Genetic polymorphisms and association of KIR-HLA system of Chinese Henan Han population and an extensive KIR gene diversity study between populations distributed worldwide

Killer-cell immunoglobulin-like receptors are expressed on the plasma membrane of natural killer cells and a minority of T cells, which can regulate the killing function of these cells by interacting with their special ligands. The major ligands for them are the human leukocyte antigen class I molecules. Combinations of human leukocyte antigen class I molecules and Killer-cell immunoglobulin-like receptor variants contribute to the intensity of acquired immune, resistance to infections, susceptibility to autoimmune disorders, complications of pregnancy, cancers and so on. In order to reveal this appropriate functional interaction of these two markers, some previous studies have revealed the co-evolution of these two markers within and across populations in disease researches. To our knowledge, the polymorphism data of two markers of Henan Han population haven't yet been available to date. In this study, we obtained their allelic frequencies of the two markers, on this basis, we obtained 26 Killer-cell immunoglobulin-like receptor genotypes, the extensive Killercell immunoglobulin-like receptor gene diversity between populations distributed worldwide, and the frequencies of the estimated main human leukocyte antigen haplotypes. And we also conducted the correlation analysis to investigate populationlevel evidence for co-evolution of the two markers based on their frequencies and the receptor-ligand pairs. This present study could provide basic and valuable polymorphism data of the two markers and their combinations for anthropological analysis and associated disease studies. In addition, it may provide some valuable clues to the co-evolution of these two complex genetic systems based on the study of the two marker pairs.


