PANIK-Netz: Subproject P6

Efficacy of CBT on fear reduction in panic disorder: The role of genetic variation

Principal Investigator
Univ.-Prof. Dr. Jürgen Deckert
Department of Psychiatry
University of Würzburg

Duration applied for
3 years

Project description
Introduction / state of the art
Panic disorder (PD/AG) with its main components of panic attacks, anticipatory anxiety and agoraphobia is a heterogeneous disorder with complex etiology. Life events, situational stress and genetic vulnerability contribute to its pathogenesis. A recent meta-analysis based on twin studies estimates the contribution of genetic factors at 48% (Hettema et al. 2001). Molecular genetic studies in families and case-control-samples have provided evidence for a role of several gene loci, e.g. on chromosomes 9, 13, 18 and 22 (e.g. Hamilton et al. 2003, Hamilton et al. 2004) and specific genetic variants, e.g. in the monoamine oxidase (Deckert et al. 1999) and in the catechol-o-methyl-transferase genes (Hamilton et al. 2002, Domschke et al. 2004). Preliminary studies suggest a role for variation in the adenosine A2A receptor gene in the mediation of anxiogenic effects of caffeine and amphetamine (Alsene et al. 2003, Hohoff et al. 2005) and variation in the serotonin transporter gene in the mediation of the therapeutic effects of SSRIs. Findings in panic disorder have been related to findings in other anxiety disorders, affective disorders and personality dimensions of anxiety and depression (Lesch et al. 1996, Deckert et al. 1997, Strobel et al. 2004, Rothe et al. 2004).. Data are needed as to the role of genetic variation for individual clinical symptoms, psychophysiology, neuroendocrinology, regional brain activity as measured by functional MRI imaging (Domschke et al. 2005) and the interaction of genetic variation with life events as well as the relevance for therapeutic outcome. Enhanced knowledge on the influence of genetic variation on pathological habituation or sensitization/ conditioning and on the therapeutic effects of cognitive behavioural therapy in PD/AG could be of utmost relevance for improving psychotherapy and psychotherapeutic outcome.

By establishing the largest panic disorder DNA sample to date in conjunction with clinical, psychophysiological and neuroimaging data we will address the following questions:
1. Which genetic variations are associated with PD/AG and its individual components panic attacks, anticipatory anxiety and agoraphobia as well as associated personality features and life events?
2. Which genetic variations modulate anticipation, arousal, habituation and fear conditioning and reconditioning as measured by clinical, psychophysiological, neuroendocrinological, and neuroimaging approaches?
3. Which genetic variations modulate the therapeutic response to cognitive behavioural therapy, either self-guided or therapist-guided?
Results of these investigations will elucidate the molecular mechanisms acting in panic disorder and identify subgroups of patients which respond less to psychotherapy. They will thus not only  rovide the basis for the development of novel and innovative pharmacotherapies, but will contribute to the development of individual therapies for subgroups of patients which need more intensive care including additional psychotherapy elements and/or pharmacotherapy.

Own previous work and resources
The principal investigator over the last ten years has performed multiple DFG-supported multicenter studies into the genetics of panic disorder employing a case-control-approach and studying categorical as well as dimensional phenotypes:
Deckert J, Nöthen MM, Franke P, Delmo C, Fritze J, Knapp M, Maier W, Beckmann H, Propping P. Systematic mutation screening and association study of the A1 and A2A adenosine receptor genes in panic disorder suggest a contribution of the A2A gene to the development of disease. Molecular Psychiatry 3, 81-85, 1998
Deckert J, Catalano M, Syagailo J, Bosi M, Okladnova O, DiBella D, Nöthen MM, Maffei P, Franke P, Fritze J, Maier W, Propping P, Beckmann H, Bellodi L, Lesch KP. Excess of high activity monoamineoxidase A gene promoter alleles in female patients with panic disorder. Human Molecular Genetics 8, 621-624, 1999.
Alsene K, Deckert J, Sand PG, de Wit H, Association between A2A receptor gene polymorphisms and caffeineinduced anxiety, Neuropsychopharmacology 28, 1694-1702, 2003
Domschke K, Freitag CM, Kuhlenbäumer G, Schirmacher A, Sand P, Nyhuis P, Jacob C, Fritze J, Franke P, Rietschel M, Garritsen HS, Nöthen MM, Lesch KP, Stögbauer F, Deckert J. Association of the functional V158M catechol-o-methyltransferase polymorphism with panic disorder in women. International Journal of Neuropsychopharmacology, 7, 183-188, 2004
Schumacher J, Abou Jamra R, Becker T, Klopp N, Franke P, Illig T, Rietschel M, Jacob C, Sand P, Fritze J, Propping P, Cichon S, Deckert J, Nöthen MM. Investigation of the DAOA/G30 locus in panic disorder. Molecular Psychiatry 10, 428-429, 2005
Hohoff C, MacDonald JM, Baune BT, Cook EH, Deckert J, de Wit H. Interindividual Variability in Response to Amphetamine: Possible Role for Adenosine A2A Receptor Gene Polymorphisms. Neuropsychiatric Genetics, in press 2005, doi 10.1002/ajmg.b.30228

