Tissue Antigens ISSN 0001-2815

High-resolution molecular characterization of the HLA class I and class II in the Tarahumara Amerindian population
J. E. Garca-Ortiz1,2, L. Sandoval-Ramrez2,3, H. Rangel-Villalobos3,4, H. Maldonado-Torres5, S. Cox5, C. A. Garca-Sepulveda5, L. E. Figuera2,3, S. G. E. Marsh5, A. M. Little5, J. A. Madrigal5, J. Moscoso6, A. Arnaiz-Villena6 & J. R. Arguello1
1 Departamento de Inmunobiologa Molecular, Centro de Investigacion Biomedica, Facultad de Medicina, Universidad Autonoma de Coahuila, 27000 Torreon, Mexico 2 Division de Genetica, Centro de Investigacion Biomedica de Occidente, CMNO-IMSS, 44340 Guadalajara, Mexico 3 Doctorado en Genetica Humana, Centro Universitario Ciencias de la Salud, Universidad de Guadalajara, 44340 Guadalajara, Mexico 4 Laboratorio de Genetica Molecular, Centro Universitario de la Cienega, Universidad de Guadalajara (CUCI-UdeG), Ocotlan, Jalisco, Mexico 5 The Anthony Nolan Research Institute, and Royal Free & UCL School of Medicine, Royal Free Campus, Hampstead, London, UK 6 Departamento de Inmunologa y Biologa Molecular, Hospital 12 de Octubre, Universidad Complutense, 28040 Madrid, Espana

Key words Amerindian; HLA alleles; MHC; RSCA; Tarahumara Correspondence J. Rafael Arguello, MD, PhD Departamento de Inmunobiologa Molecular Centro de Investigacion Biomedica Facultad de Medicina Universidad Autonoma de Coahuila Gregorio A. Garca 198 Sur, CP 27000 Torreon, Coahuila Mexico Tel: 52 871 7226470 Fax: 52 871 7226470 e-mail: rarguello@mail.uadec.mx Received 9 December 2005; revised 9 February 2006; re-revised 28 March 2006 and 11 May 2006; accepted 13 May 2006 doi: 10.1111/j.1399-0039.2006.00636.x

Abstract We describe for the first time the high-resolution profiling of HLA-A, -B, -C, -DRB1, -DQB1 and -DPB1 in a culturally and geographically distinct Mexican ethnic group, the Tarahumaras. The alleles most frequently found by reference strand-mediated conformational analysis in this population were for class I: HLA-A*240201, *020101/09, *0206, *310102, *680102; HLA-B*4002, *1501, *510201, *3501/02/03, *4005, *4801; HLA-Cw*0304, *0801, *0102, *040101; and for class II: HLA-DRB1*080201, *1402, *040701; HLA-DQB1*0402, *0301, *0302/07; HLA-DPB1*0402, *0401, *020102. In addition, a novel allele, HLA-A*0257, was found. Based on comparison of presently known HLA-DRB1 and -DQB1 allele frequencies in Amerindian groups and worldwide populations, the Tarahumaras are unexpectedly more related to the geographically and linguistically distant Aymara and Terena Amerindian groups than they are to neighbouring tribes.

Introduction

The most polymorphic genetic system in humans is the MHC, located on 6p21.31; 40% of 224 loci are related to immune function (1). Recent advances in molecular biology have revealed the extent of polymorphism at these loci (2). Amerindian populations are characterized by restricted levels of polymorphism both in HLA class I and class II alleles, but several studies have found new variants, particularly at the HLA-B locus, many of them the result of gene conversion or interallelic recombination events (3, 4). To extend and refine the analysis of HLA alleles in relation to population movement and development through time, we have carried out the first highresolution HLA profile (HLA-A, -B, -C, -DRB1, -DQB1
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and -DPB1) of a Mexican Amerindian group: the Tarahumaras. They have an estimated population of approximately 121,835 inhabitants (75,534 being monolingual and older than 5 years of age) organized in very small communities (two to five families) distributed in the mountains and canyons of the Sierra Madre Occidental in Northwest of Mexico (Figure 1). Tarahumaras call themselves Raramuri (the fleet-footed ones). They are acknowledged to be among the few tribes in North America that have preserved their traditional lifestyle almost unmodified by three and a half centuries of contact with Caucasian and Mexican mestizo populations (5), maybe due to cultural and geographic reasons. Their

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United States of America

Chihuahua State Tarahumara region

Gulf of Mexico MEXICO Pacific Ocean Central America

Figure 1 Geographic location of the Tarahumaras on the Northwest Mexico.