INTRODUCTION
Killer-cell immunoglobulin-like receptors (KIRs), which are encoded by one of the very complex and polymorphic gene families located on chromosome 19q13.4are expressed on natural killer (NK) cells and a subset of T cells and can be activated or inhibited [1].The KIR genes exhibiting substantial segmental or nearidentical sequence copy number variations show extensive variability in terms of gene structures and gene content across haplotypes, probably because of non-allelic homologous recombination occurring between pairs of homologous KIR genes which generate novel expanded and contracted haplotypes, multiple genes and formation of novel fusion genes [2,3].Nomenclature of KIRs is based on the number of the extracellular immunoglobulinlike domains (2D or 3D) and the length of the cytoplasmic tail (L for long and S for short) or the pseudogene (P).To date, 15 distinct KIR gene loci have been identified namely KIR2DL1, 2DL2/3, 2DL5A, 2DL5B, 3DL1/S1, 3DL2, 3DL3, 2DS1, 2DS2, 2DS3, 2DS4, 2DS5, 2DL4 and two pseudogenes (KIR2DP1 and 3DP1) [4].According to distinctly different gene contents, KIR gene combinations can be divided into two specific form haplotypes (KIR A and B haplotype).The KIR A haplotype has largely fixed gene content, with mostly genes encoding inhibitory KIRs and only one activating gene KIR2DS4; the KIR B haplotype has a more variable gene content [1].Two haplotypes are always maintained in every human population, but at different frequencies in most populations distributed worldwide.Based on the above principles, we distinguished between the AA and Bx (AB or BB) genotypes of our studied population.As of March 8, 2017, 573 different KIR genotypes are found in 18,783 individuals from 155 populations [5].
Within distinct regions of the KIR cluster in classic linkage disequilibrium (LD) studies, there were two distinct regions namely centromeric region and telomeric region in the KIR cluster.KIR3DL3 is located in the end of centromeric region, KIR3DL2 is located in the end of the telomeric region, the KIR3DP1 and 2DL4 are located in the middle of the KIR cluster.The KIR2DS3, 2DS5 and 2DL5 can be present in the centromeric and telomeric regions of the KIR cluster.Except for the genes described above, the centromeric region is considered to contain the KIR2DS2, 2DL2, 2DL3, 2DP1 and 2DL1; and the telomeric region is considered to contain KIR3DL1, 3DS1, 2DS1 and 2DS4.The KIR2DL3, 3DL1 and 2DS4 belong to A-motif genes; and the KIR2DS2, 2DL2, 3DS1, 2DS1, 2DS3, 2DS5, and 2DL5 belong to B-motif genes [6][7][8].In our study, we also distinguished between the centromeric motif and the telomeric motif.
The KIRs are the critical regulators for the development, activation and tolerance of NK cells.NK cells are bone marrow-derived lymphocytes, which comprise about 10-15% of all circulating lymphocytes and are crucial components of the early innate immune response system, providing a first line of defense against transformed and virus infected cells [9].NK cells play the function by KIRs binding to specific human leukocyte antigen (HLA) class I molecules and other unknown ligands on target cells.The major ligands of KIRs are the HLA class I (HLA-A, -B or -C) molecules which located on chromosome 6p21.31 is one of the other most polymorphic regions of the human genome [10,11].
The HLA-C alleles consist of two different groups of ligands C1 (HLA-C asp80 ) and C2 (HLA-C lys80 ) on the basis of a dimorphism at position 80 of the α1 domain.
In general, HLA-C1 group is the ligands for KIR2DL2/3 and 2DS2; and HLA-C2 group is the ligands for KIR2DL1 and 2DS1 loci, respectively.Recently, it has been shown that KIR2DL1 has only interacted with HLA-C2 group [12].KIR3DL2 interacts with HLA-A3 and A11 alleles.KIR3DL1 binds to HLA-Bw4 allotype that contains the Bw4 epitope, which is present on some of the HLA-A and HLA-B molecules, defined by amino acid variation at positions 77-83 [13,14].According to the difference of the amino acids encoded by the 80th position (Ile80 or Thr80) of the second exon of the HLA-B locus, HLA-Bw4 can be divided into Bw4-Ile80 and Bw4-Thr80 alleles.The previous studies have shown that Bw4-Ile80 alleles were the better ligands for KIR3DL1 than Bw4-Thr80 alleles [15,16].The ligand for KIR3DS1 is known as Bw4-Ile80, which may be due to the strong similarity of the extracellular domains of KIR3DS1 and 3DL1 [17].A recent study indicated that full-length KIR2DS4 binded specifically to the subsets HLA-C1, C2, and A11 alleles, whereas deleted KIR2DS4 was nonfunctional [18,19].But, until now, the HLA ligands for the other KIRs have not been completely identified.
Up to now, no KIR gene and HLA gene polymorphism data of the Chinese Han population from Henan province have been reported.In this study, we investigated the diversity and distributions of the 19 KIR genes (KIR2DL1, 2DL2, 2DL3, 2DL4, 2DL5A, 2DL5B, 2DS1, 2DS2, 2DS3, 2DS4*FUL, 2DS4*DEL, 2DS5, 3DL1, 3DL2, 3DL3, 3DS1, 2DP1, 3DP1*FUL and 3DP1*DEL) and five HLA loci (HLA-A, -B -C, -DRB1 and -DQB1) of 145 individuals from Henan Han population.We also evaluated the correlation and co-evolution of KIR-HLA system for the first time in the population.Furthermore, KIR gene diversity has been studied in a large number of populations distributed worldwide.
As shown in Figure 3, the principal component analysis (PCA) based on the OFs of the same 11 overlapping KIRs (KIR3DL2 and 3DL3 were removed for the OFs in all the populations were 1.0000) was conducted for the studied Henan Han population and the above 22 other populations distributed worldwide.The distance from each locus to zero point represented the relative contribution of each KIR gene frequency to the variability along the first two axes (PC1 and PC2).In Figure 3, we can observe that the groups distributed around a KIR locus have the higher or lower OFs of this KIR gene than the other groups.For example, the highest value for KIR2DL1

HLA ligand polymorphisms
Five HLA loci were genotyped using PCR-SSO method and the allelic frequencies of HLA-A, -B, -C, -DRB1 and -DQB1 loci of 145 unrelated healthy Henan Han individuals were summarized in Table 2. Twenty-four alleles were detected at HLA-A locus in the population.The HLA-A*02 group accounting for 28.61% of the total was found to be the most diverse allele family at HLA-A locus, and detected six alleles in our study:

Estimated HLA haplotype frequencies
The frequencies of the estimated main HLA haplotypes (the haplotypic frequency≧1.00%) in the Han population from Henan province, China were shown in Table 3
Correlation analysis between KIRs and their special ligands HLA-A3, A11, Bw4, C1 and C2 in the population was listed in Table 5.In KIR-C1/C2 groups, the most frequent association was 2DL3/C1, with a frequency of 63.45%.The rarest association was 2DS1/C2C2, with a frequency of 1.38%.In KIR-Bw4 groups, there were 114 individuals having the association of 3DL1/Bw4 accounting for 78.62%.The association of 3DS1+/Bw4 (80Ile)+ accounted for 17.24% of the total.In the KIR-A3/ A11 groups, there were 55 individuals with the association of 3DL2+/A3 and/or A11 accounting for 37.93% and 34 individuals with the association of 2DS4*FUL+/A11+ accounting for 23.45%.

DISCUSSION
Henan is located in the east-central part of China, along the middle and lower reaches of the Yellow River.With most parts in the history located in the south of the Yellow River, it is therefore named Henan.By the end of 2016, Henan has a resident population of 94.8 million people, ranking 3rd in China.The Han population is the main body of Henan resident population, accounting for 99.66% of the population of the whole province, while the minority population accounts for 0.34% of the province's population.Henan is the birthplace of the Chinese nation and the Chinese civilization.Among the four ancient Chinese inventions, three of them, the compass, paper and gunpowder, are invented in Henan province.There are more than 20 dynasties in the history founding capital here or moving here as the capital.Henan province is the province which has the most dynasties, the longest history and the largest number of ancient capitals.There was a long time in ancient times that Henan has always been China's political, economic, cultural and transportation  [20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36].The present study also confirmed this.In Figure 2, KIR2DL5, 3DS1, 2DS1, 2DS5, 2DL2, 2DS2 and 2DS3 clustered together which belonged to B haplotype showed higher diversity than the other 6 KIR genes which clustered together belonged to the A haplotype.The results of Figure 3, 4 and 5 showed that the Henan Han was clustered with the groups from East Asian like Shanghai Han, Northern Han, Singapore Han, Shaanxi Han, etc. which were in accordance with many previous studies on different genetic markers, such as the population genetic analysis based on the 21 or 20 STRs of Chinese Henan Han, respectively [38,39].
The sequences of human KIR genes in the extracellular, transmembrane and cytoplasmic domains were extremely conserved, but the KIR genes have evolved to be a highly polymorphic family of receptors.Genetic evidence indicated that the genes evolved through duplication and recombination, which was probably accelerated by their close proximity of headto-tail orientation within the KIR cluster chromosomal locus in human genomics [40].In addition, some alleles of individual KIR genes have produced through point mutations and minor sequence variations encoding one to several amino acids [41].The extensive genetic diversity and different combinations of KIR genes of individuals made the diversity of the NK cell repertoire in peripheral blood, and therefore the NK cell could recognize diverse HLA-I allotypes and produce varying degrees of immune function.
HLA showed extensive genetic diversities, as did the KIR gene, in populations from different geographical regions and different ethnic groups [42].The allelic frequencies of HLA-A, -B, -C, -DRB1 and -DQB1 loci were tested by using PCR-SSO method.A total of 135 alleles of HLA-A, -B, -C, -DRB1 and -DQB1 loci were detected in the study population.HLA-B locus was detected with a total of 46 alleles and found to be the most diverse locus in the five loci.According to the IMGT/HLA database (http:// www.ebi.ac.uk/imgt/hla/stats.html,March 11, 2017), 3830 HLA-A, 4647 HLA-B, 3382 HLA-C, 2011 HLA-DRB1 and 1054 HLA-DQB1 alleles have been identified at HLA class I and class II moleculars in the world, which indicates that the HLA system constitutes the most complex and highly polymorphic genetic system in the human genome.
