HLA polymorphism of the Zhuang population reflects the common HLA characteristics among Zhuang-Dong language-speaking populations

HLA polymorphism of the Zhuang population reflects the common HLA characteristics among Zhuang-Dong language-speaking populations 1 first-author 1 1 1 1 1 1 1 1 2 corresp ‡Corresponding Authors ‡Corresponding Authors †E-mail:tokunaga@m.u-tokyo.ac.jpchujy08@gmail.com †E-mail:tokunaga@m.u-tokyo.ac.jpchujy08@gmail.com tokunaga@m.u-tokyo.ac.jp 1 corresp ‡Corresponding Authors ‡Corresponding Authors †E-mail:tokunaga@m.u-tokyo.ac.jpchujy08@gmail.com †E-mail:tokunaga@m.u-tokyo.ac.jpchujy08@gmail.com tokunaga@m.u-tokyo.ac.jp Institute of Medical Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, Kunming 650118, China Institute of Medical Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, Kunming 650118, China Department of Human Genetics, Graduate School of Medicine, University of Tokyo, Tokyo 113-0033, Japan Department of Human Genetics, Graduate School of Medicine, University of Tokyo, Tokyo 113-0033, Japan A study of the human leukocyte antigen (HLA) genetic characteristics in the Zhuang, the largest ethnic population in China, would provide insight into Zhuang history and give a useful tool for disease associations, transplantation, and anthropology. In the present study, we report the comprehensive HLA-A, HLA-B, HLA-C, and HLA-DRB1 alleles and haplotypes in the Zhuang population of southern China for the first time. A total of 13 HLA-A, 24 HLA-B, 22 HLA-C, and 18 HLA-DRB1 were identified in 104 Zhuang individuals. The frequencies of HLA-A*11:01, A*02:07, A*24:02, A*02:03, and A*33:03 on A loci, B*15:02, B*58:01, B*46:01, and B*13:01 on B loci, C*03:04, C*08:01, C*01:02, C*03:02, and C*07:02 on C loci, and DRB1*15:01, DRB1*16:02, DRB1*14:01, DRB1*15:02, and DRB1*03:01 on the DRB1 loci were >10%. The A*33:03-C*03:02-B*58:01-DRB1*03:01 and A*02:07-C*01:02-B*46:01-DRB1*14:01 haplotypes were predominant in the Zhuang. The phylogenetic tree, as well as the analysis of haplotypes, suggested that the Zhuang are genetically similar to southern Chinese populations, especially the Zhuang-Dong language-speaking populations, such as the Bouyei, Dai, and Maonan. Even though the Zhuang and southern Chinese populations shared common alleles and haplotypes, the Zhuang has maintained its unique genetic characteristics. Human leukocyte antigen (HLA) alleles Haplotypes Zhuang population Introduction 1 Human leukocyte antigens (HLAs) play a central role in immune response and exhibit extensively high levels of polymorphism. As of December 2009, 965 HLA-A, 1 543 HLA-B, 626 HLA-C, and 762 HLA-DRB1 alleles had been identified worldwide (Marsh et al., 2010 2010 ). So, HLA has been used as a valuable tool for tracing the genetic background of human populations (Tokunaga et al., 1996 1996 ; 1997 1997 ). Moreover, the high-resolution molecular typing method provides the precise examination of HLA allele distribution in different populations and allows a detailed analysis of the haplotypic relationship between alleles of different loci (Bugawan et al., 1999 1999 ; Cao et al., 2004 2004 ). In addition to the Han population, the Zhuang ethnic group, with a population size of approximately 16.2 million, is the largest ethnic population in China. Most of the Zhuang live in the Guangxi Zhuang Autonomous Region in southern China; some Zhuang also live in the Yunnan, Guangdong, Guizhou, and Hunan provinces. The Zhuang people speak a language that belongs to the Zhuang-Dai branch of Zhuang-Dong (or Kam-Tai) subfamily of the Sino-Tibetan language family, which has northern and southern dialects. The