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TLR10-TLR1 -TLR6 HAPLOTYPES IN CHELYABINSK REGION POPULATIONS
Abstract
This paper is devoted to assessing the prevalence of different haplotypes in toll-like receptor genes in Chelyabinsk Region populations. Here is examined gene cluster TLR10-TLR1-TLR6, containing single nucleotide polymorphisms, effected by natural selection during development of human world populations. Differences in the frequency of certain three-locus haplotypes in populations of Russians, Bashkirs and Nagaybaks of Chelyabinsk Region are demonstrated.
Keywords
toll-like receptors, TLR10-TLR1-TLR6 cluster, Bashkirs, Nagaybaks, Russians, single nucleotide polymorphisms
Authors
Aleksandr Evdokimov
Postgraduate, Teaching Assistant Immune Microbiology and General Biology Department Chelyabinsk State University Chelyabinsk, Russia avdax@yandex. ru
Aleksandra Burmistrova
PhD in Medicine, Professor Head of Immune Microbiology and General Biology Department Dean of Biology Faculty Chelyabinsk State University Chelyabinsk, Russia burmal@csu. ru
Dariya Stashkevich
PhD in Biology, Associate Professor Immune Microbiology and General Biology Department Chelyabinsk State University
Chelyabinsk, Russia stashkevich_dary@mail. ru
Introduction. The evolutionary development of animals and plants were always in close interaction with various microorganisms, symbionts and pathogens. This interaction implies the existence of structures, involved in the recognition of microorganisms. The first level of recognition of microorganisms is associated with innate immune system, providing recognition of "images" of alien and dangerous microorganisms through its receptors. Such receptors are called pathogen- or pattern-recognition receptors (pathogen/pattern recognition receptors, PRRs), since they act as microbial sensors, that recognize molecular motifs (patterns), conservative for specific groups of microorganisms - pathogen-associated molecular patterns (pathogen-associated molecular patterns, PAMPs). As a result of binding PPRs with PAMPs the inflammatory response activates (Barreiro, Ben-Ali, Quach, et al., 2009; Casanova, Abel, Quintana-Murci, 2011).
The most studied PRRs are toll-like receptors (toll-like receptors, TLRs), located on the membranes of innate immune cells. In humans, the family of TLRs include 12 receptors (TLR1 -
TLR12), which can be subdivided into groups depending on the number of properties: 1) depending on the subcellular localization there are TLRs in intracellular vesicular compartments (endoplasmic reticulum, endosomes, lysosomes, endolysosomes) - TLR3, TLR7, TLR8, TLR9, - and on the cell surface - TLR1, TLR2, TLR4, TLR5, TLR6, TLR10; 2) depending on the nature of the ligands, binding to TLRs, - intracellular receptors, recognizing fragments of bacterial and viral nucleic acids, and surface TLRs that bind components of the cell walls of microorganisms (zymosan, lipopeptides, flagellin, glycolipids, lipopolysaccharide, flagellin, etc.) (Barreiro, BenAli, Quach, et al., 2009).
After the TLRs family discovery a large number of single nucleotide polymorphisms, located in the TLRs genes, was found. Such polymorphisms arose in the course of evolution and provide modern humans defence against many of the infectious agents. At the same time, certain genetic variations can lead to a predisposition of individuals and even different populations of modern humans to dysregulatory immune responses, underlying the high sensitivity to inflammatory and autoimmune diseases (Hawks, Wang, Cochran, 2007; Netea, Wijmenga, O'Neil, 2012). Genetic history of TLRs in humans indicates the presence of selective forces, that determine their evolution. As shown by several authors, the high frequency of certain alleles and haplotypes of the genes encoding TLRs are associated with protective effects against infection and non-adaptive genetic processes in the past (migration, genetic drift, etc.) (Netea, Wijmenga, O'Neil, 2012).