language is traditionally classified into the Yuto-Nahua trunk (6). The Yuto-Nahua, Macro-Nahua, Uto-Nahua, Yuto-Aztec or Uto-Aztec are considered as synonyms and they include a wide range of languages, stretching from Idaho, Montana and Wyoming all the way down to El Salvador in Central America. The Tarahumara language, as part of the Yuto-Nahua trunk, is considered altogether with Concho and Guarij os into the subgroup Cah ta Opata-Tarahumara, closely related to the subgroup Pima-Tepehuano and Cora-Huichol (the Pima-Cora family). Even subtle dialectal differences have been recorded in the Tarahumara language (Central, Lowland, Northern, Southeastern and Southwestern Tarahumara) there are no problems of communication among Tarahumara-speaking people, a reason why the linguistics researchers usually consider no dialectal differences (6, 7). To obtain information about MHC and other genetic systems is relevant to aid in the eventual reconstruction of the route of the peopling of the American continent. In the present work, we have (1) determined the high-resolution HLA class I and class II Tarahumara alleles by the reference strand-mediated conformation analysis (RSCA) method and (2) based on the current HLA-DRB1 and -DQB1 data, inferred relatedness between Tarahumaras with other Amerindian groups and worldwide populations.
Materials and methods
Population samples

have 23 different birthplace villages distribuited in five municipalities (the aforementioned plus Urique, Uriachi and Guazaparez municipalities) of the 10 municipalities belonging to the officially recognized Tarahumara region (by Instituto Nacional Indigenista): Guadalupe y Calvo, Morelos, Balleza, Guachochi, Batopilas, Urique, Guazaparez, Moris, Uruach Ch nipas, Maguarichi, Bocoyna, Nonoava, Carich , Ocampo, Guerrero y Temosachi; all of them at the core of the Tarahumara mountains. Each individual had a typical Tarahumara phenotype, their four grand parents were born in the Tarahumara region and that exclusively spoke the Tarahumara language. The main reason to take samples of only places such as these was to try to diminish the chance of find a possible admixture with Caucasian alleles given the fact that this population, as many of the ethnic groups in Mexico, are in continuous migratory processes developing economical activities and cultural interacting with other Amerindian groups or Mexican mestizo population; in addition to geographic location, selection based on language was made taking into account that other ethnic groups (Guarij os, Tepehuanos, Pimas and Seri) are living in the same or closer States. All samples were taken with appropriate written informed consent. Efforts were taken to ensure siblings of those already sampled were excluded. The origin of all other populations used for comparison is detailed in Table 1. In total, 14,868 chromosomes were studied, including populations from different origins (Caucasoids, Orientals, African origin, Polynesians, Micronesians, Na-Dene, Eskimos and Amerindians). In particular, the Amerindian group includes populations from the linguistic families of Macro-mixteco (Mixtecan and Zapotecan), Olmec-Otomangue (Mazatecan), Macro-Maya (Mixe), Macro-Yuma (Seri), Maya-Quiche (Mayans), Chibcha (Arsarian, Kogi, Arhuacan and Cayapan), Tupi-Guarani (Guarani), Aymara (Aymaras), Quechua (Quechuans), Arawak (Wayu and Terena), Caribe (Jaidukama) and Ge Pano Caribe (Xavantes, Mataco and Toba) (7, 9).

HLA allele typing and statistical analysis

DNA was extracted by standard protocols (8) from peripheral blood lymphocytes and collected from 44 healthy unrelated individuals from the Tarahumara ethnic group, living in the Bocoyna and Guachochi municipalities of the state of Chihuahua, in the Northwestern Mexico (Figure 1), they

Allelic HLA typing was performed by RSCA for class I (HLA-A, -B, -C) and class II (HLA-DPB1, -DRB1) as previously described (10); at least two locus-specific labelled reference strands were used for each locus. Ten HLA-A, 13 HLA-B, 10 HLA-C, five HLA-DPB1, nine HLA-DQB1 and 14 HLA-DRB1 alleles were identified. ALLELE LINKSTM software (Amersham Pharmacia Biotech, Uppsala, Sweden) was used for the normalization and analysis of the results. HLADQB1 alleles were typed using Dynal RELITM SSO (Dynal, The Wirral, UK), according to the manufacturers protocol. Sequence-based typing of some selected HLA-A, -B and -C products was performed as previously described (11). Allele frequencies were obtained by direct counting. HardyWeinberg equilibrium was tested according to the

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HLA genes in Tarahumaras

Table 1 Populations studied in the present work ID 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 Population Tarahumaras Aymaras Quechuans Mazatecans Mayans Seri Mixe Mixteco Zapotecans Mexican Mestizo Wayu Arhuaco Terena Kogi Arsario Cayapa Xavantes Guarani Toba Pilaga Mataco Wichi Eastern Toba Jaidukama Eskimos Athabaskans Tlingit Nivkhs Udegeys Koryaks Chukchi Kets Evenks Singapore Chinese Buyi Manchu Koreans Japanese n 44 102 80 89 132 100 55 103 75 99 112 123 60 67 20 100 74 32 19 49 135 39 35 124 53 32 23 92 59 22 35 71 70 50 100 493 Reference Present study
a

ID 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72

Population Khalk Mongolian Tuvins Khoton Mongolian Germans Sardinians Italians French Spaniards Spanish Basques Algerians Berbers (Souss) Moroccans Albanians Macedonians Cretans Ashkenazi Jews Non-Ashkenazi Jews Lebanese NS Lebanese KZ Moroccan Jews Danish Chuvash Russians Western Samoa Madang Rabaul New Caledonia Fidji Papua New Guinea Central Desert Ainu Yuendumu Cape York Kimberley North American Blacks South American Blacks

n 202 197 85 295 91 284 179 176 80 102 98 98 65 172 135 80 80 59 93 94 124 82 200 102 65 60 65 57 57 152 50 119 80 82 447 59

