Results of Expedicion Humana

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1 I 7i.csue Antigens 1996: 48: Printed in Denmurk. All rights rcrerved TISSUE ANTIGENS ISSNOOOI-2815 Results of Expedicion Humana I. Analysis of HLA class I1 (DRB 1-DQA1-DQB 1 -DPB 1) alleles and DR-DQ haplotypes in nine Amerindian populations from Colombia, E. A. Trachtenberg, G. Keyeux, J. E. Bernal, M. C. Rhodas, H. A. Erlich. Results of Expedicion Humana. I. Analysis of HLA class I1 (DRB 1 -DQA 1 -DQB 1 -DPB 1) alleles and DR-DQ haplotypes in nine Amerindian populations from Colombia. Tissue Antigens 1996: 48: Munksgaard, 1996 HLA class I1 variation was analyzed in nine Native American populations of Colombia using PCWSSOP typing methods. Under the auspices of the Expedition Humana, approximately 30 unrelated native Colombian Indian samples each from the Tule (W Pacific Coast), Kogui (Sierra Nevada), Ijka (Sierra Nevada), Ingano (Amazonas), Coreguaje (Amazonas), Nukak (Amazonas), Waunana (Pacific), Embera (Pacific) and Sikuani (Northeastem Plains) were collected and analyzed at the DRB 1, DQA 1, DQB I and DPBl loci. The number of different DRBl, DQA1, DQB 1 and DPB 1 alleles in the Colombian Indians is markedly reduced in comparison with neighboring African Colombian populations, which exhibit a very high degree of class I1 variability, as discussed in an accompanying paper. In the Colombian Amerindian groups, DR2 (DRB 1 * 1602), DR4 (DRB 1 *0407, *0404, *0403 and *041 l), DR6 (DRB 1 * 1402) and DR8 (DRBl"0802) comprise >95% of all DRBl alleles. We also found an absence of DR3 in all populations, and DR 1, DR7 and DR9 allelic groups were either very rare or absent. Each Colombian Amerindian population has a predominant DRBl allele (f= ) and DRB 1-DQA1-DQB 1 haplotype. Several novel DR-DQ haplotypes were also found. At the DPBl locus, DPB 1 *0402 (f= ), * 1401 (f=-0.45), and *3501 (f=-0.27), were the three most prevalent alleles, each population maintaining one of these three alleles as the predominant (f>0.26) DPB 1 allele. The reduction of diversity for the HLA class 11 alleles in the Colombian Indians is suggestive of a population bottleneck during the colonization of the Americans, with little to no subsequent admixture with neighboring African Colombian populations in the last -300 years. E. A. Trachtenberg', G. Keyeux', J. E. Bernal', M. C. Rhodas' and H. A. Erlich' 'Department of Human Genetics, Roche Molecular Systems, Alarneda and Children's Hospital Oakland Research Institute, Oakland, California, USA,%stituto de Genetica Huamana, Facultad de Medicina, Pontificia Universidad Javeriana, Bogota. Colombia Key words: Colombian Amerindian - HLA- MHC - PCR -population genetics Received 23 April, accepted for publication 14 May 1996 The recent analyses of Amerindian population genetics data from the highly polymorphic human leukocyte antigen (HLA) complex (1-9) are significnnt in: (1) clinical research, such as tissue transplantation and disease susceptibility or resistance. (2) the forensic sciences, and (3) basic research, such as molecular evolution and anthropology. The sequence-defined polymorphism in the HLA class I1 genes is located primarily in the second exon, whlch encodes the peptide binding groove of the cell surface heterodimer. Analysis of HLA polymorphism in South American Indian populations 174

2 ?-' i, 7 1 WAUNANA HLA class II in nine Amerindian populations from Colombia SlKUANl / \ COREGUAJl - INGANO "71U Figure 1. Location of the Colombian Amerindian populations analyzed at the HLA class I1 DR-DQ-DP loci. For this study, samples from six geographically diverse regions of Colombia were analyzed at the DRB 1, DQA 1, DQB 1, and DPB 1 loci using PCWSSOP typing methods. Native Colombian Amerindian populations studied include the Tule (NW Pacific Coast), Kogui (Sierra Nevada), Ijka (Sierra Nevada), Ingano (Amazonas), Coreguajes (Amazonas), Nukak (Amazonas), Waunana (Pacific), Embera (Pacific) and Sikuani (Northeastern Plains) populations. The Colombian samples analyzed in this study were collected under the auspices of the Expedicion Humana, Instituto de Genetica Humana, Facultad de Medicina. Pontifica Universidad Javeriana. Bogota, Colombia. has been particularly informative