Sophie Gabreldar, Year 2 Research
Circadian rhythms are an important yet overlooked contributor to anxiety disorders. Disruptions to circadian rhythms can have negative effects such as poor sleep quality and cardiovascular issues. Period 3 (PER3) gene mutations alter circadian period length. This altered circadian period may increase anxiety levels in individuals that have PER3 variants associated with shorter period lengths. In this study, four participants were genotyped and asked to fill out the State Trait Anxiety Inventory (STAI) to establish a baseline anxiety level. Overall, the participants with shorter period lengths had higher STAI scores in comparison to those with longer period lengths. However, due to small sample of size of this experiment, no conclusive relationship can be drawn between specific PER3 polymorphisms and STAI scores.
Although the exact causes of anxiety disorders are still being researched, genetics continue to be a large factor in the likelihood of developing one. Although anxiety is a normal and necessary basic emotion, it is considered a disorder when it arises in the absence of any threat, or in disproportionate relation to a threat, and keeps the affected individual from leading a normal life (Andreas et al. 2018). Anxiety disorders are often correlated with poor general health, low life satisfaction, and in some cases may be debilitating (Liberman et al. 2017). Disruptions to circadian rhythms may play a significant role in the development of an anxiety disorder (Liberman et al. 2017). Circadian rhythms regulate the human body’s cycle of alertness and sleepiness by responding to the amount of light present in the surrounding environment (Reedy et al. 2020). This regulation is essential to the human body as the lack of sleep or poor rest significantly impacts a variety of the body’s daily functions (Reedy et al. 2020). During the sleeping cycle, memory consolidation, healing and metabolic regulation occur (Reedy et al. 2020). The sleep-wake cycle also influences eating habits, digestion, body temperature, hormone release, and many other bodily functions (Reedy et al. 2020). Various chronic health conditions have been linked to irregular sleep-wake cycles including diabetes, depression, bipolar disorder, and seasonal affective disorder (Reedy et al. 2020).
Both positive and negative molecular feedback loops regulate circadian rhythm (Reedy et al. 2020). There are many identified clock genes such as BMAL1/BMAL2, CLOCK, CRY1/CRY2, and PER1/PER2/PER3, that regulate and control transcription and translation (Reedy et al. 2020). The circadian clock mechanism is built on a conserved negative feedback loop that generates circadian rhythms at the molecular level (Lande-Diner et al. 2013). The core positive element of the clock is the heterodimeric transcription factor, circadian locomotor output cycles kaput (CLOCK) and Arnt-like protein-1 (BMAL1), which drives transcription of Period (PER) and Cryptochrome (CRY) genes (Lande-Diner et al. 2013). The turnover of PERs and CRYs ends the negative-feedback phase of the cycle. The reactivation of CLOCK-BMAL1 transcription of circadian target genes occurs several hours after the end of the negative feedback (Lande-Diner et al. 2013).
This suggests the onset of circadian transcription in each cycle by CLOCK-BMAL1 is not a passive consequence of the turnover of negative-feedback proteins but is positively regulated and timed by unknown clock-controlled factors (Lande-Diner et al. 2013). The expression of these clock genes inside the cell influence signaling pathways which allows the cells to identify the time of day and perform the appropriate function (Reedy et al. 2020). The Period homolog 3 (PER3), is often used in mood and sleep-related studies, but the exact role it plays in regulation circadian rhythms remains unclear (Liberman et al. 2017).
The first link between PER3 and circadian-related mood phenotypes was reported in a study in which the authors described a family group in which familial advanced sleep-phase disorder (FASPD), and seasonal affective disorder (SAD) co-occur with a double mutation in the PER3 gene (Zhang et al. 2016). When mice were transfected with the same two PER3 variants, the animals also manifested symptoms of depression and poor sleep quality, suggesting a causal relationship between PER3 mutations and mood (Kayikcioglu et al. 2016). In studies of mood disorders in humans, depression and anxiety are commonly comorbid (Kayikcioglu et al. 2016). Mutations in the PER3 gene resulting, in such the presence of PER3 variants associated with shorter period lengths, have been associated with higher trait anxiety (Kayikcioglu et al. 2016). This pilot experiment aims to explore the relationship between PER3 polymorphism and anxiety disorders by using the State Trait Anxiety Inventory (STAI) and miniPCR, in morning-type (advanced phase, short period) versus evening-type (delayed phase, long period) individuals.
