Salivary alpha-amylase and cortisol responsiveness to stress in first episode, drug-naïve patients with panic disorder.

Reported findings on reactivity to stress of the sympathetic-adreno-medullar (SAM) and hypothalamic-pituitary-adrenal (HPA) systems in panic disorder (PD) are very variable. This inconsistency may be explained by differences in treatment exposure, illness duration and emotion regulation strategies. The present study examined the reactivity to mental stress of the SAM and HPA axes in a sample of first episode, drug naïve patients with PD which avoids confounds of medications exposure and illness chronicity. Activation of the SAM axis was evaluated by dosage of salivary alpha-amylase (sAA) and heart rate. Activation of the HPA axis was tested by dosage of salivary cortisol. Psychological assessments were done by the Self-Rating Depression Scale, the Self-Rating Anxiety Scale, the State-Trait Anxiety Inventory, the Cope Orientation to Problems Experienced (COPE) Inventory and the 16 Personality Factor Questionnaire (16PF). Patients showed reduced sAA stress reactivity, higher baseline cortisol levels and a more rapid decrease in stress cortisol levels as compared with controls. A significant correlation was found between active coping strategies and cortisol levels (response to stress). The findings suggest that blunted SAM stress reactivity and a rapid decrease in stress cortisol levels reflect traits that may enhance vulnerability to psychopathology in patients with PD.

Epigenetic signature of panic disorder: a role of glutamate decarboxylase 1 (GAD1) DNA hypomethylation?

Glutamate decarboxylases (GAD67/65; GAD1/GAD2) are crucially involved in gamma-aminobutyric acid (GABA) synthesis and thus were repeatedly suggested to play an important role in the pathogenesis of anxiety disorders. In the present study, DNA methylation patterns in the GAD1 and GAD2 promoter and GAD1 intron 2 regions were investigated for association with panic disorder, with particular attention to possible effects of environmental factors. Sixty-five patients with panic disorder (f=44, m=21) and 65 matched healthy controls were analyzed for DNA methylation status at 38 GAD1 promoter/intron2 and 10 GAD2 promoter CpG sites via direct sequencing of sodium bisulfate treated DNA extracted from blood cells. Recent positive and negative life events were ascertained. Patients and controls were genotyped for GAD1 rs3762556, rs3791878 and rs3762555, all of which are located in the analyzed promoter region. Patients with panic disorder exhibited significantly lower average GAD1 methylation than healthy controls (p<0.001), particularly at three CpG sites in the promoter as well as in intron 2. The occurrence of negative life events was correlated with relatively decreased average methylation mainly in the female subsample (p=0.01). GAD1 SNP rs3762555 conferred a significantly lower methylation at three GAD1 intron 2 CpG sites (p<0.001). No differential methylation was observed in the GAD2 gene. The present pilot data suggest a potentially compensatory role of GAD1 gene hypomethylation in panic disorder possibly mediating the influence of negative life events and depending on genetic variation. Future studies are warranted to replicate the present finding in independent samples, preferably in a longitudinal design.

Association study of tryptophan hydroxylase 2 gene polymorphisms in bipolar disorder patients with panic disorder comorbidity.

Frequent comorbidity between panic disorder (PD) and mood disorders has been widely reported in clinical and epidemiological studies and, recently, an increasing attention has been paid to the cooccurrence of PD and bipolar disorder (BD). Several studies have shown that an imbalance of serotonin activity could be related to panic symptoms. Tryptophan hydroxylase 2 (TPH2) are plausible candidates for the association with PD. The aim of this study is to investigate a possible association between TPH2 gene polymorphisms and the PD comorbidity susceptibility.Our sample consisted of 515 patients; 274 patients with BD (subtypes I and II), including 45 patients with lifetime panic disorder comorbidity and 241 controls. These patients were genotyped for eight tagging single nucleotide polymorphisms of the gene of human TPH2. We found significant differences between patients with BD, with panic disorder comorbidity, and controls in the allelic analysis (rs4448731, P=0.0069; rs4565946, P=0.0359; rs4760820, P=0.0079; rs1487275, P=0.0439) and genotypic analysis (rs4448731, P=0.011; rs4760820, P=0.0259). We also identified significant differences between patients with BD, with and without panic disorder comorbidity in the allelic analysis (rs4448731, P=0.004; rs4565946, P=0.011; rs11179000, P=0.031; rs4760820, P=0.018; rs1487275, P=0.038; rs10879357, P=0.023) and genotypic analysis (rs4448731, P=0.004; rs4565946, P=0.010; rs4760820, P=0.023; rs10879357, P=0.052). The haplotype analysis in the group of patients with BD, with and without panic disorder comorbidity, was also significant (rs4448731-rs4565946, P=0.0190; rs4448731-rs4565946, P=0.0220; rs10506645-rs4760820, P=0.0360). Further studies are needed to replicate the positive association that we observed.

Meta-analysis of COMT val158met in panic disorder: ethnic heterogeneity and gender specificity.