Currently there is an evidence of selection in the gene cluster TLR10-TLR1-TLR6, occurred in the recent human history (Pickrell, Coop, Novembre, et al., 2009). This cluster is located on the short arm of chromosome 4 (4p14), consists of a closely linked genes, span 60 KB and carries genetic polymorphisms, which indicate the events of positive selection (Karlsson, Kwiatkowski, Sabeti, 2014; Pickrell, Coop, Novembre, et al., 2009). It is shown, that point substitution 1805T>G in the TLR1 gene and 745C>T in the for TLR6 gene determine the haplotype H34, which is typical in Caucasian populations (occurs with a frequency >30%), but almost never occurs in populations of Mongoloid and Negroid origin. At the same time, single nucleotide polymorphism 721A>C in TLR10 gene is characterized by high frequency of occurrence in the populations of Mongoloid origin (Casanova, Abel, Quintana-Murci, 2011; Karlsson, Kwiatkowski, Sabeti, 2014; Quintana-Murci, Clark, 2013).
The territory of the Chelyabinsk Region is inhabitat by multinational people, among which the most common are populations: Russians, of Caucasian descent; Bashkirs, which is characterized by a complex of genes of the immune response, with high incidence in Mongoloid populations (Zaripova, Burmistrova, Suslova, Timofeeva, 2010; Chernova, 2014); and the population of unique immunogenetic profile - Nagaybaks, the formation of which was influenced by Turkic and Finno-Ugric peoples (Zaripova, Burmistrova, Suslova, Timofeeva, 2010; Barreiro, Ben-Ali, Quach, et al., 2009).
In connection with the foregoing, it is interesting, to estimate the distribution of gene cluster TLR10-TLR1-TLR6 haplotypes in populations of the Chelyabinsk Region in comparison with the data obtained in the study of world populations.
Objective of this study: to evaluate the three-locus haplotypes frequencies on single nucleotide polymorphisms 721A>C TLR10, 1805T>G TLR1 and 745C>T TLR6 in Chelyabinsk Region populations of Russians, Bashkirs and Nagaybaks.
Materials and Methods. The study was conducted at the laboratories of Chelyabinsk State University and the Chelyabinsk Regional Blood Transfusion Station. Study subjects: a random sample of representatives of the three ethnic groups are Russians (167 subjects), Bashkirs (152 subjects) and Nagaybaks (114 subjects). These groups were formed from healthy blood donors, ethnicity was defined according to the genealogical history in three generations (according to the recommendations of the 8th International Symposium (1980), Los Angeles, CA, USA).
Genomic human DNA was isolated from venous blood samples using the Axygen kit (Quiagen, Germany) according to the manufacturer's instructions. single nucleotide polymorphisms 1805T>G TLR1 and 721A>C TLR10 were identified by restriction fragments lengths polymorphism analysis (RFLP) using restriction endonucleases AluI and Nlalll, respectively (37°C, 15 minutes, Fermentas, USA). The definition of polymorphism 745C>T TLR6 was carried out by allele-specific PCR (Lytech, Moscow). Amplification was carried out in the amplificatior "Tercyc" (DNA-Technology, Moscow, Russia). The detection of results was carried out by electrophoresis in 3% agarose gel followed by visualization of the data in the ultraviolet.
12 Statistical processing of data carried out using the software package Arlequin (V. 3.5, Swiss
Institute of Bioinformatics) by the following algorithm: 1) check the observed distribution of gene frequencies for accordance to Hardy-Weinberg equilibrium; 2) estimation of frequencies of three-locus haplotypes TLR10-TLR1-TLR6 with the gamete phase definition by the Expectation-Maximization algorithm; 3). calculation of linkage disequilibrium coefficient (A) for three loci; 4) the establishment of significant differences in the three-locus haplotypes frequencies between populations using Pearson's x2 test with Bonferroni correction for multiple comparisons. Statistically significant differences were considered when p<0.01.
Results. Calculated frequencies of genotypes in samples of the studied populations correspond to the expected according to the Hardy-Weinberg distribution, which indicates the equilibrium state of these populations.