Zhuang’s language is related to Thai, Laos, and Dai languages. The Zhuang has recorded their history from the eastern Zhou dynasty (475 to 221 BC) of China. The Zhuang originated from the Xi’ou and Luoyue of the Baiyue ancient tribe, who had inhabited along the southeast coast of China up to the Yunnan province and the northern part of Southeast Asia from the fifth century BC (Guo and Dong, 2000 2000 ). Our previous studies based on HLA polymorphisms in several ethnic groups of Southwest China have suggested that the same linguistic populations tend to cluster together (Shi et al., 2006 2006 ; 2008 2008 ; Ogata et al., 2007 2007 ). As a typical Zhuang-Dong language-speaking population, the analyses of HLA genes and haplotypes in the Zhuang population would be helpful for indentifying the common HLA characteristics of the same linguistic family. Furthermore, it would also provide an insight into Zhuang history and give a useful tool for HLA matching in transplantation and disease associations. Materials and methods 1 A total of 104 health and unrelated individuals (unrelated at least three generations) were randomly selected in the Tiandeng county of Nanning city of Guangxi Zhuang Autonomous Region in southern China. Blood samples were collected after obtaining informed consent. Genomic DNA was extracted from peripheral lymphocytes using the QIAamp blood kit (Qiagen, Hilton, Germany) according to the manufacturer’s protocol. HLA-A, HLA-B, HLA-C, and HLA-DRB1 polymorphisms were detected using the Luminex multi-analyte profiling system (xMAP) with a WAKFlow HLA typing kit (Wakunaga, Hiroshima, Japan) as described before (Yao et al., 2009 2009 ; Shi et al., 2010b 2010b ). Genotype determination and data analyses were performed automatically using the WAKFlow typing software according to the manufacturer’s instructions. The ambiguous alleles could not be identified using this method listed in Table 1 1 . 2 n =212. No.: allele numbers; AF: allele frequencies. A*11:01=A*11:01/11:03/11:05; C*03:04=C*03:04/03:06/03:09/03:10; C*07:02=C*07:02/07:05; DRB1*14:01=DRB1*14:01/14:54 HLA-A No. AF HLA-B No. AF HLA-C No. AF HLA-DRB1 No. AF A*11:01 61 0.288 B*15:02 34 0.160 C*03:04 34 0.160 DRB1*15:01 39 0.184 A*02:07 35 0.165 B*58:01 32 0.151 C*08:01 32 0.151 DRB1*16:02 32 0.151 A*24:02 30 0.142 B*46:01 31 0.146 C*01:02 30 0.142 DRB1*14:01 30 0.142 A*02:03 28 0.132 B*13:01 29 0.137 C*03:02 29 0.137 DRB1*15:02 29 0.137 A*33:03 28 0.132 B*38:02 12 0.057 C*07:02 27 0.127 DRB1*03:01 26 0.123 A*11:02 8 0.038 B*40:01 12 0.057 C*14:02 11 0.052 DRB1*09:01 10 0.047 A*29:01 7 0.033 B*51:01 10 0.047 C*04:03 8 0.038 DRB1*11:01 10 0.047 A*02:06 6 0.028 B*55:02 7 0.033 C*04:01 6 0.028 DRB1*12:02 9 0.042 A*02:01 3 0.014 B*15:25 6 0.028 C*07:14 5 0.024 DRB1*13:03 5 0.024 A*03:01 2 0.009 B*07:05 4 0.019 C*12:03 5 0.024 DRB1*10:01 4 0.019 A*26:01 2 0.009 B*39:01 4 0.019 C*15:05 4 0.019 DRB1*04:05 3 0.014 A*24:07 1 0.005 B*40:02 4 0.019 C*03:03 3 0.014 DRB1*04:06 3 0.014 A*31:01 1 0.005 B*52:01 4 0.019 C*08:04 3 0.014 DRB1*08:03 3 0.014 B*15:01 3 0.014 C*12:02 3 0.014 DRB1*13:02 3 0.014 B*27:04 3 0.014 C*01:14 2 0.009 DRB1*14:18 3 0.014 B*48:01 3 0.014 C*03:05 2 0.009 DRB1*13:01 1 0.005 B*56:01 3 0.014 C*03:37 2 0.009 DRB1*14:04 1 0.005 B*56:02 3 0.014 C*08:03 2 0.009 DRB1*14:05 1 0.005 B*35:01 2 0.009 C*03:07 1 0.005 B*35:05 2 0.009 C*04:15 1 0.005 B*35:03 1 0.005 C*14:09 1 0.005 B*38:01 1 0.005 C*15:02 1 0.005 B*56:04 1 0.005 B*67:01 1 0.005 Allele frequencies were calculated based on the HLA genotyping results and after linkage disequilibrium (LD) was analyzed, haplotype frequencies were calculated by the