LECTURE 10: NEUROIMAGING GENETICS

Objectives: to know the strengths and weaknesses of neuroimaging genetics. What can it help to address/find out? What

questions can it not answer? to know one or two examples of a neuroimaging genetics study and how genotype can influence cognitive-

affective processing. to know how neuroimaging genetics can help in researching gene-environment interactions.

Recap: Genetics and psychopathology

What genes are commonly implicated in affective disorders?

See lecture 7: Genetics, Twin studies & Gene by environment interactions.

The important role of age in genetics research:

Endophenotypes (intermediate phenotypes):

Endophenotype = measurable risk trait that is closer to pathogenic genotype than clinical phenotype.

4 criteria:1) An endophenotype is associated with illness in population.2) It is heritable.3) It is state-independent (i.e., whether illness manifests itself or not).4) Within families, endophenotype and illness cosegregate (= are inherited together).

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The problem of psychiatry – revisited

“Psychiatric diseases are complex disorders that display low penetrance, clinical heterogeneity and variable expressivity. They involve the joint action of several disease loci and are, over time, diagnostically unstable.”

Can neuroimaging help?

Brain imaging has problems with retest stability and heritability of brain activation (vs. structure) not so well demonstrated. It’s difficult to run exactly the same experiment. (High costs, who to recruit if already based on large sample? …) But, maybe it offers insight into pathophysiological mechanisms and helps us understand underlying neurobiology.

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COMT

Val66Met polymorphism (substitution) leads to reduction of enzyme activity. less rapid dopamine degradation.

COMT genotype predicts 4% of variance in Wisconsin Sorting Task (WCST), which measures executive function. It’s role in schizophrenia is established. Why?

Met allele vs. Val allele

COMT and affective disorders

Not as much research as in schizophrenia or neuro-psychiatric disorders (Huntington's, Parkinson's) has been conducted for COMT and it’s link with affective disorders.

The Met allele is associated with Major Depressive Disorder (MDD). There is an association with rapid cycling in bipolar disorder. (Rapid cycling is defined as four or more manic,

hypomanic, or depressive episodes in any 12-month period. With rapid cycling, mood swings can quickly go from low to high and back again, and occur over periods of a few days and sometimes even hours.)

There’s no evidence (yet) for an association with anxiety disorders. The Val allele is associated with lower levels of harm avoidance and higher levels of sensation seeking in women.

Strange contradiction:

Prefrontal cortex (PFC) (executive) function favours the Met allele, but this allele is a risk allele in negative mood states.

The oddball paradigm

The oddball paradigm is an experimental design used within event-related potential (ERP) research, where presentations of sequences of repetitive audio/visual stimuli are infrequently interrupted by a deviant stimulus. The subject is asked to react either by counting or by button pressing incidents of target stimuli that are hidden as rare occurrences amongst a series of more common stimuli, that often require no response. It has been found that an event related potential across the parieto-central area of the skull that is usually around 300 ms and called P300 is larger after the target stimulus. The P300 wave only occurs if the subject is actively engaged in the task of detecting the targets. Its amplitude varies with the improbability of the targets. Its latency varies with the difficulty of discriminating the target stimulus from the standard stimuli.

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The oddball response when spit by alcohol (in)dependence:

On the left you see the grand mean event-related potential (ERP) at the central electrode (Cz) to visual oddball target processing, averaged from responses of control subjects (blue trace) and alcohol-dependent subjects (red trace). The curves show a reduced P300 amplitude in alcoholics.

Time x frequency plots for the target condition measured at the Cz electrode (central midline) location on the top of the head in control (left panel) and alcoholic (right panel) subjects:

The headplots of the time-frequency regions of interest that underlie the duration of the P300 wave indicate that the peak power in θ band waves (4 to 5 Hz) during target processing (300-500 ms after the stimulus) is present in the anterior location on the scalp, whereas the δ band waves (1 to 3 Hz) peak power is seen in the posterior regions. Alcoholics have weaker responses than control subjects in both the q and d bands during the P300 response to target stimuli.

The oddball response when spit by COMT genotype:

The figure below shows that schizophrenic patients have smaller P300 amplitudes than control subjects at fronto-central (Fz) and centro-parietal (Pz) electrode positions. As predicted, subjects with Met/Met genotype have smaller P300 amplitudes than subjects with Val/Val genotype in the frontal region at the electrode positions at fronto-central (Fz) and centro-cental (Cz). A statistically significant reduction of frontal P300 amplitude at fronto-central (Fz) electrode position was also observed for the homozygous Met carriers within the schizophrenic group, whereas a separate analysis of the healthy control sample showed a statistically nonsignificant difference in the same direction.

