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B in Tujia nationality of Zhangjiajie, Hunan Province, China
Human Immunology xxx (2016) xxx–xxx
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Allele polymorphism and haplotype diversity of MICA/B in Tujia nationality of Zhangjiajie, Hunan Province, China Y.J. Wang a,b, N.J. Zhang b, E. Chen a,c, C.J. Chen a, Y.H. Bu b, P. Yu a,⇑ a
Department of Immunology, College of Basic Medical Sciences, Central South University, 110 Xiangya Road, Changsha 410078, China Department of Blood Transfusion, The Second Xiang Ya Hospital of Central South University, 139 Ren Min Zhong Road, Changsha 410011, China c Clincal Laboratory, Jiangxi Cancer Hospital, 519 Beijing Dong Road, Nanchang 330029, China b
a r t i c l e
i n f o
Article history: Received 31 January 2015 Revised 14 June 2015 Accepted 10 March 2016 Available online xxxx Keywords: MIC Gene polymorphism Haplotype diversity Tujia nationality
a b s t r a c t Previous studies indicate the distribution of major histocompatibility complex class I chain-related genes A (MICA) and B (MICB) alleles and haplotypes varies widely between different ethnic populations and geographic areas. It is meaningful to investigate allelic frequencies and establish a genetic database. In this study, we firstly reported the polymorphic variation of MICA/B in 187 healthy, unrelated Tujia individuals in Zhangjiajie region, China. Using polymerase chain reaction-sequence specific priming (PCR-SSP) and sequencing-based typing (PCR-SBT), we identified eight MICA-sequence alleles, four MICA-short tandem repeat variants, and 13 MICB variants, of which MICA⁄008:04 (29.41%), MICA⁄A5 (29.68%), MICA⁄A5.1 (29.68%) and MICB⁄005:02 (39.57%) were the most frequent. Linkage disequilibrium analysis further revealed MICB⁄005:02-MICA⁄019 (13.10%) and MICB⁄002-MICA⁄008:04 (9.89%) as the most common two-locus haplotypes. Data comparison by neighbor-joining dendrograms and principal component analysis to verify allelic frequencies in other Chinese and Asia ethnic groups showed that the Zhangjiajie Tujias were genetically closer to the Guangdong Han population, based on MICA loci variability. Our results provide new information about the MICA/B gene polymorphism in Chinese Tujia population, which will form the basis for future studies on the potential role of MICA/B in allogeneic organ transplantation and disease susceptibility in related ethnic groups. Ó 2016 Published by Elsevier Inc. on behalf of American Society for Histocompatibility and Immunogenetics.
1. Introduction The Tujia ethnic minority is the seventh largest minority group in China with a population of about 8.35 million, mainly inhabiting the Wuling Mountain area along the shared borders of the Hunan, Hubei, Sichuan, and Guizhou Provinces. Tujia is an ancient ethnic nationality belonging to the Tibeto-Burman group [1]. Some researchers believe that the Tujias are the descendants of the ancient Ba people. During the later era Qing Dynasty (1636–1911 CE), a portion of the Ba people began to migrate eastwards from their original territory-currently, the Chongqing region-and gradually came to be called ‘‘Tujia” around 1300 BCE [2]. Communication between the Tujias and other ethnic groups began in very ancient times and grew more frequent after the late Qing dynasty. The oral Tujia language is an independent Bumar-Tibet language, which still belongs to the Chinese-Tibet language family, however, most write using traditional Chinese characters since the Tujias have ⇑ Corresponding author. E-mail address: [email protected] (P. Yu).
no written language. Currently, most Tujias can speak and write Chinese. Zhangjiajie is a main territory of the Tujia people, which is located in the northwest of Hunan Province (Fig. 1), at the juncture of the Yuan-Guizhou Plateau and the subsidence of Dongting Lake. The landform is characterized by the presence of rare sandstone quartz. This mountainous topography act as a natural barrier to prevent population movement, making this area an ideal region to research human genetic and inherited diseases. The human major histocompatibility complex (MHC) class I chain-related gene (MIC) family consists of seven genetic loci, sequentially lettered from MICA to MICG, of which only MICA and MICB act as functional genes encoding expressed transcripts [3,4]. MICA and MICB share some homology with the classical human leukocyte antigen (HLA) molecules, but are not expressed on normal circulating lymphocytes. Additionally, the MICA/B proteins function as ligands to induce activation of killer cell lectin-like receptor K1 (KLRK1, also known as the NKG2D receptor), which is expressed on human natural killer (NK) cells, cd T cells, and CD8+ T cells, and regulates the host innate immune and autoimmune responses. MICA/B upregulation can be induced by certain
http://dx.doi.org/10.1016/j.humimm.2016.03.005 0198-8859/Ó 2016 Published by Elsevier Inc. on behalf of American Society for Histocompatibility and Immunogenetics.
Please cite this article in press as: Y.J. Wang et al., Allele polymorphism and haplotype diversity of MICA/B in Tujia nationality of Zhangjiajie, Hunan Province, China, Hum. Immunol. (2016), http://dx.doi.org/10.1016/j.humimm.2016.03.005
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Y.J. Wang et al. / Human Immunology xxx (2016) xxx–xxx
Fig. 1. Map showing Tujia nationality used in the study in Human (central South of China).
