Mitochondrial DNA analysis in Tibet: Implications for the origin of the Tibetan population and its adaptation to high altitude

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<ul><li><p>AMERICAN JOURNAL OF PHYSICAL ANTHROPOLOGY 93:189-199 (1994) </p><p>Mitochondrial DNA Analysis in Tibet: Implications for the Origin of the Tibetan Population and Its Adaptation to High Altitude </p><p>ANTONIO TORRONI, JULIE A. MILLER. LORNA G. MOORE. STACY ZAMUDIO, JIANGUO ZHUANG, TAFSHI DROMA, AND DOUGLAS C. WALLACE Department of Genetics and Molecular Medicine, Emory University School of Medicine, (A.T., J.A.M., D.C. W.), and Department of Anthropology, Emory Uniuersity, (D.C. W.), Atlanta, Georgia 30322; Cardiovascular Pulmonary Research Lab, University of Colorado Health Sciences Center, and Departm,ent of Anthropology, University of Colorado at Denuer, Denver, Colorado 80262 (L.G.M., S.Z.); Tibet Institute of Medical Science, Lhasa, Tibet Autonomous Region, China, 850000 (J.Z., T.D.) </p><p>KEY WORDS Mitochondrial DNA variation, Tibetans, Asian mtDNA lineages, Haplotypes, High altitude adaptation </p><p>ABSTRACT Mitochondrial DNAs (mtDNAs) of 54 Tibetans residing at altitudes ranging from 3,0004,500 m were amplified by polymerase chain reaction (PCR), examined by high-resolution restriction endonuclease analy- sis, and compared with those previously described in 10 other Asian and Siberian populations. This comparison revealed that more than 50% of Asian mtDNAs belong to a unique mtDNA lineage which is found only among Mon- goloids, suggesting that this lineage most likely originated in Asia a t an early stage of the human colonization of that continent. Within the Tibetan mtDNAs, sets of additional linked polymorphic sites defined seven minor lineages of related mtDNA haplotypes (haplogroups). The frequency and dis- tribution of these haplogroups in modern Asian populations are supportive of previous genetic evidence that Tibetans, although located in southern Asia, share common ancestral origins with northern Mongoloid populations. This analysis of Tibetan mtDNAs also suggests that mtDNA mutations are un- likely to play a major role in the adaptation of Tibetans to high altitudes. 0 1994 Wiley-Liss, Inc </p><p>The nature and the extent of the genetic variation in the Tibetan population are par- ticularly interesting for at least two reasons. First, there is a marked paucity of genetic data concerning Tibetans because of both their geographic isolation and political events occurring in the late 1950s which fur- ther isolated them. Consequently, a limited amount of information is available to recon- struct their genetic relationships with other Mongoloid populations. Second, Tibetans are likely the largest and oldest high-alti- tude population (Zhimin, 1982), and their adaptation to high altitudes could have a genetic component (Lahiri et al., 1976). </p><p>Some of the genetic data about this popu- lation has been obtained from Tibetans who moved to India and Nepal after 1959 (Bhalla, 1972; Patel, 1971, 1973; Santachi- ara et al., 1976; Bhattacharjee et al., 1977; Roychoudhury, 1981; Sharma, 1983; Papiha et al., 1989), and, more recently, several studies of blood cell antigens and immuno- globulins have been carried out on the Tibet- </p><p>Received May 17,1993; accepted October 4,1993 Address reprint requests to Douglas C. Wallace, Department </p><p>of Genetics and Molecular Medicine, Emory University School of Medicine, Atlanta, Georgia 30322. </p><p>0 1994 WILEY-LISS, INC. </p></li><li><p>A. TORRONI ET AL. 190 </p><p>ans who still live in Tibet (Ai et al., 1987; Zhao et al., 1987; Matsumoto, 1987; Zhao and Lee, 1989). </p><p>Extensive studies of the Gm and Km allo- types, HLA antigens, and dental morphol- ogy of Mongoloid populations have sug- gested that Mongoloids are divided into two groups: a northern group encompassing northern Asia and Siberian