high-resolution molecular characterization of the hla class i and class ii in the tarahumara...

High-resolution molecular characterization of the HLA class Iand class II in the Tarahumara Amerindian populationJ. E. Garcıa-Ortiz1,2, L. Sandoval-Ramırez2,3, H. Rangel-Villalobos3,4, H. Maldonado-Torres5, S. Cox5,C. A. Garcıa-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 Inmunobiologıa 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 Inmunologıa y Biologıa 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 Inmunobiologıa Molecular

Centro de Investigacion Biomedica

Facultad de Medicina

Universidad Autonoma de Coahuila

Gregorio A. Garcıa 198 Sur, CP 27000

Torreon, Coahuila

Mexico

Tel: þ52 871 7226470

Fax: þ52 871 7226470

e-mail: [email protected]

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 lin-

guistically 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 biol-

ogy 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 develop-

ment through time, we have carried out the first high-

resolution HLA profile (HLA-A, -B, -C, -DRB1, -DQB1

and -DPB1) of a Mexican Amerindian group: the

Tarahumaras. They have an estimated population of

approximately 121,835 inhabitants (75,534 being monolin-

gual and older than 5 years of age) organized in very small

communities (two to five families) distributed in the moun-

tains and canyons of the Sierra Madre Occidental in

Northwest of Mexico (Figure 1). Tarahumaras call them-

selves 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

Tissue Antigens ISSN 0001-2815

� 2006 The AuthorsJournal compilation 68 (135–146) � 2006 Blackwell Munksgaard 135

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 Guarijıos into the subgroup Cahıta-

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 refer-

ence 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

DNA was extracted by standard protocols (8) from periph-

eral blood lymphocytes and collected from 44 healthy unre-

lated 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

have 23 different birthplace villages distribuited in five muni-

cipalities (the aforementioned plus Urique, Uriachi and

Guazaparezmunicipalities) of the 10municipalities belonging

to the officially recognized Tarahumara region (by Instituto

Nacional Indigenista): Guadalupe y Calvo, Morelos, Balleza,

Guachochi, Batopilas, Urique, Guazaparez, Moris, Uruach

Chınipas, Maguarichi, Bocoyna, Nonoava, Carichı,

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 geo-

graphic location, selection based on language was made tak-

ing into account that other ethnic groups (Guarijıos,

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 popula-

tions 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

Allelic HLA typing was performed by RSCA for class I

(HLA-A, -B, -C) and class II (HLA-DPB1, -DRB1) as pre-

viously 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 soft-

ware (Amersham Pharmacia Biotech, Uppsala, Sweden) was

used for the normalization and analysis of the results. HLA-

DQB1 alleles were typed using Dynal RELITM SSO (Dynal,

The Wirral, UK), according to the manufacturer’s 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.

Hardy–Weinberg equilibrium was tested according to the

United States of America

Central America

MEXICO

Gulf of Mexico

Pacific Ocean

ChihuahuaState

Tarahumararegion

Figure 1 Geographic location of the Tarahumaras on the Northwest

Mexico.

HLA genes in Tarahumaras J. E. Garcıa-Ortiz et al.

136� 2006 The Authors

Journal compilation 68 (135–146) � 2006 Blackwell Munksgaard

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) coeffi-

cient (D) (14) was calculated according to standard for-

mula 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)

