The cause of the “sleeping sickness” that struck many residents of Kalachi settlement in 2013–2015, despite the efforts of Kazakh scientists, has still not been identified. Human exposure of the gaseous chemical compound X in the undefeated, but sufficient to affect the nervous system concentrations appears likely. Some researchers demonstrate carbon monoxide as a compound X which, in their opinion, is periodically released from abandoned mines in the neighborhood from the village. To this end, about half of the residents showed elevated carboxyhemoglobin concentrations in their blood - a strong compound of haemoglobin and carbon monoxide. When combined with carbon monoxide, hemoglobin is unable to transport oxygen and the body, including the brain, will experience hypoxia. Under such conditions the phenotype of “sleeping sickness” can develop.
Other researchers as compound X consider an unknown agent in the water from the well of a local resident who provided most of the residents of the village of Kalachi with drinking water.
Obviously, individuals, because of their individual characteristics, will react differently to external factor X. We assumed that polymorphism in the genes of the orexinoergic system, which is the most important brain mechanism with wakefulness function can be involved in pathogenesis of “sleeping sickness”. To test this hypothesis, we did case-control study to test the possibility of association between the “sleeping sickness” and and polymorphism in the genes of the orexinoergic system. For this reason we chose four most common polymorphisms in genes HCRT, HCRTR1 и HCRTR2, that were previously shown to be associated with various diseases of the nervous system.
Polymorphism rs760282 (-909T/C) constitute the substitution T > C of position − 909 from the starting codon of the gene HCRT. The location of polymorphism in the promoter zone suggests ability to influence gene expression HCRT. Previously, rs760282 showed association with sudden falling asleep during Parkinson’s disease.16 Similarity of symptoms of “sleeping sickness” with the above-mentioned disorder supported the candidacy of this polymorphism for the role of hereditary factor of “sleeping sickness”. However, we haven’t found an association between polymorphism and falling asleep in the village of Kalachi.
Polymorphism rs2271933 (also referred to in the literature as Ile408Val and G1222A) is situated in the seventh exon of the gene HCRTR1 and is the replacement for A > G which in the amino acid resulting substitution of isoleycine by valine in position 408. This polymorphism has previously shown association with severe depression disorder,17 affective disorders,18 migraine,19 post-traumatic stress disorder20 and panic disorder agoraphobia.13 The results of our study exclude this polymorphism from the list of genetic factors affecting the manifestation of the “sleeping sickness” phenotype.
Polymorphism rs3122156 is located in the second HCRTR2 gene introns and constitutes substitution T > G. This polymorphism has previously shown association with cluster headaches (Horton migraine).14 We did not find any differences in the distribution of genotypes by rs3122156 between patients and controls, suggesting that this polymorphism does not play a prominent role in the manifestation of “sleeping sickness”.
Polymorphism rs2653349 (also referred to in the literature as Val308Ile and 1246G > A) is located in the fifth HCRTR2 gene exon and constitutes substitution A > G resulting substitution of isoleycine by valine in position 308. This polymorphism has previously shown association with Alzheimer’s disease,21, 22 cluster headaches (Horton’s migraine),23, 24 nicotine addiction, the start of methamphetamine, postoperative pain, schizotypical personality traits and goitre tendencies,25 insomnia caused by sertralin taking26 panics in women.27
Of the four polymorphisms tested only rs2653349 in gene HCRTR2 has shown the association with “sleeping sickness”. Considering allele A’s sufficiently high frequency, group of sick compared to group of healthy Kalachi residents (0.22 versus 0.15) it can be assumed that carriers of this allele are more susceptible to “sleeping sickness” than carriers of the allele G. The obstacle to this assertion is that all three AA homozygotes among the studied Kalachi villagers are in the healthy control group, though understandable that settlement residents were exposed to the of “sleeping sickness” factor not equally, were not the same age or the same physical condition.
We did not find any differences in the distribution of genotypes by rs3122156 between patients and controls, suggesting that this polymorphism does not play a prominent role in the manifestation of “sleeping sickness”.