The immune function of NK cells was achieved through the signals derived from cell surface activating and inhibitory KIR receptors interacting with their major ligands: HLA class I (HLA-A, -B and -C) molecules.KIR/ HLA ligand interactions were especially diverse.And a great number of previous studies have demonstrated associations between inheritance of certain combinations of KIR and HLA genes and susceptibility to many different diseases, including viral infections, autoimmune disorder, cancers, etc [43][44][45][46].In general, HLA-C1 was the ligand of KIR2DL2/3 and KIR2DS2, and HLA-C2 was the ligand of KIR2DL1 and KIR2DS1.The previous studies [37] have shown that KIR3DL1 bind to HLA-Bw4 allotype and Bw4-Ile80 alleles were the better ligands for KIR3DL1 than Bw4-Thr80 alleles.And the ligand of KIR3DS1 was known as Bw4-Ile80, which might be due to the strong similarity between the extracellular domains of KIR3DS1 and KIR3DL1.KIR3DL2 interacted with HLA-A3 and -A11 allele families.A recent study showed that KIR2DS4 (fulllength) bound specifically to the molecules HLA-C1, -C2 and -A11, whereas 2DS4 (14bp deleted) was nonfunctional [11][12][13][14][15][16][17][18].Several models have been proposed to explain the maintenance of this degree of diversity, including frequency dependent selection, heterozygote advantage and selection that varies in time and/or space [47,48].In the study, we also conducted correlation analysis to investigate population-level evidence for co-evolution of the KIR/HLA loci based on the frequencies of the receptorligand pairs including 2DL2/C1, 2DL3/C1, 2DS2/C1, 2DL1/C2, 2DS1/C2, 3DL1/Bw4, 3DL1/Bw4-80Ile and 3DS1/Bw4-80Ile.Among the 8 receptor-ligand pairs, 2DS1/C2 and 3DS1/Bw4-80Ile showed a strong negative correlation, but 3DL1/Bw4 and 3DL1/Bw4-80Ile showed a strong positive correlation.For the KIR2DL2 and 2DS2 loci, the correlation with C1 group was negative (r=-0.274 and -0.254) but not significant.For the KIR2DL3 locus, the correlation with C1 group was positive (r = 0.184) but not significant.The correlation of the 2DL1/C2 pair was also positive (r = 0.019) but not significant.It may show that some KIR/HLA pairs were possibly the dominant factor in forming the frequency distributions and the other KIR/HLA pairs were simply hitchhiking.Studies of the LD of the KIR/HLA pairs have confirmed the above phenomenon [37].Through the studies of many groups of KIR/HLA, the results indicated that the KIR genes were evolving at a more rapid rate than the HLA class I ligand groups because they found that some pairs of neighboring populations shared similar HLA ligand group frequencies but had highly distinct phenotypic KIR gene frequencies.And their data provided population-level evidence for the evolution of the KIR gene cluster owing to selection pressure favoring frequencies of activating KIR that suit the specific HLA ligands [37].Our data also support the above conclusions.In addition, our data will provide some immunogenetic information and supplementary data for the study of the KIR/HLA co-evolution.Studies performed over the last several years have revealed that the extensive genomic diversity of the KIR/HLA pairs and the key role of their interactions in both innate and adaptive immunity was able to explain the co-evolution of these two immunogenetics markers in order to maintain appropriate functional interaction [49,50].Evidence of HLA-KIR co-evolution within and across populations has also been suggested in disease studies [49,50].

Genomic DNA extraction
Whole blood samples containing ethylene diamine tetra aceticacid were utilized for DNA extraction with TIANamp Genomic DNA Kit (TIANGEN Biotech, Beijing, China) following the manufacturer's instructions.Genomic DNA samples were quantified by NanoDrop 2000 UV-Vis Spectrophotometer (NanoDrop Technologies, Wilmington, NC, USA).The optical density values ranged from 1.6 to 1.8, evaluating the concentration and purity of the extracted genomic DNA, and the final concentration was adjusted to approximately 50 ng/μL in distilled water (dH 2 O).All DNA samples were stored at -20°C until amplification.

