Distribution of TAP gene polymorphisms and extended MHC haplotypes in Mexican Mestizos and in Seri Indians from northwest Mexico

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1 (2002) 3, Nature Publishing Group All rights reserved /02 $ Distribution of TAP gene polymorphisms and extended MHC haplotypes in Mexican Mestizos and in Seri Indians from northwest Mexico S Balladares 1, C Alaez 1, J Pujol 2, C Duran 2, JL Navarro 2 and C Gorodezky 1 1 Department of Immunogenetics, Instituto de Diagnostico y Referencia Epidemiologicos (InDRE) SSA, Mexico City, Mexico; 2 Public Health State Laboratory; Hermosillo, Sonora, Mexico The study of the genetic structure is very useful for investigating the biological significance of polymorphism and may provide clues to understand population origins. We present TAP1/TAP2 gene analysis in the Seri indians from Sonora, and in Mestizos from the highlands of Mexico. Thirty-two Seri and 89 Mestizos were studied. TAP genes were typed using the ARMS-PCR technique. The most frequent alleles in Seri were: TAP1*0101/02, (68.8%); TAP1*02011/02012, (31.2%); TAP2*0201, (38.7%) and TAP2*0101, (29.0%). TAP1*0301, TAP1*0401, TAP2*0102 TAP2*0103 and TAP2H were absent in them. For Mestizos, the prevalent alleles were: TAP1*0101/02 (75.8%); TAP1*02011/12 (20.3%); TAP2* 0101 (45.4%) and TAP2*0201 (29.3%). These results are similar to those found in Kaingang and Caucasians from Brazil, four Mediterranean, other Caucasians, two Oriental and one African group. In Seri, the extended prevalent haplotypes are typically Amerindian, such as TAP1*0101/2-TAP2*0201-QBP3.21-DQB1*0302-QAP*3.1-DQA1*03011-DRB1*0407-B*3501- A*0201 (HF = 16.6%). Thirty-two extended haplotypes were found in Seri, although TAP contributed scarcely to diversity. Mestizos show Amerindian and Caucasian combinations. No difference was detected in the distribution of amino acids in the individual variable sites, between both groups. These findings are the basis for further anthropological studies and to explore the contribution of TAP genes to disease expression in Mexicans. (2002) 3, DOI: /sj/gene/ Keywords: TAP polymorphism; extended MHC molecular haplotypes; TAP-DNA typing Introduction The TAP1 and TAP2 genes are located in the MHC class II region in a cluster of -interferon inducible genes. 1 They encode a non-covalently associated heterodimer, which belongs to the ABC superfamily of transport proteins. TAP1 has eight transmembrane segments, whereas TAP2 has seven segments. The N and C terminus of TAP1 molecule are in the cytoplasm, while TAP2 has its N terminus in the lumen of the endoplasmic reticulum (ER). 2 The transporter heterodimer allows endogenous peptides to pass into the ER lumen where they are loaded on the MHC class I molecule, maintaining adequate levels of peptides for their presentation to the T cell. 1 The analysis of TAP genes has shown some polymorphism in both genes. Initially, only two dimorphic sites were found in the TAP1 cdna sequences resulting in amino acid substitutions at positions ILE333VAL and ASP637GLY, yielding a total of four potential alleles (Table 1). For TAP2, three dimorphic sites have been described: ILE379VAL, ALA565THR and ALA665THR; the different combinations result in eight distinct alleles, described in Table 1. 3,4 Four of them have not been Correspondence: Dr C Gorodezky, Head of The Department of Immunogenetics, InDRE, SSA, Carpio 470 1st floor. Mexico D.F., 11340, Mexico. cgorodea mailer.main.conacyt.mx Received 26 July 2001; revised 14 November 2001; accepted 15 November 2001 renamed yet with the official nomenclature: 2C, 2D, 2G and 2H. 5 Some variants of TAP genes have been sequenced and new dimorphic positions have been detected; for these sequences, the WHO Nomenclature Committee gave official names following the HLA allele nomenclature. 