Biological Determinants of Sleep Disorders

*Valery V. Gafarov, Elena A. Gromova, Vladimir N. Maksimov, Igor V. Gagulin and Almira V. Gafarova*

### **Abstract**

The purpose of the study is to research the effect of polymorphism of genes such as *CLOCK, ARNTL*, *PER2, NPAS2*, *DRD4, DAT*,*TNF-α*, and *NPSR1* on sleep disorders in an open population of 25–64-year-old men. We conducted screening studies of representative samples of men aged 25–64 years. The general examination was carried out according to the standard methods included in the WHO MONICA-Psychosocial Program (MOPSY). Carriers of the C/T genotype of the *CLOCK* gene more often than others reported having "satisfactory" or "poor" sleep. Carriers of the C/T genotype of the *ARNTL* gene were more likely to experience anxiety dreams, and they woke up exhausted. Carriers of the A/A genotype of the *PER2* gene were more likely to wake up two or more times per night, a total of four to seven times per week. In the population, C/T and T/T genotypes of the *NPAS2* gene were significantly more common in individuals with 7-hour sleep. Genotype 4/6 of the *DRD4* gene and genotype 9/9 of the *DAT* gene were significantly associated with sleep disturbances. Carriers of the heterozygous A/G genotype of the *TNF-α-308* gene, compared with carriers of all other genotypes, more often rated sleep as "satisfactory" (30%) than "good."

**Keywords:** population, men, sleep disorders, *CLOCK* gene, *ARNTL* gene, *PER2* gene, *NPAS2* gene, *DRD4* gene, *DAT* gene,*TNF-α* gene, *NPSR1* gene

### **1. Introduction**

Sleep is a complex set of brain processes that support human physiological needs [1]. Sleep is part of the sleep-wake cycle. This cycle, consisting of approximately 8 hours of sleep at night and 16 hours of daytime wakefulness in humans, is controlled by a combination of two internal factors, that is, sleep homeostasis and circadian rhythms [2]. Unlike wakefulness, sleep is a period of inactivity and restoration of mental and physical functions. Sleep is thought to provide time for inputting information gained during waking into memory and for reestablishing communication between different parts of the brain. Sleep is also the time when other body systems replenish their energy and repair their tissues [3], and it is the key to wellness and optimal health [4–6]. People who get enough quality sleep have more energy, better cognitive function, memory, alertness, attention, and performance during the day, as

well as a healthier immune system [7]. Quality healthy sleep is one of the basic needs of people and is important for their health [8].

Circadian rhythms are a system that synchronizes all processes in living organisms that provide temporary adaptation, including sleep and wakefulness. The study of circadian rhythms and biological clocks progressed slowly until the methods that anticipated the beginning of the genomic millennium came to the aid of scientists. At the end of the last century, scientists found out that there is a biological "clockwork mechanism" in the mammalian brain that coordinates the work of the entire organism. To be more precise, these clocks are located in the suprachiasmatic nucleus (SCN) of the hypothalamus. Today we know that each SCN neuron is a miniature clock counting the circadian rhythm, and all these thousands of clocks work in unison, forcing the rest of the body's systems to obey. The SCN receives information about the illumination from special receptors located on the retina of the eye and sends corresponding signals to other organs using hormones and nerve impulses. Some SCN cells, as well as cells of many other organs, have individual molecular clocks. The "gears" in these clocks are transcription factors, the activity of which changes over the day. The synthesis of several different proteins depends on the activity of these key transcription factors, which gives rise to the circadian rhythms of the vital activity of individual cells and entire organs. A bright light turned on early at night can shift the circadian rhythm, activating *PER* gene transcription, which usually occurs in the morning. However, it should be understood that a nerve impulse represents the final crescendo of the long processes unfolding in a neuron. To understand the nature of these processes, one has to descend from the cellular to the gene level [9, 10].