KIR genotyping
Genotypes for KIRs were obtained by PCR amplification with sequence specific primer methods (PCR-SSP) using the Invitrogen KIR Genotyping PCR-SSP Kit (Invitrogen Carlsbad, CA, USA), according to the manufacturer's instructions.The kit consists of panels of primer mixes where each primer mixture contains one or more specific primer pairs, i.e. the allele-and/or group-specific primers, as well as a control primer pair matching non-allelic sequences.And 16 KIR genes and 3 pseudogenes (KIR2DL1, 2DL2, 2DL3, 2DL4, 2DL5A, 2DL5B, 2DS1, 2DS2, 2DS3, 2DS4*FUL, 2DS4*DEL, 2DS5, 3DL1, 3DL2, 3DL3, 3DS1, 2DP1, 3DP1*FUL and 3DP1*DEL) were tested in the samples.The total reaction volume was 10 μl, established on the basis of the manufacturer's instructions.All amplifications were performed in a GeneAmp PCR system 9700 thermal cycler (Applied Biosystems, Foster City, CA, USA) and PCR amplification parameters included a 1 min denaturing step at 95ºC, 30 cycles of 94ºC for 20s, 63ºC for 20s, 72ºC for 90s.PCR products of all samples were analyzed for KIR genotyping according to the manufacturer's instructions by the specific presence or absence band of each KIR in 2% agarose gels, which were well-mixed with ethidium

HLA genotyping
Genotypes for HLA-A, -B -C, -DRB1 and -DQB1 loci were obtained by PCR using sequence-specific oligonucleotide (PCR-SSO) using the LABType TM HD SSO HLA typing and LABType ® SSO HLA typing Tests (One Lambda, Inc. Canoga Park, CA, USA).PCR amplifications of five HLA loci were in a GeneAmp PCR system 9700 thermal cycler (Applied Biosystems, Foster City, CA, USA), respectively.And HLA genotyping was performed using LABScan TM 100 and Luminex XY platform (One Lambda, Inc. Canoga Park, CA, USA) according to the manufacturer's instructions.

Statistical analysis
The OFs of KIRs in the group were determined from the number of positive typing reactions divided by the total number of individuals.GFs of KIRs were estimated using the formula GF = 1-(1-OF) 1/2 , where OFs were the above-mentioned observed carrier frequencies of KIRs in studied individuals.
The PCA in Figure 3 was conducted by the statistical software SPSS Version 13.0.based on 11 overlapping loci.The PCA plot in Figure 4  Based on the 13 overlapping KIR gene genotype data mentioned above, a NJ tree (shown in Figure 5) including Central Chinese Han and 22 other populations was drawn by the Phylip 3.69 (http://evolution.gs.washington.edu/phylip.html).

CONCLUSIONS
In summary, this study may provide basic and valuable polymorphism data of KIR genes, HLA genes and KIR/HLA combinations for anthropological analysis and associated disease studies.In addition, it may provide some clues of the co-evolution of these two complex genetic systems as studied the KIR/HLA pairs.

Figure 1 :
Figure 1: Killer-cell immunoglobulin-like receptor loci profiles were observed in the Chinese Henan Han population (n = 145).Genotype ID were refered to genotype classification according to www.allelefrequencies.net.