populations and a southern group encompassing southeast Asian populations (Matsumoto, 1987; Turner, 1987; Lee et al., 1988; Zhao and Lee, 1989). Interestingly, the same studies also showed that, although Tibetans inhabit a relatively southern region of Asia, they ge- netically clustered within the northern Mongoloid group. This result may be ex- plained by the peculiar orography of Tibet which makes this region more accessible from the north than the southeast. </p><p>Analysis of mtDNA is relevant to the Ti- betan population for two reasons. MtDNA studies have provided a new approach to de- termine the genetic relationships and pat- terns of migrations of human populations (Stoneking et al., 1990; Schurr et al., 1990; Vigilant et al., 1991; Ballinger et al., 1992; Torroni et al., 1992). In addition, mtDNA encodes for 37 essential genes of oxidative phosphorylation (Wallace, 1992) and their variation could represent one of the genetic factors involved in the human adaptation to hypoxia. </p><p>To clarify the origin of modern Tibetans as well as the possible role of mtDNA muta- tions in their adaptation to high altitudes, mtDNA variation of a sample of Tibetans from Tibet was determined and compared to that reported in east and southeast Asians (Ballinger et al., 1992), and Siberians (Tor- roni et al., 1993b). </p><p>This analysis showed that Tibetan mtDNA haplotypes are distributed through- out the entire range of the Asian mtDNA variation, suggesting that modern Tibetans could have experienced gene flow from pop- ulations of the surrounding regions. How- ever, Tibetans maintain particularly high frequencies of certain mtDNA haplotypes which are found only in northern Asian and Siberian populations. Consequently, this finding supports the notion that Tibetans and northern Asian populations share a </p><p>common ancestral origin. In addition, the comparison of the Tibetan mtDNA variation with that reported for other Asian popula- tions suggests that mtDNA mutations are unlikely to play a major role in the adapta- tion of Tibetans to high altitude. </p><p>SUBJECTS AND METHODS Samples </p><p>The 54 Tibetans analyzed were chosen from a sample of 171 subjects living in three different geographic regions. All samples were collected from subjects living at least 20 miles from the closest town. Twenty sam- ples were from nomads residing in the re- gion surrounding the town of Nachu (4,500 m), which is approximately 250 miles north- west of Lhasa. Nineteen samples were from farming villages located in the region of the town of Tsedang (3,600 m) which is 150 miles southeast of Lhasa. This town is lo- cated in a valley which the Tibetans con- sider to be the cradle of Tibetan civilization and the birthplace of their people. The re- maining 15 samples were from farming vil- lages in the region surrounding the town of Linchi (3,000 m), which is about 300 miles southeast of Lhasa. </p><p>Information was obtained by interviews of all individuals concerning their birthplace, current or former places of residency, and birthplace and residence of their parents and grandparents. Only subjects of Tibetan ancestry from families who were unrelated through their maternal line and who had lived in the same area for several genera- tions were analyzed for mtDNA variation. </p><p>Molecular genetic analysis </p><p>DNAs were extracted from buffy coats us- ing the method described in Torroni et al. (1992). The entire mtDNAs were amplified through polymerase chain reaction (PCR) (Saiki et al., 1985) in nine overlapping seg- ments using the primer pairs and amplifica- tion conditions described in Torroni et al. (1993a). Each PCR segment was digested with 14 restriction endonucleases (AluI, AvaII, BamHI, DdeI, HaeII, HaeIII, HhaI, HincII, HinfI, HpaI, MspI, MboI, RsaI, TaqI). The resulting fragments were re- solved through electrophoresis in NuSieve plus SeaKem agarose (FMC