due to their geographic and cultural isolation. The recent identification of new class 11 allelic and haplotypic variants in Ecuadorian (6,9), Brazilian (Steve Mack, personal communication) and Argentinian populations (4; E. Trachtenberg, L. Satz and H. Erlich, unpublished data) is consistent with a relatively rapid rate of diversification, as has been postulated for the South American Amerindian class I loci (2, 3, 10). This hypothesis of a rapid rate of HLA evolution is based on the assumption that the genetic variants unique to South American populations arose fter the colonization of the Americas by Asian groups migrating across the Beringian land bridge (-20,000-40,000 years ago). and after the separation of North and South American Amerindians (-10,000 years ago). Here, we report HLA Class I1 (DRB 1 -DQA 1 -DQB 1 -DPB 1) variation, among nine isolated Amerindian populations (N=227) from six geographically distinct regions of Colombia (Figure 1). The nine Colombian Indian groups analyzed speak indigenous languages which belong to the Chibchan branch of the Amerind family, one of the three major subdivisions (Amerind, Nadene and Aleut-eskimo) of Native American languages. The Amerind group belongs to the putative first wave of the three postulated waves of migration of Asiatic people to the Americas (11). Migrating South through North America and then via routes from either Panama or from the Caribbean Islands from Florida, Amerindian populations were dispersed throughout South America by at least 12,000 BC (12). Evidence from class I serological studies indicates genetic isolation in the Colombian Indian populations analyzed here (unpublished). Molecular analysis of HLA class 11 alleles and haplotypes in these Amerindian and neighboring African Colombian populations, discussed in an accompanying paper, would serve to pinpoint any African admixture in the Colombian Indians, or any Amerindian admixture in the African Colombian populations. Comparisons of the HLA class I1 allele and haplotype distributions for these Amerindian groups with the neighboring African Colombian groups as well as with other Amerindian groups may provide insights into selective forces operating on the HLA class I1 polymorphism as well as ancestral relationships among Amerindian groups. Material and methods Samples Blood samples from 227 unrelated Colombian Amerindians including the Kogui (N=30; Sierra Nevada), Ijka (N=30; Sierra Nevada), Sikuani (N=28; Eastern Plains), Ingano (N=ll; Amazonas), Coreguaje (N=20; Amazonas), Nukak (N=20; Eastern Amazonas), Waunana (N=30; Pacific), Embera (N=20; Pacific) and Tule (N=29; NW Pacific Coast) populations were collected under the auspices of the Expedicion Humana (U. Javeriana, Bogota, Colombia). In this study, none of the samples analyzed are presumed to be from first-degree relatives. Further details on these populations can be found elsewhere (13,14). Genomic DNA was extracted from peripheral blood leukocytes using a previously published method (15). DNAs from eleven samples were extracted and amplified from buccal swabs as previously described ( 16). 175

3 Trachtenberg et al. Table 1. ORB1 in Colombian Amerindian Population Kogui ljka Guahibo Nukak Slkuani lngano Waunana Embera Tule OR1 OR2 OR4 OR5 OR6 OR7 OR8 OR9 I I I I I 0901 N 60 Missing,drological 1,2,3,- Groups: 7,9,10 [] [] <> 0.07 [] [0.07] [] ,3,9,1 [] It3, 5, 9,lC ,5,7, I [] ,3,5, 7, 9, [] , [] [] ,3, 9,lO < >=African American admixture [ ]=Caucasian admixture HLA Class II typing Genomic DNA was amplified using the polymerase chain reaction (PCR) (17, 18) and typed using nonradioactive sequence and allele-specific oligonucleotide probe methods for DRBl (19), DQAl (20), DQB 1 (2 1,22), and DPB 1 (23-25). HLA Class It haplotype analysis of the Amerindian populations Because very high linkage disequilibrium is present among DRBl-DQA1-DQB 1 haplotypes in all human populations, and because no single case of family-ascertained recombination in this interval has been reported (l), class 11 haplotypes (DR-DQ) for the Amerindian populations studied were either inferred from known linkage disequilibrium patterns (1, 9, 26) or unequivocally determined by homozygosity. Assignment of DR-DQ haplotypes