**3.8 Association of polymorphism of the rs324981 NPSR1 gene with sleep disorders (screening IV)**

In an open population of men aged 45–64 years, the frequency of homozygous C/C genotype of the *neuropeptide S* gene (*NPSR1* rs324981 gene) was 19.4%, with a lower

### *Circadian Rhythm - New Insights into Physiological and Pathological Implications*


### **Table 8.**

Tumor necrosis factor TNF-gene α *G308A polymorphism (screening III).*


### **Table 9.**

NPSR1 *gene rs324981 polymorphism in 45–64-year-old male population (screening IV).*

frequency of T/T genotype—27.8%, the most common in men was the heterozygous C/T genotype—52.8%. The frequency distribution of alleles showed that among men the T-allele prevails—54.2% and C-allele was found in 45.8% of individuals (**Table 9**).

An associative analysis of the allele frequencies of the neuropeptide S polymorphism of the *NPSR1* rs324981 gene in men with different self-assessment of sleep revealed that T-allele carriers more frequently rated their sleep as "satisfactory"—in 69% of cases, while C-allele carriers did it only in 57.1% of cases. In addition, carriers of the C-allele are more often satisfied with their sleep, rating it as "good"—28.6% and "very good" than carriers of the T-allele who have "good" sleep only in 20.7% of cases (χ<sup>2</sup> = 15,713 df = 8, p < 0.05).

### **4. Discussion**

Sleep plays an important role in promoting health. Studies conducted over the past decade have confirmed that sleep disturbance has a powerful effect on the risk of infectious diseases, the occurrence and progression of some major diseases, including

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

cardiovascular diseases and cancer, as well as the incidence of depression [55, 56]. In Russia, about 45% of adults are dissatisfied with their sleep, and almost 20% need serious treatment for sleep disorders [57]. In our population, the level of sleep disorders turned out to be high and had the following trend—it demonstrated a decrease from 1988 to 1989 to 1994–1995 and an increase in 2003–2018. The increase in sleep disturbance in 2003–2018 was observed mainly in the older age groups (45–64 years old). Problems with sleep in the population can only get worse every year. The rapid emergence of "24/7" communities, working 24 hours a day, 7 days a week, participation in round-the-clock events, increased nighttime use of television, the internet, and mobile phones, means that an adequate uninterrupted nighttime sleep is becoming rare. The proportion of workers with circadian rhythm disturbances who are needed to service "24/7" communities is likely to increase [58], which determined the feasibility of studying biological determinants of sleep disorders.

The *CLOCK* gene encoding the positive transcription factor CLOCK is among the major circadian rhythm genes. CLOCK protein with binding partner BMAL1, *BMAL1* gene product, forms a transactivation dimer influencing the promoter of controlled genes [59]. In our study, we examined the associative relationship between sleep disorders and various polymorphic variants of the rs2412646 genotype of the *CLOCK* gene among men aged 25–44 years. The response of having "satisfactory" sleep (36.8%) and "poor" sleep (5.3%) was found to be more frequent among C/T genotype carriers than among carriers of all other genotypes (χ<sup>2</sup> = 9.44 df = 4 p < 0.05). The frequency distribution of rs2412646 genotypes of the *CLOCK* gene depending on the rs934945 genotype of the *PER2* gene is also of interest. We found that carriers of the rs2412646 genotype of the *CLOCK* C/T gene were most often carrying the A/A rs934945 genotype of the *PER2* gene in the male population of 25–44 years old. Our results are supported by the findings obtained by other researchers regarding the association of the *CLOCK* gene with insomnia [60, 61] and preference for a particular sleep-wake cycle [62–64].