The present case-control-sample includes 300 patients with panic disorder diagnosed according to DSM-IIIR or DSM-IV employing a standardized interview and an identical number of age- and gender-matched anonymous blood donor controls. The control sample is currently expanded to double its size which will be achieved by 2006. An independent control sample is available through the collaboration with the NGFN NeuroNet (Prof. Nöthen and Prof. Rietschel), where 1200 population-based controls have been screened for the presence of neuropsychiatric disorders. 2 Samples of about 200 individuals each characterized for anxiety after caffeine and 1 sample of about 100 individuals characterized for anxiety after amphetamine are available at Münster through cooperations with groups at the University of Chicago (Prof. deWit) and Bristol (Prof. Rogers and Prof. Nutt). Samples of more than 200 healthy individuals characterized for personality dimensions of anxiety are available at the cooperating DFG-supported Würzburg group (Prof. Lesch). Access to other panic disorder samples for replication studies is available through established cooperations with groups at Munich (PD Keck, Prof. Holsboer), Toronto (Prof. Kennedy), Barcelona (Prof. Martin-Santos) and Milan (Prof. Catalano). The core group at Münster consists of Prof. J. Deckert. Katharina Domschke, Dr.rer.nat. C. Hohoff, the medical technical assistant K. Schwarte and as assistant manager G. Schnieders. Prof. Deckert is the head of the laboratory of Molecular Psychiatry at the Department of Psychiatry which has all equipment necessary for DNA extraction, storage and basic genotyping. In high-throughput genotyping and complex statistical analysis of large samples it will be assisted by the High-throughput Genotyping Service Unit of the Central Laboratory of the UKMünster headed by B. Schlüter and the Department of Genetic Epidemiology headed by Prof. Dr. sc.hum. M. Stoll at the Leibniz-Institute of Atherosclerosis Research which is an associated Genetic Epidemiological Methods Center within the NGFN. For the statistical analysis of smaller samples of quantitative dimensional intermediate phenotypes and longitudinal data, support will be obtained from C. Freitag (Department of Child and Adolescent Psychiatry, University of Saarland at Homburg).