stimuli, it is expressed in response to many tumors and infection [5,6]. MICA/B genes are highly polymorphic, with 100 MICA alleles and 40 MICB alleles currently identified by the World Health Organization (WHO) Human Leukocyte Antigen (HLA) Nomenclature Committee (http://hla.alleles.org/classo.html). Similarly, extensive research on MIC gene polymorphisms in various ethnic populations has shown differential distributions of MICA/B alleles and haplotypes amongst these populations [7–9]. Importantly, some of the polymorphisms present within the MICA/B coding sequence alleles or short tandem repeats (STRs) have been previously reported associated with many diseases [10–14]. Currently, information regarding MICA/B genetic variation in the Tujia population is still lacking; thus, this study will further our understanding the genetic background of MICA/B loci alleles and their relationship to disease in the Zhangjiajie Tujia population. 2. Materials and methods 2.1. Subjects A total of 187 healthy, unrelated Tujia individuals residing in Zhangjiajie region were included in this study. Full Tujia ethnicity through both maternal and paternal grandparents and familial residence in the area for the last 3 generations was claimed by all participants. The sample panel comprised 52.4% women (98/187) and 47.6% men (89/187), mean aged 34.2 years old. Blood sample was taken from each participant once obtained informed consent. All protocols were approved by the Institutional Review Committee of local authorities. 2.2. DNA extraction Genomic DNA was extracted from whole blood containing ethylenediaminetetraacetic acid (EDTA) using a standard saltingout method. The concentration and purity of the extracted genomic DNA was quantified with a UV spectrophotometer. Genomic DNA with an A260/A280 ratio >1.8 and a concentration of approximately 30 ng/ll was used for polymerase chain reaction (PCR) amplifications. 2.3. MICA genotyping We applied the PCR-SSP with modifications [15] to genotype 55 MICA-sequence alleles (from MICA⁄001 to MICA⁄050, except MICA⁄007:01 from MICA⁄026, and MICA⁄002:01 from MICA⁄020). Once the sequence alleles were typed, we calculated the
corresponding STR genotypes based on the Anthony Nolan Trust database (HLA Informatics Group website: http://www.anthonynolan.com/HIG/seq/nuc/text/mica-nt.txt/). To validate the reliability of PCR-SSP, meanwhile in view of some unreported STR information about the known sequence alleles and some rare alleles that cannot be typed by the PCR-SSP system, PCR-SBT was applied as an authentication method [16]. Consequently, a total of 65 samples were genotyped by PCR-SBT, including some randomly selected samples, while MICA⁄007:01/026 and ⁄ MICA 002:01/020 alleles were typed by PCR-SSP. 2.4. MICB typing The PCR-SBT was applied to genotype MICB: PCR was carried out using primers 50 -GGACAGCAGACCTGTGTGTTA-30 (forward) and 50 -AAAGGAGCTTTCCCATCTCC-30 (reverse) to amplify a 2.1-kb fragment containing exons 2, 4 and 5 of MICB gene, The amplified productions were sequenced with sequencing primers (50 -GGACAG CAGACCTGTGTGTTA-30 and 50 -TGCATCCATAGCACAGGG-30 for exons 2 and 3; 50 -CAGGAGTCCACCCTTGACAT-30 and 50 -AAAG GAGCTTTCCCATCTCC-30 for exons 4 and 5) [17,18]. Primers are synthesized by BGI Co. (Shenzhen, China). The PCR reaction mixture was heated in an Eppendorf Mastercycler 5333 thermocycler (Germany) The amplified products were sequenced with sequencing primers and the BigDye Terminator v3.1 Cycle Sequence kit (Axygen Biosciences, Union City, CA, USA) on an ABI 3730XL DNA Analyzer. The sequencing was repeated at least twice with independent PCR for samples showing unexpected association with MICB. The sequence chromatograms were analyzed using program Chromas Lite 2.01 (http://www.technelysium.com.au/chromas_ lite.html). Raw sequencing data were manually reviewed and the haplotypic phase of single nucleotide polymorphisms in each exonic segment was determined on the basis of MICB gene sequence database (http://hla.alleles.org/data/txt/micb_nuc.txt), blast analysis was then performed for DNA sequences against the EMBL Release database. MICB allele was assigned on the basis of the integrated information of exons 2–5. 2.5. Statistical analysis The SHESIS software (http://analysis.bio-x.cn/SHEsisMain.htm) was used to analyze the data, including genotype frequencies of MICA and MICB, Hardy–Weinberg equilibrium (HWE) test. The haplotype frequencies were estimated based on the alleles frequencies using the expectation maximization (EM) method with the Arlequin software (cmpg.unibe.ch/software/arlequin35/). The
Please cite this article in press as: Y.J. Wang et al., Allele polymorphism and haplotype diversity of MICA/B in Tujia nationality of Zhangjiajie, Hunan Province, China, Hum. Immunol. (2016), http://dx.doi.org/10.1016/j.humimm.2016.03.005
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Y.J. Wang et al. / Human Immunology xxx (2016) xxx–xxx Table 1 MICA sequence-allele frequencies in different ethnic populations. Sequence-alleles
MICA⁄001 MICA⁄002:01/02 MICA⁄004 MICA⁄006 MICA⁄00701 MICA⁄00702 MICA⁄008:01 MICA⁄008:02 MICA⁄008:04 MICA⁄009:01 MICA⁄009:02 MICA⁄010 MICA⁄011 MICA⁄012:01 MICA⁄016 MICA⁄017 MICA⁄018 MICA⁄019 MICA⁄020 MICA⁄026 MICA⁄027 MICA⁄031 MICA⁄033 MICA⁄045 MICA⁄049 MICA⁄052 MICA⁄053 MICA⁄059 MICA⁄061 MICA⁄null
Allele frequencies (%) Zhangjiajie Tujia (2n = 374)
Northern Han [26] (2n = 208)
Hunan Han [27] (2n = 324)
Guangdong Han [28] (2n = 288)
Zhejiang Han [29] (2n = 200)
Korean [8] (2n = 278)
Japanese [30] (2n = 260)
Thai [31] (2n = 510)
0.53 21.66 0 0 0 0 0 0 29.41 0 0 9.09 0 10.70 0 0 0 20.59 0 0 0 0 0 7.75 0 0 0.27 0 0 0
0 12.02 4.81 0 1.44 0 23.08⁄ 0.48 0⁄ 12.02⁄ 0.48 18.75 0.48 0.96 0.96 1.92 0.48 3.85⁄ 0 0 5.29 0 0.48 8.17 0 0 0 0.48 0 3.85
2.8 20.1 0.6 0 0 0 37.9⁄ 2.5 0⁄ 7.7 0 17.3 2.5 4.9 0 0 3.1 0.3⁄ 0 0 0 0.3 0 0 0 0 0 0 0 0
0 18.06 0.69 0 1.74 0.35 16.32⁄ 0.69 0⁄ 7.64 0 22.22 0 4.86 0 0.35 0 14.93 0 0 2.08 0 0 7.64 0 0.35 0 0 0.35 1.74
0 15.5 0.5 0 1 0 27⁄ 1.5 0⁄ 8 0.5 18.5 0 5 0 0 0 9.5 0 0 7 0 0 1.5 3.5 0 0 0 0 1
0 17.6 8.3 1.4 4 0.4 14.7⁄ 0.4 0⁄ 3.6 0.4 19.4 1.1 9.4 0 0.4 0 0.4⁄ 0 0 5 0 0 3.2 7.8 0 0 0 0 2.5
0 14.6 9.2 0 1.2 0 30.8⁄ 0 0⁄ 16.5⁄ 0 10.8 0 12.3 0 0 0 3.5⁄ 0 0 0 0 0 0 0 0 0 0 0 1.2
0 17.6 3.5 0 0 0 21.4⁄ 0 0⁄ 2.4 0 18.2 0 3.1 0.2 2.2 6.1 15.3 0.4 1.4 0 0 0 0 0 8.2 0 0 0 0
MICA sequence-allele frequencies between Zhangjiajie Tujias and other populations, *P < 0.05.