Reference
a a a a a a a a a a

(57)
a a a a a a

Vargas-Alarcon et al., unpublished

a a a

a a

Arnaiz-Villena et al., unpublished

a a a a a a a a a

a a a a a a a a

Martinez-Laso et al., unpublished

a a a a a a a a a a a a a a

a a a a a a a a a a a a a a

Terena Indians, from Mato Grosso do Sul (South Central Brazil), originally from Paraguay; Western Samoa, from Central Polynesia; Madang, Melanesians from the North New Guinea mainland coast; Rabaul, Melanesians from New Britain; New Caledonia, Melanesians from this island; Fidji, from the largest Fidjian island; Central desert, Yuendumu, Kimberley and Cape York, Australian aborigines; Ainu, inhabitants of Hokkaido, Japans northernmost islands. They are believed to be the first Japanese coming from the Asian continent. a Original references are cited in (23) and (46). A total of 14,868 chromosomes were analysed and geographic locations are represented in reference (31)

Monte Carlo method (12). Haplotype frequencies of two, three and six different loci combinations were estimated by the maximum likelihood algorithm described by Excoffier and Slatkin (13). The linkage disequilibrium (LD) coefficient (D) (14) was calculated according to standard formula for joint probability of a set of events (15), standardized D (D) was calculated according to Lewontin (14). Total LD in each locus by D (total D0 ) and D2 (total

D2) were estimated for all the haplotypes. The software CACTUS for Population Genetic Analysis Version 0.0.1b was employed for all these purposes (Maldonado Torres, unpublished data). To compare genotype and haplotype HLA frequencies with other populations, the reference tables of the 11th and 12th International HLA Workshops were used (16, 17), also see Table 1. Phylogenetic trees (dendrograms)

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were constructed with the allelic frequencies by the neighbour-joining (NJ) method (18) with the standard genetic distances (SGD) (19), by using the software DISPAN, which contained the programs GNKDST and TREEVIEW (20). Correspondence analysis in three dimensions and its bi-dimensional representation was carried out by using the VISTA v.5.02 software (21) (http://forrest. psycho.unc.edu), with the Nei genetic distance (20, 21). Correspondence analysis consists of a geometric technique that may be used for displaying a global view of the relationships among populations according to HLA (or other) allele frequencies. This methodology is based on the allelic frequency variance among populations (similar to the classical components methodology) and on the display of the statistical visualization of the differences.
Results
HLA alleles in the Tarahumara population

RSCA typing of 44 Tarahumara DNA samples provided a high-resolution pattern of HLA class I and class II (except HLA-DQB1, which was performed by reverse dot blot, see Material and methods). The correlation between RSCA and other low- and medium resolution methods has been previously demonstrated (22). Ten samples were sequenced four samples for HLA-A: confirming the presence of the alleles A*0206, A*020101, A*0240 and identifying the new allele A*0257; five samples for HLA-B: confirming alleles B*350101, B*3503, B*390602, B*4005 and B*4002; and one sample for HLA-C: confirming the presence of the allele Cw*0304. Table 2 shows the HLA class I and class II allele frequencies found. All the genotype distributions for HLA class I and class II were in HardyWeinberg equilibrium (P > 0.05; data not shown). For HLA class I, five of 10 HLA-A alleles included almost 95% of the sample: HLA-A*240201, *020101/09, *0206, *310102 and *6801. All of those alleles have been observed in other Amerindian populations (3, 4, 23, 24). Seven out of 16 alleles in HLA-B comprised 91% of samples: HLAB*4002, *1501, *510201, *3501/02/03, *4005, *4801 and *270502, all of them previously described in several Amerindian populations (3, 4, 24, 25). Additionally, three more alleles observed in Amerindian groups (25) were found at frequencies below 5%: HLA-B*3905, *390602 and *520102; B*390602 has been described only in North American native populations (25) and it has been proposed that it was derived from a recombination between a B*39 allele and the hypothetical donor allele, B*520102; each allele was observed in one individual, respectively. Compared with South American native populations, which have a significant number of novel HLA-B alleles (4, 26), no new HLA-B allele was identified in our

Tarahumara sample. However, a novel HLA-A allele, now named HLA-A*0257, which differs from A*0206 at two nucleotides: Val95Leu (C355G) and Arg97Met (T362G) was discovered in this population (27). It was also noticeable that alleles virtually absent in some South American ethnic groups and more indicative of North American native populations were present in the Tarahumara population, such as HLA-B*5102 (14.7%) and -B*4005 (11.4%) (28). The presence of HLA class I alleles such as HLA-B*2705, *3501, *5102, *4801 and *4002, this population resembles the geographically close related native North American populations (4). For HLA-C, four of the 10 alleles observed had frequencies near or above 10%: HLA-Cw*030401 (39.8%), *0801 (22.72%), *0102 (13.63%) and *040101 (9.09%). For HLA class II, of five HLA-DPB1 alleles, three comprised 95% of the sample: HLA-DPB1*0402, *0401 and *020102; all of them reported in Amerindian populations amongst others (29). HLA-DPB1*0501 has been reported with high frequencies in Japanese and Indonesians (29), but also in South American Indians and Mexican Amerindians (24). Four of nine HLADQB1 alleles comprised 93%: HLA-DQB1*0402, *0301, *0302/07 and *030302; all of them commonly found in Amerindian populations (30, 31). For HLA-DRB1, four out of the five HLA-DRB1 allelic lineages described in Amerindian populations (DRB1*04, DRB1*08, DRB1*09, DRB1*14, except DRB1*16) (24, 26), comprised 90.7% of the total: HLA-DRB1*040301, *040701, *0411, *080201, *090102 and *1402 (Table 2). However, some striking differences with other Mexican Amerindian populations were observed such as a less diverse DRB1*04 lineage, presence of DRB1*09 alleles, and a noticeable absence of DRB1*16 alleles in the Tarahumara population (24, 31); absence of DRB1*16 has also been reported in some South Amerindian populations (32). The following are some remarkable allele findings: HLADRB1*0411, present in only two Tarahumara individuals, has been reported also in Australian aborigines, Eastern Toba and Bari Amerindians and in the Zuni tribe from North America (33); DRB1*140101 is uncommonly reported in Native Americans, but present in Athabascans, Penutians and Ainu, and its presence in a low frequency might be considered as evidence of a distant common relationship (34); finally, DRB1*1501, identified also in two Tarahumara individuals, is considered as marker of Caucasian admixture, but has been also reported in isolated populations, such as Athabascan, Kogi or Asian subgroups (3436).
Haplotype analysis