In the 1960s and early 1970s, Seymour Benzer at the California Institute of Technology [11] and Ronald Konopka, one of his students, studied the genetics of Drosophila behavior and the latter discovered the first circadian rhythm gene localized in the X chromosome [12]. The gene was named *period*, or *per* (the protein encoded by this gene was respectively named PER). Scientists have found three mutant alleles for *per*, in addition to the normal "wild type." With one of them, the daily cycle of the fly was shortened to 19 hours, with the other, it was lengthened to 29 hours, while the carriers of the third "did not observe hours" at all, that is, their periods of rest and activity were of random duration [12]. The 2017 Nobel Prize in physiology or medicine was awarded to Jeffrey C. Hall, Michael Rosbash, and Michael W. Young for cloning and sequencing the *period* gene in 1984 [13, 14]. Michael Rosbash and his colleagues also noticed that the concentration of messenger RNA (mRNA) of the *per* gene increases and decreases within 24 hours. In mutants, these fluctuations accelerated or slowed down [13]. In the 90s*, new details of the mechanism were discovered*—*the timeless* genes, or *tim* genes, *doubletime* (Michael W. Young) [15], as well as *Clock*, *cycle,* and *Cryptochrome* genes (Michael Rosbash group) [16].

The relationship between these genes and their products is seen in **Figure 1** [17]. The clock mechanism is based on two proteins—CLOCK (CLK) and BMAL1 (- ARNT-like protein 1 in the brain and muscle; also known as ARNTL or MOP3). By dimerizing, CLOCK and BMAL1 activate the transcription of the Period *(PER*) and Cryptochrome *(CRY*) genes. In nocturnal rodents, as well as in some diurnal animals, the transcription of the *PER1* and *PER2* genes in the SCN peaks in the morning or afternoon, while for *CRY1* and *CRY2* genes, the peak is observed closer to the evening. An increase in the concentration of PER and CRY proteins in the cell triggers a feedback mechanism that blocks further synthesis of these proteins. According to recent studies, the main inhibitor of the CLOCK-BMAL1 complex is CRY, but it works only when combined with PER. During the night, cellular enzymes gradually degrade

### *Biological Determinants of Sleep Disorders DOI: http://dx.doi.org/10.5772/intechopen.101765*

**Figure 1.**

*Direct and indirect outputs of the core clock mechanism [17].*

PER and CRY, and when their concentration reaches a critically low point, transcription is reactivated. The duration of the cycle depends on the degradation rate of PER and CRY [18, 19].

An important question is what molecular mechanisms provide a link between the light signal and the genes of the biological clock? Until recently, it was believed that phototransduction—the conversion of a light signal into an electrical signal transmitted through neurons—can only take place in the retina of the eye and only through the retinal, an active component of rhodopsin. In 2011, Fogle K.J., Parson K., et al. found that the CRY protein has the same ability, and it uses a mechanism independent of TIM and PER [20]. If the neuron in which CRY is expressed is illuminated by blue light, a complex cascade of reactions involving potassium membrane channels is triggered and an action potential is generated, that is, the neuron produces an electric signal under the direct influence of light. Control experiments have shown that this reaction has nothing to do with opsin, the visual pigment in Drosophila. However, if in the course of an experiment the other neurons not previously possessing photosensitivity are forced to synthesize CRY, they also start generating signals in response to flashes of light [20].

Mention should also be made of the *NPAS2* gene, which is located on chromosome 2 in 2q11.2, one of the most important circadian genes. NPAS2 forms heterodimers with BMAL1 and then activates the circadian genes *PER* and *CRY*, which are essential for maintaining biological rhythms in many organisms. Animal studies have shown that loss of normal function of the *NPAS2* gene can cause defects in several aspects of the circadian system, such as sleep patterns and behavior [21].

According to the current understanding of the neurophysiology of sleep, monoamines, one of which is dopamine, also play an important role [22]. Damage to central dopaminergic synaptic transmission plays an important role in the onset of severe neuropsychiatric disorders [23]. People with these conditions have serious sleep disturbances such as excessive daytime sleepiness [24], rapid eye movements disrupting sleep behavior [25], a decrease in the period of paradoxical sleep, and disrupted sleep architecture [26]. In general, all these observations suggest that dopamine plays an important role in the regulation of the sleep-wake cycle [27]. The evidence that increased extracellular dopamine is one of the key mechanisms of wakefulness activation also associates reduced dopamine metabolism with sleep disturbances [28].

TNF-α (tumor necrosis factor-) is a pro-inflammatory cytokine that contributes to the formation of atherosclerotic plaque. Although early reviews showed a contradictory association with CHD (coronary heart disease) [29], the replacement of G (guanidine) by A (adenine) at position 308 in the promoter region is now associated with increased TNF-α production [30], as well as with increased inflammatory response after cardiac surgery [31], insulin resistance [32], CHD, in individuals with type II diabetes [33] and increased C-reactive protein in individuals with vital exhaustion, defined as a condition with excessive fatigue, difficulty falling asleep, general malaise, apathy, irritability, and lack of energy [34]. In addition, it was suggested that variability in location 308 may affect the development of depressive symptoms, for example, allele A is more common in depressed patients than in controls [35]. There is an association between obstructive sleep apnea and the presence of the A allele [36].