6 The new and old names are included in Table 1. At least three different approaches provide information on the significance of TAP polymorphism: 7 investigation on the spectrum of peptides generated, transported and presented; 8 11 disease association studies; and population genetic analysis. 15 Evidence for the first phenomenon comes from studies showing many variations in rat TAP genes that have been associated with differences in the spectrum of MHC class I peptides transported and presented to the T cell receptor (TCR). 16 In contrast, human TAP genes carry relatively limited polymorphism and when different TAP allelic combinations were analysed, 17,18 no difference in the spectrum of transported peptides was shown. However, it has been suggested that alleles carrying Asp/637 (charged polar residue) appeared to be more restrictive in transporting peptides than those carrying Gly/637 (nonpolar residue). 19 Thus, it has been claimed that this position and position 648 lie within the region believed to make direct contact with peptides 20,21 being the most likely candidates for disease associations. In fact, this has been shown for HIV infection. 22,23 The results on the contribution of these genes to the

2 TAP genes in Mestizos and Seri Indians from Mexico Table 1 Distribution of allele frequency of TAP1 and TAP2 genes in Seri Indians and in Mexican Mestizos 79 Previous equivalents, N = number of individuals; UD = undefined; AF = Allele frequency. expression of certain autoimmune diseases have been controversial. This is the case for rheumatoid arthritis, 24,25 multiple sclerosis, type I diabetes mellitus; myasthenia gravis, 39 systemic lupus erythematosus, and some chronic infections, such as leprosy and tuberculosis. 43 Most of the studies claim there is no primary association with TAP genes, but it is due to linkage disequilibrium with DR and DQ genes. Finally, the analysis of the genetic profile in different ethnic groups and in some species such as chimpanzee, gorilla, rat and mouse 44 was essential to unravel the origin of polymorphism and to understand the evolution mechanisms of these loci. 45 Analyses in these species demonstrated that TAP1*0101 is not an ancestral human allele although it is prevalent in most populations. 44 The investigation of TAP gene polymorphism using a combination of PCR-based techniques including single-strand conformation polymorphisms (SSCP), restriction fragment length polymorphism (RFLP), nucleotide sequencing and PCR with sequence-specific primers (SSP) in different populations suggest there are some new polymorphic positions in addition to those described here. 15,44,46,47 However, these have not yet been included in the official nomenclature. The distribution of TAP genes in Mexican groups is unknown; therefore we considered it interesting to investigate the genetic profile in two populations: one Native group and one admixed population. The Seri Indians belong to the Hokanno-Coahuitleca linguistic family, which is related to the Macroyuma trunk. 48 They have been semi-nomads and live along the coast of the State of Sonora. The Seri found refuge during the Spanish conquest in the 16th century in inhospitable territories such as Isla Tiburón ( Shark Island ) located in the Gulf of California. They returned to the mainland of Sonora in Due to an isolated lifestyle, they are an inbred group and only 619 individuals are left who preserve their cultural and genetic identity. 48 The prevalent diseases in them are respiratory and viral infections, hypertension, parasitic diseases, type II diabetes, rheumatic and urinary diseases. The Mestizos constitute about 95% of the total Mexican population. They are the result of a tri-racial admixture of Caucasian, Amerindian and African populations. The Amerindian component in the Mestizos comes from the indigenous groups that inhabited the territory when the Spaniards arrived in Mexico has nowadays 68 different Indian groups classified from a linguistic point of view and they preserve their cultural and geographic identity. The European component came mainly from three provinces of Spain; Andalucía, Castilla and Extremadura. The African genes were introduced in Mexico during the 17th century by the slaves. The Africans were brought into the country by the Spanish conquerors, from different parts of Africa, mainly from the Occidental Coast between the Senegal and Portuguese Angola located in the South. Therefore, different degrees of admixture are found in the Mestizos distributed along

