Inflammation is characterized as a protective and adaptive homeostatic reaction of the body to damage or the action of an irritant of a physical, chemical, or biological, including allergic, nature [1]. The inflammatory response is aimed at eliminating the products and agents of damage and maximizing recovery. However, inflammation can play an important role in the induction of various diseases including oncological diseases, cardiovascular diseases, autoimmune diseases, and others [2–8].

The inflammation process is regulated by pro- and anti-inflammatory cytokines—small protein molecules that provide intercellular interaction, determine cell survival, stimulate or suppress cell growth, differentiation, functional activity and apoptosis, and also ensure the coordination of the immune, endocrine and nervous systems under normal conditions and in response to pathological influences. Interleukin-6 (IL-6) is a pro-inflammatory cytokine and functions as a regulator of the immune response and mediator of inflammation [9, 10].

Another important role of IL-6 was identified in connection with the COVID-19 pandemic. The level of IL-6 in the blood of patients began to be considered as a significant prognostic marker of the severity of the disease with the development of the so-called “cytokine storm” [11, 12].

The severity of inflammation, its nature, course and outcome depend not only on the pathogenic potential of the stimulus, but also on the reactivity of the organism [1], which is largely determined by genetic factors. Gene IL6 is localized on the short arm of chromosome 7 (7p15.3) and contains 5 exons and 4 introns, with a total length of 1183 bp. The most studied polymorphic loci are ‒174G/C (rs1800795) and ‒572C/G (rs1800796) in the promoter region of the IL6 gene.

Allele ‒174G (rs1800795) IL6 leads to a significant increase in promoter activity compared to the ‒174C variant and, as a consequence, increased gene expression [13]. IL6 allele ‒572C (rs1800796) is also associated with increased levels of IL6 expression compared to allele ‒572G [14, 15].

Variants ‒174G and ‒572C, which provide increased production of IL-6, are associated with a number of diseases whose pathogenesis is based on inflammation. It has been shown that their carriers are more likely to suffer from liver diseases [16] and have an increased risk of tuberculosis [15, 17]. Allele ‒174G is considered a risk factor for squamous cell lung cancer and chronic obstructive pulmonary disease [18], cancer and fundic atrophy of the stomach [19], ovarian cancer [20], intrauterine infection of the fetus [21], development of cerebral arteriovenous malformation [22] and is associated with the risk of developing type 2 diabetes [23, 24]. Allele ‒572С has also been associated with ischemic stroke in indigenous West African men [25].

However, data on the association of the ‒174G/C polymorphism of the IL6 gene with the risk of developing malignant neoplasms remain controversial [26], and interpopulation differences in allele frequencies may be one of the reasons for the existing contradictions [27]. It has been shown that for people of Tatar ethnicity of postmenopausal age, the homozygous ‒174G/‒174G genotype serves as a marker of a reduced risk of developing ovarian cancer [28], while in Caucasians with non-alcoholic steatohepatitis and hepatocarcinoma, the frequency of IL6 ‒174G is significantly lower than in healthy people [5, 29, 30]. This variant may be protective against undifferentiated connective tissue dysplasia [31]. The protective role of the IL6‒572C allele has been established for the development of endocarditis [32]. It is possible that there are interethnic differences in the effects of polymorphic IL6 gene variants on the development of diseases associated with features of the distribution of allele frequencies of other genes, the products of which mediate the action of IL-6, for example, the gene for the IL-6 transmembrane receptor - IL6R [16, 33].

Knowledge of the nature of the geographical distribution of alleles IL6 ‒174G and IL6 ‒572C is important for understanding the process of formation of population gene pools and the influence of the adaptive value of alleles on it in various living conditions [34]. Environmental changes associated with the departure from the traditional way of life, as well as the anthropogenic impact on nature, can modulate the adaptive significance of alleles, in connection with which population studies in this area become especially relevant. Currently, a large amount of knowledge has been accumulated on the distribution of polymorphic variants of the IL6 gene in different countries and among different ethnic groups, including Russia [34–38]. However, for the indigenous populations of Siberia, this issue is poorly understood.

This work presents the results of the study of IL6 ‒174G and IL6 ‒572C allele frequencies in Siberian samples in comparison with Russians, as well as with some other populations.