Table 2 MICA STR-alleles frequencies in different ethnic populations. MICA STR alleles
A4 A5 A5.1 A6 A9 STR-null
Allele frequencies (%) Zhangjiajie Tujia (2n = 374)
Guangxi Zhuang [32] (2n = 418)
Guangdong Han [28] (2n = 288)
Northern Han [26] (2n = 208)
Zhejiang Han [29] (2n = 200)
Hainan Li [44] (2n = 688)
18.98 29.68 29.68 0 21.66 0
25.12 34.93 20.10 2.63 15.31 1.74
14.58 39.58 17.01 8.33 18.75 1.74
11.54 29.33 23.56 17.79⁄ 13.94 3.85
7.50 35.00 28.5 12.50⁄ 15.5 1.00
35.61⁄⁄ 26.16 17.73 2.04 17.59 0.87
MICA-STR allele frequencies between Tujias and different populations, *pc < 0.05, **pc < 0.001.
linkage disequilibrium (LD) analysis was restricted to MICA-MICB haplotypes with observed frequencies >2.0%. D, D0 and r2 were computed for each individual haplotype as described previously [19]. Phylip 3.695 (http://evolution.genetics.washington.edu/phylip/getme.html) was used to assess Nei’s standard genetic distance of different ethnic groups [20]. Phylogenetic tree were constructed based on allelic frequencies using neighbour-joining method with Nei’s distance using Mega 6.06. Principal component analysis was processed using the SPSS 16.0 software package [21].
3. Results
respectively. The MICA allelic distributions were consistent with Hardy-Weinberg equilibrium (HWE) proportions (p value = 0.0568). 3.2. MICB allele frequencies We totally observed 13 MICB alleles in Zhangjiajie Tujia people, with MICB⁄005:02 (39.57%) presenting the highest frequency (Table 3). The other two alleles showed frequencies exceeding 10% [MICB⁄002:01 (24.87%) and MICB⁄014 (10.16%)]. The three alleles counted a cumulative frequency of 74.60%. Allele frequencies for MICB were in Hardy-Weinberg equilibrium (p value = 0.99932).
3.1. MICA-sequence and MICA-STR alleles frequencies 3.3. LD analysis between MICA and MICB As shown in Table 1, 2 and 8 MICA and 4 MICA-STR alleles were observed in Zhangjiajie Tujia population, amongst which MICA⁄008:04, MICA-STR allele MICA⁄A5 and A5.1 were the most prevalent alleles, with frequencies of 29.41%, 29.68% and 29.68%,
As shown in Table 4 and 14 kinds of MICA-MICB haplotypes with a frequency >2.0% were observed in Zhangjiajie Tujia population. Amongst the 4 haplotypes with significant LD,
Please cite this article in press as: Y.J. Wang et al., Allele polymorphism and haplotype diversity of MICA/B in Tujia nationality of Zhangjiajie, Hunan Province, China, Hum. Immunol. (2016), http://dx.doi.org/10.1016/j.humimm.2016.03.005
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Y.J. Wang et al. / Human Immunology xxx (2016) xxx–xxx
Table 3 MICB allele frequencies in different ethnic populations. MICB⁄alleles
MICB⁄002:01 MICB⁄003 MICB⁄004:01 MICB⁄005:02 MICB⁄005:03 MICB⁄005:04 MICB⁄005a MICB⁄008 MICB⁄007 MICB⁄006 MICB⁄009N MICB⁄012 MICB⁄013 MICB⁄014 MICB⁄024 MICB⁄016 MICB⁄023 MICB⁄026 MICB⁄028
Allele frequencies (%) Zhangjiajie Tujia (2n = 374)
Zhejiang Han [29] (2n = 800)
Northern Han [7] (2n = 208)
Southern Chinese [9] (2n = 402)
Korean [8] (2n = 278)
Spanish [25] (2n = 200)
Welsh [48] (2n = 332)
24.87 1.34 0 39.57 2.67 4.55 0 9.09 0.53 0.27 1.87 0.27 0 10.16 4.28 0.53 0 0 0
12.25 1.75 8.37 57.38 5.625 0 0.625 7.875 0 0 0.875 0 0.125 3.875 0 0 0.75 0.125 0.375
16.35 1.75 1.44 51.44 4.33 0 0 1.92 0 0 3.37 0 0 8.65 0 0 0.48 0 0
12.44 1.44 0.25 64.93 3.23 0 0.25 3.73 0 0 0.25 0 0 7.21 0 0 0 0.25 0
11.5 2.5 2.5 57.2 8.3 0 0 6.8 0 0 2.2 0 0 3.2 0 0 0 0 0
17 3.5 3.5 48 4.5 0 0 12.5 0 0 0 0 0 0⁄ 0 0 0 0 0
15.7 4.8 4.8 35.5 0.9 0 0 15.4 0 0 0 0 0.3 1.8 0 0 0 0 0
MICB allele frequencies between Guangxi Zhuang and other populations, *P < 0.05. a In the populations list here it could also be 005:05/06/07.
Table 4 Linkage disequilibrium (LD) analysis between MICA and MICB. Haplotypesa ⁄
⁄
MICA 019-MICB 005:02 MICA⁄008:04-MICB⁄002 MICA⁄008:04-MICB⁄005:02 MICA⁄002:01-MICB⁄005:02 MICA⁄010-MICB⁄005:02 MICA⁄012:01-MICB⁄002 MICA⁄002:01-MICB⁄008 MICA⁄019-MICB⁄002 MICA⁄008:04-MICB⁄014 MICA⁄002:01-MICB⁄002 MICA⁄012:01-MICB⁄005:02 MICA⁄045-MICB⁄005:02 MICA⁄045-MICB⁄014 MICA⁄002:01-MICB⁄005:04
HF
D
D0
R2
p-Value
0.1310 0.0989 0.0963 0.0615 0.0562 0.0481 0.0428 0.0401 0.0374 0.0348 0.0267 0.0240 0.0241 0.0214
0.0495 0.0258 0.0201 0.0242 0.0202 0.0215 0.0231 0.0138 0.0076 0.0191 0.0156 0.0066 0.0162 0.0115
0.3982 0.1469 0.1729 0.2822 0.3674 0.2677 0.3243 0.2168 0.1053 0.3548 0.3684 0.2152 0.2325 0.3236
0.0628 0.0171 0.0082 0.0144 0.0206 0.0279 0.0380 0.0040 0.0030 0.0115 0.0107 0.0025 0.0402 0.0181
0.1087 0.7169 0.2134 0.0383 0.0209 0.2669 0.0029⁄ 0.2060 0.6230 0.3048 0.5085 0.1992 0.0013⁄ 0.1846
a There were 14 kinds of MICA-MICB haplotypes showing frequency 2%. P values < 0.00357 (0.05 after Bonferroni’s correction) are bold. HF, haplotype frequencies. Significant difference (p-value < 0.05).
*
MICA⁄019-MICB⁄005:02 (13.1%), MICA⁄008:04-MICB⁄002 (9.9%) and MICA⁄008:04-MICB⁄005:02 (9.6%) were the most prevalent ones.
clustered in another clade. The Guangxi Zhuang population was closest to the Tujia population (see Fig. 4). 4. Discussion
3.4. Phylogenetic tree analysis The phylogenetic tree in Fig. 2 was constructed using the allelic frequencies at MICA locus in the Tujia population and its neighboring ethnic groups. The neighboring ethnic groups used in the phylogenetic tree construction were Hunan Han, Northern Han, Zhejiang Han, Guangdong Han, Thai, Japanese, Korean. According to the phylogenetic tree, the Tujia population showed the closest genetic structure to the Guangdong Han populations.