3 80 TAP genes in Mestizos and Seri Indians from Mexico the country. 49 This was shown by us in a recent paper where we reported the distribution of class II MHC genes in three different populations from Mexico, one of the north, one of the center and one of the highlands of Mexico. 50 The aim of this study was to analyse the genetic profile of TAP1 and TAP2 genes in two different Mexican populations: the Seri and the Mestizos from the highlands of the country. We also incorporated the results of class I and class II MHC typing in order to build extended haplotypes, to add new information on the MHC profile of Mexican populations. Results Because the typing methodology does not allow the unequivocal assignment of alleles in individuals who are heterozygous at more than one dimorphic residue, 10 Seri individuals could not be assigned for TAP2. Because the family members were also typed for TAP1 and TAP2 as well as for Class I (A and B) and Class II loci (DQB1, DQA1, DRB1 and QAP/QBP promoters, manuscript in revision), it was possible to get the exact typing through the analysis of extended haplotypes looking at family segregation. It was not possible to assign TAP1 individual alleles in 27 Mestizos and TAP2 alleles in 15 samples of them. The subjects with the ambiguous combination TAP1* 0101/02/TAP1*02011/12 or TAP1*0301/TAP1*0401 are most likely TAP1*0101/02/TAP1*02011/12; this is supported by two facts; TAP1*0101/02 was the most frequent allele and TAP1*0301 was not detected in Mestizos (Table 1). The ambiguities were broader for TAP2 genes, therefore it was not possible to assign the individual allele: 2A/2D or 2C/2E, 2B/2D or 2A/2F or 2C/2G or 2E/2H, 2A/2G or 2B/2E, 2B/2C or 2A/2H, and 2A/2H or 2B/2C (Table 1). These samples were assigned as undefined (UD). The prevalent TAP1 alleles in Seri were TAP1*0101/02 (68.8%) and TAP1*02011/12 (31.2%); the most frequent TAP2 alleles were: TAP2*0201 (38.7%), and TAP2*0101 (29.0 %). TAP1*0301, *0401, TAP2*0102, *0103 and 2H were absent. The most prevalent alleles in Mestizos were: TAP1*0101/02 (75.8%) and TAP1*02011/12 (20.3%); TAP2*0101 (45.4%), and TAP2*0201 (29.3%). TAP1*0301, TAP2*0103 and TAP2G were absent in this group (Table 1). As already mentioned, the family members of the Seri included in the study, were previously typed for class I and class II genes. With this data, extended haplotypes were built, since segregation family analysis of TAP1- TAP2-QBP-DQB1-QAP-DQA1-DRB1-B-A. was confirmed. Forty-eight haplotypes were obtained. Haplotype frequencies were assessed by direct counting (Table 2). The most frequent haplotypes in Seri were the following: TAP1*0101/02-TAP2*0201-QBP3.21-DQB1*0302-QAP3.1- DQA1*03011-DRB1*0407-B*3501-A*0201, (HF = 16.6%); TAP1*0101/02-TAP2*0101-QBP3.21-DQB1*0302-QAP3.1- DQA1*03011-DRB1*0407-B*3501-A*0201, (HF = 6.2%); TAP1*0101/02-TAP2*0101-QBP3.21-DQB1*0302-QAP3.1- DQA1*03011-DRB1*0407-B*51-A*31 (HF = 6.2%); TAP1* 02011/12-TAP2*0201-QBP4.1-DQB1*0402-QAP4.2-DQA1 *0401-DRB1*0802-B*51-A*31 (HF = 6.2%). The remaining haplotypes had a frequency under 5% (Table 2). Since we did not have family data in the Mestizos to build extended haplotypes, a bilocus analysis of linkage disequilibrium was performed between TAP1 and TAP2 loci (Table 3). The only significant value found was for TAP1*0401/TAP2*0201 ( = 0.037, P = 0.04) and the most frequent combination was TAP1*0101/02/TAP2*0101 (HF = 39.9%), reflecting