Two-locus, three-locus and extended haplotype frequencies, as well as LD for HLA class I and class II were

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Table 2 Allelic distribution of HLA class I and class II in the Tarahumara sample Allele HLA-A A*240201 A*020101/09 A*0206 A*310102 A*6801 A*3001 A*0251 A*0257 A*030101 A*0240 HLA-B B*4002 B*1501 B*510201 B*3501/02/03 B*4005 B*4801 B*270502 B*0702/09 B*4006 B*520102 B*1302 B*3905 B*390602 HLA-DQB1 DQB1*0402 DQB1*0301 DQB1*0302/07 DQB1*030302 DQB1*0602/11 DQB1*0604 DQB1*0603/14 DQB1*0503 DQB1*0201/02 Number observed Frequency Allele HLA-C Cw*0304 Cw*0801 Cw*0102 Cw*040101 Cw*070201 Cw*020202 Cw*1502 Cw*0303 Cw*0602 Cw*1203 HLA-DPB1 DPB1*0402 DPB1*0401 DPB1*020102 DPB1*0501 DPB1*010101 Number observed Frequency

33 22 11 9 8 1 1 1 1 1 18 13 13 12 10 8 6 2 2 1 1 1 1 31 27 19 5 2 1 1 1 1

0.3750 0.2500 0.1250 0.1022 0.0909 0.0113 0.0113 0.0113 0.0113 0.0113 0.2045 0.1477 0.1477 0.1363 0.1136 0.0909 0.0681 0.0227 0.0227 0.0113 0.0113 0.0113 0.0113 0.3522 0.3068 0.2159 0.0568 0.0227 0.0113 0.0113 0.0113 0.0113

35 20 12 8 4 3 2 2 1 1

0.3977 0.2272 0.1363 0.0909 0.0454 0.0340 0.0227 0.0227 0.0113 0.0113

56 19 9 3 1

0.6363 0.2159 0.1022 0.0340 0.0113

HLA-DRB1 DRB1*080201 DRB1*1402 DRB1*040701 DRB1*090102 DRB1*040301 DRB1*150101 DRB1*0411 DRB1*140101 DRB1*110101 DRB1*1406 DRB1*130201 DRB1*130101 DRB1*110201 DRB1*030101

31 28 10 4 3 2 2 2 1 1 1 1 1 1

0.3522 0.3181 0.1136 0.0454 0.0340 0.0227 0.0227 0.0227 0.0113 0.0113 0.0113 0.0113 0.0113 0.0113

computed (12 HLA-A, -B; eight HLA-C, -B; seven HLADQB1, -DPB1; six HLA-DRB1, -DQB1; and 11 HLA-A, -C, -B, -DRB1, -DQB1, -DPB1) and the most frequent haplotypes are shown in Tables 35. These are the first reported extended HLA haplotypes for the Tarahumara population. It can be observed that few of the haplotypes have frequencies above 10%. Table 3 shows the HLA-A-B haplotypes found in the Tarahumara population. We were able to demonstrate the presence of some common previously described Mexican Amerindian haplotypes such as HLA-A*240201-B*1501, -A*020101/09B*4005, -A*240201-B*3501/02/03, -A*310102-B*510201, -A*020101/09-B*4002 and -A*020101/09-B*3501/02/03 (31). HLA-A*020101-B*4801 has been also reported in Mexican Mestizo population (37). The haplotypes HLACw*0304-B*4002, -Cw*0304-B*4005, -Cw*040101B*3501/02/03 and -Cw*0801-B*4801 have been also

reported previously in other populations, including the Mexican Mestizo (37), HLA-Cw*040101-B*3501/02/03 is common in Nahua Amerindian population (38). HLACw*0102-B*1501 and -Cw*020202-B*270502 have been also reported in the Yupik Alaskan natives, the first one with a frequency of 0.6%, compared with 13.6% in the Tarahumara population, and the second one with 10.7% compared with 3.6% (39). Only the haplotype HLACw*0801-B*510201 would be considered as a marker of the Tarahumara. In class II HLA haplotypes (Table 3), the two-locus analysis for HLA-DRB1-DQB1 shows that the three most frequent haplotypes in the Tarahumara population: HLADRB1*080201-DQB1*0402, -DRB1*1402-DQB1*0301 and -DRB1*040701-DQB1*0302/07, are commonly found in Amerindian populations throughout the Americas (23, 31, 32, 35, 4043). DRB1*1402-DQB1*0301 and DRB1*0802-