Table 1 Populations studied in the present work

ID Population n Reference ID Population n Reference

1 Tarahumaras 44 Present study 37 Khalk Mongolian 202 a

2 Aymaras 102 a 38 Tuvins 197 a

3 Quechuans 80 (57) 39 Khoton Mongolian 85 a

4 Mazatecans 89 a 40 Germans 295 a

5 Mayans 132 a 41 Sardinians 91 a

6 Seri 100 a 42 Italians 284 a

7 Mixe 55 a 43 French 179 a

8 Mixteco 103 a 44 Spaniards 176 a

9 Zapotecans 75 a 45 Spanish Basques 80 a

10 Mexican Mestizo 99 Vargas-Alarcon et al.,

unpublished

46 Algerians 102 a

11 Wayu 112 a 47 Berbers (Souss) 98 a

12 Arhuaco 123 a 48 Moroccans 98 a

13 Terena 60 a 49 Albanians 65 Arnaiz-Villena et al.,

unpublished

14 Kogi 67 a 50 Macedonians 172 a

15 Arsario 20 a 51 Cretans 135 a

16 Cayapa 100 a 52 Ashkenazi Jews 80 a

17 Xavantes 74 a 53 Non-Ashkenazi Jews 80 a

18 Guarani 32 a 54 Lebanese NS 59 a

19 Toba Pilaga 19 a 55 Lebanese KZ 93 a

20 Mataco Wichi 49 a 56 Moroccan Jews 94 a

21 Eastern Toba 135 a 57 Danish 124 a

22 Jaidukama 39 Martinez-Laso et al.,

unpublished

58 Chuvash 82 a

23 Eskimos 35 a 59 Russians 200 a

24 Athabaskans 124 a 60 Western Samoa 102 a

25 Tlingit 53 a 61 Madang 65 a

26 Nivkhs 32 a 62 Rabaul 60 a

27 Udegeys 23 a 63 New Caledonia 65 a

28 Koryaks 92 a 64 Fidji 57 a

29 Chukchi 59 a 65 Papua New Guinea 57 a

30 Kets 22 a 66 Central Desert 152 a

31 Evenks 35 a 67 Ainu 50 a

32 Singapore Chinese 71 a 68 Yuendumu 119 a

33 Buyi 70 a 69 Cape York 80 a

34 Manchu 50 a 70 Kimberley 82 a

35 Koreans 100 a 71 North American Blacks 447 a

36 Japanese 493 a 72 South American Blacks 59 a

Terena Indians, from Mato Grosso do Sul (South Central Brazil), originally from Paraguay; Western Samoa, from Central Polynesia; Madang, Mela-

nesians 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, Japan’s north-

ernmost islands. They are believed to be the first Japanese coming from the Asian continent.aOriginal references are cited in (23) and (46).

A total of 14,868 chromosomes were analysed and geographic locations are represented in reference (31)

J. E. Garcıa-Ortiz et al. HLA genes in Tarahumaras


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 dimen-

sions 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 pre-

sence of the alleles A*0206, A*020101, A*0240 and iden-

tifying 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 geno-

type distributions for HLA class I and class II were in

Hardy–Weinberg 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: HLA-

B*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 popu-

lations, 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 notice-

able that alleles virtually absent in some South American

ethnic groups and more indicative of North American

native populations were present in the Tarahumara popu-

lation, 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 popu-

lation 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 popula-

tions 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 HLA-

DQB1 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), com-

prised 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 popula-

tion (24, 31); absence of DRB1*16 has also been reported

in some South Amerindian populations (32). The follow-

ing are some remarkable allele findings: HLA-

DRB1*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 mar-

ker of Caucasian admixture, but has been also reported in

isolated populations, such as Athabascan, Kogi or Asian

subgroups (34–36).

Haplotype analysis

Two-locus, three-locus and extended haplotype frequen-

cies, as well as LD for HLA class I and class II were




computed (12 HLA-A, -B; eight HLA-C, -B; seven HLA-

DQB1, -DPB1; six HLA-DRB1, -DQB1; and 11 HLA-A, -C,

-B, -DRB1, -DQB1, -DPB1) and the most frequent

haplotypes are shown in Tables 3–5. 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 hap-

lotypes such as HLA-A*240201-B*1501, -A*020101/09-

B*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 HLA-

Cw*0304-B*4002, -Cw*0304-B*4005, -Cw*040101-

B*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). HLA-

Cw*0102-B*1501 and -Cw*020202-B*270502 have been

also reported in the Yup’ik 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 HLA-

Cw*0801-B*510201 would be considered as a marker of

the Tarahumara.

In class II HLA haplotypes (Table 3), the two-locus ana-

lysis for HLA-DRB1-DQB1 shows that the three most

frequent haplotypes in the Tarahumara population: HLA-

DRB1*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, 40–43). DRB1*1402-DQB1*0301 and DRB1*0802-

Table 2 Allelic distribution of HLA class I and class II in the Tarahumara sample

