.0 (-6.0;14.8)

HTR3B exon 4

IVS4+12G>A

Patients-controls

<0.1 (-13.0 13.5)

HTR3B exon 4

IVS4+11C>T

Patients-controls

-1.3 (-7.8;6.3)

HTR3B exon 5

386A>C (Tyr129Ser)

Patients-controls

6.9 (-5.0;19.2)

HTR3B exon 6

IVS6+72A>G

Patients-controls

9.2 (-1.8;20.5)

Mutational analysis of HTR3B

We determined the exon-intron organisation of HTR3B using the SIM4 algorithm, which enabled us to establish primers for the subsequent mutational analysis. In HTR3B, we screened the complete coding region of nine exons using exon-flanking primers. The sizes of the analysed PCR products ranged from 195 to 521 bp (Table 3). All exons were investigated by dHPLC analysis. Since exon 4 failed to provide clear data due to indistinguishable dHPLC elution profiles, we additionally screened this exon by SSCP. The analysis covered the described samples of 48 fibromyalgia patients and 62 control individuals. We identified seven known HTR3B sequence variations: a deletion within the 5’ UTR (100_-102delAAG, heterozygous), two missense mutations [(386A>C, Tyr129Ser), heterozygous; (466A>C, Ser156Arg), heterozygous], the silent mutation 462G>A (Ala154Ala, heterozygous) and three intronic exchanges [(IVS4+11C>T), heterozygous], [(IVS4+12G>A), homo- and heterozygous], [(IVS6+72A>G), heterozygous] [28, 29]. In addition, one novel missense mutation (547G>A; Val183Ile, heterozygous) could be detected (Table 2 and Fig. 1). The silent mutation was found in one control sample, but in none of the patients. The variants 386A>C (Tyr129Ser) and 466A>C (Ser156Arg) were detected in the same control individual both affecting the same allele. The p value calculation for the respective variants suggested no significance with regard to fibromyalgia (Table 2).

Note

HTR3A –42C>T and 1377A>G have already been described by Niesler et al. [24, 27] as C178T and A1596G. HTR3B Ser156Arg and 462G>A (Ala154Ala) have already been mentioned by Gutiérrez et al. [28] as Ser533Arg and 529-G/A.

Discussion

The aim of the present study was to investigate a potential contribution of the serotonin receptor subunit genes HTR3A and HTR3B to the aetiology of fibromyalgia syndrome. SSCP and dHPLC analysis were used to screen a cohort of 48 FMS patients for point mutations, small deletions, insertions as well as other small rearrangements. dHPLC analysis required preceding determination of HTR3B exon-intron boundaries for subsequent primer design. The mutational analyses revealed genetic alterations in HTR3A and HTR3B. Regarding HTR3A, we detected six polymorphisms (Fig. 1) two of which are already present in SNP databases (http://www.ncbi.nih.gov/SNP/). Five of them had already been described [24, 27] and seemed to be non-disease related since they were not found in significantly different frequencies in patients and control subjects (Table 1, Fisher’s exact test/χ2 calculation, p values ranging from 0.18 to 0.69). However, -42C>T (C178T) has been formerly reported to be associated with bipolar affective disorder [27]. The novel genetic variant Ala33Thr (97G>A) was identified in two fibromyalgia patients. The frequency of Ala33Thr comes to 2.1% in patients vs 0.4% in control individuals, yet it does not reach statistical significance (p=0.18). Of course, we cannot exclude additional mutations that have not been detected due to the following reasons. First, the sensitivity of the SSCP technique has been deemed to be about 90% in PCR products up to 200 bp and approximately 80% in those of 400 bp in size [30]. Furthermore, SSCP analysis fails to detect micro-deletions encompassing one or more exons. It is known that up to 15% of disease-related mutations reside in splice sites [31]. However, except for exons 1 and 3, exon-intron junctions just as primer binding sites were not part of the analysis [24]. It also cannot be excluded that HTR3A as well as HTR3B promoter regions may reveal additional variants. The dHPLC analysis—used for HTR3B screening—is much more sensitive than the SSCP analysis and rests with 95–100%. In spite of a very high transfer rate, homozygous mutations are not detectable with this very method.

In HTR3B, one novel sequence variant could be detected (Fig. 1). Seven variants have already been described in cancer patients suffering from chemotherapy-induced emesis as well as in schizophrenic patients [28, 29]. Four of those have already been listed in public SNP databases. According to Fisher’s exact test as well as χ2 calculation, none of the alluded HTR3B alterations reached statistical significance (Table 1).

Our results show that unlike other serotonin receptor genes [32, 33, 34, 35, 36, 37, 38], HTR3A and HTR3B show a rather high genetic variability. Despite its genetic variability, HTR3A is highly conserved as the variants described do not affect amino acid coding and most likely do not interfere with codon usage. HTR3B, however, shows more missense mutations within the open reading frame (ORF) indicating a weaker conservation. The genetic complexity of FMS is reflected by contradictory and inconclusive research findings. For instance, the possible association of a serotonin transporter gene polymorphism has been frequently discussed [18, 39]. Further, recent studies suspect linkage of FMS to the HLA region [40]. According to previous investigations, the silent T102C mutation of the 5-HT2A receptor gene in fibromyalgia patients revealed a significantly different genotype distribution in patients suggesting a putative impact on the complex circuits of nociception [19]. Altered nociception is a consistent characteristic feature of FMS with serotonergic neurotransmission playing a significant role. Fibromyalgia patients exhibit disturbances in serotonin metabolism and transmission together with disturbances in several chemical pain mediators such as nerve growth factor, dynorphin and substance P (SP) [41]. The release of SP e.g. is thought to be limited in consequence of 5-HT3 receptor antagonists [42]. Thus, a study of fibromyalgia tropisetron-treated patients proved to be efficacious and well tolerated [43]. Although a contribution of HTR3A and HTR3B variants to the pathophysiology of the disease cannot be totally ruled out, explicit evidence remains to be supplied. The involvement of HTR3A/B variants in the putative pathomechanism of the disease might now be proven in pharmacogenetic studies. Nevertheless, the detected variants (SNPs, single nucleotide polymorphisms) represent valuable tools for future investigations.

Notes

Acknowledgements

We thank Nadja Muncke, Rüdiger J. Blaschke and Stefan Kirsch for helpful discussions and the Deutsche Forschungsgemeinschaft for support.

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Copyright information

© Clinical Rheumatology 2004

Authors and Affiliations

  • Bernd Frank
    • 1
  • Beate Niesler
    • 1
  • Brigitta Bondy
    • 2
  • Michael Späth
    • 3
  • Dieter E. Pongratz
    • 3
  • Manfred Ackenheil
    • 2
  • Christine Fischer
    • 1
  • Gudrun Rappold
    • 1
  1. 1.Institute of Human GeneticsUniversity of HeidelbergHeidelbergGermany
  2. 2.Psychiatric HospitalUniversity of MunichMunichGermany
  3. 3.Friedrich-Baur-InstituteUniversity of MunichMunichGermany

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