3.5. Principal component analysis Principal component analysis (PCA) was performed based on the allelic frequencies of the MICA locus. As shown in Fig. 3, all the ethnic groups can be divided into two clusters. The Tujia population was clustered in one clade, while the remaining 8 populations were
In this study, we investigated the distribution of MICA/B variants and haplotypes in the Zhangjiajie Tujia population. The SBT and SSP PCR protocols used in this study provide an accurate and reliable methodology to identify MICA/B alleles. Our results revealed that the eight identified MICA alleles in the Tujia population is smaller than those previously reported in other geographically related populations [8,22–25], and might be due to founder effects or/and bottlenecks in Tujia population. The three predominant alleles, MICA⁄008:04, MICA⁄002:01 and MICA⁄019, accounted for 71.66% of the MICA gene pool. A comparison of the Tujia people with similar populations in China and Asia highlighted differences in the distribution of the sequence allelic MICA variants (Table 1) [8,26–32]. For instance, the MICA⁄008:04 and MICA⁄019 alleles were observed at higher frequencies (29.41% and 20.59%, respectively) in the Tujia, while these were much lower or absent in other populations. Alternatively, neither the MICA⁄008:01 nor
Please cite this article in press as: Y.J. Wang et al., Allele polymorphism and haplotype diversity of MICA/B in Tujia nationality of Zhangjiajie, Hunan Province, China, Hum. Immunol. (2016), http://dx.doi.org/10.1016/j.humimm.2016.03.005
Y.J. Wang et al. / Human Immunology xxx (2016) xxx–xxx
MICA⁄009:01 alleles were detected in the Tujias, but both were commonly found in other populations (2.4–37.9%). Similar discrepancies were observed in the distribution of MICA STR-alleles (Table 2), for which the frequency of MICA⁄A5 and MICA⁄A5.1 represented the two highest variances (both 29.68%). Because of the proximity of MICA to the autoimmune-related HLA-B loci, Gonzalez et al. [33] reported that MICA⁄002 associates with psoriatic arthritis in Spanish patients independently of the other HLA class I alleles commonly associated with the disease. Additionally, MICA acts as an immune ligand for NK cells; thus, the association between MICA polymorphisms and immunosurveillance has been extensively described. For instance, the MICA⁄A9 and SNPMICA-129val variants in the Chinese Han [34] and Tunisian populations [35], respectively, associate with an increased risk of nasopharyngeal carcinoma, whereas MICA⁄A5.1 correlates with oral squamous cell carcinoma (OSCC) in the Japanese [36,37]. Interestingly, MICA⁄A5.1 also seems to confer susceptibility to breast cancer in Spanish women [38]. Thus, the unique MICA allelic distribution observed in the Zhangjiajie Tujia population may be valuable for the further study of the association between ethnic background and disease susceptibility. Though limited reports on MICB polymorphism exist, accumulating evidence suggests that allelic variation can be associated with various diseases and may play an important role in the outcome of allogeneic organ transplantation [39–41]. Our data showed that MICB⁄005:02 (39.57%), MICB⁄002:01 (24.87%), and MICB⁄014 (10.16%) were the most common alleles and accounted for 74.6% of the MICB gene pool in the Zhangjiajie Tujia population. Moreover, an inter-population comparison indicated that MICB distribution varied amongst the different Chinese populations. For instance, the MICB⁄002:01 allele showed a higher frequency in Zhangjiajie Tujias (24.87%) than other populations (11.5–16.35%) (Table 3). Since HLA-MICA/B haplotypes have been reported in different populations [7–9,42,43], it was presumed that the wide distribution of haplotypes have been conservatively maintained via selective advantage. Accordingly, our study revealed that MICA⁄045 was strongly linked with MICB⁄014 in the Zhangjiajie Tujias, as well as the Guangxi Zhuang and Hainan Li populations [44]. This finding is highly indicative of potential recombination hotspots between the MICA and MICB genes, particularly since we have also found that all the MICB⁄009N carriers were also HLA⁄B48:01 carriers, while these data will likely aid in characterizing the potential pathogenic mechanisms of MIC polymorphisms in the Zhangjiajie Tujia population, further studies are necessary to investigate the effects of HLA-A, B, and DR polymorphism in these individuals. Furthermore, we also identified the origins of genetic heterogeneity in the Zhangjiajie Tujias and constructed a phylogenetic tree of the different ethnic groups based on MICA and MICB allelic variation (Figs. 2 and 3). Interestingly, while relatively limited genetic variability exists amongst the Chinese populations as a whole, the MICA/B allele distributions in the Tujia population were relatively distant comparatively. As shown in Fig. 2, Tujias are most closely related to the Guangdong Han population, indicating that the two population groups have a similar lineage. The origin of the Tujia nationality is controversial, Xie et al. [45] analyzed the distribution of haplotypes in the Tujia Y chromosome and found that the genetic structure was similar to that observed in the Han population. Additionally, Zhang et al. [46] and Qiu et al. [47] reported on the HLA-A and HLA-DRB1 loci frequencies, respectively, and showed the Tujias living in the Wufeng region had the closest genetic relationship with the central Han population. However, principal component analysis showed the Tujias were genetically distant from other Chinese populations, which might be due to the relative genetic isolation and different origin branch of the Zhangjiajie Tujias.
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Fig. 2. Phylogenetic tree constructed by the neighbor-joining method showing the relationship between Zhangjiajie Tujias with other populations based on the frequencies of MICA locus.
In conclusion, our study showed a unique distribution of MICA/ B gene alleles in the Zhangjiajie Tujia population and uncovered interesting findings with respect to MICA/B allelic and haplotypic configurations. This analysis is likely to facilitate further understanding of the genetic background, evolution, and origination of the Tujia ethnicity, as well as enrich the genomic data resources for the Chinese population as a whole. Funding This study was supported by grants from the National Natural Science Foundation of China (project No: 81371864) and the Science and Technology Plan Project of Hunan Province (project No: 2014SK3271). Competing interests The authors declare that they have no conflict of interests or commercial links pertinent to the manuscript.
Please cite this article in press as: Y.J. Wang et al., Allele polymorphism and haplotype diversity of MICA/B in Tujia nationality of Zhangjiajie, Hunan Province, China, Hum. Immunol. (2016), http://dx.doi.org/10.1016/j.humimm.2016.03.005
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Fig. 3. Phylogenetic tree constructed by the neighbor-joining method showing the relationship between Zhangjiajie Tujia population with other populations based on the frequencies of MICB locus.
Fig. 4. Principal component analysis between Zhangjiajie Tujia population with other 9 ethnic groups based on the allelic frequencies of MICA locus.
Acknowledgments We would like to thank Xinfeng Zhang (The People’s Hospital of Zhangjiajie City) for his providing kindly some of blood samples and background data.