EXPERIMENTAL

Samples of study participants. The genetic material for this study was collected during the expeditions of the Laboratory of Population Ethnogenetics in 2000‒2019. The study involved volunteers, healthy at the time of the study, who gave informed consent. Blood was taken from the subjects in compliance with the WHO International Rules (https://apps.who.int/ iris/bitstream/handle/10665/44298/9789241599252_ eng.pdf?sequence=1). Before donating blood, each subject filled out a specially designed demographic questionnaire, in which they specified the nationality of their ancestors up to 3–4 generations. Based on the collected information, 8 samples of the population of Southern and Eastern Siberia were formed. Persons of Buryat nationality who do not have any other ethnic ancestors, living in the villages of Alkhanai and Orlovsky, Aginsky Buryat Okrug (ABO) of the Zabaykalsky Krai, were included in the group of Eastern Buryats (N = 133). Ethnic Buryats of the Ekhirit-Bulagatsky District of the Ust-Ordynsky Buryatsky Okrug (UOBO) of the Irkutsk Region (N = 273) made up the Western Buryat group. The study included the Teleuts of the Belovsky District of the Kemerovo Region (N = 117). Two ethnic groups of Yakuts were also formed: Nyurbinskaya (from those living in the villages of Nyurbachan and Syultsy of the Nyurbinsky Ulus; N = 109) and Ust-Aldanskaya (from the inhabitants of the village of Dyupsya, Ust-Aldan Ulus; N = 99). Residents of the city of Dudinka, the settlements of Volochanka and Ust-Avam of the Taimyr Dolgano-Nenetsky District of the Krasnoyarsky Krai, identifying themselves as the Dolgan ethnos, made up the Dolgan group (N = 179). The seventh sample of the indigenous inhabitants of Siberia included ethnic Tuvinians from the city of Kyzyl (N = 301). Finally, the eighth group included persons who call themselves Russians and live in the Zabaykalsky Krai (N = 65), Irkutsk Region (N = 67) and Tuva (N = 24). Most of the sample consisted of representatives of the Russian old-timer population, who have lived in Siberia for more than one generation. It should be noted that some people, who make up 5% of the sample, indicated among their ancestors, in addition to Russians, also representatives of other European nationalities: Ukrainians, Belarusians, Poles, Germans, etc. The described group did not include the descendants of mixed marriages of Russians with representatives of the peoples of the Caucasus or indigenous Siberian ethnic groups.

Genotyping of single nucleotide polymorphisms: real time PCR. Total DNA was isolated from the leukocyte fraction of venous blood by the standard method of phenol–chloroform extraction [39] using a kit for DNA isolation from whole blood (LLC BioSilica, Russia). Genotyping of single nucleotide substitutions ‒174G/C (rs1800795) and ‒572C/G (rs1800796) was performed by real-time PCR using competing TaqMan probes complementary to polymorphic DNA regions. The structure of primers and probes was selected according to the sequences available in the NCBI database (http://www.ncbi.nlm.nih.gov/) using the programs UGENE (version 1.14, http://ugene.unipro.ru/) and Oligo Analyzer (version 1.0.3, https://eu.idtdna. com/pages/tools/oligoanalyzer) (Table 1).

Table 1.   Structures of primers and probes used for genotyping single nucleotide substitutions in the IL6 gene
Full size table

Amplification was carried out in a volume of 25 µL, the PCR mixture included: 300 nM of each primer, 100 nM TaqMan probes, 65 mM Tris-HCl (pH 8.9), 16 mM (NH4)2SO4, 2.5 mM MgCl2, 0.05% Tween-20, 0.2 mM dNTPs, 0.5‒10 ng DNA, and 0.5 U Taq-DNA polymerase (hot-start, Biosan, Latvia). PCR was performed under the following conditions: initial denaturation 3 min at 96°C; then 46 cycles, including denaturation at 96°C for 5 s, primer annealing and subsequent elongation at 61°C for 30 s (each step was accompanied by the registration of a fluorescent signal at the emission wavelength of the fluorophores Fam (517 nm) and Hex (549 nm).

Statistical processing of results. The population frequencies of allelic variants were determined based on the observed genotype frequencies. The correspondence of the empirically observed distribution of genotype frequencies to the theoretically expected distribution, which is equilibrium according to the Hardy–Weinberg law, was checked using the Pearson test (χ2; at p (H–W) > 0.05 equilibrium is satisfied, where p (H–W) is the value of the probability of deviation from the equilibrium Hardy–Weinberg distribution). Assessing the significance of differences in allele frequencies between the studied samples was carried out according to the criterion χ2 using the Yates correction for continuity; at p < 0.025 (corrected for multiplicity comparison: 0.025 = 0.05/2) the results were considered statistically significant.

RESEARCH RESULTS

Genotyping results IL6 ‒174G/C (rs1800795) and IL6 ‒572C/G (rs1800796) in samples of Buryats, Teleuts, Yakuts, Dolgans, Tuvinians, and Russians are given in Table 2.