The *ARNTL* gene is a key element of the positive feedback loop of the molecular circadian oscillator [65]. According to the activity of some genes, Jun Z. Li, et al. 2013 [66] were able to determine that in individuals suffering from affective disorders circadian rhythms are interrupted, and "night" genes were expressed during the day. It has been suggested that desynchronization may occur due to a disruption in the connection between individual circadian genes. Considering the association of genotypes of the rs2278749 *ARNTL* gene with affective disorders, we have identified several components that have shown the strongest association with polymorphic variants of the gene under study. It turned out that carriers of the genotype C/T had problems with sleep, especially since they had much more anxiety dreams during the month. Men, carriers of the T allele, both homozygotes (T/T genotype) and heterozygotes (C/T genotype) were more likely to wake up tired or exhausted. Thus, our results confirm the data of other researchers concerning the identification of individual polymorphic variants of the *ARNTL* gene, leading to possible desynchronization and disruption of the circadian rhythm and, accordingly, leading to affective disorders [66].

Examining the association of *PER2* gene genotypes with sleep characteristics, we found that among carriers of the A/A genotype there is a tendency to have more anxiety dreams in comparison with carriers of other genotypes. Moreover, A/A genotype carriers were more likely to wake up during the night, and the tightest sleep was observed in men who were A/G and G/G genotype carriers. Sleep deprivation (5 hours or less) also occurred more frequently in individuals whose genotype contained the

homozygous A allele. Our results overlap in part with those obtained by Ojeda D.A., et al. [67], who studied the association of the *PER2* gene (rs934945) with circadian rhythms in healthy individuals, students at Columbia University. The *PER2* gene (rs934945) showed a statistically significant association with two subscales of the morning sleepiness scale, that is, "activity planning" and "morning alertness." The association of rs934945 with "morning restlessness" was first shown.

The most common in the population was the heterozygous genotype of the candidate gene *NPAS2 C/T*—53.3%, followed by the homozygous genotype T/T, with both variants of the candidate gene more common among men who had enough sleep for only 7 hours a day. The C/C genotype of the *NPAS2* candidate gene was significantly more common in those who slept for at least 8 hours (33.3%) or 9 hours (33.3%) per day. The major T allele of the candidate gene *NPAS2* was 4.5 times more common in men who sleep 6 hours a day and four times more common in men with 7 hours of sleep. Thus, the obtained data indicate that rs4851377 of the candidate NPAS2 gene determines whether men are night owls or early birds [21].

According to the literature devoted to genetic research, it has been established that some mental and emotional characteristics of a person are associated with polymorphism of the 3rd exon of the gene of the neurotransmitter system of the dopamine receptor, subtype 4 (*DRD4*) [68] and the dopamine transporter gene (*DAT*) [69].

When considering the occurrence of identified polymorphic variants of *DRD4* candidate genes in people with sleep disorders, it was found that carriers of the genotype 4/4 were more likely to believe that they had either good or satisfactory sleep. Carriers of genotypes 2/4 and 3/4 were more likely to rate their sleep positively, while men with genotypes 2/6 and 4/6, on the contrary, were dissatisfied with their sleep and rated it as "poor" more often.

When comparing the "short" and "long" alleles of the *DRD4* gene, we observed approximately the same pattern: carriers of the "long" allele 6 more often evaluated their sleep as "poor," carriers of allele 2 believed that their sleep was "very good," and carriers of the most common in the population allele 4 mostly reported having "good" sleep.

According to the current understanding of the biosynthesis of dopamine, it is known that just one sleepless night is enough for its level in the brain to increase [70]. The findings of the study conducted by Nora Volkow et al. suggest that dopamine in the human brain is involved in the so-called adaptation process, which leads to sleep disturbance. The researchers also found that the amount of dopamine in the brain is associated with feelings of fatigue and physical ability to perform cognitive tasks. However, the study also found that increased levels of dopamine in the brain cannot compensate for cognitive disorders caused by lack of sleep. On the other hand, according to the literature, people with the "long" form of the *DRD4* gene (six or more repeat units) have a lowered affinity of dopamine receptors and a reduced number of receptors. These individuals are less sensitive to dopamine. This means that they need more stimulation to get the same reaction than people with a "short" gene [71]. It can be assumed that "stimulating wakefulness," for example, a sleepless night, is one of the "ways" to naturally raise the level of dopamine, to receive a "reward" by the brain, for which we later have to pay with insomnia.