Allele Number observed Frequency Allele Number observed Frequency

HLA-A HLA-C

A*240201 33 0.3750 Cw*0304 35 0.3977

A*020101/09 22 0.2500 Cw*0801 20 0.2272

A*0206 11 0.1250 Cw*0102 12 0.1363

A*310102 9 0.1022 Cw*040101 8 0.0909

A*6801 8 0.0909 Cw*070201 4 0.0454

A*3001 1 0.0113 Cw*020202 3 0.0340

A*0251 1 0.0113 Cw*1502 2 0.0227

A*0257 1 0.0113 Cw*0303 2 0.0227

A*030101 1 0.0113 Cw*0602 1 0.0113

A*0240 1 0.0113 Cw*1203 1 0.0113

HLA-B

B*4002 18 0.2045 HLA-DPB1

B*1501 13 0.1477 DPB1*0402 56 0.6363

B*510201 13 0.1477 DPB1*0401 19 0.2159

B*3501/02/03 12 0.1363 DPB1*020102 9 0.1022

B*4005 10 0.1136 DPB1*0501 3 0.0340

B*4801 8 0.0909 DPB1*010101 1 0.0113

B*270502 6 0.0681

B*0702/09 2 0.0227

B*4006 2 0.0227 HLA-DRB1

B*520102 1 0.0113 DRB1*080201 31 0.3522

B*1302 1 0.0113 DRB1*1402 28 0.3181

B*3905 1 0.0113 DRB1*040701 10 0.1136

B*390602 1 0.0113 DRB1*090102 4 0.0454

HLA-DQB1 DRB1*040301 3 0.0340

DQB1*0402 31 0.3522 DRB1*150101 2 0.0227

DQB1*0301 27 0.3068 DRB1*0411 2 0.0227

DQB1*0302/07 19 0.2159 DRB1*140101 2 0.0227

DQB1*030302 5 0.0568 DRB1*110101 1 0.0113

DQB1*0602/11 2 0.0227 DRB1*1406 1 0.0113

DQB1*0604 1 0.0113 DRB1*130201 1 0.0113

DQB1*0603/14 1 0.0113 DRB1*130101 1 0.0113

DQB1*0503 1 0.0113 DRB1*110201 1 0.0113

DQB1*0201/02 1 0.0113 DRB1*030101 1 0.0113



Tab

le3

Tw

o-locus

haplo

type

dis

trib

ution

of

HLA

cla

ss

Iand

cla

ss

IIin

the

Tara

hum

ara

popula

tion

Haplo

type

Fre

quency

by

MLE

DD0

P-v

alu

eH

aplo

type

Fre

quency

by

MLE

DD0

P-v

alu

e

A*240201

B*1501

0.1

25

0.0

696

0.7

538

0.0

055

Cw

*0304

B*4002

0.1

80.0

987

0.8

0097

0.0

012

A*020101/0

9B

*4005

0.0

795

0.0

511

0.6

0.0

044

Cw

*0801

B*510201

0.1

477

0.1

141

1<

0.0

01

A*240201

B*4002

0.0

774

0.0

007

0.0

053

0.9

816

Cw

*0102

B*1501

0.1

364

0.1

162

1<

0.0

01

A*240201

B*3501/0

2/0

30.0

774

0.0

262

0.3

08

0.2

762

Cw

*0304

B*4005

0.1

136

0.0

6844

10.0

025

A*310102

B*510201

0.0

568

0.0

417

0.4

785

0.0

015

Cw

*040101

B*3501/0

2/0

30.0

909

0.0

785

1<

0.0

01

A*240201

B*270502

0.0

568

0.0

312

0.7

333

0.0

668

Cw

*0801

B*4801

0.0

795

0.0

589

0.8

382

<0.0

01

A*020101/0

9B

*4002

0.0

476

�0.0

035

�0.0

689

0.8

838

Cw

*020202

B*270502

0.0

341

0.0

317

1<

0.0

01

A*020101/0

9B

*3501/0

2/0

30.0

476

0.0

135

0.1

322

0.4

921

Cw

*0304

B*3501/0

2/0

30.0

341

�0.0

201

�0.3

714

0.4

171

A*6801

B*510201

0.0

455

0.0

32

0.4

133

0.0

095

Com

ple

teLD

by

D2:

0.0

689

A*020101/0

9B

*4801

0.0

411

0.0

184

0.2

70.2

52

Com

ple

teLD

by

D0 :

0.7

369

A*0206

B*4002

0.0

341

0.0

085

0.0

857

0.6

171

A*0206

B*510201

0.0

341

0.0

156

0.1

467

0.2

807

Com

ple

teLD

by

D2:

0.0

222

Com

ple

teLD

by

D0 :

0.4

529

DR

B1*080201

DQ

B1*0402

0.3

523

0.2

282

1<

0.0

01

DQ

B1*0402

DP

B1*0402

0.2

775

0.0

533

0.4

16

0.2

911

DR

B1*1402

DQ

B1*0301

0.2

727

0.1

751

0.8

37

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01

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B1*0302/0

7D

PB

1*0402

0.1

56

0.0

186

0.2

371

0.6

375

DR

B1*040701

DQ

B1*0302/0

70.1

136

0.0

891

1<

0.0

01

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B1*0301

DP

B1*0402

0.1

461

�0.0

492

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519

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964

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70.0

455

�0.0

232

�0.3

386

0.4

055

DQ

B1*0301

DP

B1*0401

0.0

956

0.0

294

0.1

964

0.2

839

DR

B1*090102

DQ

B1*030302

0.0

455

0.0

429

1<

0.0

01

DQ

B1*0402

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B1*0401

0.0

748

�0.0

012

�0.0

164

0.9

661

DR

B1*040301

DQ

B1*0302/0

70.0

341

0.0

2673

10.0

035

DQ

B1*0301

DP

B1*020102

0.0

651

0.0

337

0.4

757

0.0

741

Com

ple

teLD

by

D2:

0.1

3D

QB

1*030302

DP

B1*0402

0.0

568

0.0

207

10.3

081

Com

ple

teLD

by

D0 :

0.8

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Com

ple

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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 pre-

sent in our sample (indeed, represented by two haplotypes:

HLA-DRB1*040701-DQB1*0302/07 and -DRB1*0411-

DQB1*0302/07). The most commonly found HLA-

DRB1-DQB1 haplotype in the Tarahumara population,

-DRB1*080201-DQB1*0402 (see Table 3), is found in LD

inMexicanMestizo population,NorthAmericanAmerindians

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 4 Three-locus haplotype distribution of HLA class I and class II in the Tarahumara population

Haplotype Frequency by MLE D D0 P-value

A*240201 Cw*0102 B*1501 0.1136 0.1061 0.8236 <0.001

A*020101/09 Cw*0304 B*4005 0.0795 0.0683 0.6669 <0.001

A*240201 Cw*0801 B*4801 0.0568 0.0491 0.5901 <0.001

A*310102 Cw*0801 B*510201 0.0568 0.0534 0.5401 <0.001

A*240201 Cw*0304 B*4002 0.0511 0.0206 0.1185 0.2679

A*6801 Cw*0801 B*510201 0.0455 0.0424 0.4826 <0.001

A*020101/09 Cw*0304 B*4002 0.0455 0.0251 0.1363 0.0985

A*240201 Cw*040101 B*3501/02/03 0.0455 0.0408 0.4731 <0.001

A*020101/09 Cw*040101 B*3501/02/03 0.0341 0.0310 0.3529 <0.001

A*0206 Cw*0801 B*510201 0.0341 0.0299 0.2475 <0.001

A*0206 Cw*0304 B*4002 0.0341 0.0239 0.2083 0.0261

Complete LD by D 2: 0.0347


DRB1*080201 DQB1*0402 DPB1*0402 0.2785 0.1995 0.7301 <0.001

DRB1*1402 DQB1*0301 DPB1*0402 0.1192 0.0571 0.2334 0.0316

DRB1*040701 DQB1*0302/07 DPB1*0402 0.1136 0.0980 1.0000 <0.001

DRB1*1402 DQB1*0301 DPB1*0401 0.0967 0.0756 0.3880 <0.001

DRB1*080201 DQB1*0402 DPB1*0401 0.0738 0.0470 0.2485 0.0071

DRB1*1402 DQB1*0301 DPB1*020102 0.0568 0.0468 0.5075 <0.001

DRB1*090102 DQB1*030302 DPB1*0402 0.0455 0.0438 1.0000 <0.001

DRB1*040301 DQB1*0302/07 DPB1*0402 0.03410 0.0294 0.9999 <0.001



LD, linkage disequilibrium.

Table 5 Extended haplotype distribution of HLA class I and class II in the Tarahumara population

Haplotype Frequency by MLE D D0 P-value

A*240201 Cw*0102 B*1501 DRB1*1402 DQB1*0301 DPB1*0402 0.0795 0.0791 0.5819 <0.001

A*020101/09 Cw*0304 B*4005 DRB1*080201 DQB1*0402 DPB1*0402 0.0511 0.0502 0.4456 <0.001

A*310102 Cw*0801 B*510201 DRB1*080201 DQB1*0402 DPB1*0402 0.0455 0.0452 0.4430 <0.001

A*240201 Cw*0304 B*4002 DRB1*080201 DQB1*0402 DPB1*0402 0.0341 0.0317 0.1567 <0.001

A*240201 Cw*0102 B*1501 DRB1*1402 DQB1*0301 DPB1*0401 0.0341 0.0339 0.2491 <0.001

A*240201 Cw*0304 B*270502 DRB1*1402 DQB1*0301 DPB1*0401 0.0341 0.0339 0.4984 <0.001

A*020101/09 Cw*0304 B*3501/02/03 DRB1*080201 DQB1*0402 DPB1*0402 0.0341 0.0330 0.2441 <0.001

A*240201 Cw*0801 B*4801 DRB1*080201 DQB1*0402 DPB1*0401 0.0341 0.0339 0.3736 <0.001

A*020101/09 Cw*0304 B*4002 DRB1*1402 DQB1*0301 DPB1*020102 0.0341 0.0339 0.3320 <0.001

A*6801 Cw*0801 B*510201 DRB1*080201 DQB1*0402 DPB1*0402 0.0341 0.0339 0.3733 <0.001



LD, linkage disequilibrium.