Fz = fronto-central electrode position. Cz = centro-central electrode position. Pz = centro-parietal electrode position.

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When there is a Met/Met genotype, the differences between healthy and schizophrenic subjects are larger.

Do we believe these data blindly?

There is a smaller sample for schizophrenic patients. It is normal that there are more schizophrenic men than women, though.There are age differences between the healthy control subjects and the schizophrenic patients!

The duration of illness is shorter for patients with the Val/Val genotype, and the age of onset is later.The number of episodes is the smallest for patients with the Val/Val genotype, and is largest for patients with the Met/Met genotype.

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Is COMT really only involved in prefrontal cortex (PFC) supported cognitive function?

In a study, 35 right handed volunteers were genotyped for COMT. Participants viewed unpleasant, pleasant, and neutral pictures (18 of each).

Below, you can see the correlation between the COMT Met allele dosage (0 = Val/Val, 1 = Val/Met, or 2 = Met/Met) and the activation by unpleasant stimuli of…

a …

the ventrolateral prefrontal cortex (ventrolateral PFC)

b …the right amygdala

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c …left dorsal hippocampus.

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5HT

Polymorphism (5HTTLPR); location 17q11.2

44 base pair insertion (L allele) or deletion (S allele) In L allele there is 2-fold increase in 5HTT expression and thus serotonin reuptake.

5HT: Structural Magnetic Resonance Imaging (MRI)

A study revealed a significant effect of genotype for hippocampal white matter in patients with depression, whereas no significant effect was found for healthy controls.

Furthermore, significantly smaller right hippocampal white matter volumes and a trend toward smaller left hippocampal white matter volumes were found in patients with the L/L genotype as compared with those who carried the L/S or S/S genotype.

Scattergrams of left and right hippocampal white matter showing serotonin transporter polymorphisms in patients with depression:

(Bars depict the mean for each group.)

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5HT: Functional Magnetic Resonance Imaging (MRI)

Are genetic effects additive?

This study used an imaging genomics approach to investigate amygdala activity in Major Depression as a function of common functional polymorphisms in the serotonin transporter gene (5-HTTLPR) and the serotonin receptor 1A gene (5-HT1A-1019C/G). In 27 medicated patients with Major Depression, amygdala responses to happy, sad and angry faces were assessed using functional magnetic resonance imaging at 3 Tesla. Patients were genotyped for the 5-HT1A-1019C/G and the 5-HTTLPR polymorphism, including the newly described 5-HTT-rs25531 single nucleotide polymorphism. Risk allele carriers for either gene showed significantly increased bilateral amygdala activation in response to emotional stimuli, implicating an additive effect of both genotypes.

The effect of the 5-HT polymorphism on left amygdala activation:

Below you see amygdala activity as a function of risk alleles. Bilateral amygdala activity in response to angry, sad and happy faces is dependant on the number of risk alleles (S and LG-allele for 5-HTTLPR/5-HTT-rs25531, G-allele for 5-HT1A-1019C/G). The number of risk alleles covaries significantly with amygdala activity, indicating an additive effect.

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5HT: Subgenual anterior cingulate cortex (sgACC) and genetic/familial risk

Boys (but not girls) with subclinical scores of depression had lower subgenual anterior cingulate cortex (sgACC) volume than those without.

There’s a negative correlation between subgenual anterior cingulate cortex (sgACC) volume and depressive symptoms. This was particularly robust for individuals with family history of depression. This suggests subgenual anterior cingulate cortex (sgACC) is a biological marker of vulnerability OR trait marker for depression.

In the next study, a multimodal neuroimaging in a large sample of healthy human subjects was used. Morphometrical analyses showed reduced gray matter volume in short-allele carriers in limbic regions critical for processing of negative emotion, particularly perigenual cingulate and amygdala. Functional analysis of those regions during perceptual processing of fearful stimuli demonstrated tight coupling as a feedback circuit implicated in the extinction of negative affect. Short-allele carriers showed relative uncoupling of this circuit. Furthermore, the magnitude of coupling inversely predicted almost 30% of variation in temperamental anxiety. These genotype-related alterations in anatomy and function of an amygdala-cingulate feedback circuit critical for emotion regulation implicate a developmental, systems-level mechanism underlying normal emotional reactivity and genetic susceptibility for depression.