the association of the prevalent alleles present in the Mestizos. A bilocus analysis was also done considering the prevalent class II haplotypes described previously 50 in combination first with TAP1 and then with TAP2 alleles (Table 4). Linkage disequilibria between class II and TAP1 in Mestizos showed significant values for TAP1* 0401/DQB1*0301-DQA1*0501-DRB1*1602 ( = 0.034, P = 0.004); TAP1*02011/12/DQB1*0201-DQA1*0201-DRB1* 0701 ( = 0.032, P = 0.01); TAP1*02011/12/DQB1*0301- DQA1*0501-DRB1*1602 ( = 0.023, P = 0.04); and the most frequent combinations were TAP1*0101/02/DQB1*0402- DQA1*0401-DRB1*0802 (HF = 12.6%); and TAP1*0101/ 02/DQB1*0302-DQA1*03011-DRB1*0407 (HF = 11.4%). Significant values for class II-TAP2 genes were observed for TAP2*0201/DQB1*0302-DQA1* DRB1*0407 which was also the most frequent combination (P = 0.002, HF = 12.9%), together with TAP2* 0101/DQB1*0402-DQA1*0401-DRB1*0802 (P = NS, HF = 12.6%). Other combinations such as TAP2C/DQB1*0301- DQA1*0501-DRB1*1406 (P = 0.04, HF = 2.8%) and TAP2C/DQB1*0301-DQA1*0501-DRB1*1104 (P = 0.03, HF = 1.9%) were also in, although not very frequent. Frequency of individual variable sites was estimated and compared between Mestizo and Seri. The prevalent amino acid substitutions in both populations were ILE/333/TAP1, ASP/637/TAP1, VAL/379/TAP2, ALA/ 565TAP2 and THR/665/TAP2. The distribution was similar among them (Table 5). Finally, as shown in Table 6, the obtained frequencies in Mestizos and Seri were compared with other populations: Guarani, Kaingang, Caucasians from Paraná- Brazil, 7 French, 29,51 German, 52 Sardinian, 38 Spaniards, 53 Caucasians from USA, 12,54 Tunisian, 55 Japanese, 56,57 British, 40 and Italians. 36 The results showed that the distribution of TAP1 and TAP2 alleles was very similar in all the groups excepting for Guarani. TAP1, TAP1*0101/02 and TAP1*02011/12 were most frequent in all populations with the former being the most prevalent, but in Guarani, the proportion was inverted. TAP1*0301 was not very common, and it was absent in Mexican Mestizo, Seri, Kaingang and Japanese. TAP1*0401, previously described as a rare allele, had a very low frequency and was only present in Japanese (9.0%), Tunisian (5%), Mexican Mestizo (3.9%), German and French populations (1.5% and 1.2% respectively). TAP2*0101 was the most frequent TAP2 allele followed by TAP2*0201, differing from the distribution found in Guarani, in which TAP2*0201 and TAP2*0102 were highly prevalent (AF = 40.3% and 30.1% respectively). TAP2C had a frequency ranging from % and was absent in Guarani. TAP2D was not detected in Sardinians, Spaniards, Japanese and Italians; Kaingang showed an increased frequency (AF = 18.1%), while in the rest it was low ( %). TAP2*0102 was absent in Seri, Kaingang and Spaniards and had an unexpected high frequency (30.1%) in Guarani, remaining variable ( %) in the others. TAP2*0103 was only present in French, Germans, Americans and Italians. TAP2G was only shown in Seri, French and Caucasians of USA (AF = %)

4 TAP genes in Mestizos and Seri Indians from Mexico Table 2 TAP1-TAP2-QBP-DQB1-QAP-DQA1-DRB1-B-A haplotype frequency in Seri 81 TAP1* TAP2* QBP DQB1* QAP DQA1* DRB1* B* A* n = 48 HF (%) *0101/02 * *0101/02 * *0101/02 * *0101/02 * *0101/02 * *0101/02 * *0101/02 * *0101/02 * *0101/02 * *0101/02 * *0101/02 * *0101/02 * *0101/02 * *0101/02 * *0101/02 * *0101/02 * *0101/02 * *0101/02 2C *0101/02 2C *0101/02 2C *0101/02 2D *0101/02 2D *0101/02 2D *02011/12 * *02011/12 * *02011/12 * *02011/12 * *02011/12 * *02011/12 * *02011/12 * *02011/12 * *02011/12 2G n = Number of haplotypes; HF = Haplotype frequency. Table 3 Linkage disequilibrium of TAP1/TAP2 haplotypes in Mexican Mestizos TAP1* TAP2* a b c d HF P Fisher *0101/02 * NS *0101/02 * NS *0101/02 2C NS *0401 * n = 51, = Linkage disequilibrium, HF = Haplotype frequency. and TAP2H in the Mexican Mestizos and in Japanese (AF = %). Discussion Mexico is a very interesting area, because the ecological conditions and the epidemiology are distinct from the conditions found in South America. 