The neuropeptide S receptor (NPSR1) is a metabotropic G protein receptor with seven transmembrane helices [37]. The receptor was first described in 2002, and in 2004 neuropeptide S (NPS) was identified as a ligand [38, 39]. NPS refers to neuropeptides, a diverse group of neurons expressing signaling molecules involved in various brain functions. Studies in rats have shown that administration of NPS greatly induces wakefulness and reduces the occurrence of all stages of sleep [38, 40]. NPS appears to be expressed only in certain brain regions [41–46]. The highest concentration of NPS precursor mRNA was found in brainstem neurons adjacent to the locus coeruleus, in the parabrachial nuclei of the variolar bridge, and the sensory nucleus of the trigeminal nerve [39, 40]. The locus coeruleus and parabrachial nuclei belonging to the ascending activating reticular system, as well as the sensory nucleus of the trigeminal nerve, are known for their contribution to modulating the sleep/wake cycle [42, 43]. Compared to NPS, the expression pattern of NPSR1, a precursor of mRNA, is more widely distributed in the brain. It covers important centers of the sleep/wake system in the hypothalamus and thalamus and is also present in the cortex and amygdala. In particular, it can be found in hypothalamic regions such as the peripheral region (red band) and the tuberomammillary nucleus, which are known for their expression of orexin and histamine, respectively [44–46]. In addition, NPSR1 mRNA was found in key regions responsible for sleep induction. At the molecular level, the receptor activates protein kinases and increases intracellular cAMP and Ca2+ levels [38]. Thus, NPS is believed to modulate the neurotransmission of the expressing neurons NPSR1. Although the NPS system appears to play a critical role in modulating sleep, most of the conclusions have been drawn from rodent studies, and data on its effect on sleep in humans are limited. A single rs324981 nucleotide polymorphism (lying at triplet position 107 of the *NPSR1* gene on chromosome 7p14.3) provides an opportunity for a noninvasive study of the effect of NPS/NPSR1 in the human body. The SNP T allele leads to the exchange of amino acids in the active center of the receptor binding site (Asn ≥ Ile) [47]. This, in turn, leads to an approximately tenfold increase in the sensitivity of neuropeptide S [47]. The T allele has already been identified as a risk factor for the development of asthma and panic disorder [48–50]. Gottlieb et al. [51] conducted a whole-genome

*Biological Determinants of Sleep Disorders DOI: http://dx.doi.org/10.5772/intechopen.101765*

sequencing that examined sleep parameters—falling asleep time and sleep duration. They found a connection between rs324981 and the time a person goes to bed. This study showed a delay in the moment of falling asleep among T-allele carriers.

Thus, our study aimed to investigate the effect of polymorphism of circadian rhythm genes (i.e., *CLOCK*, *ARNTL*, *PER2,* and *NPAS2*), dopamine receptor genes (i.e., *DRD4, DAT*), pro-inflammatory cytokines (*TNF-α* gene), and the *NPSR1* gene on sleep disorders in an open population of 25–64-year-old men.

## **2. Materials and methods**

We conducted screening studies of representative samples of the population aged 25–64 years in one of the districts of Novosibirsk city (the budget theme No. АААА17–117112850280-2):

at screening II in 1988–1989, 725 men were examined, average age—43.4 0.4 years, the response rate was 71.3%;

at screening III in 1994–1995, 647 men were examined, average age—44.3 0.4 years, the response rate was 82.1%;

at screening IV in 2003–2005, 576 men were examined, average age—54.23 0.2 years, the response rate was 61%;

at screening V in 2013–2016, 427 men were examined, the average age—34 0.4 years, the response rate was 71%;

at screening VI in 2016–2018, 275 men were examined, average age—49 0.4 years, the response rate was 72%.

The general examination in 1988–1989, 1994–1995, 2003–2005, 2013–2016, and 2016–2018 was conducted according to standard methods included in the WHO MONICA-Psychosocial Program (MOPSY) [52].

A standard Jenkins questionnaire was used to study sleep disorders. Statistical analysis was performed using the SPSS software package version 20 [53].