When analyzing the frequency distribution according to the conjugation tables of genotypes and alleles of the VNTP polymorphism of the *DAT* gene in a population of 25–64-year-old men, with different sleep self-assessment, no significant differences were found. There was only a tendency toward an increased number in the positive assessments of sleep-in carriers of genotype 9/10 and genotype 10/10 of the *DAT*

gene. Negative sleep assessments were slightly more frequent in carriers of genotype 9/9 of the *DAT* gene. There was a tendency for an increase in the number of those who reported their sleep to be "very good" among the carriers of allele 10 and "poor" among the carriers of allele 9. Dopamine uptake is carried out through active transmembrane transfer using the dopamine protein transporter, it has been experimentally established that disabling the *DAT* gene in mice leads to a reduction in the paradoxical sleep phase (REM sleep) and promotes early awakening [28]. In individuals containing a short variant of the *DAT* gene in the genome, the reuptake of dopamine is altered [72], and there is reason to believe that an increase in free dopamine contributes to an increase in the period of wakefulness, but, as mentioned above, does not contribute to either physical or mental rest [70]. Probably, for this reason, men carrying the genotype 9/9 of the *DAT* gene are *more* likely to evaluate their sleep negatively.

In the Novosibirsk population, the most frequent genotype-G308A of the tumor necrosis factor *TNF-α* gene polymorphism was the G/G genotype—it was observed in almost 80% of men. Moreover, the same genotype was predominant in all groups differing in sleep quality. Comparative analysis showed that individuals with homozygous genotype G /G are much more likely to give positive assessments of their sleep, unlike carriers of all other genotypes combined, who evaluate sleep as satisfactory or even poor. In addition, heterozygous carriers with the A/G genotype are less likely to positively assess sleep than carriers of the homozygous G/G genotype. Although there are no direct analogous studies in the world literature, nevertheless, studies devoted to the study of obstructive sleep apnea can provide indirect evidence. According to various authors, insomnia occurs in 42%–54.9% of patients with sleep breathing disorders [73]. A meta-analysis published in 2012 showed an association between 308G/A and obstructive sleep apnea—the presence of the 308 A allele increases the risk of developing obstructive sleep apnea by 65%, compared with individuals with a homozygous G/G genotype (OR = 1.65, 95% CI = 1.02–2.68, p = 0.04) [36].

In the study population, we found an association between some polymorphic variants of the *NPSR1* gene and sleep self-assessment among men. The presence of the T allele in the genotype was found to contribute to poorer sleep quality among men. Our results are confirmed by the results obtained by Spada J et al. [74], who proved that the study participants with the homozygous T/T genotype had significantly shorter sleep/rest times than individuals carrying the C allele in the genotype. These findings are confirmed by the studies by G.W.A. Gottlieb et al. [51], who found associations between rs324981 and sleep. Our conclusion about the relationship between sleep quality and rs324981 is also consistent with studies on rats since the direct introduction of NPS into the rat brain strongly affects sleep architectonics. Already within the first hour after injecting NPS into the brain, wakefulness time was lengthened, while the rapid eye movement phase, as well as the slow sleep phase, were shortened [39]. Similar results were obtained by Zhao et al. [40], who reported a decrease in sleep phases. These studies indicate that NPS can inhibit different sleep phases that were previously thought to be independently regulated [75]. However, the causal relationship can be much more complicated, as it is still unknown how rs324981 polymorphism acts in ontogeny. There is a hypothesis that various compensatory mechanisms may be induced simultaneously with the loss of allele function, or there may be interaction with other unknown genetic or environmental factors, which may explain the lack of association with sleep disturbances in heterozygotes [76, 77].
