Circadian timing system (CTS) characteristics in bipolar disorder: investigation of possible endophenotypes

Bipolar disorder (BD), also known as manic-depressive illness, is a brain
disorder that causes unusual shifts in a person*s mood, energy, and ability to
function. BD typically develops in late adolescence or early adulthood and is
present in roughly two percent of the population aged 18 and older in any given
year. BP is highly heritable, based on the results of several twin studies.
First-degree relatives of BP probands have a 20-fold increased risk for BD
compared with relatives of healthy individuals. Adoption studies show that
increased familial
aggregation is not simply due to environmental factors. Despite the fact that
increased risk for BP inherited, the disorder's genetic basis remains elusive.

Importance of endophenotypes
Due to the genetic heterogeneity, it is hard to successfully link genetic loci
to bipolar disorder. Therefore it is important to, in addition to GWA studies,
also consider other approaches to investigate the etiology of bipolar disorder.
One of these approaches is to make use of traits that often accompany bipolar
disorder, known as endophenotypes. Biological endophenotypes are measurable
intermediate phenotypes that are generally closer to the action of the gene and
thus exhibit higher genetic signal-to-noise ratios. Endophenotypes involved in
BD might be abnormal neurophysiological, biochemical, endocrinological,
neuroanatomical, cognitive or neuropsychological findings.

Endophenotypes in BD
Despite the overlap and comparable prevalence and public health importance
between BD and schizophrenia there has been less study of possible
endophenotypes for BD than for schizophrenia. Traits of interest for the use as
endophenotypes in BD are abnormal regulation of circadian rhythms, response to
sleep deprivation, P300 ERP*s, response to medication and increase in white
matter hyperintensities.

Rhythms and activity as endophenotypes of BD
For this study we will focus on the regulation of circadian rhythms and
activity, as altered circadian and infradian rhythmicity is a key aspect of
recurrent mood disorders and has been proposed as a possible endophenotype.
Euthymic BD twins show greater seasonal changes in sleep length and mood and
higher global seasonality-scores than unaffected co-twins. Family and twin
studies point to genetic components in seasonal affective disorder and
seasonality (seasonal variations in mood and behavior) and there is
moderate-high heritability for diurnal preference, sleep length and sleep
quality. Bipolar patients show less stable and more variable activity patterns
then controls and variability of the rest-activity rhythm alone is a
significant independent predictor of diagnostic group.

Study of cultured fibroblasts
Fibroblast cell cultures can be relatively easily obtained from small skin
biopsies. As they originate from the same embryonic layer as neurons they
provide a valuable paradigm for cellular research of neuropsychiatric
disorders. Several studies have shown that circadian oscillators in fibroblasts
are similar to those operative in the brain. Fibroblasts are therefore a valid
in vitro model for molecular oscillators in the brain. Similar to the
circadian pacemaker in the mammalian brain, cultured cells, including
fibroblasts, harbor self sustained and cell-autonomous circadian clocks that
persist even during cell division. The periods for the circadian gene
expression in fibroblasts have been found to correspond closely with the human
circadian physiology and mouse behavior. Altered rhythmic expression patterns
have been found in cultured fibroblasts of bipolar patients in several clock
genes.

Summary
BD is a complex psychiatric trait with unknown susceptibility factors. It is
important to investigate possible endophenotypes for bipolar disorder, like
deviations in circadian rhythmicity in vivo by using actimetry as well as in
vitro, using fibroblast cell cultures.

The objective of this study is to include 200 cases, 200 unaffected first and
second degree family members and 200 unrelated healthy controls to participate
in the assessment of variability in activity and light exposure, as these
domains are critical features of the neurobiological underpinnings of BD. Based
on prior evidence for heritability of several of these features (Freimer,
unpublished), we expect that they will be important in the Quantitative Trait
Locus (QTL) analyses.

For this study we will recruit 200 cases, 200 unaffected first and second
degree family members and 200 unrelated healthy controls to collect circadian
rest-activity measurements for comparison. These subjects will be part of the
same homogeneous Dutch population that have earlier participated in the study
*the genetics of bipolar disorder* (CCMO nr NL62946.041.10). Data collection is
completed in year 4.

The timeline of the proposed 5-year project is provided in table below. Four
years are needed to complete recruitment and sample collection of 3 groups of
200 participants. Statistical analyses are initiated in the fourth year and
continue in year 5. Data will be released to the scientific community starting
at the end of year 4 until the end of the proposed project.

For this study we will use the following data from the *Genes of bipolar
disorder* study (protocol 10-285): demographic data, genetic information from
the GWAS. For this study five additional questionnaires will be used; the
current mood state will be assessed with the ASRM and IDS, the Seasonal Pattern
Assessment Questionnaire (SPAQ), the Munich Chronotype Questionnaire (MCTQ), a
questionnaire on physical activity (IPAQ), and a questionnaire on current use
of medication. Also, waist circumference will be measured. Supplemented with
Actimetry, using the Actiwatch-2, this is a reliable and well-validated
approach to collect activity data.


Fibroblast cell lines
The fibroblast cell lines are obtained by a skin biopsy at the upper arm using
a 3 mm punch (Stiefel). Circadian rhythm will be assed in cell lines with
genetic variants of interest using standard target RNA expression profiling
(qPCR of selected transcripts using TaqMan technology) at regular 2 hours
intervals for 48 hours.

Wearing the Actiwatch has no risks for the participant. The only potential
burden is that participants have to wear the watch on their wrist for two
weeks. Future benefits may include development of better treatment of symptoms
or even prevention of developing BD. As the project involves minimal risk to
participants and the potential benefits in terms of knowledge gained are quite
large, the benefits clearly outweigh the risks.
Risk of skin biopsy is neglectable and consists of minor risk for infection or
scar tissue. To minimize this, biopsies will be taken by qualified medical
personal in dedicated medical clinic rooms. To limit cosmetic disadvantages
biopsies will be taken at places indicated by the participants (generally
behind the elbow).