There is strong evidence for a genetic contribution to the pathogenesis of panic disorder, with the functional catechol-O-methyltransferase (COMT) val158met polymorphism having been suggested as a potential susceptibility factor. In the present study, a meta-analysis of six available case-control studies (557 patients with panic disorder and 763 healthy controls in total) on the role of the COMT val158met polymorphism in panic disorder was conducted in an attempt to reconcile previous conflicting results and to facilitate evaluation of the role of COMT gene variation in panic disorder. Overall, no significant association, but strong between-study heterogeneity, was discerned. Analysis of studies pooled by ancestry yielded a significant association of the COMT 158val allele with panic disorder in Caucasian samples and, conversely, a trend towards association of the COMT 158met allele with the disorder in Asian samples. Interestingly, stratification for gender as well as ethnicity revealed that association of the 158val allele in Caucasians and, reciprocally, the 158met allele in Asian samples was restricted to females. The present meta-analysis provides tentative support for the COMT val158met polymorphism as a possible risk factor for panic disorder, with differential effects in Caucasian and Asian populations, and suggests a female-specific effect. However, given the relatively small number of case-control studies presently available, several more association studies, preferably including a larger number of family-based studies, are warranted for conclusive evaluation of the COMT val158met polymorphism as a vulnerability factor in panic disorder.

Serotonin-mediated phosphorylation of extracellular regulated kinases in platelets of patients with panic disorder versus controls.

Phosphorylation of extracellular signal-regulated kinases (ERK 1/2) represents a converging intracellular signalling pathway which is involved in the modulation of gene transcription and may contribute to the feed-back regulation of neurotransmitter receptor functioning. The purpose of the current study was to investigate the serotonin-mediated phosphorylation of ERK 1/2 in platelets from patients (n = 17) with panic disorder, with respect to healthy volunteers (n = 17). Patients presented a severe symptomatology as assessed by the self-report rating scales for panic-agoraphobic (PAS-SR) and mood (MOOD-SR) spectrum, and by Clinical Global Impression Severity Scale (CGI-S). In platelets from healthy volunteers, serotonin induced a rapid increase of ERK 1/2 phosphorylation with a transient monophasic kinetic. The dose-response curves showed this effect was concentration dependent with an average of the EC(50) value of 22.8 +/- 2.4 microM. Platelet pre-incubation with 5HT(1A) and 5HT(2A) antagonists, pindobind and ritanserin, significantly inhibited serotonin-mediated kinase activation with an EC(50) of 3.2 +/- 0.2 and 1.99 +/- 0.08 nM, respectively, suggesting an involvement of these specific receptor subtypes in serotonin-mediated response. Furthermore, the 5HT(1A) and 5HT(2A) agonists, 8-hydroxy-N,N-dipropyl-aminotetralin (8OH-DPAT) and 1-(2,5-dimethoxy)-4-iodophenyl-2-aminopropane (DOI), were able to modulate ERK 1/2 phosphorylation in a concentration-dependent manner with an EC(50) value of 3.1 +/- 0.2 and 76 +/- 4.5 nM, respectively. ERK 1/2 phosphorylation was not observed after serotonin treatment of platelets from drug-free panic disorder patients, suggesting an alteration in intracellular phosphorylative pathways. Since ERK 1/2 responsiveness to other stimulus, such as collagen and thrombin, was comparable in platelets from healthy volunteers and patients, our results suggested that a specific alteration of serotonergic system occurred in panic disorder. Further studies to investigate 5HT(1A) and 5HT(2A) receptor expression and threonine phosphorylation levels showed that, nevertheless no significant differences in the receptor expression levels were detected, an increase of both 5HT receptor phosphorylation, on threonine residues, occurred in platelet from panic patients with respect to controls, suggesting that a reduction of serotonin receptor functioning was involved in the loss of serotonin responsiveness in panic.

Association of the functional V158M catechol-O-methyl-transferase polymorphism with panic disorder in women.

Panic disorder is an anxiety disorder with an estimated heritability of up to 48%. Pharmacological and genetic studies suggest that genes coding for proteins involved in the catecholaminergic system might be relevant for the pathogenesis of the disease. In the present study, we genotyped a single nucleotide polymorphism (472G/A=V158M) in the coding region of the catechol-O-methyl-transferase (COMT) gene in 115 patients with panic disorder and age- and sex-matched controls. Association analysis revealed a significant excess of the more active COMT allele (472G=V158) in patients with panic disorder (p=0.04), particularly in female patients (p=0.01), but not in male patients (p=1.0). The assessment of a possible interaction of the COMT polymorphism with a previously reported functional 30-bp VNTR in the monoamine oxidase A promoter (MAOALPR) in female patients did not yield significant results. Our data support a role of the 472G/A (V158M) COMT polymorphism or a nearby locus in the pathogenesis of panic disorder in women.

The protein kinase A in platelets from patients with panic disorder.