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Allele polymorphism and haplotype diversity of MICA/B in Tujia nationality of Zhangjiajie, Hunan Province, China Y.J. Wang a,b, N.J. Zhang b, E. Chen a,c, C.J. Chen a, Y.H. Bu b, P. Yu a,⇑ a
Department of Immunology, College of Basic Medical Sciences, Central South University, 110 Xiangya Road, Changsha 410078, China Department of Blood Transfusion, The Second Xiang Ya Hospital of Central South University, 139 Ren Min Zhong Road, Changsha 410011, China c Clincal Laboratory, Jiangxi Cancer Hospital, 519 Beijing Dong Road, Nanchang 330029, China b
a r t i c l e
i n f o
Article history: Received 31 January 2015 Revised 14 June 2015 Accepted 10 March 2016 Available online xxxx Keywords: MIC Gene polymorphism Haplotype diversity Tujia nationality
a b s t r a c t Previous studies indicate the distribution of major histocompatibility complex class I chain-related genes A (MICA) and B (MICB) alleles and haplotypes varies widely between different ethnic populations and geographic areas. It is meaningful to investigate allelic frequencies and establish a genetic database. In this study, we firstly reported the polymorphic variation of MICA/B in 187 healthy, unrelated Tujia individuals in Zhangjiajie region, China. Using polymerase chain reaction-sequence specific priming (PCR-SSP) and sequencing-based typing (PCR-SBT), we identified eight MICA-sequence alleles, four MICA-short tandem repeat variants, and 13 MICB variants, of which MICA⁄008:04 (29.41%), MICA⁄A5 (29.68%), MICA⁄A5.1 (29.68%) and MICB⁄005:02 (39.57%) were the most frequent. Linkage disequilibrium analysis further revealed MICB⁄005:02-MICA⁄019 (13.10%) and MICB⁄002-MICA⁄008:04 (9.89%) as the most common two-locus haplotypes. Data comparison by neighbor-joining dendrograms and principal component analysis to verify allelic frequencies in other Chinese and Asia ethnic groups showed that the Zhangjiajie Tujias were genetically closer to the Guangdong Han population, based on MICA loci variability. Our results provide new information about the MICA/B gene polymorphism in Chinese Tujia population, which will form the basis for future studies on the potential role of MICA/B in allogeneic organ transplantation and disease susceptibility in related ethnic groups. Ó 2016 Published by Elsevier Inc. on behalf of American Society for Histocompatibility and Immunogenetics.
1. Introduction The Tujia ethnic minority is the seventh largest minority group in China with a population of about 8.35 million, mainly inhabiting the Wuling Mountain area along the shared borders of the Hunan, Hubei, Sichuan, and Guizhou Provinces. Tujia is an ancient ethnic nationality belonging to the Tibeto-Burman group [1]. Some researchers believe that the Tujias are the descendants of the ancient Ba people. During the later era Qing Dynasty (1636–1911 CE), a portion of the Ba people began to migrate eastwards from their original territory-currently, the Chongqing region-and gradually came to be called ‘‘Tujia” around 1300 BCE [2]. Communication between the Tujias and other ethnic groups began in very ancient times and grew more frequent after the late Qing dynasty. The oral Tujia language is an independent Bumar-Tibet language, which still belongs to the Chinese-Tibet language family, however, most write using traditional Chinese characters since the Tujias have ⇑ Corresponding author. E-mail address: [email protected] (P. Yu).
no written language. Currently, most Tujias can speak and write Chinese. Zhangjiajie is a main territory of the Tujia people, which is located in the northwest of Hunan Province (Fig. 1), at the juncture of the Yuan-Guizhou Plateau and the subsidence of Dongting Lake. The landform is characterized by the presence of rare sandstone quartz. This mountainous topography act as a natural barrier to prevent population movement, making this area an ideal region to research human genetic and inherited diseases. The human major histocompatibility complex (MHC) class I chain-related gene (MIC) family consists of seven genetic loci, sequentially lettered from MICA to MICG, of which only MICA and MICB act as functional genes encoding expressed transcripts [3,4]. MICA and MICB share some homology with the classical human leukocyte antigen (HLA) molecules, but are not expressed on normal circulating lymphocytes. Additionally, the MICA/B proteins function as ligands to induce activation of killer cell lectin-like receptor K1 (KLRK1, also known as the NKG2D receptor), which is expressed on human natural killer (NK) cells, cd T cells, and CD8+ T cells, and regulates the host innate immune and autoimmune responses. MICA/B upregulation can be induced by certain
http://dx.doi.org/10.1016/j.humimm.2016.03.005 0198-8859/Ó 2016 Published by Elsevier Inc. on behalf of American Society for Histocompatibility and Immunogenetics.
Please cite this article in press as: Y.J. Wang et al., Allele polymorphism and haplotype diversity of MICA/B in Tujia nationality of Zhangjiajie, Hunan Province, China, Hum. Immunol. (2016), http://dx.doi.org/10.1016/j.humimm.2016.03.005
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Y.J. Wang et al. / Human Immunology xxx (2016) xxx–xxx
Fig. 1. Map showing Tujia nationality used in the study in Human (central South of China).