Subgenual anterior cingulate cortex volume is markedly reduced (by >25%) in s allele carriers in comparison to l/l individuals.

This statistical map of structural covariance analysis displays different degrees of positive correlation between bilateral amygdala volume and perigenual cingulate volumes, with two local peaks located supra- and subgenually.

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Short allele carriers show significantly reduced functional connectivity between the subgenual cingulate and amygdala compared to l/l genotype, which explains harm avoidance scores. Plot represents extracted peak results normalized to the mean absolute functional connectivity, relative to the l/l genotype group.

Statistical functional connectivity maps between bilateral amygdala and perigenual anterior cingulate cortex representing degree of functional coupling between these structures:

long allele

Subgenual cortical regions in left hemisphere (top) and right hemisphere (bottom) correlate positively with amygdala activity during the perception of threatening faces, whereas supragenual regions correlate negatively.

(The color bars represent t-scores.)

short allele

5-HTTLPR s allele carriers show significantly less functional coupling between amygdala and perigenual anterior cingulate cortex than l/l individuals, particularly in the subgenual region.

(The color bar represents absolute t-scores.)

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BDNF

learning, neurogenesis, pruning, … hippocampus

BDNF: Structural Magnetic Resonance Imaging (MRI)

Study 1

A variation in the BDNF gene (val66met) affects the function of BDNF in neurons, predicts variation in human memory, and is associated with several neurological and psychiatric disorders. Here, we show that, in magnetic resonance imaging (MRI) scans of a large sample (n = 214) of normal individuals, this polymorphism affects the anatomy of the hippocampus and prefrontal cortex, identifying a genetic mechanism of variation in brain morphology related to learning and memory.

The figure shows that the met-BDNF group has significantly reduced cortical volume (red and yellow areas) compared with val/val-BDNF carriers being found predominantly at the lateral convexity of the frontal lobe. Peak differences (red) are also found within dorsolateral prefrontal cortical areas, which are related to memory function.

On this figure you can see mean differences in hippocampal volume reduction in met-BDNF carriers relative to val/val-BDNF subjects within regions of statistical significance.

This statistical map of t-transformed hippocampal volume differences derived by morphometry in met relative to val/val-BDNF carriers, shows bilateral significant hippocampal volume reduction in met-BDNF carriers.

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Study 2

How about these data, do we believe them?

Study 3: BBDF in pediatric anxiety

Associations of BDNF and clinical anxiety with regional gray matter volume (GMV) were examined in 39 affected (17 Met allele carriers, 22 Val/Val homozygotes) and 63 non-affected adolescents (27 Met allele carriers, 36 Val/Val homozygotes).

Amygdala and anterior hippocampal regional gray matter volume (GMV) were significantly smaller in patients than healthy adolescents, with a reverse pattern for the insula. Post-hoc regression analyses indicated a specific contribution of social phobia to the regional gray matter volume (GMV) reductions in the amygdala and hippocampus. Additionally, insula and dorsal anterior cingulate cortex (dorsal ACC) regional gray matter volumes (GMV) were modulated by BDNF genotype. In both regions, regional gray matter volumes (GMV) were larger in the Val/Val homozygote patients than in those carrying the Met allele.

These results implicate reduced regional gray matter volumes (GMV) in the amygdala and hippocampus in pediatric anxiety, particularly social phobia. In addition, the data suggest that genetic factors may modulate differences in the insula and dorsal anterior cingulate cortex (dorsal ACC).

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Main effect of group (comparison > patients): Figure displays the decreased gray matter volume in the amygdala and anterior hippocampus for the patient relative to the comparison group regardless of BDNFVal66Met polymorphism. Scatterplots for the amygdala cluster show significant differences in the post-hoc tests. So: anxious participants had smaller amygdala and anterior hippocampus volumes.

Main effect of group (patients > comparison): Figure displays insula gray matter volume increases in the patients relative to the comparison group. Scatterplots show significant differences in the post-hoc tests. So: anxious participants had larger insula volume.

Interaction of BDNF polymorphism by group: Significant gray matter volume changes in the dorsal anterior cingulate cortex (dorsal ACC). Adjacent scatterplot illustrates the significant interactions in the post-hoc tests.

