Association of P10L Polymorphism in Melanopsin Gene with Chronic Insomnia in Mexicans

Insomnia is characterized by the difficulty to initiate and/or maintain sleep, which results in either chronically non-restorative or poor quality sleep. It is a serious health problem with a prevalence of 10–50% in the general population. Patients with insomnia have a positive family history of ~35% [1]. Many factors are involved in insomnia—environmental as well as genetic—even though these components have not been clearly defined. The global literature contains only one case report of a patient with chronic insomnia, which was a heterozygous carrier of nonsense mutation in the GABRB3 gene. This mutation is located in exon six and consists of the substitution of the amino acid arginine with histidine at position 192 (R192H). The patient had a family history of sleep problems [2].

Melanopsin is a photosensitive protein present in a subset of the ganglion cells in the retina of all vertebrates, including humans. The melanopsin-containing cells are known as intrinsically photosensitive retinal ganglion cells (ipPRG). ipPRG act as an additional photoreceptor to the classical rod and cone cells—reviewed in [3]. They directly project into the suprachiasmatic nuclei (SCN), the pacemaker of biological rhythms. Melanopsin mediated phototransduction, performed by the ipRGCs, is complementary to the activity of cones and rods for SCN photosynchronization, but only the ipPRG axons constitute the retinohypothalaic tract that conducts the nerve impulse required for SCN photosynchronization [3]. It is then understandable that a large number of investigations have focused on the relationship of melanopsin and ipPRG in behavioral disorders of the circadian rhythm—reviewed in [4]). In humans, the participation of melanopsin has already been demonstrated in different behavioral disorders, such as seasonal affective disorder [5] and delayed sleep/wake timing [6] (see below).

There are three isoforms of protein opsin 4 consisting of 478, 489 and 495 amino acid residues, they have a molecular weight of 52.6, 53.8 and 54.5 kDa [7]. Opsin 4 is encoded by the OPN4 gene, located on chromosome 10q23.2, with a length of 12,292 bases (Genecards). Functional single nucleotide polymorphisms (SNPs) identified in the OPN4 gene are: P10L (rs2675703), I394T (rs1079610), and D444G (rs12262894). See supplementary Figure S1. The P10L polymorphism is a change from proline to a leucine at position 10, it seems to not interfere with the correct distribution of melanopsin on the plasma membrane. However, SNP P10L has been associated with seasonal affective disorder, a condition where depressive states are experienced in autumn and winter, when days are shorter [8,9]. The I394T polymorphism is a change of isoleucine by tyrosine at position 394 and has been associated with a pupillary light response under various photic conditions including different intensities and wavelengths [10]. The D444G polymorphism consists of the replacement of aspartic acid by glycine at position 444, it is frequent in African populations (4.9%, and has been classified as a benign variant with no associated phenotype [11]. These different associations of the OPN4 gene SNPs show how melanopsin mediated photransduction and ipRGCs activity are associated with several light-driven functions not related to vision.

Affective and sleep disorders are related. Correct sleep homeostasis is dependent upon an efficient sensing of environmental light. The P10L SNP associated with affective disorder, could be also linked to a defective sensing of environmental light leading to perturbance in sleep homeostasis, manifesting most severely as difficulty initiating and/or maintaining this process. Hence, we wanted to seek an association between the P10L polymorphism OPN4 gene and insomnia. Thus, the aim of this study was to determine whether there was an association between the OPN4 gene P10L polymorphism and chronic insomnia of uncertain etiology in a Mexican population.

Chronic insomnia is the most common primary sleep disorder in the population. Among the risk factors are advanced age, female sex, stress, substance abuse, as well as medical and psychiatric illnesses. There are few genetic studies regarding genetic variations involved in the susceptibility to insomnia. Genetic information can contribute to our understanding of its pathophysiology, the screening of risk factors and the development of an effective treatment. The participation of the ipPRG in the regulation of the sleep wake cycle and therefore the participation of the OPN4 in the integrity of this cycle is evident. Table 4 shows a brief summary of studies reported to date on the genetic contribution of OPN4 gene polymorphisms to sleep and their disturbances which corroborate the implication of melanopsin in the regulation of the photo response, chronotype and sleep cycles. Noteworthy, one study was performed in a Japanese population and two more performed in Caucasian populations, thus, our study is the first report analyzing OPN4 SNPs in a Hispanic/Latin American population sample.