Work program
EDTA-blood of patients taking part in the core clinical network trial as well as patients and controls taking part in the associated psychophysiological, neuroendocrinological and neuroimaging studies, who have given their informed consent, will be collected and send to the laboratory of Molecular Psychiatry at the University of Münster. Collection and shipment of the blood will be taken care of by members of the cooperating psychiatry departments. DNA will be extracted employing an established automated procedure (ABI 6100; MTA K. Schwarte). Genotyping (Tecan-Genesis 150, ABI Prism 7000 SDS and 7900HT, ABI Prism 3700 and 3730; Dr. Hohoff, Prof. Deckert) will be performed for candidate genes on the basis of drug mechanisms (e.g. serotonergic genes), challenge paradigms (e.g. adenosine and cholecystokinin receptor genes), neuroendocrinological findings (e.g. HPA axis and ANP/BNP genes), linkage study findings (positional candidates) in humans and mice (e.g. G30/G72 and RGS genes) as well as on the basis of knock-out mice models. Functionally characterized genetic variants will be investigated as well as tagging single nucleotide polymorphisms developed employing HapMap. At Münster, SNPs will be genotyped primarily by means of 5’ exonuclease allelic discrimination assays using fluorescent probes specific for the individual alleles on an ABI Prism 7900HT or an ABI Prism 7000 Sequence Detection System. Genotyping for some SNPs may have to be performed by direct sequencing with the ABI Prism 3730 DNA analyzer. For this, PCR reactions will be controlled by separating PCR reactions on an agarose gel. PCR products will be purified using the vacuum manifold system with MultiScreen-96PCR filtration plates (Millipore GmbH) for pre-sequencing PCR purification. Sequencing reactions will then be performed forward and reverse using the BigDyeTM Terminator v3.1 Kit (Applied Biosystems). Post-sequencing PCR products will be purified using MultiScreen HV plates filled with sepharose. Purified products will then be separated on an ABI Prism 3700 or 3730 DNA analyzer using automated laser fluorescence capillary electrophoresis. To determine the respective allele frequencies of repeat polymorphisms, PCR-based automated fragment length analysis will be applied. After PCR amplification using one 5’-FAM labeled primer, the diluted PCR product will be mixed and incubated with denaturing solution and TAMRA or ROX standard size marker (Applied Biosystems). Separation of the PCR generated alleles will be performed by the ABI Prism 3700 or 3730 DNA analyzer and analyzed using the GeneScan analysis software 3.5. All genotyping assays will be performed blind with respect to the phenotypic characteristics of the probands, and at least in triplicate. Genotypes will be assessed for Hardy-Weinberg equilibrium. Haplotypes will be individually assigned by PHASE, version 2.1. Statistical analyses (Prof. Stoll, Dr. Freitag) will be performed to address the questions listed under 4.2. Classical association analysis for genotypes will be performed by chi2-tests; haplotype analysis will be done by permutation tests implemented in Haploview or FastEH. Logistic and linear regression as well as generalized linear mixed models for longitudinal data will be performed to control for possible confounding variables, and to assess genetic effects on dimensional intermediate phenotypes as well as on repeated measures. Utilizing the NGFN NeuroNet control sample, classical case-control studies with high power can be performed allowing for the identification of novel candidate genes. The functional relevance of these in addition to those of already established candidate genes for the intermediate phenotypes provided by the study can then be investigated. An exploratory analysis of the various intermediate phenotypes obtained from the physiological, neuroendocrinological and neuroimaging projects aims to generate hypotheses about the relevance of genetic variation for the various measures of anticipation, arousal, habituation, conditioning and extinction. Finally, the relevance of genetic variation for the clinical outcome data from the trial (i.e. response, remission, improvement) will be evaluated. Results of these analyses will not only help identify susceptibility genes for panic disorder, but also their functional consequences for individual symptoms and intermediate phenotypes as well as their prognostic value for CBT treatment outcome.

Time Frame:
Year 1 EDTA blood collection and DNA extraction
Year 2 Genotyping
Year 3 Statistical analysis of genotype and phenotype variation

Cooperation and add-on
EDTA-blood will be obtained at the first lab test time point of the clinical core project. In the network as DNA will be obtained from all consenting participating patients and control, the genetic module will be linked not only with the core therapy project (P2), but with all associated scientific projects (P4-P7). It will thus be possible to look for genetic variations relevant at the categorical clinical level as well as for genetic variations relevant at the dimensional intermediate phenotype level, be it psychophysiological, neuroendocrinological or imaging genomics as well as therapeutic outcome. Part of the genotyping and the statistical analyses will be performed in cooperation with the NGFN-associated Dept. of Psychiatry and Institutes of Human Genetics (Prof. Maier, Dr. Zobel, Prof. Propping, Dr. Schumacher) and the NGFN National Genotyping Platform at the Life&Brain Center, University of Bonn, (Prof. Nöthen, Dr. Cichon), with the Dept. of Psychiatry at the Univ. of Würzburg and the Depts. of Child and Adolescent Psychiatry at the Univ. of Würzburg and Homburg (Prof. Lesch, PD Mößner, Dr. Jacob, Prof. Warnke, Dr. Freitag). Cooperations are established with the Max-Planck Institute of Psychiatry at Munich (Prof. Holsboer, PD Keck), the CAMH at Toronto (Prof. Kennedy, Dr. Rothe), the Depts of Psychiatry at the Univ. of Milan (Prof. Catalano) and Univ. of Barcelona (Prof. Martin-Santos) and the Depts. of Psychiatry at the Univ. of Chicago (Prof. deWit) and the Univ. of Bristol (Prof. Rogers and Prof. Nutt). The cooperations with Bonn and Würzburg will allow to genotype more genetic variations as the limiting factor is the establishment of genotyping assays which will be split between the cooperation partners. The cooperations with Bonn and Würzburg as well as those with the other departments and institutes will allow us to investigate the specificity of the obtained findings by studying samples with different ethnic and age background as well as additional categorical and dimensional anxiety-related phenotypes (African, Asian, children and adolescents, drug-induced, personality dimensions).

Ethical and legal considerations
The main consideration of this research is the protection of private data. This will be ensured by an anonymization procedure. Ethical votes and informed consent will be obtained according to the Helsinki principles. The principle investigator and the cooperating centers in the past have successfully obtained consenting ethical votes for comparable studies.