Interaction of BDNF polymorphism by group: Significant gray matter volume changes in the anterior insula. Adjacent scatterplot illustrates the significant interactions in the post-hoc tests. So: anxious met-participants had smaller anterior insula volume than anxious val-participants.

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A meta-analysis & some observations

(When the confidence interval lays totally left from the striped line, there’s a negative effect. When the confidence interval lays totally right from the striped line, there’s a positive effect.)

So there are all these BDNF findings… This means there is an effect, right? Well… There’s only a small effect, and…

… It depends on the used method!

Manual tracing Automated tracing

Often, there’s an effect when using manual tracing. In contrary, there’s often no effect when using automated tracing! However, with automated tracing, researchers are able to work with larger samples, resulting in smaller confidence intervals.

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What are we actually talking about here?

A and B: coronal sections through occipitorostral parts of the hippocampusC: section through the posteromedial part of the amygdalaD: section through the anteromedial part of the amygdala

Methods have become more strict over the years

Scatter plot showing the relation between effect size and year of publication for the association of the hippocampal volume and BDNF:

The size of the shapes indicates the sample size of each study.

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Squares represent the studies that traced the hippocampus manually.- Circles represent the studies that

measured the hippocampus automatically.

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So what does this mean for patient studies?

Main effect of diagnosis (depressed vs. non-depressed) and genotype No genotype x diagnosis interaction(N=120 total)

Scattergram of left and right hippocampal gray matter volumes showing BDNF polymorphisms of patients with depression and of healthy control subjects:

A significant main diagnosis effect was found indicating smaller hippocampal gray and white matter volumes in patients with major depression compared with healthy controls. Moreover, there was a significant effect of BDNF allele on the hippocampal gray matter volume.

BDNF: Functional Magnetic Resonance Imaging (MRI)

Significant diagnosis-by-genotype interactions emerged in amygdala and anterior hippocampal regions:

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To decompose these diagnosis-by-genotype interactions, the effects of genotype were examined in patients and healthy comparisons separately. Genotypic differences on neural responses in each diagnostic group are illustrated for each region. The significant two-way interactions were driven by genotypic differences in patients: Met-carriers showed greater neural responses than Val-homozygotes. Effect sizes indexed by Cohen’s d for these group differences were 1.61 and 0.71 for the left and right amygdala respectively, and 1.14 and 0.80 for the left and right anterior hippocampus respectively. Such genotypic differences were not observed in the healthy comparison group.

We still know very little…

Chronic treatment with antidepressants (fluoxetine, imipramine) restores social functioning and increases BDNF activity.

However, blockade of BDNF activity in ventral tegmental area (VTA) also exerts antidepressant effects.

Contradiction !!!

Both administration of BDNF and blockage of BDNF can have antidepressant effects, and we have no idea why!

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DISC1 – differentiating bipolar disorder from schizophrenia

Bipolar disorder and schizophrenia are two distinct disorders BUT share quite a few common features (e.g. jumping of ideas, delusions of grandeur, prefrontal cortical function deficits).

In both disorders genetic factors are important. In DISC1 a common substitution is overrepresented in patients. Hayling sentence completion test (1997) – e.g., “I find this lecture particularly . . . “

A comparative study:

The researchers sought to examine the effects of the DISC1 Leu607Phe polymorphism on brain activation in young healthy individuals at familial risk of bipolar disorder (n = 84), in a group of controls (n = 78), and in a group at familial risk of schizophrenia (n = 47), performing a language task. They assessed whether genotype effects on brain activation differed according to risk status. There was a significant genotype × group interaction in a cluster centered on the left pre/postcentral gyrus, extending to the inferior frontal gyrus. The origin of this genotype × group effect originated from a significant effect of the presumed risk variant (Phe) on brain activation in the control group, which was absent in both high-risk groups.

The cluster centered on the left precentral gyrus (BA 6) – which extended to the left inferior frontal gyrus and encompassed part of the postcentral gyrus (BA9 + 44), cluster-corrected across whole brain – was significant on on testing the main effect of genotype and genotype × group interactions with the schizophrenia high-risk group included in the analysis. The figure demonstrates significant group × genotype interaction for DISC Leu607Phe for sentence completion versus baseline.

Within group comparisons indicated that the DISC1 group difference stemmed from a significant effect in the controls (Phe carriers > LeuLeu homozygotes), an effect not seen in the high-risk groups (Phe carriers < or > LeuLeu homozygotes: n/s). This can be seen in the graph of the extracted data for cluster of significant interaction. Further statistical comparison of the groups revealed a significant difference between the Phe carrier controls versus bipolar high-risk Phe carriers and versus the schizophrenia high-risk Phe carriers.