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Frequency by MLE 0.125 0.0795 0.0774 0.0774 0.0568 0.0568 0.0476 0.0476 0.0455 0.0411 0.0341 0.0341 0.0689 0.7369 0.0222 0.4529 0.3523 0.2727 0.1136 0.0455 0.0455 0.0341 <0.001 <0.001 <0.001 0.4055 <0.001 0.0035 0.13 0.8091 0.2282 0.1751 0.0891 0.0232 0.0429 0.02673 1 0.837 1 0.3386 1 1 DQB1*0402 DQB1*0302/07 DQB1*0301 DQB1*0301 DQB1*0402 DQB1*0301 DQB1*030302 Complete LD by D 2: Complete LD by D 0 : DPB1*0402 DPB1*0402 DPB1*0402 DPB1*0401 DPB1*0401 DPB1*020102 DPB1*0402 0.2775 0.156 0.1461 0.0956 0.0748 0.0651 0.0568 0.0136 0.4054 0.0533 0.0186 0.0492 0.0294 0.0012 0.0337 0.0207 0.416 0.2371 0.2519 0.1964 0.0164 0.4757 1 0.2911 0.6375 0.2964 0.2839 0.9661 0.0741 0.3081 0.032 0.0184 0.0085 0.0156 0.4133 0.27 0.0857 0.1467 0.0095 0.252 0.6171 0.2807 Complete LD by D 2: Complete LD by D 0 : 0.0696 0.0511 0.0007 0.0262 0.0417 0.0312 0.0035 0.0135 0.7538 0.6 0.0053 0.308 0.4785 0.7333 0.0689 0.1322 0.0055 0.0044 0.9816 0.2762 0.0015 0.0668 0.8838 0.4921 Cw*0304 Cw*0801 Cw*0102 Cw*0304 Cw*040101 Cw*0801 Cw*020202 Cw*0304 B*4002 B*510201 B*1501 B*4005 B*3501/02/03 B*4801 B*270502 B*3501/02/03 0.18 0.1477 0.1364 0.1136 0.0909 0.0795 0.0341 0.0341 D D0 P-value Haplotype Frequency by MLE D 0.0987 0.1141 0.1162 0.06844 0.0785 0.0589 0.0317 0.0201 D0 0.80097 1 1 1 1 0.8382 1 0.3714 P-value 0.0012 <0.001 <0.001 0.0025 <0.001 <0.001 <0.001 0.4171

HLA genes in Tarahumaras

Table 3 Two-locus haplotype distribution of HLA class I and class II in the Tarahumara population

Haplotype

A*240201 A*020101/09 A*240201 A*240201 A*310102 A*240201 A*020101/09 A*020101/09

B*1501 B*4005 B*4002 B*3501/02/03 B*510201 B*270502 B*4002 B*3501/02/03

A*6801 A*020101/09 A*0206 A*0206

B*510201 B*4801 B*4002 B*510201

Complete LD by D 2: Complete LD by D 0 :

DRB1*080201 DRB1*1402 DRB1*040701 DRB1*1402 DRB1*090102 DRB1*040301

DQB1*0402 DQB1*0301 DQB1*0302/07 DQB1*0302/07 DQB1*030302 DQB1*0302/07

Complete LD by D 2: Complete LD by D 0 :

LD, linkage disequilibrium. J. E. Garca-Ortiz et al.

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Table 4 Three-locus haplotype distribution of HLA class I and class II in the Tarahumara population Haplotype A*240201 A*020101/09 A*240201 A*310102 A*240201 A*6801 A*020101/09 A*240201 A*020101/09 A*0206 A*0206 Cw*0102 Cw*0304 Cw*0801 Cw*0801 Cw*0304 Cw*0801 Cw*0304 Cw*040101 Cw*040101 Cw*0801 Cw*0304 Complete LD by D 2: Complete LD by D 0 : DRB1*080201 DRB1*1402 DRB1*040701 DRB1*1402 DRB1*080201 DRB1*1402 DRB1*090102 DRB1*040301 DQB1*0402 DQB1*0301 DQB1*0302/07 DQB1*0301 DQB1*0402 DQB1*0301 DQB1*030302 DQB1*0302/07 Complete LD by D 2: Complete LD by D 0 : LD, linkage disequilibrium. B*1501 B*4005 B*4801 B*510201 B*4002 B*510201 B*4002 B*3501/02/03 B*3501/02/03 B*510201 B*4002 0.0347 0.3782 DPB1*0402 DPB1*0402 DPB1*0402 DPB1*0401 DPB1*0401 DPB1*020102 DPB1*0402 DPB1*0402 0.2785 0.1192 0.1136 0.0967 0.0738 0.0568 0.0455 0.03410 0.0851 0.6454 0.1995 0.0571 0.0980 0.0756 0.0470 0.0468 0.0438 0.0294 0.7301 0.2334 1.0000 0.3880 0.2485 0.5075 1.0000 0.9999 <0.001 0.0316 <0.001 <0.001 0.0071 <0.001 <0.001 <0.001 Frequency by MLE 0.1136 0.0795 0.0568 0.0568 0.0511 0.0455 0.0455 0.0455 0.0341 0.0341 0.0341 D 0.1061 0.0683 0.0491 0.0534 0.0206 0.0424 0.0251 0.0408 0.0310 0.0299 0.0239 D0 0.8236 0.6669 0.5901 0.5401 0.1185 0.4826 0.1363 0.4731 0.3529 0.2475 0.2083 P-value <0.001 <0.001 <0.001 <0.001 0.2679 <0.001 0.0985 <0.001 <0.001 <0.001 0.0261