BioProducts) </p></li><li><p>MtDNA VARIATION IN TIBETANS 191 </p><p>TABLE I. MtDNA haplotypes observed in Tibetans </p><p>+ 10394d Haplotype N2 +10397a Haplogroup Altitude3 </p><p>AS56* AS62* AS65 </p><p>AS123 AS124 AS125 AS126 AS127 AS128 AS129 AS130 AS131 AS132 AS133 AS134 AS135 AS136 AS137 AS138 AS139 AS140 AS141 AS142 AS143 AS144 As145 AS146 AS147 AS148 AS149 As150 AS151 AS152 AS153 AS154 AS155 AS156 AS157 AS158 AS159 AS160 </p><p>~ ~ 1 1 8 4 </p><p>1 2 2 2 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1 1 2 1 1 3 1 1 1 1 1 1 1 1 2 5 1 1 1 1 1 1 1 </p><p>Total 54 </p><p>A Other </p><p>C Other </p><p>A A A A A B B B D D D D D D D D E E E F F F F F F G G G </p><p>Other Other Other Other Other Other Other Other Other Other </p><p>1 1 </p><p>2 (l), 3 (1) 3 3 1 1 1 3 3 3 1 1 3 </p><p>3 2 3 1 3 2 1 3 2 </p><p>1(1) , 2 (31, 3 (1) 1 1 3 3 2 1 1 </p><p>2 (2), 3 (1) </p><p>The nomenclature ofthe haplogroups corresponds to that reported in Fig. 1. The polymorphic restriction sites which define the 42 haplo- types observed in the Tibetans (AS56, AS62, AS65, AS118, AS123- AS1601 are listed the Appendix The haplotype of the African outgroup is described in Torroni et al. (1993a). N = number of subjects. Altitudes of 3,000 m, 3,700 m and 4,500 m indicated by 1, 2 and 3, respectively. Haplotypes already described in Asian populations and numbered according to Ballinger et al. (1992). </p><p>gels and visualized by W-induced fluores- cence. This restriction analysis probes about 15-20% of the mtDNA nucleotides and per- mits definition of mtDNA haplotypes (Ta- ble 1). </p><p>Phylogenetic analysis </p><p>The haplotypes observed in the Tibetans were compared to those previously reported </p><p>Koreans </p><p>TIBETANS -%&amp; Malaysian Chinese --B_-_--1 Taiwanese Han Vietnamese Malay Aborigines </p><p>Fig. 1. Geographic location of the Asian and Siberian populations whose mtDNA haplotypes were compared to those observed in the Tibetans. Data from east Asian and Siberian populations are from Ballinger e t al. (1992) and Torroni e t al. (1993b1, respectively. </p><p>in the other Asian and Siberian populations analyzed with the same set of restriction en- zymes (Fig. 1) (Ballinger et al., 1992; Tor- roni et al., 1993b), and their evolutionary relationships were inferred using parsi- mony analysis (PAUP 3.0s [Swofford 19921). All dendrograms were rooted using a Sene- galese mtDNA haplotype (African out- group [Torroni et al., 1993a,b]). This haplo- type was obtained from a mtDNA characterized by the presence of an HpaI site at np 3592. This site defines haplotypes which are African specific and observed in 70-100% of the sub-Saharan Africans but are absent in Asians and Caucasians (HpaI morph-3) (Denaro et al., 1981; Cann et al., 1987; Scozzari et al., 1988, in press). Maxi- mum parsimony (MP) trees were generated through random addition of sequences using the Tree Bisection and Reconnection (TBR) algorithm. Because of the large number of terminal taxa, thousands of MP trees could be obtained. We terminated our searches at 3,000 trees after 1,148 replications and saved no more than 10 MP trees for each replication. Consequently, shorter trees </p></li><li><p>192 A. TORRONI ET AL </p><p>could exist, although none were observed in our analyses. </p><p>RESULTS Haplotype analysis </p><p>Forty-two haplotypes (AS56, AS62, AS65, AS118, AS123-160) were observed among the 54 Tibetans whose mtDNA variation was determined (Table 1). Four of these hap- lotypes, AS56, AS62, AS65 and AS118, were previously described in other Asian popula- tions (Ballinger et al., 1992) whereas the re- maining thirty-eight (AS123-160) were ob- served for the first time in this analysis. A total of 71 polymorphic restriction sites and the 9-base pair (9-bp) COII-tRNALys inter- genic deletion (Cann and Wilson, 1983; Ho- rai and Matsunaga, 1986; Wrischnik et al., 1987; Hertzberg et al., 1989) defined