However, anxiety disorders represent a heterogenous group of disorders, with no single unifying etiology. There is a higher chance of an anxiety disorder in the parents, children, and siblings of a person with an anxiety disorder than in the relatives of someone without an anxiety disorder (Gottschalk et al. 2017). It may also be difficult to identify an anxiety disorder since it may be a concomitant symptom of several major psychiatric diseases such as schizophrenia and dementia (Bandelow et al. 2015).
Materials and Methods
Four participants were chosen using convenience sampling. They ranged in age from 16 to 50 and had no family history of anxiety disorders.
First, DNA samples were collected by asking participants to scrape the insides of their cheek using a flat ended toothpick. The DNA from the samples was extracted by swirling the toothpick in 50 µl of X-Tract DNA Extraction Buffer from miniPCR bio™ in a 200 µl thin-walled PCR tube. The DNA samples were prepped for the PCR following the procedure outlined in Sleep Lab™ – Morning lark or night owl (2019). In brief, the procedure included adding 20 µl of Sleep Lab Primers, 5 µl of 5X EZ PCR Master Mix and 3 µl of the DNA extract sample into a new PCR tube. The of 5X EZ PCR Master Mix is composed of Taq DNA polymerase, 5X reaction buffer, magnesium chloride, dNTP (nucleoside triphosphate), blue dye, yellow dye and a compound that increases sample density. The PCR protocol parameters were as follows: initial denaturation for 30 seconds, denaturation for 20 seconds, annealing for 20 seconds, and extension for 20 seconds. This cycle was repeated 30 times. The PCR machine used in the experiment was the miniPCR mini8 thermal cycler.
While the PCR machine was running, the agarose gels needed for the gel electrophoresis was poured. The gels that were prepared were a 2.0% agarose gel using 1X TBE buffer following the gel pouring procedure outlined in Sleep Lab™ – Morning lark or night owl (2019). The 1X TBE buffer is a solution consisting of tris base, boric acid, and EDTA (Ethylenediaminetetraacetic acid). When the gels had solidified, they placed in the blueGel™ electrophoresis system and covered in 1X TBE Buffer. In the first lane of the gel 10 µl of bp DNA Ladder was pipetted in. The following lanes were filled with 14 µl of the participants PCR sample, one lane was used per sample.
Once the lanes were filled, the gel ran for approximately 45 minutes and the observed results were recorded. While the gel was running, participants filled out the State Trait Anxiety Inventory (STAI) and the answers were recorded in Table 1.
Table 1: STAI Scores of four participants and their corresponding PER3 genotype
Figure 1: Gel electrophoresis results for PER3 genotypes of four participants
This pilot experiment aims to explore the relationship between PER3 polymorphism and anxiety disorders. Specifically, variable number tandem repeats (VNTR), which are short, repetitive sequences in succession within a gene, were analyzed. In the PER3 gene there is a 54-base pair sequence that is repeated 4 times in one allele, and 5 times in another variant. Since polymorphisms in this repeat change the length of this gene, the difference between 4- repeat and 5-repeat Per3 genes can be seen in gel electrophoresis (Figure.1.).
On the STAI, scores are commonly classified as “no or low anxiety” (20-37), “moderate anxiety” (38-44), and “high anxiety” (45-80) (Kayikcioglu et al. 2016). The genotypes were determined using the “expected results” section of the Sleep Lab™ – Morning lark or night owl (2019). Participant 1 had the lowest STAI score and the longest circadian period length with a genotype. Alternatively, Participant 2 had the highest STAI score and the shortest circadian period length with a genotype. The remaining participants had genotypes, with a moderate STAI score.
