DEVELOPMENT OF MULTIPLEX GENOTYPING METHOD OF POLYMORPHIC MARKERS FOR GENES INVOLVED IN HUMAN ADAPTATION TO COLD CLIMATE

Dispersal of the human population out of the place of origin in East Africa over the globe proceeded rapidly (on the evolutionary scale) and was associated with change of climatic zones and associated changes in habitat parameters temperature, humidity, insolation, and infectious load. The aim of the study is to develop a method for genotyping of genetic markers associated with adaptation to climate according to the literature data and functional analysis of genes; and identification of signals of adaptation to cold climate in two indigenous Siberian populations. In the course of study, genes and genetic markers were selected, which show reliable signals of natural selection in populations living in cold arctic and subarctic climates in previously published papers and which are involved in biological processes having a cold adaptation potential. A panel of 28 single nucleotide markers (SNP) was selected, and a method of their multiplex genotyping was developed based on multiplex PCR and separating DNA fragments by MALDI-TOF mass spectrometry. Allele frequencies of 28 SNPs in two indigenous Siberian populations (Yakuts and Kets) were determined. A low level of intrapopulation diversity in these populations and significant genetic differences between them were found. Loci under natural selection conditions were detected by analyzing the distribution of the observed Fst values in comparison with the expected distribution, obtained in the simulation calculations based on the hierarchical island model of population structure. The possible role of selection (p<0.1) in differentiation of populations between allele frequencies was determined for 2 markers rs133036 in MKL1 gene and rs2305508 in CPT1A gene, which are candidate in terms of climate change adaptation.


INTRODUCTION
Dispersal of the human population out of the place of origin in East Africa over the globe proceeded rapidly (on the evolutionary scale), over the past 50-100 thousand years, and was associated with change of climatic zones from tropical to temperate and Arctic and associated changes in habitat parameters -temperature, humidity, insolation, and infectious load.Modern humans entered Eurasian arctic and sub-arctic zones relatively recently (on the evolutionary scale) -30-40 thousand years ago.Human survival in cold climates could involve mechanisms and processes of different scale and natureshort-term acclimatization, long-term adaptation, and socio-cultural changes.Correlation and role of these mechanisms in survival in extreme climates remain unclear, but phenotypic features of contemporary indigenous populations of northern Eurasia and characteristics of their gene pool suggest a possible role of directional selection in adaptation of indigenous populations to extreme climatic conditions [1,2].
A number of current studies aimed at finding signals of natural selection in the human genome and gene pools of individual populations, revealed a large number of polymorphic positions in the genome which were under the influence of selection during human dispersal [3][4][5][6].Some of these markers could be associated with adaptation of Eurasian North population genetic structure to cold climate as a result of natural selection [6].
The above papers are based on genome-wide approaches and on populations of different regions of the world that do not include, as a rule, samples of indigenous populations of Siberia and Northern Asia.The main goal of the study is to select the most significant genetic markers associated with adaptation to cold climate (low temperature) according to genomewide studies; to develop a method of their multiplex analysis and pilot genotyping of the marker panel in samples from two indigenous Siberian populations.