We found no differences between chronotypes of patients when compared with chronotypes of controls. To our knowledge, no previous study has attempted to sort patients suffering with chronic insomnia into chronotypes, namely, whether individuals could be classified as morning (morningness), intermediate, or evening (eveningness) types. As surprising as this is, for us, the results of particular noteworthiness—regardless of the difficulty to initiate/maintain the sleep—individuals still show a diurnal preference similar to control group.

We demonstrated several statistically different behavioral disturbances in the insomnia group when compared with the control group. From Table 1, the data evidently show that insomnia patients are more susceptible to apnea, bad quality of sleep, lack the feeling of having a restful sleep, difficulty in concentration, alteration in mood, muscular tension, seasonal depression and anxiety. In this way, our analysis reports additional features that lead to a lower quality of life in insomnia patients.

Furthermore, we also detected a trend for the insomnia group to present overweight and obesity: almost one half of the patients showed overweight (44.8%), and considering both type 1 and type 2 altogether, 20.7% of individuals were obese (13.8% and 6.9%, respectively). That makes a total proportion of more than 65% patients of chronic insomnia suffering some metabolic disfunction. According to this, it has been demonstrated that the insomnia phenotype is associated with a significant risk of cardiometabolic and neurocognitive morbidity and mortality [22]. Additionally, several studies have demonstrated a strong association between persistent complaints of difficulty initiating or maintaining sleep with an increased risk of hypertension [23], acute myocardial infarction [24] and type 2 diabetes [25].

We performed the genotyping of control and insomnia subjects seeking for the occurrence of OPN4 alleles variants. Our analysis regarding alleles and genotypes strongly reveals a higher risk of insomnia when the T allele is present in all the genetic models tested. The contribution of allele T was statistically significant (p ≤ 0.001, OR = 9.73, CI = 4.78–19.93), suggesting an almost ten-fold risk of occurrence of insomnia in individuals with the T allele. Therefore, the T allele is a risk factor for chronic insomnia, based on all three inherited models including dominant (p ≤ 0.001, OR = 37.38, CI = 8.18–335.66), recessive (p = 7.5 × 10⁻⁵, OR = 9.37 CI = 2.70–34.29) and codominant (p = 7.12 × 10⁻⁴, OR = 4.8, CI = 1.8–12.71). It is important to highlight that when the genotype TT is present, this conveys a risk of being 37.38 times more likely to suffer from chronic insomnia.

Additionally, there is no correlation between genotypes with chronotype and daytime sleepness severity, but interestingly, the chronotype was correlated with daytime sleepiness severity. Individuals with an eveningness chronotype had worse prognosis in terms of severity and sleepiness during the day than those with a morning or intermediate chronotype. Is well known that an evening chronotype carries negative consequences for sleep and reports of higher levels of daytime sleepiness [26] and more maladaptive beliefs about sleep compared to morning types [27]. We found that the chronic insomnia group has >68% more patients with the eveningness chronotype than controls. It is probable that the chronotype shift towards eveningness could be a pre-insomnia state, if insomnia is seen as a threshold disease. If this is true, this result would lead to an opportunity to prevent insomnia cases in future approaches. However, this remains to be demonstrated in a longitudinal and larger study. We demonstrated that in all the genotype and allele comparisons the statistical power was above 94.3%, in spite of a low sample size. The high statistical power in our result can be explained due to (i) the marked differences in allele and genotype frequencies (exposures) between cases and controls and to (ii) and the high control/case ratio (3.38).

Evening chronotype has also been associated with clinical depression [28,29], psychopathology in adolescents [30], bipolar disorder [31], subclinical mania [32], and insomnia [33]. Taken together, these studies and ours suggest that poor sleep and eveningness could be a predictive marker of insomnia severity. Therefore, insomnia patients with an evening chronotype are particularly high-risk group for sleepiness severity and susceptibility of other mental disorders.