58 Because this country is a subtropical area where different parasites and infectious agents exist, 59 the analysis of as many loci as possible within the MHC, including TAP genes will provide valuable information on the current, genetic structure of the population. Interestingly, it is well known from several studies that the genetic profile of some class I alleles found in North American Indians shows that ancestral alleles are present in all groups, but new alleles emerged or were selected in Mexican and South Amer- ican Natives. These alleles are being analysed in the context of peptide anchoring sites. 45,60,61 This is the first study looking at TAP polymorphism in a Mexican Indian population and in the admixed population, the Mestizos, who are the prevalent ethnic group in Mexico. We also compared the TAP gene profile in Mexicans with the one found in other populations of the American Continent, to get a better understanding on the origin of these genes in Mexicans and on their contribution to the MHC pattern. It is important to emphasise that extended nine loci haplotypes (TAP1- TAP2-QBP-DQB1-QAP-DQA1-DRB1-B-A) are reported for the first time. This information is a valuable tool for future anthropologic studies, for the analysis of migration patterns, and disease association analysis. Indian populations in all America have demonstrated a restricted class II pattern probably due to factors such as genetic drift or founder effect. The well documented history of the different peoples that settled along the American Continent demonstrated that these groups were relatively small in number. 45,49,59,60 This may have been the major cause of the restricted class II pattern. The extended haplotypes shown herein, were highly informative to look at the loci involved in diversity, because as demonstrated with the Seri data, in spite of the limited polymorphism, TAP variations can contribute to haplotypic diversity since no preferential combinations with class I and or class II haplotypes was observed. Only four QBP-DQB1-QAP-DQA1-DRB1 haplotypes were

5 TAP genes in Mestizos and Seri Indians from Mexico 82 Table 4 Linkage disequilibrium of TAP1/TAP2 with DRB1-DQA1-DQB1 haplotypes in Mexican Mestizos DQB1*DQA1* DRB1* a b c d HF P Fisher TAP1* *0101/ NS *0101/ NS *0101/ NS *02011/ *02011/ * TAP2* * NS * NS * NS * NS * NS * C C n = 51, = Linkage disequilibrium, HF = Haplotype frequency; TAP2C has no official equivalent in the current nomenclature. Table 5 Analysis of individual variable sites of TAP1 and TAP2 genes Mestizos Seri TAP1 n = 178 AF (%) n = 64 AF (%) 333 ile val asp gly TAP2 n = 174 AF (%) n = 62 AF (%) 379 val ile ala thr ala thr n = number of alleles, AF = Allele frequency. found in the Seri, but when class I data (A and B genes) were added, 23 different haplotypes were shown. Finally, 32 haplotypes were obtained, after inclusion of TAP1/TAP2 results. Diversity was mainly due to class I alleles, with the contribution of TAP being limited. Moreover, the same QBP-DQB1-QAP-DQA1-DRB1-B-A haplotype may be found in combination with different TAP1 and/or TAP2 alleles, suggesting lack of directional/ conservative selection over HLA-TAP haplotypes. This probably means that differences in TAP do not represent biological advantage. Thus, TAP genes may have evolved separately from these genes. All the combinations found in Seri are mainly of Amerindian ancestry, with the exception of one subject that carry the haplotype QBP2.1-DQB QAP2.1-DQA1* 0201-DRB1*0701 which is beyond doubt, a Caucasian haplotype introduced by the Spaniards, indicating the presence of certain degree of admixture. It is well known there is little linkage disequilibrium between TAP1 and TAP2 loci suggesting high rates of recombination between them. 62 In fact, only one significant value was found TAP1*0401-TAP2*0201 ( = 0.037, P = 0.04) in Mestizos. Interestingly, TAP1*0401 is either absent or very infrequent in most populations, except in a group of Japanese, who had a frequency of 9%. 