MATERIALS AND METHODS
We used two samples of indigenous Siberian populations -Yakuts and Kets.Yakuts live in the territory of the Republic of Sakha (Yakutia) and speak Turkic language of the Altaic language family.The sample of 102 individuals was collected in the village Byadi, Ust-Aldan region of Yakutia.Kets living in the upper course of the Yenisei River, are one of the smallest indigenous Siberian populations and speak an isolated language, which is the only survived language of the Yenisei family [7,8].Sample of Kets (N = 48) was collected in the village of Kellog in Turukhansk District of the Krasnoyarsk Territory.The samples included unrelated individuals non-admixed in three generations, who have signed the informed consent for the study.The study was approved by the ethical committee of the Institute of Medical Genetics.
DNA was isolated by standard methods.Primers were selected using «Sequenom Assay Design» software suite as explained below.Genotyping was performed by multiplex PCR coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF mass spectrometry), with the use of «Sequenom MassARRAY 4» mass spectrometer as described earlier [9].Multiplex PCR to obtain amplicons containing the analyzed SNPs, was carried out in 96-well plates in a volume of 5 microliters with the use of «Thermo Scientific» and «Applied Biosystems» amplifiers in the presence of 5 ng of genomic DNA matrix and a mixture of PCR primers of the corresponding multiplex.PCR procedure: initial denaturation at 94°C (5 min), then 42 cycles of amplification under the following conditions: denaturation at 94°C (20 s), annealing at 56°C (40 seconds), and elongation at 72°C (60 s), after which samples were incubated for 5 min at 72°C.
Statistical analysis of the results obtained was carried out with the use of standard modern population genetic approaches, implemented in «Arlequin» software suite [10] and described previously [11].Compliance of genotypes distribution to Hardy-Weinberg equilibrium was assessed using an exact Guo and Thompson test.Intra-and interpopulation genetic diversity was assessed by the method of molecular dispersion (AMOVA).Molecular dispersion analysis means that the total molecular diversity (total dispersion) in the group of populations is made up of independent and additive components characterizing dispersion of alleles (haplotypes) within populations, dispersion, which is due to molecular differences within population groups and dispersion, which is due to differences between population groups.The coefficient of gene differentiation, obtained in AMOVA, is an analogue of Fst.
Loci under natural selection were detected by analyzing the distribution of the observed Fst values in comparison with the expected distribution, obtained in the simulation calculations (20,000 simulations) based on the hierarchical island model of the population structure.The model assumes that distribution of locus-specific genetic differentiation values (Fst) is a function of interpopulation heterozygosity, formed by factors considered in the island hierarchical model (isolation by distance and gene flow).Loci, which Fst values are on the edges of the distribution are distribution outliers; level of interpopulation differences for such loci was formed by other factors (in particular, by natural selection) against the background of migration and genetic drift [12,13].

Selection of markers associated with adaptation to cold climate (low temperature) according to the literature.
Physiological mechanisms of adaptation to cold climate of Northern Eurasia, whose main characteristics are extremely low winter temperatures and a wide range of winter and summer temperatures, affect biological processes such as metabolism of lipids and carbohydra tes, thermoregulation, blood pressure regulation, regulation of smooth muscle contractility, response to stress etc. Obviously, genes involved in these biological processes, are prime candidates for a role of targets for natural selection during human dispersal in the North.Several recent studies have identified some genetic systems, genes and markers correlating with climatic parameters (such as temperature) or demonstrating selection signals in northern populations [3][4][5][6].

Development of a multiplex SNP genotyping method.
One of the efficient modern methods of genotyping of panels of markers, consisting of dozens of SNPs, is the method of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF mass spectrometry).Previously, we have developed a number of multiplex panels of SNPs, based on MALDI-TOF, with capacity of 30-70 markers for the purposes of human population genetics, associative genetic research and DNA identification [9,[14][15][16].Based on this experience, we chose mass spectrometry of DNA molecules in order to develop a method of multiplex genotyping of markers associated with adaptation to cold climate.
We formed a multiplex of 28 SNPs in 23 genome regions out of 100 SNPs sampled at the first phase with the use of «Sequenom Assay Design» software suite available on-line at website www.sequenom.co.List of selected markers, their genes (genomic regions) and their characteristics are given in Table 1.(F ST Further we selected two PCR primers (forward and reverse) and extension primer for IPLEX reaction for each marker.Multiplex PCR to obtain amplicons containing analyzed SNPs, was carried out with the use of «PCR Accessory Set» reagent kit, which is a part of the kit for genotyping by MALDI-TOF mass spectrometry from «Agena Bioscience» (USA).Subsequent stages of the experiment -SAP reaction, iPLEX reaction, transfer of samples to spectro-chip, samples ionization and mass spectra analysis was carried out as described earlier [9].SAP-reaction is the dephosphorylation of deoxynucleotide triphosphates (dNTPs) not included in amplicons with alkaline phosphatase (SAP).IPLEX reaction involves multiplex extension of prolongin primers by adding a modified dideoxytriphosphate (ddNTP) with modified mass, complementary to nucleotide located in polymorphic position of each SNP.The result is a mixture of short PCR products corresponding to alleles represented in multiplex of polymorphic markers.Reaction mixture purification of salts accumulated in the course of multiplex PCR and iPLEX reaction was performed by cationic resin «SpectroCLEAN» («Agena Bioscience», Germany) treatment.Sample transfer to spectro-chip and ionization of samples was carried out at «Sequenom MassARRAY 4».Real-time mass spectra analysis, primary processing and experiment results registration were carried out with the use of «MassARRAY TYPER 4.0» software suite («Agena Bioscience», Germany).
An example of mass spectrum for multiplex consisting of 28 SNPs is shown in Fig. 1., Fig. 2 shows a fragment of mass spectrum of three samples, demonstrating three different genotypes for one SNP (rs3741135).Fig. 3 shows clustering of samples between three different genotypes at genotyping of rs3741135.