DQB1*0402 are also common haplotypes in the Na-Dene and Ainu populations, and indicate a common origin (35). Only one of the most commonly found haplotypes in the Mestizo population (HLA-DRB1*04-DQB1*0302) was present in our sample (indeed, represented by two haplotypes: HLA-DRB1*040701-DQB1*0302/07 and -DRB1*0411DQB1*0302/07). The most commonly found HLADRB1-DQB1 haplotype in the Tarahumara population, -DRB1*080201-DQB1*0402 (see Table 3), is found in LD in Mexican Mestizo population, North American Amerindians

and South American Amerindians (31, 35, 37, 44). One of the most fixed HLA-DRB1-DQB1 haplotype in Amerindian populations (DRB1*1602-DQB1*0301) (43), was absent in the Tarahumara sample; however, its absence has also been observed in some South American ethnic groups from Colombia and Argentina (32). The most frequent three-locus haplotypes for HLA class I and class II are shown in Table 4, almost all of them have been previously described in Amerindian and Asian populations (45). The extended haplotypes in Tarahumara

Table 5 Extended haplotype distribution of HLA class I and class II in the Tarahumara population Haplotype A*240201 A*020101/09 A*310102 A*240201 A*240201 A*240201 A*020101/09 A*240201 A*020101/09 A*6801 Cw*0102 Cw*0304 Cw*0801 Cw*0304 Cw*0102 Cw*0304 Cw*0304 Cw*0801 Cw*0304 Cw*0801 B*1501 B*4005 B*510201 B*4002 B*1501 B*270502 B*3501/02/03 B*4801 B*4002 B*510201 DRB1*1402 DRB1*080201 DRB1*080201 DRB1*080201 DRB1*1402 DRB1*1402 DRB1*080201 DRB1*080201 DRB1*1402 DRB1*080201 DQB1*0301 DQB1*0402 DQB1*0402 DQB1*0402 DQB1*0301 DQB1*0301 DQB1*0402 DQB1*0402 DQB1*0301 DQB1*0402 Complete LD by D 2: Complete LD by D 0 : LD, linkage disequilibrium. DPB1*0402 DPB1*0402 DPB1*0402 DPB1*0402 DPB1*0401 DPB1*0401 DPB1*0402 DPB1*0401 DPB1*020102 DPB1*0402 Frequency by MLE 0.0795 0.0511 0.0455 0.0341 0.0341 0.0341 0.0341 0.0341 0.0341 0.0341 0.0273 0.1185 D 0.0791 0.0502 0.0452 0.0317 0.0339 0.0339 0.0330 0.0339 0.0339 0.0339 D0 0.5819 0.4456 0.4430 0.1567 0.2491 0.4984 0.2441 0.3736 0.3320 0.3733 P-value <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001

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population shown in Table 5, are the first Amerindian ones reported with high-resolution typing techniques; they differ with previously extended haplotypes, mainly due to differences in allele frequencies (higher frequencies of HLA-A*2402, B*1501 and DRB*1402, and lower frequencies or absence of the B39 and DRB16 subgroups). Only two extended haplotypes (A*020101/09-Cw*0304B*4005-DRB1*080201-DQB1*0402-DPB1*0402 (0.0511) and A*020101/09-Cw*0304-B*3501/02/03-DRB1*080201DQB1*0402-DPB1*0402 (0.0341) can be inferred from previously reported extended haplotypes in Aymaras, Mayans, Peruvian Indians, Quechuas, Nahuas, Seris and Yupik Alaskan natives (23, 39, 44, 46) and might suggest the existence of a possible relationship between them (46).
Population comparisons

Inter-population analysis shows that all human populations are related according to a smooth geographic gradient (31), but Amerindians are separated from all the other populations, and they are included in a separate branch (Figures 2 and 3). Current available high-resolution data on loci with limited polymorphisms, as DRB1 and DQB1 loci, may show spurious relationships due to low frequencies or absence of some haplotypes and higher frequencies of others, and mainly based on the sharing of alleles. The Tarahumaras cluster with South American Amerindians, both in the NJ and the correspondence analysis. Particularly, Tarahumaras are genetically closer to South American Amerindians (Terena and Aymara, Figures 2 and 3) than to other Mesoamerican Amerindians, who now live geographically closer. Thus, it is not possible to create a correlation between geography and genetics, as observed before for other Amerindian groups (23, 31) (Table 1). Also, the genetic analysis highlights a close relatedness of the probably more ancient American inhabitants. It contrasts with the fact that Eskimo and North American Na-Dene ethnic groups are closer to Siberians (Figures 2 and 3), and have probably come into the Americas in latter times.
Discussion
HLA alleles in the Tarahumara population