Allelic frequencies analysis revealed the presence of linkage disequilibrium for the studied polymorphic sites of TLR10, TLR1 and TLR6 genes in all samples. Table 1 presents the frequency distribution of three-locus haplotypes TLR10-TLR1-TLR6 (HF) in the studied populations and the linkage disequilibrium coefficients (A) for haplotypes.
Table 1. Three-locus haplotypes in Chelybinsk Region (CR) populations
Haplotypes Russians CR (n=334) Bashkirs CR (n=304) Nagaybaks CR (n=228)
HF, abs. ( %) A HF, abs. ( %) A HF, abs. ( %) A
TLR10 721*A-TLR1 1805*T-TLR6 745*C 11 (3,3) -0,06 73 (24,0) -0,05 30 (13,2) -0,04
TLR10 721*A-TLR1 1805*T-TLR6 745*T 1 (0,3) -0,05 3 (1,0) -0,04 1 (0,4) -0,07
TLR10 721*A-TLR1 1805*G-TLR6 745*C 80 (24,0) -0,06 35 (11,5) -0,02 33 (14,5) -0,07
TLR10 721*A-TLR1 1805*G-TLR6 745*T 121 (36,2) 0,18 41 (13,5) 0,11 63 (27,6) 0,18
TLR10 721*C-TLR1 1805*T-TLR6 746*C 66 (19,8) 0,14 130 (42,8) 0,14 70 (30,7) 0,17
TLR10 721*C-TLR1 1805*T-TLR6 746*T 1 (0,3) -0,03 2 (0,7) -0,05 0 (0,0) -0,06
TLR10 721*C-TLR1 1805*G-TLR6 746*C 50 (15,0) -0,02 17 (5,6) -0,08 27 (11,8) -0,06
TLR10 721*C-TLR1 1805*G-TLR6 746*T 4 (1,2) -0,09 3 (1,0) -0,02 4 (1,8) -0,06
According to the table 1, the TLR10 721*A-TLR1 1805*G-TLR6 745*T and TLR10 721*C-TLR1 1805*T-TLR6 746*C haplotypes are characterized by positive value of the linkage disequilibrium coefficient (A, p<0.001). This indicates, that the alleles, included in these haplotypes, are coupled together and with high probability are inherited together.
When comparing frequencies of three-locus haplotypes distribution between populations, statistically significant differences were found. The relevant data are shown in table 2.
According to the table 2, the haplotype TLR10 721*A-TLR1 1805*G-TLR6 745*T is significantly more common in the Russian population (36,2%) and less frequent in the population of Bashkirs (13,5%). This haplotype contains alleles with replacement in the loci of genes TLR1 and TLR6, and in TLR10 gene locus is wild-type allele. Haplotype TLR10 721*C-TLR1 1805*T-TLR 6 745*C is rare in Russian (19,8%), but more often in the population of Bashkirs (42,8%) and is characterized by wildtype alleles in the loci TLR1 and TLR6, but contains the allele with the substitution in the locus of TLR10. The frequency of TLR10 721*A-TLR1 1805*G-TLR6 745*T and TLR10 721*C-TLR1 1805*T-TLR 6 745*C haplotypes in the population of Nagaybaks are mean values with respect to the data, characterizing the population of Russians and Bashkirs.
