Genetic regulation of stress responsivity as a risk factor for schizophrenia and bipolar disorder. ;Subtitle: The association of (epi)genetic factors on the hormonal and autonomic stress response after psychosocial stress

Exposure to stress is a risk factor for the development of psychiatric
disorders including schizophrenia and bipolar disorder. In support, altered
stress responsivity has been reported in patients with schizophrenia and
bipolar disorder. To date, it is unclear which factors determine this
susceptibility, although some evidence suggests that (epi)genetic factors
regulate altered stress functionality. So far, genetic variation per se only
explains a minority of the increased genetic risk in schizophrenia and bipolar
disorder, and it has been suggested that gene x environment interaction may be
of added value. Therefore, it would be of great interest to study whether
changes in stress responsivity in patients and subjects with an increased
vulnerability to develop schizophrenia and bipolar disorder exist, and whether
these changes are associated with genetic risk factors for these psychiatric
disorders as well as stress genes involved in the autonomic and hormonal stress
response. Considering the partial overlap between genetic risk factors for
schizophrenia and bipolar disorder, we aim to compare these two disorders.
In addition to the effects of genetic variations, epigenetic
modifications of the stress response are likely to exist. Epigenetics is the
study of inherited changes in phenotype caused by mechanisms other than changes
in the underlying DNA sequence. Recent results from our group studying DNA
methylation show that epigenetic modification of stress response and
inflammatory genes is associated with the susceptibility to bipolar disorder.
To date, it is unknown whether changes of DNA methylation have functional
consequences.
In conclusion, we aim to investigate whether the autonomic and hormonal
stress response is altered in subjects with schizophrenia/bipolar disorder and
subjects with (epi)genetic vulnerability to develop schizophrenia/bipolar
disorder compared to healthy controls. To this end, patients with these
disorders, subjects with an increased genetic susceptibility but non-present
schizophrenia and bipolar disorder and healthy controls are exposed to a
standardized psychosocial stress test, the Trier Social Stress Test for Groups.
Moreover, genotype and DNA methylation of selected candidate genes are
determined. After stress exposure, subjects will also be asked to perform a
number of social decision tasks in order to assess any differential effects of
stress on task performance and its dependence on (epi)genetic factors. In
addition, stress induces changes in immune response will be measured to detect
differences between healthy volunteers, bipolar patients and schizophrenic
patients.

Primary objective: Are changes in stress-induced autonomic and hormonal
parameters associated with (epi)genetic vulnerability for schizophrenia and
bipolar disorder?

Secondary objectives:
1. Are changes in autonomic and hormonal parameters after stress exposure
associated with genotype or DNA methylation levels of genes involved in the
stress response?
2. Does stress influence performance on a number of cognitive tasks, and does
this depend on the (epi)genetic background?
3. Does stress influence the immune response, and does this
depend on disease status?

In a case-control study stratified for a family history of psychiatric
diagnosis, we will investigate the stress response induced by the Trier Social
Stress Test for Groups (TSST-G), and aim to associate the stress response with
gene expression and DNA methylation levels. Adult patients with either bipolar
disorder or schizophrenia will be compared to healthy controls with no family
history of any psychiatric disorder. For the discordant twins, the unaffected
twin will be compared to the co-twin with bipolar disorder as well as healthy
controls. For any participant (bipolar disorder and schizophrenia patients,
discordant twins and healthy controls) the total duration of participation is
around 3,5 hours during which they will be exposed to 15-min lasting
standardized and validated social stress test (the Trier Social Stress Test for
Groups). The stress test consists of a short public speech test and an
arithmetic test and does not lead to extreme perceived stress levels.
Autonomic, immune and hormonal parameters will be measured before, during and
after the stress test, and anxiety questionnaires will be filled in. After
stress exposure, subjects will be asked to participate in a number of social
decision tasks.

1 Urine sample
A urine sample will be obtained at arrival to conduct a drug test (MDMA,
barbiturates, cannabinoids, benzodiazepines, cocaine and opiates) and to test
for nicotine use.

2 Skin temperaturesensor
All participants receive telemetry skinesnsors (iButtons ) on the day of the
stress test, at least 1 hour before commencing the TSST-G. Immediately after
attaching the patch, temperature data will be checked to establish a good
functioning of the telemetry pill.

3 Heart rate monitor
All volunteers will be equipped with an automatic heart rate monitor at least 1
hour before
commencing the TSST-G which will measure the heart rate continuously

4 Saliva samples
All volunteers will be asked to donate saliva at discrete intervals before,
during and after the experiment.
Saliva samples will be collected in tubes after arrival (-60 min), immediately
before the preparation phase
(pre-stress levels, -1 min), directly and at more discrete samples. Saliva
samples are kept at room temperature throughout the test session and are then
stored at -20 ° C. Free cortisol, the biologically active fraction of cortisol
will be assayed. Samples will be coded with the participant number. After
analysis, saliva samples will be destroyed. Saliva samples will only be used to
determine cortisol levels.

5 Questionnaires
All participants will be asked to fill in several questionnaires before the
Trier Social Stress Test for Groups to establish psychopathology and
symptomatology . Questionnaires that will be taken are the Clinical Global
Impression Scale (CGI), Positive and Negative Syndrome Scale (PANSS), Young
Mania Rating Scale (YMRS), Inventory of Depressive Symptomatology (IDS-SR), the
BIS/BAS test, Jeugd Trauma Vragenlijst (JTV) and Life Symptom Checklist Revised
(LSC-R).

6 Stress questionnaires
All participants will be asked to fill in several stress questionnaires twice.
The first time, questionnaires have to be completed as a baseline before the
Trier Social Stress Test for Groups. The second time, questionnaires have to be
completed just after the Trier Social Stress Test for Groups and indicate a
stress-induced state. Questionnaires that will be taken are the STAI-score
(state-trait anxiety inventory) (Spielberger 1989) and visual analog scales.

7 Cognitive Tasks
A number of cognitive tasks will be taken after the stress test: the Trust
Game, Ultimatum Game, Dictator Game, Prisoner*s Dilemma, Social Ranking Task,
the Iowa gambling task (risk taking) and a delay discounting task (reward).

8 Trier Social Stress Test for Groups
The Trier Social Stress Test for Groups will be taken in the afternoon since
body temperature and cortisol levels are subject to circadian rhythmicity. The
Trier Social Stress Test for Groups consists of a standardized public speaking
test as well as a short arithmetic task performed in groups of 4 participant,
which will take around 15 minutes (excluding 2 minute explanation and 3 minute
preparation time).

Risks for participants are minimal. The stress test consists of a short public
speech test and an arithmetic test and does not lead to extreme perceived
stress levels. The time spent in the laboratory is limited, and participants
have sufficient time for breaks. Participants receive a lunch, ¤35,- as token
of appreciation. Moreover, the actual gain of one task trial is paid out
(between ¤ 10,- and ¤20,-) in order to ensure sufficient economic incentives,
in line with previous studies using these tasks.