A similar association of this SNP has been found for patients suffering with seasonal affective disorder [5], and also for healthy individuals in predicting their sleep timing and chronotype: earlier bedtimes in shorter days and later bedtimes in longer days [6]. There is no correlation so far between the substitution of proline 10 by leucine in the melanopsin protein and the alteration of its function, even more, it seems that the peptide with the substitution has normal sorting to the plasma membrane [6]. It is therefore difficult to hypothesize on a feasible role for this allele variation leading to a disturbance in light perception and thus in the etiology of insomnia, joined to the fact that P10L did not show melanopsin loss-of function in vitro, with no evidence of protein misfolding in cellular localization. The only reported effect is that cells expressing P10L showed a reduced amplitude response compared with the wild type [34]. Proline to leucine mutations in transmembrane proteins can lead to altered chaperone-mediated stabilization and proteasome-dependent degradation in the central nervous system [35]. Another probable explanation is that P10L could be in linkage with a functional variant. In this regard, additional in silico evidence suggests that P10L SNP (rs2675703) is in complete linkage with the intron SNP rs2254051, having both the same population frequency of 12.5%, as is expected according to 1kGP in Mexicans in the Los Angeles population. However, SNP rs2254051 has no predicted functional effect nor does it overlap with any regulatory features (See Supplementary Figure S1, [36])

Regardless of the extensively documented role of melanopsin and ipPRG with photoentrainment, there are no studies on the association of the P10L melanopsin SNP with sleep disorders, beyond Seasonal affective disorder (SAD), sleep/awakening time and chronotype reported by Rocklein et al., [5,37] and Lee et al. [6]. Hence, we do not have a source of comparison for our data, nevertheless, it is clear that we have identified an association for P10L with insomnia. Our results suggest that melanopsin is involved in sleep disorders, at least in chronic insomnia. We encourage research groups to conduct larger studies in populations with different ethnic backgrounds in order to reinforce this hypothesis.

Melanopsin and ipPRG have a primary role in non-visual retinal light responses. Thus, an alteration of melanopsin signaling or ipPRG signaling to encephalic nuclei could also lead to disturbances in sleep control. Several single nucleotide polymorphisms have been reported for the OPN4 gene, and some of these have been related to alterations in extravisual light responses. We analyzed—by genotyping—a group of insomnia patients to look for an association of the P10L polymorphism OPN4 gene. We found a higher risk of suffering with insomnia to be associated with the occurrence of this allele variant. Our preliminary results add evidence to the role of melanopsin and ipPRG in the control of light driven behaviors involved in the sleep–awake cycle, but further research is needed to demonstrate the biological impact of this SNP or its associated functional variant.

Administrative support from Academic Development Coordination (CADAC) of Biomedical Sciences Institute of Autonomous University of Ciudad Juárez is acknowledged.

The following are available online at https://www.mdpi.com/1660-4601/18/2/571/s1↗, Figure S1: Sequence of isoform 1 of melanopsin protein (478 residues, 10 exons). Missense variants are depicted in yellow. Synonymous variants are depicted in green. Different exons are represented with alternate black and blue residues. Red residues are overlapping splicing sites. Figure was made by the authors using the sequences of OPN4 reported in [34]

Conceptualization, B.E.G.-A. and J.A.P.-L.; data curation, B.E.G.-A.; formal analysis, B.E.G.-A., E.P.M.d.O., J.P.G.-A. and J.A.P.-L.; funding acquisition, M.A.R. and J.A.P.-L.; investigation, B.E.G.-A., J.P.G.-A., O.D.C., C.H.R.-M., K.P.C.-A., R.P.C. and J.A.P.-L.; project administration, J.A.P.-L.; resources, M.A.R. and J.A.P.-L.; supervision, B.E.G.-A., M.A.R. and J.A.P.-L.; visualization, E.P.M.d.O.; writing—original draft, B.E.G.-A., E.P.M.d.O., J.P.G.-A. and J.A.P.-L.; writing—review and editing, B.E.G.-A., E.P.M.d.O., J.P.G.-A., O.D.C., C.H.R.-M., K.P.C.-A., R.P.C., M.A.R. and J.A.P.-L. All authors have read and agreed to the published version of the manuscript.

Bianca Ethel Gutiérrez-Amavizca was a recipient of a postdoctoral scholarship from the National Council of Science and Technology (CONACYT). Jorge Pérez-León received financial support from Programa al Desarrollo Profesional Docente-SEP-2019.

The study was conducted according to the guidelines of the Declaration of Helsinski and approved by the Institutional Committee of Ethics and Bioethics of the University Autonomous of Juarez City (CIBE-2016-1-40) September 2016.

Informed consent was obtained from all subjects involved in the study.

The data presented in this study are available on request from the corresponding author.

We declare that the authors have no conflict of interests that might be perceived to influence the results and/or discussion reported in this paper.