56 It has been claimed that TAP1*02011, the ancestral allele 63 evolved through two point mutations to TAP1*0101 resulting one of them in TAP* Nevertheless, there is no explanation for the low worldwide frequency of this allele; its presence in American Indians has probably a Mongoloid origin because it is present in some Oriental populations. 56 Seri, Kaingang and Mestizos have a similar distribution for both, TAP1 and TAP2 alleles differing from the profile in Guarani. In a previous paper Petz-Erler et al claimed that Kaingang and Guarani were the most divergent among several South-American Indian groups, although living close to one to another for centuries. 64 Based on TAP polymorphism the results of this paper are concordant with those observed by them, because Guarani have a unique TAP pattern that differs from Mexicans as well as from all the other groups (Table 6). No difference was found in the distribution of individual variable sites in these populations. However, it is worth mentioning that TAP1 sequences involving nonconservative amino acid substitutions are located at position 637. It was suggested that alleles carrying Asp/637 appeared to be more restrictive for peptide transport than those carrying Gly/637, 21 therefore the investigation of this position will be very valuable for disease associations analysis. 22,23 Moreover, even though not directly related to these sites, the importance of TAP defects in disease has been pointed out in certain immune abnormalities. It has been shown that defect in the TAP1 gene leads to the absence of a functional TAP1 subunit and the expression of the TAP2 subunit is also impaired. These abnormalities were found in patients with bare lymphocyte syndrome and, chronic bacterial lung infections have been found in TAP1/2 deficient patients. 65 In conclusion, HLA haplotypes are very valuable to interpret population relationships and migratory movements worldwide. It has been predicted that a large num-

6 TAP genes in Mestizos and Seri Indians from Mexico Table 6 Allele frequency of TAP1 and TAP2 genes in Mexican Mestizos and Seri, compared with other populations 83 MEX SER GUA 7 KAI 7 CPR 7 FRE 29 FRE 51 GER 52 SAR 38 SPA 53 USA 12 TUN 55 JPN 56 n = 89 n = 32 n = 92 n = 240 n = 91 n = 41 n = 162 n = 101 n = 79 n = 106 n = 80 n = 81 n = 35 TAP1*0101/ TAP1*02011/ TAP1* TAP1* MEX SER GUA 7 KAI 7 CPR 7 FRE 29 GBR 40 GER 52 SAR 38 SPA 53 USA 54 JPN 57 ITA 36 n = 87 n = 31 n = 93 n = 240 n = 91 n = 41 n = 156 n = 101 n = 81 n = 106 n = 209 n = 106 n = 62 TAP2* TAP2* TAP2C TAP2D TAP2* TAP2* TAP2G TAP2H MEX = Mexican Mestizos, SER = Seri, GUA = Guarani, KAI = Kaingang, CPR = Caucasians from Paraná, FRE = French, GER = German, SAR = Sardinian, SPA = Spanish, USA = USA Caucasians, TUN = Tunisian, JPN = Japanese, GBR = British, ITA = Italian. ber of informative loci and more sophisticated analytical methods are needed for the genetic classification of the Native American groups, due to the effect of intertribal migrations, and other factors, that affect the origin of this groups. Regarding the Mestizos, admixture has been the major contributor to the growth of cities, and possibly this mechanism is responsible for the large diversity found in urban populations. Materials and methods Study population The Seri: Thirty-two unrelated individuals belonging to the Seri population of Desemboque and Punta Chueca were recruited for this study. The samples were previously typed for class I 66 and class II genes. 