NPSR Gene & Panic Disorder

NPSR A/T (Asn107Ile) variant (rs324981)

Timothy M. Hanna, MS

Overview O Animal studies suggest NPS and NPSR involved in

pathogenesis of anxiety-related behavior.O This study attempts to test animal model findings

with human subjects.O The functional NPSR A/T (Asn107Ile) variant

(rs324981) investigated to study potential association with: 1. Panic Disorder with and without agoraphobia2. Dimensional anxiety traits3. Autonomic arousal level during a behavioral

avoidance test4. Brain activation correlates of anxiety-related

emotional processing in panic disorder.

Review Fo You and You and You

O Evidence from animal model studies and human molecular genetic studies suggests that NPS and NPSR are involved in the mediation of anxiety-related behavior and associated disorders.

O NPS: 20 amino-acid peptide, acts as an agonist at the G-protein-coupled NPSR increasing free intracellular calcium and cyclic adenosine monophosphate accumulation.

Review (cont.)Rodent Models

O NPS or NPSR agonists observed to elicit arousal 1. Hyperlocomotion2. Righting reflex3. Wakefulness

In animal models increased arousal appears to be paralleled by an anxiolytic-like effect!

Review (cont.)NPS on the Anatomical Level

(Rodent Models)O Expressed in the amygdaloid complex O Exerts a modulatory effect on both afferent and

intrinsic transmission in amygdala networks

Review (cont.)NPS on the Cellular Level

O NPS glutamatergic transmission to intercalated GABAergic neurons in the amygdala

O inhibitory influence on the central amygdaloid nucleus and anxiety-like responses.

O Indicates the importance of NPS in processing acute fear

O Can selectively inhibit the release of serotonin and norepinephrine in the frontal cortex (another possible way that NPS controls for anxiety and arousal behaviors).

Caffeine Administration(Tie in between main paper and paper #2)

O Caffeine= Potent anxiogenic and arousal

O Been observed to induce a marked decrease in mRNA levels of NPS. However, it has been shown to upregulate NPSR expression levels in the brain stem

Human NPSR GeneO Located on chromosome 7p14.3O NPSR gene possible functional and

positional candidate geneO 7p14-15 previously linked to panic

disorder

rs324981O A/T single-nucleotide polymorphism

at position 107 of the NPSR gene amino-acid exchange from Asn to Ile (N107I) relevant to T allele (Ile107) NPSR expression and NPS efficacy at NPSR about tenfold.

O Prior study found a correlation between T (Ile107) allele and risk of panic disorder.

Research QuestionO Multilevel approach applied to elucidate the role of

NPS in the pathogenesis of human anxiety. O Is the functional NPSR A/T (Asn107Ile) variant

(rs324981) associated with: 1. The categorical phenotype of panic disorder with and

without agoraphobia?2. Participants’ scores on dimensional anxiety traits

such as anxiety sensitivity?3. Automatic arousal level (measured with heart rate

and symptom ratings) during a behavioral avoidance test (BAT)?

4. Brain activation correlates of anxiety-related emotional processing in patients with and without agoraphobia (fMRI)?

Panic Disorder with Agoraphobia

NIH Agoraphobia Info Sheet

M & MAssociation Studies with Panic Disorder and

Dimensional Anxiety Traits

SamplesO Discovery Sample (MMWBG Sample)=499 patients with panic disorder (female n=312; male n=187). Patients with panic disorder and agoraphobia comprised 75.7% of the sample. O Control Group=499 unrelated anonymous blood donors of German decent (female n=312; male n=187). Could not be controlled for mental disease or defect. O Replication Sample= 277 patients enrolled in the mechanisms of action in cognitive-behavioral therapy (MAC) study in patients with panic disorder and agoraphobia (female n=209; male n=68).

M & MAssociation Studies with Panic Disorder and

Dimensional Anxiety Traits (cont.)

O 35% of replication sample suffered from a 12-month comorbid diagnosis of depression.

O All patients free of psychotropic medicationO A chemical substance that crosses the blood-brain barrier and acts primarily upon the CNS where

it affects brain function, resulting in alterations in perception, mood, consciousness, cognition,

and behavior.

O For categorical association studies control subjects were gender matched 1:1 (n=277, female 209, male 68) and drawn from screened healthy controls (n=519).

O Complete control (n=519) was used to test for association of NPSR rs324981 with dimensional anxiety traits.

O Patient and Control groups were evaluated for panic fear and anxiety sensitivity using Agoraphobic Cognitions Questionnaire (ACQ) and the Anxiety Sensitivity Index (ASI) [German version]

M & MAssociation Studies with Panic Disorder and

Dimensional Anxiety Traits (cont.)

GenotypingO Functional NPSR rs324981 A/T (Asn107Ile) polymorphism determined through amplification of DNA isolated from venous blood samples by PCR (polymerase chain reaction) using the primers F:5’-GAAGGAAAAAAATTAAAAATGAACCTCCCCAGGATTTCAT and R:5’-TTCTACCCAGGAGAAAGCGGGCAGTTTGATGCA resulting in an amplicon size of 353 bp. O Genotyping failed in 10 patients. Total Discovery Sample available for further analysis post genotyping: n=489 (female=305, male=184).

M & MAssociation Studies with Panic Disorder and

Dimensional Anxiety Traits (cont.)

Statistical AnalysisO Statistical analyses of allele and genotype distributions were performed by means of X2-test (implemented in Haplowview 3.2 and Armitage’s trend test). O Grouped genotype (AA vs AT/TT) distribution statistically analyzed based upon results.