Genotyping of the studied polymorphisms of circadian rhythm genes (*CLOCK, ARNTL*, *PER2, NPAS2* genes) (screening V), as well as those related to the dopaminergic system (*DRD4, DAT* genes) (screening III) and inflammatory pro-cytokines (*TNF-α*) (screening III) and *NPSR1* gene (screening IV), was performed in the Laboratory of Molecular Genetic Studies of the Research Institute of Therapy and Preventive Medicine—Branch of ICG SB RAS, Novosibirsk [Vladimir N. Maksimov, Doctor of Sciences (Medicine), is the head of the laboratory].

The distribution of traits and their quantitative characteristics was analyzed. Simple relations between variables were analyzed (contingency tables). The hypothesis of independence of factors A and B or homogeneity of factor B in relation to the levels of factor A was tested using the method of contingency table construction. The reliability of the independence of the factors was assessed by the criterion χ<sup>2</sup> [54]. Reliability was accepted at a significance level of p ≤ 0.05.

### **3. Results**

In the male population under study, the rate of sleep disturbances ("poor" and "very poor" sleep) in the group aged 25–34 years in 1988–1989 was 5.4%; in 1994–1995, it went down to 3.6%; and in 2013–2016, it reached 4.3%. In the group aged 35–44 years, the rate of sleep disorders in 1988–1989 was 9.5%; in 1994–1995, it was 9.3%; in 2013–2016, it decreased to 4.2%; and later in 2016–2018, it grew dramatically to 11%. In the group aged 45–54 years, the rate of sleep disorders was 11% in 1988–1989, 9.8% in 1994–1995, in 2003–2005, it rose to 12.5%, and fall to 4.9% in 2016–2018. Among men in the older age group of 55–64 years, the rate of sleep disturbance was the highest in 1988–1989—2020.8%; in 1994–1995, it plummeted to 12.1%; in 2003–2005, it showed a slight decrease to 11.8%; and finally, in 2015–2018, it rose to 19.7% (**Table 1**).

### **3.1 Association of polymorphism of the rs2412646 CLOCK gene with sleep disorders**

**Table 2** shows the frequency distribution of rs2412646 genotypes of the *CLOCK* gene among men aged 25–44 years in Novosibirsk. In the studied population of 25–44-year-old men, the most common was the homozygous C/C genotype of the *CLOCK* gene—50.3%, the heterozygous C/T genotype was found in 42.5% and the T/T genotype in only 7.2%.

The respondents were asked how well they sleep. Among carriers of the C/T genotype, the response "satisfactory" (36.8%) and "poorly" (5.3%) sounded more often than among carriers of all other genotypes (χ<sup>2</sup> = 9.44 df = 4 p < 0.05). Carriage of the T/T and C/T genotype of rs2412646 *CLOCK* gene was most frequently combined with carriage of the A/A genotype of rs934945 *PER2* gene among men aged 25–44 years in Novosibirsk (30.8% and 46.2%, respectively). The carriers of the C/C genotype of rs2412646 *CLOCK* gene most often had A/G and G/G genotypes of rs934945 *PER2* gene (68.4% and 68.9%, respectively) (χ<sup>2</sup> = 27.18 df = 4 p = 0.001).

### **3.2 Association of the polymorphism of the rs2278749 ARNTL gene with sleep disorders**

**Table 3** shows the frequency distribution of rs2278749 genotypes of the *ARNTL* gene among 25–44-year-old men in Novosibirsk.

The most common rs2278749 genotype of the *ARNTL* gene was the homozygous C/C genotype—74.9%, the second most common was the heterozygous C/T genotype—22.3%, while only 2.8% of individuals in the population had the homozygous T/T genotype.

The question was asked: "How often in the last month have you had disturbing dreams while sleeping?" Only C/T genotype carriers experienced such disturbances for 22 days or more, that is, 7.5%; 27.5% of C/T genotype and 20% of T/T genotype carriers reported having disturbing dreams 4–7 days per month, while C/C genotype carriers more often than others responded that they had no disturbing dreams at all, that is, 42.5% (χ<sup>2</sup> = 16.35 df = 8 p < 0.05).

The question, "During the past month, how often did you wake up after a normal sleep, feeling tired or exhausted?", showed that 40% of T/T genotype carriers experienced such problems one to three times a month, while C/T genotype carriers (7.5%) experienced this problem more often than others, from 15 days to 3 weeks (χ<sup>2</sup> = 18.71 df = 8 p < 0.01). Men carrying the heterozygous C/T genotype (57.3%) were more likely than others to wake up feeling exhausted and tired (χ<sup>2</sup> = 19.52 df = 4 p = 0.001).