the 42 haplotypes (Appendix). Virtually all of the Tibetan haplotypes were characterized by sets of linked polymorphic sites already re- ported in Mongoloid populations (Stoneking et al., 1990; Ballinger et al., 1992; Torroni et al., 1992, 1993a,b; Passarino et al., 1993). These linked polymorphic sites define groups of haplotypes which share a common ancestral origin. </p><p>Tibetan mtDNAs, as well as mtDNAs from all Mongoloid populations, can be di- vided into two major lineages (Ballinger et al., 1992; Torroni et al., 1993a,b; Passarino et al., 1993). One is characterized by the con- comitant presence of a DdeI site at np 10394 (A to G at np 10398) and an Nu1 site at np 10397 (C to T at np 10400). The DdeI site is generated by an A to G transition at np 10398. The AluI site is generated by the same A to G transition at np 10398 plus an additional C to T transition at np 10400. Of the 42 Tibetan haplotypes, 24 belong to the lineage that possesses these two sites (Table 1). Conversely, the second haplotype lineage lacks these two sites. The remaining 18 Ti- betan haplotypes are members of this sec- ond major lineage. </p><p>Within these two major lineages, several additional restriction sites define smaller groups of related haplotypes or haplogroups. Seven of these haplogroups (named A, B, C, </p><p>D, E, F, and G1) are common in Tibetans and all together represent approximately 60% of their mtDNAs. Haplogroups C, D, E, and G belong to the + 10394 DdeI\+ 10397 AluI lin- eage, whereas haplogroups A, B, and F are members of the lineage lacking these two sites (Table 1). </p><p>Tibetan haplotypes AS56 and AS123- AS127 belong to haplogroup A which is de- fined by a HaeIII site at np 663 (Torroni et al., 1992). Haplotype AS56 was previously reported in the Han from Taiwan (Ballinger et al., 1992) and in Native Americans and is one of the four Asian mtDNAs which colo- nized the Americas (Torroni et al., 1993a). In contrast, haplotypes AS123-AS127 have not been observed in any other human popu- lation. </p><p>Haplotypes AS128-AS130 belong to hap- logroup B which is defined by the 9-bp COII- tRNALy" intergenic deletion. Haplotypes of this haplogroup have been observed in nu- merous Asian, Melanesian, Polynesian, and Amerind populations (Horai and Mat- sunaga, 1986; Hertzberg et al., 1989; Schurr et al., 1990; Stoneking et al., 1990; Ballinger et al., 1992; Harihara et al., 1992; Torroni et al., 1992, 1993a; Passarino et al., 1993). However, haplogroup B haplotypes are ab- sent in Siberian and NaDene populations (Shields et al., 1992; Torroni et al., 1993a,b). </p><p>Haplotype AS65 is the only group C haplo- type observed in the Tibetans. Haplogroup C is defined by the HincII np 13259 site loss and the AluI np 13262 site gain (Torroni et al., 1992). This haplotype has been reported at low frequencies in Taiwanese Han (Bal- linger et al., 1992) and at high frequencies in Aboriginal Siberian and Amerind popula- tions (Torroni et al., 1993a,b). Similar to haplotype AS56, haplotype AS65 is sug- gested as being one of the four mtDNA hap- lotypes which migrated to the Americas dur- ing the first human migration into that continent (Torroni et al., 1993a,b). </p><p>Haplotypes AS131-138 belong to haplo- group D which is defined by a AluI site loss </p><p>'Nomenclature of haplogroups A-D is according to Torroni et al. (1992,1993a,b). Haplogroups A, B, C, D, and F correspond to haplotype groupings H, D,+C, R, L, and A (Ballinger et al., 19921, respectively. Haplogroups E and G are new haplotype groupings. </p></li><li><p>APPENDIX. Polymorphic restriction sites observed in 42 distinct Tibetan mtDNA halotypes (AS56, AS62. AS65. AS1 18, AS1234S160) </p><p>Sites Haplotypes </p><p>1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 5 6 6 1 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 4 4...</p></li></ul>

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