Expectation-Maximization algorithm using PyPop software (Lancaster et al., 2003 2003 ; 2007 2007 ). For each locus, the Hardy-Weinberg equilibrium test was performed as the method by Guo and Thompson ( 1992 1992 ) and the Ewens-Watterson homozygosity test of neutrality was performed using PyPop software too (Ewens, 1972 1972 ; Watterson, 1978 1978 ). Nei’s standard genetic distances among 18 populations were calculated based on HLA-A, HLA-B, and HLA-DRB1 allele frequencies and a neighbor-joining tree was constructed using Mega4.0 software (Tamura et al., 2007 2007 ). Frequency data for other Asian populations were obtained from previous studies, as follows: Wa and Jinuo (Shi et al., 2008 2008 ), Bouyei (Imanishi et al., 1992 1992 ; Chen et al., 2007 2007 ), Maonan (Ogata et al., 2007 2007 ), Han in Taiwan (Wen et al., 2008 2008 ), Han in Hongkong (Hardy et al., 1997 1997 ), Han in Guangdong (Trachtenberg et al., 2007 2007 ), Menba (Zhang et al., 2005 2005 ), Tibetan (Lai et al., 1999 1999 ; Chen et al., 2006 2006 ), Hui (Yu et al., 2002 2002 ; Hong et al., 2007 2007 ), Han in Beijing (Sun et al., 1997 1997 ; Deng et al., 2005 2005 ), Han in Northern (Yang et al., 2006 2006 ), Dai (Shi et al., 2010a 2010a ), Han in Yunnan (Yao et al., 2009 2009 ), and Bulang and Hani (Shi et al., 2010b 2010b ). Fig. 1 1 shows the geographic distribution of these 17 populations (including the Zhuang) in China. Han1: Han in Yunnan; Han2: Han in Hongkong; Han3: Han in Guangdong; Han4: Han in Taiwan; Han5: Han in Beijing; Han6: Han in Northern Results 1 Allele frequencies 2 The frequencies of HLA-A, HLA-B, HLA-C, and HLA-DRB1 alleles in the Zhuang population are shown in Table 1 1 . A total of 13 HLA-A, 24 HLA-B, 22 HLA-C, and 18 HLA-DRB1 in the Zhuang were identified. The HLA-A locus was dominated by A*11:01, which had a frequency of 0.288. The next four most common alleles, A*02:07, A*24:02, A*02:03, and A*33:03, were each observed at frequencies of > 10%, and these four alleles accounted for 85.9% of the total for HLA-A in this population. For the HLA-B locus, B*15:02, B*58:01, B*46:01, and B*13:01 were common in the Zhuang at frequencies of > 10% and accounted for 59.4% of the total. Similarly, for the HLA-C locus, C*03:04, C*08:01, C*01:02, C*03:02, and C*07:02 occurred at frequencies > 10% and accounted for 71.7% of the total. For the HLA-DRB1 locus, DRB1*15:01, DRB1*16:02, DRB1*14:01, DRB1*15:02, and DRB1*03:01 were at frequencies > 10% and accounted for 73.7% of the total. All of the alleles in this population were common and existed in other East Asia populations, except a rare allele, DRB1*14:18 (0.014), which was only identified in the Han in Yunnan (0.005), Lahu (0.018) and Yao (0.024) in Yunnan province (Jia et al., 2002 2002 ; Liu et al., 2006 2006 ; Yao et al., 2009 2009 ). Hardy-Weinberg equilibrium test and Ewens-Watterson homozygosity test of neutrality 2 The distribution of genotypes at each locus did not show significant deviations from those expected under the Hardy-Weinberg equilibrium (HLA-A, P =0.518; HLA-B, P =0.657; HLA-C, P =0.330; HLA-DRB1, P =0.165). Ewens-Watterson homozygosity test of neutrality selection for all the loci could not reject the neutral model (Table 2 2 ). F O : observed F ; F E : expected F ; Fnd: normalized deviate of the homozygosity P value Locus F O F E Fnd Watterson F * * A 0.169 0.249 −0.884 0.156 B 0.102 0.129 −0.644 0.266 C 0.111 0.142 −0.658 0.265 DRB1 0.119 0.178 −0.950 0.109 Haplotype frequencies 2 The likelihood ratio test of linkage disequilibrium demonstrated that all pair-wise associations were statistically significant in the Zhuang ( P < 0.0001). Therefore, we