The purpose of this study was to determine if there was a correlation between certain PER3 polymorphisms and higher STAI scores. The participants with the highest STAI scores, indicating high trait anxiety, had the genotypes, and , that are associated with shorter circadian period lengths. In humans and other organisms, the timing of 24-hour behavior is governed by the period length of one’s circadian rhythm (Pagani et al. 2013). This period is approximately 24 hours long and has a reported population range of 23.47–24.64 in laboratory conditions (Pagani et al. 2010). Short periods lead to behavior occurring at an earlier clock time in some individuals, which makes the individual more active in the early morning, and long periods to later timing of behavior (Pagani et al. 2010). Mutations in clock genes, such as PER3, affect period length, and can lead to circadian rhythm sleep disorders (Pagani et al. 2010). The extreme shifts in circadian period lengths could play a role in the development of an anxiety disorder due to a misalignment of the functions that are typically regulated by the circadian rhythm (Brown et al. 2008).
The first link between PER3 and circadian-related mood phenotypes occurred in a study that described a family group in which FASPD and SAD co-occur with a double mutation in the PER3 gene. When mice were transfected with the same two PER3 variants, the animals manifested symptoms of depression and poor sleep quality, suggesting a relationship between PER3 mutations and mood (Zhang et al. 2016). Although there are few studies regarding the relationship between PER3 polymorphism and anxiety specifically, many other mood disorders have been studied such as bipolar disorder. Furthermore, mood disorders are often comorbid with anxiety disorders and represent a risk factor for developing an anxiety disorder.
The participants in this piolet study were also administered the State-Trait Anxiety Inventory (STAI), which is commonly used to measure state and trait anxiety. The Trait Anxiety Scale (T-Anxiety) measures relatively stable aspects of “anxiety proneness,” which includes general states of calmness, confidence, and security (Viena et al. 2016). One study genotyped and surveyed a total of 205 healthy women for PER3 allele status and were asked to respond to previously validated psychological questionnaires, including the STAI (Viena et al. 2016). The study found that the women with no self-reported sleep dysfunction, relative to the longer allele carriers, the allele carriers were at greater risk for temporary psychological effects when they reported reduced sleep (Viena et al. 2016). It showed that a reduced sleep duration, combined with the genotype, was associated with greater mood disturbances and increased state anxiety (Viena et al. 2016). This was not the case for depressive symptomatology or trait anxiety; there were no interactions between PER3 allele status and sleep duration on these measures (Viena et al. 2016). This suggests that the influence of PER3 polymorphism on mood is related to some type of sleep deprivation (Viena et al. 2016). This could indicate that PER3 polymorphism would only affect STAI scores if the participants were suffering from poor rest.
Another study attempted to find an association between PER3 variants and established measures of depression (Beck Depression Inventory) and anxiety (STAI) in a population of undergraduates (Liberman et al. 2017). In the sample, the those with the genotype were significantly associated with depression in this sample, and individuals homozygous for the reported significantly higher anxiety (Liberman et al. 2017). These extremes are caused because of the change in the timing of circadian oscillations ( Brown et al. 2008). This can cause misalignments between the functions that are regulated by the clock and the external environment ( Brown et al. 2008). Delayed or advanced phases in clock gene oscillations are also characteristic of individuals with extreme diurnal preference, which may help explain why depression and SAD have been previously linked to extreme morning- or evening-type individuals (Liberman et al. 2017). However, the exact relationship between PER3 polymorphism and STAI remains unclear.
However, since extreme morning individuals may be at higher risk for developing an anxiety disorder, certain life-style changes may be able to lesson to the risk. There is no way to change an individual’s circadian type since it is genetically determined. If an individual’s circadian rhythm misaligns with their desired schedule, they can try to shift their social life to match their circadian rhythm. It is also important to note that exposure to bright light at night, including bright artificial lights and screen time on laptops, tablets, and phones, can cause disruption in circadian rhythm and may contribute to worsening mood and negative consequences for health (Wahl et al. 2019).
The administration of the STAI itself may also have impacted the results. As with any survey, response bias may be a cause for concern. Another source of error related to PCR and gel electrophoresis was the visualization of the dye. Initially the DNA dye that was used was standard food colouring, but no results would appear in the gel. Throughout the experiment, there were many difficulties trying to make the equipment work and as such the results that were recorded may not be the accurate. Although they may be an association between PER3 polymorphisms and high STAI scores based due to the results obtained in this study, due to the complexities of anxiety disorders, no definite correlation can be drawn.
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