stimuli, it is expressed in response to many tumors and infection [5,6]. MICA/B genes are highly polymorphic, with 100 MICA alleles and 40 MICB alleles currently identified by the World Health Organization (WHO) Human Leukocyte Antigen (HLA) Nomenclature Committee (http://hla.alleles.org/classo.html). Similarly, extensive research on MIC gene polymorphisms in various ethnic populations has shown differential distributions of MICA/B alleles and haplotypes amongst these populations [7–9]. Importantly, some of the polymorphisms present within the MICA/B coding sequence alleles or short tandem repeats (STRs) have been previously reported associated with many diseases [10–14]. Currently, information regarding MICA/B genetic variation in the Tujia population is still lacking; thus, this study will further our understanding the genetic background of MICA/B loci alleles and their relationship to disease in the Zhangjiajie Tujia population. 2. Materials and methods 2.1. Subjects A total of 187 healthy, unrelated Tujia individuals residing in Zhangjiajie region were included in this study. Full Tujia ethnicity through both maternal and paternal grandparents and familial residence in the area for the last 3 generations was claimed by all participants. The sample panel comprised 52.4% women (98/187) and 47.6% men (89/187), mean aged 34.2 years old. Blood sample was taken from each participant once obtained informed consent. All protocols were approved by the Institutional Review Committee of local authorities. 2.2. DNA extraction Genomic DNA was extracted from whole blood containing ethylenediaminetetraacetic acid (EDTA) using a standard saltingout method. The concentration and purity of the extracted genomic DNA was quantified with a UV spectrophotometer. Genomic DNA with an A260/A280 ratio >1.8 and a concentration of approximately 30 ng/ll was used for polymerase chain reaction (PCR) amplifications. 2.3. MICA genotyping We applied the PCR-SSP with modifications [15] to genotype 55 MICA-sequence alleles (from MICA⁄001 to MICA⁄050, except MICA⁄007:01 from MICA⁄026, and MICA⁄002:01 from MICA⁄020). Once the sequence alleles were typed, we calculated the
corresponding STR genotypes based on the Anthony Nolan Trust database (HLA Informatics Group website: http://www.anthonynolan.com/HIG/seq/nuc/text/mica-nt.txt/). To validate the reliability of PCR-SSP, meanwhile in view of some unreported STR information about the known sequence alleles and some rare alleles that cannot be typed by the PCR-SSP system, PCR-SBT was applied as an authentication method [16]. Consequently, a total of 65 samples were genotyped by PCR-SBT, including some randomly selected samples, while MICA⁄007:01/026 and ⁄ MICA 002:01/020 alleles were typed by PCR-SSP. 2.4. MICB typing The PCR-SBT was applied to genotype MICB: PCR was carried out using primers 50 -GGACAGCAGACCTGTGTGTTA-30 (forward) and 50 -AAAGGAGCTTTCCCATCTCC-30 (reverse) to amplify a 2.1-kb fragment containing exons 2, 4 and 5 of MICB gene, The amplified productions were sequenced with sequencing primers (50 -GGACAG CAGACCTGTGTGTTA-30 and 50 -TGCATCCATAGCACAGGG-30 for exons 2 and 3; 50 -CAGGAGTCCACCCTTGACAT-30 and 50 -AAAG GAGCTTTCCCATCTCC-30 for exons 4 and 5) [17,18]. Primers are synthesized by BGI Co. (Shenzhen, China). The PCR reaction mixture was heated in an Eppendorf Mastercycler 5333 thermocycler (Germany) The amplified products were sequenced with sequencing primers and the BigDye Terminator v3.1 Cycle Sequence kit (Axygen Biosciences, Union City, CA, USA) on an ABI 3730XL DNA Analyzer. The sequencing was repeated at least twice with independent PCR for samples showing unexpected association with MICB. The sequence chromatograms were analyzed using program Chromas Lite 2.01 (http://www.technelysium.com.au/chromas_ lite.html). Raw sequencing data were manually reviewed and the haplotypic phase of single nucleotide polymorphisms in each exonic segment was determined on the basis of MICB gene sequence database (http://hla.alleles.org/data/txt/micb_nuc.txt), blast analysis was then performed for DNA sequences against the EMBL Release database. MICB allele was assigned on the basis of the integrated information of exons 2–5. 2.5. Statistical analysis The SHESIS software (http://analysis.bio-x.cn/SHEsisMain.htm) was used to analyze the data, including genotype frequencies of MICA and MICB, Hardy–Weinberg equilibrium (HWE) test. The haplotype frequencies were estimated based on the alleles frequencies using the expectation maximization (EM) method with the Arlequin software (cmpg.unibe.ch/software/arlequin35/). The
Please cite this article in press as: Y.J. Wang et al., Allele polymorphism and haplotype diversity of MICA/B in Tujia nationality of Zhangjiajie, Hunan Province, China, Hum. Immunol. (2016), http://dx.doi.org/10.1016/j.humimm.2016.03.005
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Y.J. Wang et al. / Human Immunology xxx (2016) xxx–xxx Table 1 MICA sequence-allele frequencies in different ethnic populations. Sequence-alleles
MICA⁄001 MICA⁄002:01/02 MICA⁄004 MICA⁄006 MICA⁄00701 MICA⁄00702 MICA⁄008:01 MICA⁄008:02 MICA⁄008:04 MICA⁄009:01 MICA⁄009:02 MICA⁄010 MICA⁄011 MICA⁄012:01 MICA⁄016 MICA⁄017 MICA⁄018 MICA⁄019 MICA⁄020 MICA⁄026 MICA⁄027 MICA⁄031 MICA⁄033 MICA⁄045 MICA⁄049 MICA⁄052 MICA⁄053 MICA⁄059 MICA⁄061 MICA⁄null
Allele frequencies (%) Zhangjiajie Tujia (2n = 374)
Northern Han [26] (2n = 208)
Hunan Han [27] (2n = 324)
Guangdong Han [28] (2n = 288)
Zhejiang Han [29] (2n = 200)
Korean [8] (2n = 278)
Japanese [30] (2n = 260)
Thai [31] (2n = 510)
0.53 21.66 0 0 0 0 0 0 29.41 0 0 9.09 0 10.70 0 0 0 20.59 0 0 0 0 0 7.75 0 0 0.27 0 0 0
0 12.02 4.81 0 1.44 0 23.08⁄ 0.48 0⁄ 12.02⁄ 0.48 18.75 0.48 0.96 0.96 1.92 0.48 3.85⁄ 0 0 5.29 0 0.48 8.17 0 0 0 0.48 0 3.85
2.8 20.1 0.6 0 0 0 37.9⁄ 2.5 0⁄ 7.7 0 17.3 2.5 4.9 0 0 3.1 0.3⁄ 0 0 0 0.3 0 0 0 0 0 0 0 0
0 18.06 0.69 0 1.74 0.35 16.32⁄ 0.69 0⁄ 7.64 0 22.22 0 4.86 0 0.35 0 14.93 0 0 2.08 0 0 7.64 0 0.35 0 0 0.35 1.74
0 15.5 0.5 0 1 0 27⁄ 1.5 0⁄ 8 0.5 18.5 0 5 0 0 0 9.5 0 0 7 0 0 1.5 3.5 0 0 0 0 1
0 17.6 8.3 1.4 4 0.4 14.7⁄ 0.4 0⁄ 3.6 0.4 19.4 1.1 9.4 0 0.4 0 0.4⁄ 0 0 5 0 0 3.2 7.8 0 0 0 0 2.5
0 14.6 9.2 0 1.2 0 30.8⁄ 0 0⁄ 16.5⁄ 0 10.8 0 12.3 0 0 0 3.5⁄ 0 0 0 0 0 0 0 0 0 0 0 1.2
0 17.6 3.5 0 0 0 21.4⁄ 0 0⁄ 2.4 0 18.2 0 3.1 0.2 2.2 6.1 15.3 0.4 1.4 0 0 0 0 0 8.2 0 0 0 0
MICA sequence-allele frequencies between Zhangjiajie Tujias and other populations, *P < 0.05.