Elevated allele A content in test group was achieved by significantly more heterozygotes than in the control group (0.44 versus 0.15). In the control group, there was a clear heterozygote dicyte (0.15 instead of the expected 0.24, HWE p = 0.028). These data indicate that among Kalachi residents to “sleeping sickness” were exposed heterozygotes. Indeed, when all the tested residents were grouped together (sick and healthy) genotype distribution aligns Hardy-Wynberg equilibrium (p = 0.76).
When testing an ultra-dominant inheritance model, OR scores were highest compared to those of other models.
When testing a super-dominant inheritance model OR (4.33, 95%CI = 1.58–11.90) were the highest for others however, the lowest values of the Akaike information criterion (AIC = 124.1), and the lowest values of p (0.0011) were received for the codominant model, which supports this model of inheritance as the most likely for a given locus. The worst scores are for models implying a risky mortar allele: the dominant model showed significant indicators, obviously because of heterozygote; the recessive model was not applicable due to the lack of minor homozygotes in the test group.
Almost all described effects on the locus rs2653349 were saved when switching from a mixed sample to a sample of Russians. When comparing the Russian test group with the independent Russian control group confirmed the association under the super-dominant inheritance model.
Associative analysis between the locus rs2653349 and the degree of “sleeping sickness” (number of cases of falling asleep patient) among Kalachi population, has shown that there are credible differences in super-dominant and codominant models and, to a lesser extent, the dominant model.
Associative analysis between the locus rs2653349 and the degree of “sleeping sickness” (number of cases of falling asleep patient) among Kalachi population, has shown that there are credible differences in super-dominant and codominant models and, to a lesser extent, the dominant model. Degree of expression of “sleeping sickness” among Kalachi residents with heterozygous GA genotype was on average higher than those with homozygous genotype GG. This data provides additional proof of case-control results.
These data demonstrate that degree of “sleeping sickness” among Kalachi population with heterozygous GA genotype were on average higher, than those with homozygous genotype GG. So, despite the association we’ve identified, due to the lack of homozygote on minor allele A in the test group we can’t point out this allele as a risky for “sleeping sickness”. in locus rs2653349. Instead, we highlight the heterozygous genotype GA as risky. We probably didn’t find AA homozygotes in the test group due to the relatively small sample.
Interestingly, both alleles rs2653349 has been considered a risk in various studies. In the works21 (Alzheimer’s disease) and25 (nicotine addiction) the risk allele was A, whereas in work20 (post-traumatic stress disorder),22 (Alzheimer’s disease),23, 24 (cluster headaches) and26 (insomnia caused by the take of sertralin) on the contrary, the risk allele is G. This is probably due to the multidirectional role of the hypocrite system in pathogenesis of various neurodiseases: in diseases, related to the redundancy of the system, the wild allele will have to reinforce the manifestation of the phenotype, and vice versa, for diseases related to system deficiency, the phenotype will be amplified by the already mutant (weakened) allele.
The neurobiological mechanism underlying the association between rs2653349 and “sleeping sickness” in the village of Kalachi are unknown to us. However, there is no doubt that the primary role in pathogenesis of “sleeping disease” must play a factor specific to the Kalachi village, depressing the nervous system of people, probably chemical nature. Earlier computer analysis found that the replacement of isoleucine by valine in position 308 does not alter the receptor shape of the binding site, i.e. does not affect its affinity, but influences the dimerization of receptors, which can change the hypocretinergic transfer.14 Given this model and our data, in a heterozygote genotype, it can be assumed that the interaction of two allele proteins adversely affect the transmission of the orexin signal, and thus increases the risk of the phenotype of falling asleep in the village of Kalachi.
When the sample is divided by sex, the association between rs2653349 and “sleeping sickness” appeared only in the female sample. This may be due to differences in performance of the orexinergic system in women and men.28 In support of this, an association between the locus and panic disorder among women but not men.27
It should be noted that the association has been able to identify despite a relatively small sample. This is particularly true for the sample of healthy Kalachi residents serving as a verification. The small sample size is difficult in the search for research candidates in a densely populated in the past and uninhabited village now from where, after the excitement connected with the “sleeping sickness”, the mass exodus of the population began.