Table 2.   Distribution of genotypes IL6 in samples of indigenous peoples of Siberia and Russians
Full size table

The observed distribution of genotype frequencies of both polymorphic loci in the studied samples corresponded to the expected distribution under the Hardy–Weinberg equilibrium. Allele frequencies ‒174G and ‒572C in the studied samples, as well as in some ethnic groups described in the literature [35], and comparison of populations (p-value) are presented in Tables 3 and 4.

Table 3.   Allele frequency ‒174G IL6 in some populations (ethnic groups) and comparison of populations
Full size table
Table 4.   Allele frequency ‒572C IL6 in some populations (ethnic groups) and comparison of populations
Full size table

It has been shown that the IL6‒174G allele frequency in a sample of Russians corresponds to its frequency in European groups [35]. The indigenous peoples of Siberia are statistically significantly different in the higher frequency of occurrence of this polymorphism compared to the Russians and European populations described in the literature and lower in comparison with a number of East Asian populations: the Chinese, Japanese and Vietnamese, in which this figure is close to 100%.

In the samples of Siberian ethnic groups studied by us, the IL6-572C allele frequency is statistically significantly higher than in the groups of Russians and Europeans. Statistically significant differences between Siberian samples and East Asian populations, in which this indicator is even higher, were demonstrated.

Thus, according to the frequency of occurrence of Alleles IL6‒174G and IL6‒572C in Siberian indigenous populations are located between Europeans and samples from East Asia. The same trend was revealed by us earlier in studies of polymorphisms in other functionally important genes [40, 41].

RESULTS AND DISCUSSION

Understanding the features of the frequency distribution of the polymorphic variants ‒174G/C (rs1800795) and ‒572C/G (rs1800796) of the IL6 gene, encoding the pro-inflammatory cytokine IL-6 in the indigenous population of various regions of the world is important both for fundamental population genetic studies and in the medical aspect. As a result of our screening in the ethnic groups of Buryats, Teleuts, Yakuts, Dolgans and Tuvinians, the allele frequencies of IL6 ‒174G and IL6 ‒572C associated with an increase in the production of IL-6, an enhanced inflammatory response, and a number of diseases were determined for the first time. It is shown that in samples of indigenous Siberian ethnic groups, the frequencies of both studied variants are statistically significantly higher than among Russians, and lower than among East Asian peoples, that is, they occupy an intermediate position.

Interestingly, in the indigenous population of Africa, the frequency of the IL6‒174G variant is 100%, it is close to this value in Asian countries. In the populations of Europe, it is lower - up to the predominance of the allelic variant -174С of the gene IL6, which is “responsible” for the reduced production of the pro-inflammatory cytokine [35]. S.A. Borinskaya et al. [34] suggested that in European populations, a decrease in the frequency of the IL6‒174G allele was the result of adaptation to new living conditions: a temperate climate with a reduced load of pathogens, that is, with a decrease in selection pressure. There is a similar geographic distribution of the IL6‒572C allele, determining the high level of transcription of the gene. In African countries, the frequency of this allele is about 10%, it is even lower in the indigenous populations of Europe—up to 5%, but significantly increased in the peoples of Asia—80% and higher [35].

Based on the publication of summary pathogen load indices calculated from historical data for nine infectious diseases (leishmaniasis, trypanosomiasis, malaria, schistosomiasis, filariasis, leprosy, dengue, typhoid, and tuberculosis) [42], S.A. Borinskaya et al. [34] found that the frequency of the IL6‒174G, which determines high-level protein expression and a strong inflammatory response, positively correlates with the pathogen load index. Probably, in the process of human settlement from the African continent to Asia, both options IL6: ‒174G and ‒572C—had adaptive advantages.

It is known that in some cases, especially after the “exit” of a person from reproductive age, an increased response of the body to a pathogenic stimulus can also have negative effects, triggering pathological processes (including carcinogenesis), maintaining chronic inflammation underlying metabolic, cardiovascular vascular, neurodegenerative and neoplastic diseases in the elderly [43]. It can be assumed that due to the increase in anthropogenic environmental pollution, which provokes inflammatory reactions, the risk of inflammation-associated diseases will also increase in ethnic groups with increased IL6‒174G and ‒572C allele frequencies gene. However, to test this hypothesis, additional medical genetic studies are needed in various populations with a large sample size, as well as the study of the frequencies of allelic variants, not only of IL6, but also of other functionally significant inflammatory genes.