Table 2 MICA STR-alleles frequencies in different ethnic populations. MICA STR alleles
A4 A5 A5.1 A6 A9 STR-null
Allele frequencies (%) Zhangjiajie Tujia (2n = 374)
Guangxi Zhuang [32] (2n = 418)
Guangdong Han [28] (2n = 288)
Northern Han [26] (2n = 208)
Zhejiang Han [29] (2n = 200)
Hainan Li [44] (2n = 688)
18.98 29.68 29.68 0 21.66 0
25.12 34.93 20.10 2.63 15.31 1.74
14.58 39.58 17.01 8.33 18.75 1.74
11.54 29.33 23.56 17.79⁄ 13.94 3.85
7.50 35.00 28.5 12.50⁄ 15.5 1.00
35.61⁄⁄ 26.16 17.73 2.04 17.59 0.87
MICA-STR allele frequencies between Tujias and different populations, *pc < 0.05, **pc < 0.001.
linkage disequilibrium (LD) analysis was restricted to MICA-MICB haplotypes with observed frequencies >2.0%. D, D0 and r2 were computed for each individual haplotype as described previously [19]. Phylip 3.695 (http://evolution.genetics.washington.edu/phylip/getme.html) was used to assess Nei’s standard genetic distance of different ethnic groups [20]. Phylogenetic tree were constructed based on allelic frequencies using neighbour-joining method with Nei’s distance using Mega 6.06. Principal component analysis was processed using the SPSS 16.0 software package [21].
3. Results
respectively. The MICA allelic distributions were consistent with Hardy-Weinberg equilibrium (HWE) proportions (p value = 0.0568). 3.2. MICB allele frequencies We totally observed 13 MICB alleles in Zhangjiajie Tujia people, with MICB⁄005:02 (39.57%) presenting the highest frequency (Table 3). The other two alleles showed frequencies exceeding 10% [MICB⁄002:01 (24.87%) and MICB⁄014 (10.16%)]. The three alleles counted a cumulative frequency of 74.60%. Allele frequencies for MICB were in Hardy-Weinberg equilibrium (p value = 0.99932).
3.1. MICA-sequence and MICA-STR alleles frequencies 3.3. LD analysis between MICA and MICB As shown in Table 1, 2 and 8 MICA and 4 MICA-STR alleles were observed in Zhangjiajie Tujia population, amongst which MICA⁄008:04, MICA-STR allele MICA⁄A5 and A5.1 were the most prevalent alleles, with frequencies of 29.41%, 29.68% and 29.68%,
As shown in Table 4 and 14 kinds of MICA-MICB haplotypes with a frequency >2.0% were observed in Zhangjiajie Tujia population. Amongst the 4 haplotypes with significant LD,
Please cite this article in press as: Y.J. Wang et al., Allele polymorphism and haplotype diversity of MICA/B in Tujia nationality of Zhangjiajie, Hunan Province, China, Hum. Immunol. (2016), http://dx.doi.org/10.1016/j.humimm.2016.03.005
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Table 3 MICB allele frequencies in different ethnic populations. MICB⁄alleles
MICB⁄002:01 MICB⁄003 MICB⁄004:01 MICB⁄005:02 MICB⁄005:03 MICB⁄005:04 MICB⁄005a MICB⁄008 MICB⁄007 MICB⁄006 MICB⁄009N MICB⁄012 MICB⁄013 MICB⁄014 MICB⁄024 MICB⁄016 MICB⁄023 MICB⁄026 MICB⁄028
Allele frequencies (%) Zhangjiajie Tujia (2n = 374)
Zhejiang Han [29] (2n = 800)
Northern Han [7] (2n = 208)
Southern Chinese [9] (2n = 402)
Korean [8] (2n = 278)
Spanish [25] (2n = 200)
Welsh [48] (2n = 332)
24.87 1.34 0 39.57 2.67 4.55 0 9.09 0.53 0.27 1.87 0.27 0 10.16 4.28 0.53 0 0 0
12.25 1.75 8.37 57.38 5.625 0 0.625 7.875 0 0 0.875 0 0.125 3.875 0 0 0.75 0.125 0.375
16.35 1.75 1.44 51.44 4.33 0 0 1.92 0 0 3.37 0 0 8.65 0 0 0.48 0 0
12.44 1.44 0.25 64.93 3.23 0 0.25 3.73 0 0 0.25 0 0 7.21 0 0 0 0.25 0
11.5 2.5 2.5 57.2 8.3 0 0 6.8 0 0 2.2 0 0 3.2 0 0 0 0 0
17 3.5 3.5 48 4.5 0 0 12.5 0 0 0 0 0 0⁄ 0 0 0 0 0
15.7 4.8 4.8 35.5 0.9 0 0 15.4 0 0 0 0 0.3 1.8 0 0 0 0 0
MICB allele frequencies between Guangxi Zhuang and other populations, *P < 0.05. a In the populations list here it could also be 005:05/06/07.
Table 4 Linkage disequilibrium (LD) analysis between MICA and MICB. Haplotypesa ⁄
⁄
MICA 019-MICB 005:02 MICA⁄008:04-MICB⁄002 MICA⁄008:04-MICB⁄005:02 MICA⁄002:01-MICB⁄005:02 MICA⁄010-MICB⁄005:02 MICA⁄012:01-MICB⁄002 MICA⁄002:01-MICB⁄008 MICA⁄019-MICB⁄002 MICA⁄008:04-MICB⁄014 MICA⁄002:01-MICB⁄002 MICA⁄012:01-MICB⁄005:02 MICA⁄045-MICB⁄005:02 MICA⁄045-MICB⁄014 MICA⁄002:01-MICB⁄005:04
HF
D
D0
R2
p-Value
0.1310 0.0989 0.0963 0.0615 0.0562 0.0481 0.0428 0.0401 0.0374 0.0348 0.0267 0.0240 0.0241 0.0214
0.0495 0.0258 0.0201 0.0242 0.0202 0.0215 0.0231 0.0138 0.0076 0.0191 0.0156 0.0066 0.0162 0.0115
0.3982 0.1469 0.1729 0.2822 0.3674 0.2677 0.3243 0.2168 0.1053 0.3548 0.3684 0.2152 0.2325 0.3236
0.0628 0.0171 0.0082 0.0144 0.0206 0.0279 0.0380 0.0040 0.0030 0.0115 0.0107 0.0025 0.0402 0.0181
0.1087 0.7169 0.2134 0.0383 0.0209 0.2669 0.0029⁄ 0.2060 0.6230 0.3048 0.5085 0.1992 0.0013⁄ 0.1846
a There were 14 kinds of MICA-MICB haplotypes showing frequency 2%. P values < 0.00357 (0.05 after Bonferroni’s correction) are bold. HF, haplotype frequencies. Significant difference (p-value < 0.05).
*
MICA⁄019-MICB⁄005:02 (13.1%), MICA⁄008:04-MICB⁄002 (9.9%) and MICA⁄008:04-MICB⁄005:02 (9.6%) were the most prevalent ones.
clustered in another clade. The Guangxi Zhuang population was closest to the Tujia population (see Fig. 4). 4. Discussion
3.4. Phylogenetic tree analysis The phylogenetic tree in Fig. 2 was constructed using the allelic frequencies at MICA locus in the Tujia population and its neighboring ethnic groups. The neighboring ethnic groups used in the phylogenetic tree construction were Hunan Han, Northern Han, Zhejiang Han, Guangdong Han, Thai, Japanese, Korean. According to the phylogenetic tree, the Tujia population showed the closest genetic structure to the Guangdong Han populations.