estimated HLA haplotypes bearing 2–4 loci. Tables 3 3 and 4 4 show 2–4 loci haplotypes in which the frequencies were > 2%. n =106. HF: haplotype frequency A-B HF C-B HF B-DRB1 HF A-C-B-DRB1 HF 33:03-58:01 0.123 08:01-15:02 0.151 58:01-03:01 0.113 33:03-03:02-58:01-03:01 0.075 11:01-15:02 0.107 03:02-58:01 0.137 46:01-14:01 0.099 02:07-01:02-46:01-14:01 0.066 02:07-46:01 0.099 01:02-46:01 0.132 15:02-15:01 0.070 11:01-08:01-15:02-15:01 0.047 11:01-13:01 0.070 03:04-13:01 0.122 13:01-15:01 0.054 11:01-03:04-13:01-16:02 0.033 02:03-38:02 0.038 07:02-38:02 0.052 13:01-16:02 0.051 24:02-08:01-15:02-15:02 0.028 11:01-51:01 0.035 14:02-51:01 0.042 15:02-15:02 0.045 02:03-07:02-38:02-16:02 0.025 24:02-13:01 0.033 04:03-15:25 0.024 40:01-16:02 0.032 02:06-01:02-46:01-14:01 0.024 02:03-15:02 0.025 07:02-40:01 0.024 51:01-11:01 0.028 11:01-14:02-51:01-11:01 0.024 02:06-46:01 0.024 38:02-16:02 0.025 24:02-03:04-13:01-15:01 0.023 11:01-15:25 0.024 15:02-16:02 0.022 11:01-03:04-13:01-15:01 0.021 24:02-15:02 0.023 02:03-55:02 0.021 02:07-13:01 0.021 n =106. HF: haplotype frequency A-B-DRB1 HF A-C-B HF C-B-DRB1 HF 33:03-58:01-03:01 0.085 33:03-03:02-58:01 0.113 03:02-58:01-03:01 0.099 02:07-46:01-14:01 0.070 11:01-08:01-15:02 0.100 01:02-46:01-14:01 0.089 11:01-15:02-15:01 0.057 02:07-01:02-46:01 0.094 08:01-15:02-15:01 0.059 11:01-13:01-16:02 0.043 11:01-03:04-13:01 0.055 03:04-13:01-15:01 0.054 11:01-51:01-11:01 0.028 02:03-07:02-38:02 0.038 08:01-15:02-15:02 0.046 24:02-15:02-15:02 0.028 24:02-03:04-13:01 0.035 03:04-13:01-16:02 0.040 11:01-13:01-15:01 0.025 11:01-14:02-51:01 0.033 07:02-38:02-16:02 0.026 02:03-38:02-16:02 0.025 24:02-08:01-15:02 0.027 14:02-51:01-11:01 0.024 02:06-46:01-14:01 0.024 02:06-01:02-46:01 0.024 08:01-15:02-16:02 0.023 24:02-13:01-15:01 0.023 11:01-04:03-15:25 0.024 Some predominant haplotypes even occurred at frequencies >10%, and included A*33:03-B*58:01 (12.3%), A*11:01-B*15:02 (10.7%), C*08:01-B*15:02 (15.1%), C*03:02-B*58:01 (13.7%), C*01:02-B*46:01 (13.2%), C*03:04-B*13:01 (12.2%), B*58:01-DRB1*03:01 (11.3%), A*33:03-C*03:02-B*58:01 (11.3%), and A*11:01-C*08:01-B*15:02 (10.0%). Other predominant haplotypes occurred at frequencies >5%, and included A*33:03-C*03:02-B*58:01-DRB1*03:01 (7.5%), A*02:07-C*01:02-B*46:01-DRB1*14:01 (6.6%), A*33:03-B*58:01-DRB1*03:01 (8.5%), A*02:07-B*46:01-DRB1*14:01 (7.0%), A*11:01-B*15:02-DRB1*15:01 (5.7%), C*03:02-B*58:01-DRB1*03:01 (9.9%), C*01:02-B*46:01-DRB1*14:01 (8.9%), C*08:01-B*15:02-DRB1*15:01 (5.9%), and C*03:04-B*13:01-DRB1*15:01 (5.4%). Phylogenetic tree analysis 2 We constructed a neighbor-joining tree using HLA-A, HLA-B, and HLA-DRB1 allele frequencies in 18 populations because HLA-C genotyping results were not found in some populations of interest (Fig. 2 2 ). On this tree, the Han in Northern and Han in Beijing were clustered together with the Hui, Menba, and Tibetan as the northern branch. The southern Han, which included the Han in Yunnan, Han in Hongkong, Han in Singapore, Han in Guangdong, and Han in Taiwan, was clustered together and close to the Zhuang, Bouyei, Dai, and Maonan, while the Wa, Bulang, Hani, and Jinuo were clustered together and showed some genetic distance to the other southern Chinese populations. Discussion 1 The study of the HLA characteristics of the Zhuang is useful for medical and forensic applications, as well as in anthropology. This is the first report on the distributions of HLA-A, HLA-B, HLA-C, and HLA-DRB1 alleles and haplotypes at high resolution in the Zhuang. As the largest ethnic group in China, the Zhuang has had a close cultural affiliation with the Han for centuries (Guo and Dong, 2000 