67 Expert anthropologists of the Institute of Anthropological Research (UNAM) in Mexico City designed a demographic questionnaire that was applied to every individual included in the study. Due to the extensive inbreeding of the population, the Seri are highly endogamic. The subjects included herein were unrelated individuals; the spouses of each of the nine families plus 14 truly unrelated subjects were included to determine allele frequencies; the family pedigrees were shown in the class I paper. 66 The Mestizos: A group of 89 unrelated individuals was included. The group was formed by volunteers, students and blood bank donors. All of them were born and living in Mexico City and surroundings. Although they are an admixed population, they have an important native background as shown by their ABO and Rh distribution: 11.1% were A1, 2.1% were A2, 5.6% were B and 81.2% typed as group O. Ninety-eight percent are Rh DNA typing of TAP genes From every person under study, 30 ml of peripheral blood in EDTA were withdrawn and mixed with RPMI medium. For the Seri, the personnel of the Public Health Laboratory in Sonora prepared and sent the samples to the Department of Immunogenetics in Mexico City. The mononuclear cells were isolated and DNA was extracted using the proteinase K and phenol/ chloroform protocol. 68 Dimorphisms at TAP1 codons 333 and 637 and TAP2 codons 379, 565 and 665 were typed according to the XIIth IHW protocols using the ARMS-PCR method. 5 PCR was done using the conditions stated in the protocol. The amplifications resulted in a constant control product together with one or two allele-specific products depending on weather the individual was homozygous or heterozygous. The PCR products were separated in a 2% agarose gel and were stained with ethidium bromide. Homozygous individuals were clearly distinguished from heterozygous. TAP1 and TAP2 alleles were assigned based upon the different combinations between dimorphic positions. However, ARMS-PCR did not allow the unequivocal assignment of alleles in individuals who were heterozygous at more than one dimorphic residue. Statistical analysis The Hardy-Weinberg law was used to analyse all the possible genotype frequencies according to the classical method 69 to assess if the system is in genetic equilibrium and if there was a significant excess of homozygosity. Allele frequency was calculated by direct counting in both groups. Unrelated Seri individuals were included taking one or two parents and the subjects with no relatives. A total of 32 unrelated Seri were used to calculate allele frequencies and 24 to ascertain extended haplotype frequencies. The latter were determined by direct counting and by segregation analysis of families to avoid duplication of the results. An unrelated individual was presumed homozygous when typing results showed the presence of only one haplotype, and by segregation analysis in families where conclusive evidence was obtained for homozygosity. In the Mestizos, who were selected at random and who were all unrelated individuals, linkage disequilibrium and haplotype frequencies were calculated according to

7 84 TAP genes in Mestizos and Seri Indians from Mexico Mattiuz et al, 70 using a computer program developed in our laboratory for analysis of two-point loci. In an unrelated population the haplotypes must be deduced because it is not possible to assemble them directly since no family members were available. FH AB = FG A FG B + AB = d/n b+d N c+d N Where FG A is the allelic frequency of the allele at one locus and FG B is the allelic frequency of the allele at the other locus; is the linkage disequilibrium obtained using a 2 2 contingency table. Acknowledgements We are truly grateful to A Hernandez for his help in DNA extraction and to M Vazquez for her valuable comments on the manuscript. We are especially indebted to E Infante for his very valuable help in the statistical analysis. References 1 Heemels MT, Ploegh H. Generation, translocation, and presentation of MHC class I-restricted peptides. Ann Rev Biochem 1995; 64: Vos JC, Spee P, Momburg F, Neefjes J. 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