There is one previous report from the Tarahumara population describing only 24 class I and 10 class II HLA alleles at a low-resolution level (47), here we report 32 class I and 24 class II HLA alleles. A related work on Tarahumara population has been recently published as an abstract in the 35th ASMASI and 14th IHIW [Tissue Antigens 2005: 66: 343604] typed 110 non-related individuals for intermediate resolution typing of the HLA class I and class II reporting 21 class I and 15 class II HLA alleles. All these alleles and class I haplotypes were found in similar

proportions than our results shown on Tables 2 and 4, confirming the homogeneity in terms of HLA alleles of the Tarahumara population. The authors recognized that differences exist between the Tarahumaras and other Mexican ethnic groups as the Seri (linguistically and geographically related), Lacandon, Maya descendants, Mixteco, Mixe and Zapoteco groups. Despite the small size of the present sample, the use of RSCA allowed us to measure levels of polymorphism in the HLA system more accurately, and the present range of polymorphic loci clearly shows the dominant presence of alleles previously reported in Amerindian populations for all the analysed loci. For HLA class I: HLA-A*240201, *020101/09, *0206, *310102 and *6801; HLA-B*4002, *4005, *1501, *270502, *3501, *3503, *3905, *390602, *4801, *510201 and *520102; HLA-Cw*0304, *0801, *0102, *04 0101, *0702, *0303 and *1502; and for HLA class II: HLA-DPB1*0402, *0401, *020102; HLADQB1*0402, *0301, *0302/07 and *030302; HLADRB1*080201, *1402, *040701, *040301 and *0411. Some authors have established the range of alleles in Amerindian populations, at least for HLA class I: three to six for HLAA, 620 for HLA-B and three to five for HLA-C (48), and also have proposed ancestral alleles for the Amerindian populations HLA-A: A*020101/09, *2402, *310102 and *6801; HLA-B: B*1501, *270502, *3501, *390101, *4002, *4801, *510101 and *520102; HLA-C: Cw*0102, *02022, *0304, *0401, *0702, *0801 and *1502 (3, 4). Our population sample was in the range for HLA-B, but above in HLA-A and HLA-C. It was noteworthy that allelic frequencies of some commonly observed alleles in Amerindians, such as B*3905, B*390101 or DRB1*1602, were present in very low frequency (B*3905: 1.3%) or absent in the Tarahumara sample, respectively. It was also noteworthy that of the 20 hypothetical ancestral Amerindian HLA alleles (28), considered as part of a minimum allelic set brought to America by the migrant population that gave rise to the Paleo-indians, eight of nine ancestral alleles and three of 11 novel Amerindian alleles were present in the Tarahumara sample, including HLA-B*4005, the first novel Amerindian allele defined in a North American population (the Pima of Arizona) (49). The allelic distribution in this populations resembles the pattern found in North American populations such as the Havasupai (4), which tends to remain static if compared with South American indigenous populations. Additionally, until now, data have indicated that Northern tribes (including the Tarahumara population) are more likely to bear ancestral alleles of the original founder populations, whereas southern groups frequently show new alleles in the HLA system, mainly in HLA-B locus for class I and DRB1 for class II (50). Several suggestions have been proposed to explain the high proportion of these new alleles in South American Amerindian populations such as random genetic drift and balancing, as well as pathogen-driven selection,

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Germans Russians

Figure 2 Neighbour-joining dendrogram showing relatedness between Tarahumaras and other Amerindian, Na-Dene, Eskimo, Asian, African origin and European populations. Genetic distances between populations (DA) were calculated using high-resolution HLADRB1 and -DQB1 genotyping (four digits). Data from other populations were taken from references detailed in Table 1. The tree was unrooted and genetic distances are proportional to branch lengths. North Am. Blacks, North American Blacks; South Am. Blacks, South American Blacks.

Wayu

Chuvash Danish Khoton-Mongolian Tuvinians Khalk-Mongolian Japanese Korean Manchu Buyi Singapore Chinese Evenks Kets Tlingit Nivkhs Athabaskan Udegeys Koryaks Chukchi Eskimos Guarani

Quechuas Mayans

Toba-Pilaga Mataco-Wichi Eastern-Toba Arhuaco Tarahumara Aymaras Terena Zapotecans Jaidukama Cayapa Mazatecans Kogi Arsario Seri Mixteco Xavantes Mixe

leading to preferential maintenance of new alleles (generated mainly by gene conversion) (4, 25, 48). However, in this instance, we were able to detect and identify a new allele in our sample: HLA-A*0257 (1.13%). Due to the small sample size analysed, it would be impossible to discern if its low frequency is caused by selective pressure, genetic drift or recent origin. More wide application of high-resolution typing methods will help to see how the views about numbers of new alleles may change in Amerindian populations. Nonetheless, it has been observed that identification of unique alleles into a population suggests that selection has occurred since the founding populations established themselves or alternatively, the founding population consisted of the entire ancestral population which brought the novel alleles with them (51).