2000 ). On the neighbor-joining tree in the present study, the Zhuang belonged to the southern Chinese population. Most of the alleles identified in this study have been frequently found in other southern Chinese populations. The predominant alleles in the Zhuang, such as A*11:01, A*02:07, A*02:03, B*15:02, B*46:01, B*13:01, DRB1*16:02, and DRB1*14:01, were predominant in the Chinese, especially in southern Chinese populations. In addition to alleles, the predominant haplotypes in the Zhuang were also predominant in the southern Chinese population and southern East Asia populations. For example, the predominant A-C-B haplotype, A*02:07-C*01:02-B*46:01, was predominant in Vietnamese as well as in some southern Chinese populations, such as the Dai, Hani, Miao, Bouyei, Shui, Han in Yunnan, and Han in Guangdong, but existed at low frequencies in Chinese Hui, Japanese, and Koreans. Another predominant haplotype, A*11:01-C*08:01-B*15:02, was also predominant in Vietnamese and some Chinese populations, such as the Bulang, Dai, Hani, Shui, Han in Yunnan, and Han in Guangdong, who were located in southern China (Tokunaga et al., 1997 1997 ; Middleton et al., 2004 2004 ; Liu et al., 2006 2006 ; Chen et al., 2007 2007 ; Hong et al., 2007 2007 ; Ogata et al., 2007 2007 ; Trachtenberg et al., 2007 2007 ; Hoa et al., 2008 2008 ; Shi et al., 2008 2008 ; 2010a 2010a ; 2010b 2010b ; Wen et al., 2008 2008 ; Yao et al., 2009 2009 ). Based on the HLA alleles, haplotypes, and neighbor-joining tree analysis, we concluded that the Zhuang is a typical southern Chinese population. Despite the Zhuang and southern Chinese populations sharing the common alleles and haplotypes, the Zhuang still keeps its unique genetic characteristics. For example, A*11:01-B*15:02-DRB1*12:02 was common in southern populations of East Asia (Ogata et al., 2007 2007 ; Trachtenberg et al., 2007 2007 ; Hoa et al., 2008 2008 ; Shi et al., 2008 2008 ; 2010a 2010a ; 2010b 2010b ; Wen et al., 2008 2008 ; Gendzekhadze et al., 2009 2009 ; Yao et al., 2009 2009 ); however, it was not identified in the Zhuang. Instead, A*11:01-B*15:02-DRB1*15:01 was predominant in the Zhuang, but was not identified in other East Asian populations. Except the different frequencies of predominant haplotypes in different populations, some unique alleles were only indentified in the Zhuang, such as A*02:06-B*46:01-DRB1*14:01, A*11:01-B*51:01-DRB1*11:01, A*24:02-B*13:01-DRB1*15:01, A*24:02-B*15:02-DRB1*15:02, and A*02:06-C*01:02-B*46:01. Therefore, the Zhuang is a typical southern Chinese population, but maintains its unique HLA characteristics as well. From the neighbor-joining tree, we also found that the Zhuang clustered together with the Bouyei (Buyi or Bouyi), Dai, and Maonan, who all speak the Zhuang-Dong language. Our previous studies based on HLA distributions suggested that the same linguistic-speaking populations were genetically similar to each other (Shi et al., 2006 2006 ; 2008 2008 ; Ogata et al., 2007 2007 ). The other studies of Y chromosomes, mitochondrial DNA, as well as short tandem repeat, have also demonstrated that the Zhuang-Dong language-speaking population showed close affinity (Yao et al., 2002 2002 ; Shi et al., 2005 2005 ; Tian et al., 2008 2008 ). For example, the analysis of the hypervariable segment I sequences of the mitochondrial DNA control region in different Chinese ethnic populations and Thai has showed that the haplotype diversities of three Zhuang-Dong language-speaking populations including the Zhuang, Dai and Thai were similar, and the genetic distance among them even showed negative estimated values (Yao et al., 2002 2002 ). When we compared the distribution of the HLA alleles in the Zhuang, Bouyei, Dai, Maonan, and Shui, we found that the HLA characteristics were very similar to each other. Not only were the predominant alleles with high frequencies similar, but also the common alleles with moderate frequencies did. For example, on the HLA-A locus, the predominant alleles with frequencies > 10% commonly existed in these four populations, including A*02:07, A*02:03, A*11:01, and A*24:02, while the common alleles with frequencies around 1%–5% included A*02:01, A*02:06, and A*11:02, and all of these alleles comprised 80.66%–95.37% of the total HLA-A alleles, respectively. On the HLA-B locus, the commonly predominant alleles included B*13:01, B*15:02, B*38:02, B*40:01, B*46:01, and B*58:01, while the alleles with moderate frequencies included B*15:01, B*15:25, B*27:04, B*39:01, B*40:02, B*51:01, B*55:02, and B*56:01, and all of them comprised 84.27%–94.45% of the total HLA-B alleles. On the HLA-C locus, the commonly predominant alleles included C*01:02, C*03:02, C*03:04, C*07:02, and C*08:01, while the alleles with moderate frequencies included C*03:03, C*04:03, C*12:02, C*12:03, and C*15:02, and all of them comprised 84.91%–97.30% of the total HLA-C alleles (Chen et al., 2007 2007 ; Ogata et al., 2007 2007 ; Shi et al., 2010a 2010a ). When we compared the HLA-DRB1 locus, two Zhuang-Dong language-speaking populations (Tai Dam and Dai Lue in Thailand) were added. Similarly, the commonly predominant alleles existed in these six populations including DRB1*14:01, DRB1*16:02, DRB1*15:01, and DRB1*15:02, while the alleles with moderate frequencies included DRB1*04:05, DRB1*04:06, DRB1*08:03, DRB1*11:01, and DRB1*13:03. However, DRB1*03:01 was predominant in the Zhuang but moderate in other populations, while DRB1*09:01 and DRB1*12:02 were moderate in the Zhuang but predominant in other populations (Imanishi et al., 1992 1992 ; Chandanayingyong, 1997 1997 ; Stephens et al., 2000 2000 ). The haplotypes were also similar among the Zhuang-Dong language-speaking populations. For example, A*02:07-B*46:01, A*11:01-B*15:02, A*33:03-B*58:01, B*46:01-DRB1*14:01, and B*58:01-DRB1*03:01 were predominant in the Zhuang, Dai, and Maonan; however, A*11:01-B*40:01, the most common haplotype existing in other Chinese populations, was not identified in the Zhuang, Dai, and Maonan. A*02:07-C*01:02-B*46:01 and A*11:01-C*08:01-B*15:02 were predominant in the Zhuang, Dai, Bouyei, and Shui (Chen et al., 2007 2007 ; Ogata et al., 2007 2007 ; Shi et al., 2010a 2010a ). Zhuang-Dong language-speaking populations include the Zhuang, Bouyei, Dai, Dong, Shui, Mulam (Mulao), Maonan, and Li, who live in southern China. According to the historical record, all of them originated from the ancient Baiyue tribe, who had inhabited along the southeast coast of China up to the Yunnan province and the northern part of Southeast Asia from the fifth century BC (Guo and Dong, 2000 2000 ; You, 1994 1994 ). Some Pai-Yuei people later migrated to the North Thailand and contributed to the ancient gene pool of the Thai (Chandanayingyong, 1997 1997 ; Stephens et al., 2000 2000 ). The Zhuang-Dong language-speaking populations used to live in semi-tropic areas and live on planting rice. The similar surrounding, similar diet shift, similar language, together with similar origination make these populations to be genetically close to each other even though they live in different geographic places. In conclusion, according to our HLA data, we observed that the Zhuang was genetically close to southern Chinese populations. 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