Table 2. Comparison of CR populations in frequencies of linked haplotypes
Haplotypes Russians CR (n=334) / Bashkirs CR (n=304) Bashkirs CR (n=304) / Nagaybaks CR (n=228) Nagaybaks CR (n=228) / Russians CR (n=334)
HF, abs. (%) Evaluation of differency significance HF, abs. (%) Evaluation of differency significance HF, abs. (%) Evaluation of differency significance
TLR10 721*A-TLR1 1805*G-TLR6 745*T 121 (36,2) / 41 (13,5) x2[1]=58,18 p<0,001 41 (13,5) / 63 (27,6) x2[1]=17,16 p<0,001 63 (27,6) / 121 (36,2) x2[1]=8,86 p=0,003
TLR10 721*C-TLR1 1805*T-TLR6 745*C 66 (19,8) / 130 (42,8) 130 (42,8) / 70 (30,7) 70 (30,7) / 66 (19,8)
Discussions. According to literature it is known that allele linkage TLR1 1805*G-TLR6 745*T determine the haplotype H34, which is typical for Europeans, found among them with a frequency of 30% or more and almost never occurs in populations of Mongoloid and Negroid origin (Barreiro, Ben-Ali, Quach, et al., 2009). This distribution is due to polymorphism 1805T>G TLR1, leading to the substitution of isoleucine for serine at position 602 (I602S) of TLR1 molecule. This replacement occurs in the transmembrane domain of the receptor, which is itself quite rare for TLRs phenomenon: most amino acid substitutions occur in the extracellular domain. The functional consequence of the I602S replacement is the deterioration of embedding the synthesized receptor molecules in the membrane, resulting in decreased synthesis of proinflammatory cytokines to 60% (Barreiro, Ben-Ali, Quach, et al., 2009). In this regard, interesting results are obtained in respect of TLR10 (Oosting, Cheng, Bolscher, 2014), according to which activation of this receptor induces the synthesis of anti-inflammatory cytokines of innate immunity (primarily IL-Ra) (Oosting, Cheng, Bolscher, 2014). It is assumed, that single nucleotide polymorphism 721A>C in TLR10 gene, causing a substitution of asparagine for histidine at position 241 of the TLR10 molecule, leads to weaker activation through TLR10, resulting in increased proinflammatory response. It is interesting that, in our study allele 721*C TLR10 frequency in Russian population is relatively low, and dominated by the wild-type allele 721*A. Several studies of various authors convincingly show that such changes have occurred in the relatively recent periods of human evolution and may reflect the pressure of infectious diseases, which went along with the successful development of agricultures, which began approximately 10,000 years ago (Barreiro, Quintana-Murci, 2010; Hawks, Wang, Cochran, et al., 2007; Raj, Kuchroo, Replogle, et al. 2013). The presence of alleles, which determine a low level of inflammatory reactivity, had, apparently, an adaptive character for Caucasian populations in conditions of frequent epidemics of infectious diseases.
We have shown that the frequency of haplotype TLR10 721*A-TLR1 1805*G-TLR6 745*T in the studied population of Russians corresponds to the value characteristic for the Caucasians (36%). According to the data, presented by different researchers (Zaripova, Burmistrova, Suslova, Timofeeva, 2010; Chernova, 2014), the population of Russians in the frequency distribution of HLA (human leucocyte antigen), mitochondrial DNA, Y-chromosome gene haplotypes differ not to much from the average European level. At the same time, the frequency of TLR10 721*A-TLR1 1805*G-TLR6 745*T haplotype in the population of Bashkirs more than 2.5 times less than that of the Russians, which is associated with significant Mongoloid component in their gene pool.
For a small ethnic group of Nagaybaks it was found that the frequencies of the haplotypes had intermediate values compared with those in populations of Russians and Bashkirs, which may be due to genetic heterogeneity of the ancestors of Nagaybaks and other non-adaptive factors.
Conclusion. Chelyabinsk Region is one of the most multicultural and multi-religious regions of Russia. Since ancient times this area was experienced by the peoples who came from the East and West. As a result of their interaction was not only the interpenetration of cultural influences, but also genetic mixing and the formation of new ethnic groups. The process of globalization, started in the XX century, blurs the boundaries between previously separate populations. Freedom of movement and disappearance of the necessity of intrapopulation mate choosing lead to the exchange of genes between previously isolated peoples that makes it very difficult to carry out
population-genetic studies. These events explain the necessity of such research at present (Chernova, 2014).
In addition, the study of TLRs genetic polymorphisms frequencies in different populations can not only improve our understanding of the adaptation processes in the human genome, but also have practical significance, in particular, may be used to search for candidate genes, responsible for risk of common multifactorial diseases (Takeuchi, Akira, 2010).
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