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 fre-

quencies or absence of the B39 and DRB16 subgroups).

Only two extended haplotypes (A*020101/09-Cw*0304-

B*4005-DRB1*080201-DQB1*0402-DPB1*0402 (0.0511)

and A*020101/09-Cw*0304-B*3501/02/03-DRB1*080201-

DQB1*0402-DPB1*0402 (0.0341) can be inferred from

previously reported extended haplotypes in Aymaras,

Mayans, Peruvian Indians, Quechuas, Nahuas, Seris and

Yu’pik 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 popula-

tions are related according to a smooth geographic gradi-

ent (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 spur-

ious 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 popu-

lation 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: 343–604] typed 110 non-related individuals for inter-

mediate 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 geo-

graphically 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 pre-

viously 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; HLA-

DQB1*0402, *0301, *0302/07 and *030302; HLA-

DRB1*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 HLA-

A, 6–20 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), consid-

ered 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,




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 populationstudies

Analysing HLA class II genetic relatedness among

Amerindians, we observed the following striking facts:

(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 Nahua-

Cuitlateco group, Pima Cora Family (6) cluster with

Bolivian Aymaras and Brazilian – Paraguayan Terenas

(52) (Table 1). One might think that Amerindian popula-

tions 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.

North Am BlacksSouth Am Blacks

Lebanese-NSLebanese-KZ

Moroccan JewsAshkenazi Jews

Non Ashkenazi JewsItalians

SardiniansMacedonians

CretansMoroccans

FrenchSpaniards

Spanish BasquesAlgeriansBerbers

GermansRussians Chuvash

DanishKhoton-MongolianTuvinians

Khalk-MongolianJapanese

KoreanManchu Buyi

Singapore ChineseEvenks

KetsTlingit

NivkhsAthabaskan

UdegeysKoryaks

ChukchiEskimos

GuaraniWayu

QuechuasMayans Toba-Pilaga

Mataco-WichiEastern-Toba

Arhuaco TarahumaraAymaras

TerenaZapotecans

JaidukamaCayapa

MazatecansKogi

ArsarioSeri

MixtecoXavantes

Am

erin

dian

sS

iber

ians

/E

skim

osO

rient

als

Cen

tral

Eur

ope

Med

iterr

anea

nsA

fric

ans

Mixe

Figure 2 Neighbour-joining dendrogram show-

ing 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 HLA-

DRB1 and -DQB1 genotyping (four digits). Data

from other populations were taken from refer-

ences 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.



(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 lan-

guages 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 Greenberg’s, have been previously sug-

gested 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 impor-

tant 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 frequen-

cies 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 impos-

sible 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 presum-

ably 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 mole-

cules 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, contradict-

ing 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

0.4

Berbers MoroccansAlbanians

Non-Ashkenazi Jews

Ashkenazi JewsDanishChuvash

SardiniansSpanish BasquesTuvinians

Khoton Mongolian Khalk Mongolian

KoreanKets

EvenksMadangJapanese

Rabaul

PapuaKimberley

Central Desert

YuendumuCape York

Ainu Nivkhs

Koryaks

Chukchi

TlingitUdegeys

Athabaskan

Eskimos

Terena

Cayapa XavantesMixteco

Guarani

Jaidukama

Toba-Pilaga

Mataco-WichiTarahumara

Aymaras

MixeArsario

SeriKogi

Mazateco

WayuMexican Mestizo Quechuas

Mayans

Arhuaco

Eastern-TobaZapoteco

Singapore Chinese

Manchu

New Caledonia

Buyi

Fidji

W Samoa

North American BlacksSouth American Blacks

Lebanese KZLebanese NS

SpaniardsFrench

CretansItaliansAlgeriansRussians

MacedoniansGermans

Dim

ensi

on 2

–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).




population sample used for this paper should be taken into

account when interpreting the obtained extended haplo-

types, and that those conclusions would be interpreted as

premature and speculative, it is also important to empha-

size 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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high-resolution molecular characterization of the hla class i and class ii in the tarahumara...

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