Psychophysiological Assessment by way of Behavioral Avoidance Test

(BAT)

Chamber of Death Not really, but it does seem pretty unpleasant…

O 264 patients suffering from panic disorder and agoraphobia were investigated (MAC/ Replication Sample). 59 patients excluded leaving an n=205. O 55 patients were homozygous NPSR rs324981 A allele carriers. 105 patents were T allele carriers. O ECG recording at T1, T2, and T3.

BAT (cont.)O T1=Anticipation Phase: Patients in front of a small,

dark chamber with the door open for 10 min. O T2=Exposure Phase: Patients locked in chamber

and told to stay as long as possible for a maximum of 10 min.

O T3=Recovery Phase: Patients in front of opened chamber for 8 min.

O Patients instructed to rate intensity of 13 panic symptoms and subjective indices of anxiety on a scale of 1-10 after each period.

O Additionally, mean heart rate was calculated for each period.

BAT (cont.)

Effects of Genotype on Symptom Reports and Autonomic Arousal

O Mixed-model analysis of variance including genotype (AA vs AT/TT) as between-subjects factor. O Block (T1 vs T2 vs T3) as a within-subjects factor. O All stats used P<0.05

Figure 1NPSR rs324981 and panic disorder: Forest plot

showing analysis of TT/TA vs AA in (a) the combined sample, (b) females, and (c) males

fMRI During Presentation of Emotional Stimuli

O 20 patients with panic disorder were investigated (female 12, male 8).

O 10 patients treated with 5-HT reuptake inhibitor; remaining 10 medication-free.

fMRI Procedure O Subjects viewed 30s blocks of

alternating emotional faces (fearful, angry, happy, neutral) or a no-face control stimulus (gray rectangle).

O Emotional stimuli presented twice per second in a random sequence for 500 ms.

O fMRI= 3 Tesla

fMRI StatsO Statistical analysis = General Linear

Model modeling the different stimuli as variables.

O Standard hemodynamic response function (SHRF) = Control

O Subject specific contrasts, reflecting emotion-related activation entered into a random-effects analysis.

fMRI Stats (cont.)Voxel= Volumetric Picture Element/ Volumetric Pixel

A single sample or data point on a regularly spaced 3D grid.

Shaded area=one voxel

fMRI voxels are typically = 27 mm3

(cube with 3mm sides). [mad props to Wikipedia]

fMRI Stats (cont.)O Voxel values of 5 x 2 predefined regions

of interest (ROIs) based upon a priori hypotheses of emotional stimuli processing.

O Amygdala, orbitofrontal cortex, ventromedial PFC, dorsolateral PFC, ACC.

O Depiction of activation with Wake Forrest PickAtlas for same regions P<0.001

O Clusters defined by ≥ 10 contiguous voxels of significant response

fMRI Stats (cont.)O Genotype effects on activation in ROIs

tested using a MANOVA. Genotype=between subjects variable; four contrast values (happy, angry, fearful, neutral vs no faces)= within subjects factor.

O X2 test=No statistically significant difference in medication with SSRI, sex, and comorbidity with major depression or social phobia across NPSR genotype groups.

Results

Association Studies with Panic Disorder

O AA genotype significantly underrepresented in patients with panic disorder and especially pronounced in female sample.

O Meta-association=766 patients & 776 controls.

O Highly significant association of the NPSR rs324981 risk genotypes containing at least one T allele with disease. Meta-analysis confirmed result.

Table 1

Association Study with Dimensional Anxiety Traits

O Does NPSR rs324981 T allele also influence dimensional constructs associated with panic disorder and agoraphobia? MAC study + 519 healthy control.

O ACQ did not yield significant results. However, ACI score was significantly associated with genotype.

O NPSR rs324981 AA carriers ASI score 27.5 ± 11.1. O NPSR rs324981 T allele carriers ASI score 32.1 ±

11.7.O P=0.002O Difference due to female subsample (fig. 2).

Figure 2

Closed Bars=patientsOpen Bars=controlsP=0.002

Genetic Modulation of Heart Rate and Symptom Reports: Behavioral Avoidance

Test

O Genotype groups did not differ significantly in duration of exposure.

O Mean HR significantly increased from anticipation to exposure and then decreased during recovery. This shows that the BAT reliably induced intense fear in these patients.

O + NPSR rs324981 genotype modulated response patternsO AA=significantly lower overall HR than T allele

carriersO No difference during anticipation; significant

difference during exposure (self-report). T allele carriers reported significantly more intense symptoms during exposure.

Figure 3Panel A= Mean HRPanel B= Mean symptom intensity

Genetic Modulation of Fear Circuit Activation

Fearful Faces vs No-Face (Gray Rectangle)

O Protective AA= Increased activation in the Dorsolateral PFC and Lateral Orbitofrontal CortexO T risk Allele= Decreased activity in the ACCO No significant impact of NPSR rs324981 on responsiveness in any of the ROIs to happy, angry, or neutral faces.

Figure 4NPSR rs324981 A/T (Asn107Ile) effects on brain activation during processing of fearful faces vs no-face condition. (a) Contrast values analyzed by MarsBaR; (b)

ROI analysis of same areas using Wake Forrest University PickAtlas.

Take HomeO AA=Resilient O T carriers=Susceptible O AT/TT Overrepresented in female

subjects associated with panic disorder

O Explainable discrepancies between study findings and animal-model studies (945), and between this study and prior Japanese study.

Take Home (cont.)O Patients who carry the T risk allele have

enhanced sympathomimetic activation and thus arousal level

O These patients are more prone to panic symptoms when faced with challenging situations due to increased arousal over an already elevated baseline.

O Conditioned anxious apprehension + genetic risk factors contribute to increased arousal