Language and genetic conflict in ascribing population studies

Analysing HLA class II genetic relatedness among Amerindians, we observed the following striking facts:
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(1) They appear separated from other world ethnic groups. This could be earlier because of a limited number of alleles in the founder populations, or later due to a tight bottleneck after 1492 AD (it has been estimated that about 60 million American Indians died as a consequence of infectious diseases brought to the Americas from Europe). (2) Meso-, South and also North American Amerindians cluster according to HLA with no discernible geographic correlation (52) (Table 1). For example, in our Figure 2 (NJ), North American Tarahumaras from the NahuaCuitlateco group, Pima Cora Family (6) cluster with Bolivian Aymaras and Brazilian Paraguayan Terenas (52) (Table 1). One might think that Amerindian populations would cluster in the genetic analysis according to degree of admixture. However, the Maya (23) (Table 1) and Aymara ethnic groups show little admixture and are placed quite distant from one to another (Figures 2 and 3). Also, Terenas have been more isolated than Aymaras, with less resultant admixture, but cluster with the Aymaras.

Amerindians
143

Siberians/ Eskimos

Orientals

Central Europe

North Am Blacks South Am Blacks Lebanese-NS Lebanese-KZ Moroccan Jews Ashkenazi Jews Non Ashkenazi Jews Italians Sardinians Macedonians Cretans Moroccans French Spaniards Spanish Basques Algerians Berbers

Mediterraneans

Africans

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J. E. Garca-Ortiz et al.

0.4

Dimension 2

Moroccans Albanians Non-Ashkenazi Jews Spaniards Mexican Mestizo Lebanese KZ French Lebanese NS Cretans Ashkenazi Jews Italians Chuvash Danish Algerians North American Blacks Russians Sardinians South American Blacks Macedonians Spanish Basques Germans Tuvinians Berbers Khoton Mongolian Khalk Mongolian Korean Tlingit Kets Evenks Madang Japanese Rabaul

Mayans Arhuaco Quechuas Eastern-Toba Toba-Pilaga Zapoteco Seri Mazateco Mixe Mataco-Wichi Kogi Arsario Tarahumara Aymaras Mixteco Jaidukama Cayapa Xavantes Guarani Terena Wayu

Singapore Chinese Buyi Manchu New Caledonia Fidji

Udegeys Athabaskan Chukchi Eskimos Koryaks Ainu Nivkhs

W Samoa

Kimberley Yuendumu Papua Cape York Central Desert

0.4 0.6 Dimension 1 0.6

Figure 3 Correspondence analysis showing a global view of the relationship among Amerindian, Na-Dene, Eskimo, Asian, African origin, European, Australian and Polynesian populations according to HLA-DRB1 high-resolution (four digits) allele frequencies (a two-dimensional representation).

(3) Language and genetics do not correlate among Amerindian groups. Tarahumara indians belong to the Nahua linguistic group, Aymaras to the unrelated Andean group, and Terena to the altogether different Arawak linguistic group (7). This raises the possibility that other (HLA) genetically close Amerindians may show similar concordance with language subgroups. These results offer a challenge to further research. Currently, the possible explanations have severe problems. For example, one might suggest that Amerindian languages are not yet properly classified, but the formal linguistic rules that have been applied have been found effective in many other cases, and no alternative approach has been suggested. Inconsistencies in current linguistic classifications as Greenbergs, have been previously suggested by genetic analysis of the Y chromosome (53), mtDNA (54) and HLA system (23, 46), in addition to the effect of genetic drift, genic flow or selection (4). (4) Interestingly, the HLA analysis in the Tarahumara population suggests that genetic drift has been an important factor in the configuration of the current genetic structure, particularly indicated by high frequencies of HLA-B*510201 and B*4005 alleles and very low frequencies or absence of HLA-B*3905, B*390602 or DRB1*16 alleles. Even this observation has been also previously suggested for HLA class II (35) in Amerindians, and for

STRs/VNTRs for the Tarahumara population amongst other Mexican Amerindian populations (55); however, given the small size of the population studied, it is impossible to rule out the possibility of sampling error.
Conclusions

The critical importance of infectious diseases arriving with Europeans as a decisive selective force in the devastation of Amerindian populations is incontrovertible. However, the differential response to the disease challenge is presumably of at least secondary importance because it would help to determine selective survival. Likely to be relevant are recent studies demonstrating a wide range of peptides presented by the restricted number of HLA class I molecules present within Amerindian populations (4, 26). Our data further support the uniqueness of the Amerindian populations and may also point towards a more complex pattern of peopling of the American continent, contradicting the simplistic hypothesis that America was populated by a wave of individuals who came across the Bering strait (48) and moved monotonically south. Perhaps movements also occurred in the opposite direction, from South America towards North America and Asia (23). We have observed an instance related to the conjectures of Boas (56). Even a caveat about the relatively small Tarahumara

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population sample used for this paper should be taken into account when interpreting the obtained extended haplotypes, and that those conclusions would be interpreted as premature and speculative, it is also important to emphasize that current genetic distance estimations with previous published data are problematic because differences in allele resolution exist between studies; so initial steps toward a more complete drawing of the HLA variability of the Amerindian populations have to be done, and future work will be helpful to better determine the whole picture now distorted by the lack of information about high-resolution HLA class I and class II in Amerindian populations. In summary, high-resolution HLA profile for HLA-A, -B, -C, -DRB1, -DQB1 and -DPB1 has been established for the Tarahumara tribe. HLA allele frequencies were largely in agreement with previous reports in Amerindians, but HLA genetic relatedness among Amerindians was clearly discrepant from geographic and linguistic expectations.
Acknowledgments

To Bambos Soteriou, Anila Shah and Karla Lam-Haces for technical assistance, and to Lizette M. Cortes-Prieto and David Schlessinger for a critical review of the manuscript.

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