3.5. Principal component analysis Principal component analysis (PCA) was performed based on the allelic frequencies of the MICA locus. As shown in Fig. 3, all the ethnic groups can be divided into two clusters. The Tujia population was clustered in one clade, while the remaining 8 populations were
In this study, we investigated the distribution of MICA/B variants and haplotypes in the Zhangjiajie Tujia population. The SBT and SSP PCR protocols used in this study provide an accurate and reliable methodology to identify MICA/B alleles. Our results revealed that the eight identified MICA alleles in the Tujia population is smaller than those previously reported in other geographically related populations [8,22–25], and might be due to founder effects or/and bottlenecks in Tujia population. The three predominant alleles, MICA⁄008:04, MICA⁄002:01 and MICA⁄019, accounted for 71.66% of the MICA gene pool. A comparison of the Tujia people with similar populations in China and Asia highlighted differences in the distribution of the sequence allelic MICA variants (Table 1) [8,26–32]. For instance, the MICA⁄008:04 and MICA⁄019 alleles were observed at higher frequencies (29.41% and 20.59%, respectively) in the Tujia, while these were much lower or absent in other populations. Alternatively, neither the MICA⁄008:01 nor
Please cite this article in press as: Y.J. Wang et al., Allele polymorphism and haplotype diversity of MICA/B in Tujia nationality of Zhangjiajie, Hunan Province, China, Hum. Immunol. (2016), http://dx.doi.org/10.1016/j.humimm.2016.03.005
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MICA⁄009:01 alleles were detected in the Tujias, but both were commonly found in other populations (2.4–37.9%). Similar discrepancies were observed in the distribution of MICA STR-alleles (Table 2), for which the frequency of MICA⁄A5 and MICA⁄A5.1 represented the two highest variances (both 29.68%). Because of the proximity of MICA to the autoimmune-related HLA-B loci, Gonzalez et al. [33] reported that MICA⁄002 associates with psoriatic arthritis in Spanish patients independently of the other HLA class I alleles commonly associated with the disease. Additionally, MICA acts as an immune ligand for NK cells; thus, the association between MICA polymorphisms and immunosurveillance has been extensively described. For instance, the MICA⁄A9 and SNPMICA-129val variants in the Chinese Han [34] and Tunisian populations [35], respectively, associate with an increased risk of nasopharyngeal carcinoma, whereas MICA⁄A5.1 correlates with oral squamous cell carcinoma (OSCC) in the Japanese [36,37]. Interestingly, MICA⁄A5.1 also seems to confer susceptibility to breast cancer in Spanish women [38]. Thus, the unique MICA allelic distribution observed in the Zhangjiajie Tujia population may be valuable for the further study of the association between ethnic background and disease susceptibility. Though limited reports on MICB polymorphism exist, accumulating evidence suggests that allelic variation can be associated with various diseases and may play an important role in the outcome of allogeneic organ transplantation [39–41]. Our data showed that MICB⁄005:02 (39.57%), MICB⁄002:01 (24.87%), and MICB⁄014 (10.16%) were the most common alleles and accounted for 74.6% of the MICB gene pool in the Zhangjiajie Tujia population. Moreover, an inter-population comparison indicated that MICB distribution varied amongst the different Chinese populations. For instance, the MICB⁄002:01 allele showed a higher frequency in Zhangjiajie Tujias (24.87%) than other populations (11.5–16.35%) (Table 3). Since HLA-MICA/B haplotypes have been reported in different populations [7–9,42,43], it was presumed that the wide distribution of haplotypes have been conservatively maintained via selective advantage. Accordingly, our study revealed that MICA⁄045 was strongly linked with MICB⁄014 in the Zhangjiajie Tujias, as well as the Guangxi Zhuang and Hainan Li populations [44]. This finding is highly indicative of potential recombination hotspots between the MICA and MICB genes, particularly since we have also found that all the MICB⁄009N carriers were also HLA⁄B48:01 carriers, while these data will likely aid in characterizing the potential pathogenic mechanisms of MIC polymorphisms in the Zhangjiajie Tujia population, further studies are necessary to investigate the effects of HLA-A, B, and DR polymorphism in these individuals. Furthermore, we also identified the origins of genetic heterogeneity in the Zhangjiajie Tujias and constructed a phylogenetic tree of the different ethnic groups based on MICA and MICB allelic variation (Figs. 2 and 3). Interestingly, while relatively limited genetic variability exists amongst the Chinese populations as a whole, the MICA/B allele distributions in the Tujia population were relatively distant comparatively. As shown in Fig. 2, Tujias are most closely related to the Guangdong Han population, indicating that the two population groups have a similar lineage. The origin of the Tujia nationality is controversial, Xie et al. [45] analyzed the distribution of haplotypes in the Tujia Y chromosome and found that the genetic structure was similar to that observed in the Han population. Additionally, Zhang et al. [46] and Qiu et al. [47] reported on the HLA-A and HLA-DRB1 loci frequencies, respectively, and showed the Tujias living in the Wufeng region had the closest genetic relationship with the central Han population. However, principal component analysis showed the Tujias were genetically distant from other Chinese populations, which might be due to the relative genetic isolation and different origin branch of the Zhangjiajie Tujias.
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Fig. 2. Phylogenetic tree constructed by the neighbor-joining method showing the relationship between Zhangjiajie Tujias with other populations based on the frequencies of MICA locus.
In conclusion, our study showed a unique distribution of MICA/ B gene alleles in the Zhangjiajie Tujia population and uncovered interesting findings with respect to MICA/B allelic and haplotypic configurations. This analysis is likely to facilitate further understanding of the genetic background, evolution, and origination of the Tujia ethnicity, as well as enrich the genomic data resources for the Chinese population as a whole. Funding This study was supported by grants from the National Natural Science Foundation of China (project No: 81371864) and the Science and Technology Plan Project of Hunan Province (project No: 2014SK3271). Competing interests The authors declare that they have no conflict of interests or commercial links pertinent to the manuscript.
Please cite this article in press as: Y.J. Wang et al., Allele polymorphism and haplotype diversity of MICA/B in Tujia nationality of Zhangjiajie, Hunan Province, China, Hum. Immunol. (2016), http://dx.doi.org/10.1016/j.humimm.2016.03.005
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Fig. 3. Phylogenetic tree constructed by the neighbor-joining method showing the relationship between Zhangjiajie Tujia population with other populations based on the frequencies of MICB locus.
Fig. 4. Principal component analysis between Zhangjiajie Tujia population with other 9 ethnic groups based on the allelic frequencies of MICA locus.
Acknowledgments We would like to thank Xinfeng Zhang (The People’s Hospital of Zhangjiajie City) for his providing kindly some of blood samples and background data.
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Please cite this article in press as: Y.J. Wang et al., Allele polymorphism and haplotype diversity of MICA/B in Tujia nationality of Zhangjiajie, Hunan Province, China, Hum. Immunol. (2016), http://dx.doi.org/10.1016/j.humimm.2016.03.005