Figure 2 :
Figure 2: A Heatmap drawn using Package 'pheatmap' containing the Chinese Henan Han and 22 other populations distributed worldwide illustrated the molecular evolutionary structure of the 13 overlapping KIR genes.The deeper color indicated the higher OFs.
HLA-A*0201, A*0203, A*0205, A*0206, A*0207 and A*0211.The most common allele belonging to HLA-A*02 group was HLA-A*0201, which accounted for 18.28% of the total.However, HLA-A*0205, A*0211, A*2420, A*3004, A*0302, A*6824 and A*6901 showed the lowest frequency of 0.34%.HLA-B locus was detected with a total of 46 alleles and found to be the most diverse one of the five loci.HLA-B*13 group accounting for 17.23% of the total was detected with the highest frequency in the locus and included two alleles: HLA-B*1301 and B*1302.The most common allele belonging to HLA-B*13 group was HLA-B*1302, accounted for 12.41% of the total.The HLA-B*15 group accounting for 12.39% of the total was also found to be one of the most diverse allele family at HLA-B locus, and observed seven alleles: HLA-B*1501, B*1502, B*1507, B*1511, B*1517, B*1518 and B*1527.Nevertheless, there were 14 HLA-B alleles showing the lowest frequency of 0.34%.Twenty-one alleles were detected at HLA-C locus in the population.HLA-C*3 group accounting for 19.31% of the total was detected with the highest frequency and contained three alleles: HLA-C*0302, C*0303 and C*0304.The most common allele belonging to HLA-C*6 group was HLA-C*0602 accounting for 15.17% of the total.The two other common HLA-C alleles with the frequencies higher than 10% were HLA-C*0102 and C*0702.At HLA-DRB1 locus, twenty-nine alleles were detected in the population.HLA-DRB1*15 group accounting for 17.93% of the total was detected with the highest frequency, and observed three alleles: HLA-DRB1*1501, DRB1*1502 and DRB1*1506.The HLA-DRB1*4 group was found to be the most diverse allele family at HLA-DRB1 locus and accounted for 12.06% of the total and consisted of six alleles: HLA-DRB1*0401, DRB1*0403, DRB1*0405, DRB1*0406, DRB1*0407 and DRB1*0410.The three most common HLA-DRB1 alleles with frequencies higher than 10% were HLA-DRB1*1501, DRB1*0701 and DRB1*0901 alleles, respectively.HLA-DQB1 locus was detected with a total of 15 alleles and found with the lowest polymorphism in the five HLA loci.HLA-DQB1*3 group accounting for 38.97% of the total was detected with the highest frequency and contained three alleles: HLA-DQB1*0301, DQB1*0302 and DQB1*0303.And the HLA-DQB1*6 group accounted for 25.17% of the total including five alleles: HLA-DQB1*0601, DQB1*0602, DQB1*0603, DQB1*0604 and DQB1*0609 which was found to be the most diverse alleles at HLA-DQB1 locus.HLA-DQB1*0301 was found to be the most common allele, accounting for 20.69% of the total.

Figure 4 :
Figure 4: The principal component analysis plot was constructed to study the genetic relationships of the Chinese Henan Han and 22 other populations distributed worldwide based on the 13 overlapping KIR gene frequencies.

Figure 5 :Figure 6 :
Figure 5: A Neighbor-Joining tree was constructed between the Chinese Henan Han and 22 other populations distributed worldwide based on genotype data of the 13 overlapping KIR gene.
Blood samples were obtained from 145 unrelated healthy individuals of the Han population from Henan province in central China.All the individuals provided their written informed consent for the collection of the samples and subsequent analysis.And the investigation and study were conducted in accordance with humane and ethical research principles of Henan Provincial People's Hospital and Xi'an Jiaotong University Health Science Center, China, and approved by the Ethics Committee of Henan Provincial People's Hospital and Xi'an Jiaotong University Health Science Center, China.

The principal component analysis was constructed to study the relationships between the populations and KIR genes based on OFs of the 11 overlapping KIR genes in Chinese Henan Han and 22 other populations distributed worldwide.
KIR gene between the Chinese Henan Han and 22 other populations.As shown in the figure, Henan Han was first clustered with Singapore Han and Shanghai Han, followed by other East Asian populations, and then by Pacific and Cook Islands populations, finally with the four European populations.

Table 5 : Distribution of the KIRs and their special HLA ligands in Henan Han population, China
Number of individuals; PI: Percentage of individuals.www.impactjournals.com/oncotarget

Table 6 : Correlation analyses between KIR and HLA ligand based on allelic frequencies KIR-HLA-C1/C2 groups Correlation r values
Statistically significant p values (p < 0.05) are indicated in boldface type.bromide.Each lane of the gel, containing a loaded PCR sample product, should be a control band, and a positive reaction band if there was presence of KIR, and vice versa, except for a negative control well.The false reaction, displaying no control band, was repeated.