- C = controls.- BHR = bipolar high-risk.- SHR = schizophrenia high-risk.

What happens when we increase the sample size?

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Allele distributions for 13 single-nucleotide polymorphisms (SNPs) in the DISC1 gene in patients with Major Depressive Disorder (MDD), those with schizophrenia and controls:

(Minor allele frequencies in controls are shown. SZ = schizophrenia. Significant results (P<0.05) are indicated by italics.)

Possible association between genetic variants in the DISC1 gene and Major Depressive Disorder (MDD) or schizophrenia was examined. The results suggest that DISC1 is associated with Major Depressive Disorder (MDD) and with schizophrenia and that Ser704Cys single-nucleotide polymorphism (SNP), in particular, is associated with Major Depressive Disorder (MDD) in our sample. The Cys allele frequency of SNP12 (Ser704Cys) was greater in patients with Major Depressive Disorder (MDD) when compared with controls.

Brain morphology according to Ser704Cys SNP in healthies:

Cys carriers have reduced volumes relative to Ser carriers.

Impact on the brain morphology of the Ser704Cys SNP in healthy subjects:

Cys-DISC1 carriers had reduced volumes in the bilateral anterior cingulate cortex (bilateral ACC), cingulate gyrus and the posterior cingulate gyrus compared with ser/ser-DISC1 individuals.

Ser/Ser-DISC1 individuals demonstrated decreased volumes of the lateral ventricle, interhemispheric fissure and bilateral Sylvian fissure, indicating an expansion of the CSF space in cys-DISC1 carriers.

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N-acetyl aspartate (NAA)

NAA is a highly concentrated molecule in the brain. NAA is present in neurons and “indicates marker of density of viable neuronal tissue and reversible changes in

neuronal health”. NAA gives large signal in magnetic resonance spectroscopy (MRS) and abnormal reductions in levels have been

found in many disorders including Alzheimer's, stroke, but also... bipolar disorder, depression and PTSD.

Switching levels: NAA and anxiety (and genetics)

There’s a negative correlation between hippocampal NAA concentrations and trait anxiety scores (STAI).

Scatterplot of hippocampal NAA concentrations and trait anxiety scores (STAI):

People with the l/l variant of the 5-HT have a higher hippocampal NAA concentration than people with the s/l or s/s variant.

l = long allel, which is the protective allel.s = short allel, which is the risk allel.

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Combining levels: true ‘multimodal imaging’

The biological approach to behavioral genetics, with on the right an example:

(STAI = State-Trait Anxiety Inventory.)(SHS = septo-hippocampal system.)

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Anxiety

SSAT and suicide

Do your genes define your risk of suicide?

SSAT gene is implicated in polyamine stress response (at the molecular level). Post-mortem study of brains: Differential expression of SSAT in frontal cortex in suicide completers and

depressed suicide completers relative to comparisons was found.

Graphical representation of the relative SSAT messenger RNA (mRNA) levels in controls (C), suicide completers (SC), and depressed suicide completers (DSC) (percentage of β-actin):

BA4 = Motor cortex.BA8/9 = Dorsolateral prefrontal cortex (but in the lecture named as superior frontal gyrus).BA11 = Orbital cortex (but in the lecture named as inferior frontal gyrus).

(Asterisks indicate significant differences compared with the C group.)

Differential expression of SSAT in the motor cortex (BA4), dorsolateral prefrontal cortex (BA8/9), and orbital cortex (BA11) was confirmed. Lower expression of SSAT was confirmed in the motor cortex (BA4) for the suicide completers and depressed suicide completers compared with the controls. In the dorsolateral prefrontal cortex (BA8/9), SSAT expression was lower among the suicide completers and depressed suicide completers compared with the controls. Similarly, in the orbital cortex (BA11), SSAT expression was 1.5-fold lower in the suicide completers and 1.4-fold lower in depressed suicide completers than in the controls.

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A final comment

Remember: we still know very little…

As stated before: Chronic treatment with antidepressants (fluoxetine, imipramine) restores social functioning and increases BDNF

activity. However, blockade of BDNF activity in ventral tegmental area (VTA) also exerts antidepressant effects.

Contradiction !!!

Both administration of BDNF and blockage of BDNF can have antidepressant effects, and we have no idea why!

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