Allele frequencies of SNPs, localized in climate adaptation candidate genes and genetic diversity in populations of Yakuts and Kets.
Allele frequencies of 28 SNPs, potentially associated with adaptation to cold climate (low temperature) in populations of Yakuts and Ket are listed in Table 2. Genotype frequencies are available from the authors upon request.Distribution of genotypes in both populations complies with the Hardy-Weinberg equilibrium except for rs6724627 and rs809812 for Kets and rs908394 for Yakuts.A number of deviations from the equilibrium (3 of 56 distributions) does not exceed the expected number of random deviations.
Both studied populations demonstrate sufficiently low intrapopulation genetic diversity -0.36-0.37 (Table 2) that corresponds with data for other systems of genetic markers for these populations [17][18][19].Analysis of genetic differentiation of Kets and Yakuts by 28 markers revealed significant differences between populations with respect to allele frequencies.The overall level of genetic differentiation was 4.3% = 0.04313, p = 0.00000).Locus-specific values of genetic difference index range from -0.09 for rs2216163 in the gene region of HNRNPA1P70 / LOC100509370 to 0.34 for rs908394 in the LOC105369791 locus (Table 2).In addition to last one, a very high genetic heterogeneity (10%) was found for rs133036 in MKL1 gene, rs1800849 in UCP3 gene and rs2305508 in CPT1A gene.
Loci under natural selection were detected by analyzing the distribution of the observed values of F ST in comparison with the expected distribution, obtained in the simulation calculations (20.000 simulations) based on the hierarchical island model of the population structure [13,[20][21][22].The possible role of selection (p<0.1) in differentiation of allele frequencies between populations was revealed for 2 markers -rs133036 in MKL1 gene (p=0.01)and rs2305508 in CPT1A gene (p=0.09).The functional characteristic of one of these genes, CPT1A, whose product is involved in the processes of thermoregulation and energy metabolism, makes it an obvious target for selection in northern    The multiplex panel genotyping method was developed, which was tested in the analysis of allele frequencies and genetic diversity in two indigenous Siberian populations -Kets and Yakuts.Comparison of the populations studied revealed a high level of inter-population differences against the background of relatively low intra-population diversity.The possible role of natural selection in formation of the allele frequency spectrum through markers of two genes involved in mechanisms that ensure resistance to low temperatures is revealed.

Fig. 2 .
Fig. 2. Three different genotypes for one rs3741135.Homozygote CC (top), heterozygote CT (in the middle), homozygote TT (at the bottom).Positions of the peaks for rs3741135 presence in the sample are highlighted in blue dotted line.Brown color dotted line indicates the prolonging primer peak position and peak positions for rs3741135 alleles missing in the sample.

2 Fig. 3 .
Fig. 3. Samples clustering between three different genotypes for rs3741135 genotyping.Blue triangles -СС homozygotes, green squares -CT heterozygotes, brown triangles -TT homozygotes The zero position sample (red circle)the control sample without DNA.

Table 1 .
Markers included in the multiplex and characteric of their genomic regions

Table 1 .
Continued.Markers included in the multiplex and characteric of their genomic regions

Table 1 .
End. Markers included in the multiplex and characteric of their genomic regions

Table 2 .
Allele frequencies of 28 SNPs, genetic diversity (average expected heterozygosity, H ) and genetic differentiation (F ) parameters and significance level of inter-population differences (F P) in the Ket and Yakut populations e Science Evolution, 2016, vol. 1, no. 2

Table 2 .
End. Allele frequencies of 28 SNPs, genetic diversity (average expected heterozygosity, H ) and genetic differentiation (F ST ) parameters and significance level of inter-population differences (F ST P) in the Ket and Yakut populations., whereas a mechanism of possible adaptive importance of variants of the second gene, MKL1, is not clear.Thus, this paper presents a new panel of genetic markers associated with adaptation of human populations to low temperatures and a cold climate. populations