Although previous studies suggested that dysfunctions in the protein kinase A (PKA) and in some of its substrates are associated with several psychiatric disorders, there is no evidence regarding the possible involvement of such components in panic disorder (PD). Thus, the aim of the present study was to investigate the levels of PKA and Rap1 in platelets from patients with such disorder. Twenty-four drug free patients with PD and 24 healthy volunteers participated to the study. Employing the Western Blot analysis, immunostaining and computer-assisted imaging, the levels of the regulatory (R, type I and type II) and the catalytic (C) subunits of PKA, and those of Rap1 were assessed in platelets from the two groups. The data show that patients with PD have significantly higher levels of platelet RI and C subunits of PKA than controls, whereas the levels of RII were unchanged. No significant differences were found in the immunolabelling of Rap1 between groups. These findings may provide clues toward understanding the involvement of cAMP signalling in anxiety disorders.

Antioxidant enzyme and malondialdehyde levels in patients with panic disorder.

There is evidence of an etiopathogenetic role of free radicals (FRs) in some neuropsychiatric disorders. The aim of the present study was to determine whether the activity levels of some antioxidant enzymes [glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and catalase (CAT)] and malondialdehyde (MDA), a product of lipid peroxidation, were associated with panic disorder (PD). Twenty patients diagnosed with PD and 20 healthy controls were enrolled in this study. A clinical evaluation and measurements of GSH-Px SOD, CAT and MDA were performed. Additionally, all patients were assessed by the Panic Agoraphobia Scale (PAS). The mean GSH-Px, SOD and MDA levels of the patient group were significantly higher than those of the controls. There was a significant positive correlation between PAS scores and GSH-Px, SOD and MDA levels and between the duration of illness and SOD, CAT and MDA levels in the patient group. In conclusion, our results suggest that FRs may be involved in PD.

No association between anxiety disorders and catechol-O-methyltransferase polymorphism.

Several studies have shown that the morbidity risk for anxiety disorders is increased among the relatives of patients with obsessive-compulsive disorder (OCD). Recently, it was reported that a polymorphism of the catechol-O-methyltransferase (COMT) gene is significantly associated with OCD. The purpose of this study was to determine the association, if any, between the COMT polymorphism and anxiety disorders. We undertook an association study of the COMT polymorphism in 108 patients who met DSM-IV criteria for anxiety disorders and 135 healthy controls. All subjects were unrelated Japanese. The subdiagnostic groups did not differ significantly from the control group in either the genotypic or allelic frequencies. There were no statistically significant differences between the genotype and males, females, or a family history. The mean age of onset did not significantly differ among the genotypes. Our results suggest this functional COMT polymorphism does not make an important contribution to anxiety disorders in the Japanese population.

An association of NAG levels and a mutation of the CCK gene in panic disorder patients.

Levels of the enzyme N-acetyl-beta-glucosaminidase (NAG) and a mutation of cholecystokinin (CCK) gene appear to be independently associated with panic disorder. We explored whether there was an association of NAG levels and a CCK mutation identified in a group of panic disorder patients. NAG was measured in 12 panic disorder patients who had a mutation of the CCK gene and 17 who did not. Urine for NAG was collected at baseline and after 3 and 6 weeks of treatment. NAG levels were lower at all three times in the patients that did not have the CCK mutation. The difference between the two groups was significant at week 6 (P < 0.02), and showed a trend toward a difference at baseline (P < 0.15).

Platelet sulfotransferase in different psychiatric disorders.

The aim of our study was to measure and compare platelet phenolsulfotransferase (PST) activity in patients affected by different psychiatric disorders and in healthy volunteers. The results showed that the activity of both of the two forms of PST was significantly higher in 30 patients with obsessive-compulsive disorder and in 25 manic patients than in 20 healthy volunteers. On the contrary, normal values were found in 11 dysthymic patients, 12 bipolar depressives, and 14 patients with panic disorder, whereas lower values were found in 12 unipolar depressives and 30 patients with migraine. It would therefore seem that different neuropsychiatric disorders are associated with different levels of PST activity.

Association of levels of N-acetyl-beta-glucosaminidase with severity of psychiatric symptoms in panic disorder.

Urinary levels of N-acetyl-beta-glucosaminidase (NAG) were measured in 58 patients with panic disorder. NAG levels were found to be significantly related to the severity of 23 of 72 mood states, measured by the Profile of Mood States, which were grouped in three categories: hostility or irritability, sadness, and panic. A similar result was found in a previous study of bipolar patients. NAG levels were also related to scores on the Hamilton Rating Scale for Anxiety and the Sheehan Patient-Rated Anxiety Scale. It is speculated that NAG could be a marker for serotonin.

An association between the lysosomal enzyme NAG and urinary free cortisol and platelet imipramine binding.

Urinary levels of the lysosomal enzyme N-acetyl-beta-glucosaminidase (NAG) were significantly correlated to levels of 24-hour urinary free cortisol in a group of panic disordered patients. In a separate study of healthy controls NAG levels showed a trend toward association with platelet imipramine binding density. A hypothesis is proposed that NAG levels may be proportional to the density of postsynaptic serotonin receptors.