in the Colombian Amerindians needs to be confirmed through a haplotype frequency estimation computer program (27). Assignment of DR-DQ haplotypes in the Colombian African populations, whose HLA class I1 distribution is much more diverse and is discussed in an accompanying paper, also requires analysis and confirmation through this haplotype estimation program. Comprehensive DR-DQ linkage disequilibrium analysis in the Colombian Amerindian and African American populations is the subject of a forth- 176 coming paper (E. Trachtenberg, W. Klitz, G. Keyeux, J. Bernal and H.Erlich, in preparation). Results and discussion DRBl All nine of the Colombian Indian populations showed significantly reduced DRB 1 diversity. The following serotypes or allelic groups: DR1,3,5,7,9 and 10, were absent or in very low frequencies in every population (Table 1). This result is consistent with results obtained for other South American Amerindian groups (4,6-9), but is in striking contrast to the high level of diversity exhibited in the Colombian African populations studied and discussed in an accompanying paper. Overall, the most common Colombian Amerindian alleles were DR2 (* 1602), DR4 (*0403, *0404, *0407, *0411), DR6 (*1402) and DR8 (*0802). The distribution of alleles differed significantly, however, among these nine groups. Each populations has one or two very common DRBl alleles. The highest allele frequency (f=0.65) was for DRB1*1402 found in the Nukak. The lowest frequencies of predominant alleles included f=0.22 for DRB 1*0403 and f=0.26 for DRB 1 * 1402, both in the Tule. Geographically isolated populations such as the Kogui (Sierra Nevada), the Coreguaje (Central Plains), and the Nukak (Eastern Amazonas), have only 4 or 5 DRB 1 alleles and, in addition, a low level of heterozygosity due to the high frequency of one al-,

4 ~ ~~ Table 2. DQAl in Colombian Amerindian Population HLA class I1 in nine Amerindian populations from Colombia Kogui ljka Guahibo Nukak Sikuani lngano Waunana Ernbera Tule N Table 3. DQBl in Colombian Amerindian Population Kogui ljka Guahibo Nukak Sikuani lngano Waunana Embera Tule N lele (Table 1). The other six populations carry between 7 to 10 of the 20 different DRB 1 alleles found in total. The low frequency of DRB 1 *0901 (f=) in two populations is not unexpected, as DR9 has been found in other isolated South American Amerindian populations (4,9). Given that the frequency of DR9 is very high among Asian populations, finding DR9 in these two populations may reflect the Asian ancestry of the Amerindians. Other rare alleles, however, may reflect admixture. Traces of Caucasian or African (DRl31*0102, *1501, *1101, *1102, *I 104, *0701 and *0801) and of uniquely African (DRB 1 * 1503) alleles at very low frequencies (f=o.o2-) could be seen in a few populations, and may be attributed to admixture. Overall, however, the Amerindian populations show very little admixture. The most admixture is found in the Ijka, a group that trades frequently with the Mestizo (Caucasian in origin) civilization. The Ijka, however, live very close to the Kogui, a population which is more traditional ( wisemen group) and stays deliberately isolated from the Mestizo civilization. Consistent with this cultural difference, the Kogui show no admixture. Although DRB I * 1402 is found in a11 Amerindians, DRB1*1406, a position 86 variant (Val) of DRB1*1402 (Gly), which was found at high frequencies in Argentinian Amerindians (4; Trachtenberg, Satz and Erlich, unpublished), was not found in Amerindians from Colombia or Ecuador (9: Tra- chtenberg, Watkins and Erlich, unpublished). DRBl 1406 was, however, found in some North Amerindians (i.e., Zuni) groups (5). This difference in DRB 1 * 1406 frequencies may reflect disparate migratory routes from North America into western or eastern South America, or perhaps selection acting to increase the frequency of this allele in the Argentinian population. In the Colombian Indians, 7 different DR4 alleles were found. This increase in diversity of DR4 alleles, also noted in other Amerindian groups, is in striking contrast to the overall lack of DRBl diversity and may reflect strong directional selection, perhaps in response to pathogenic agents, acting to augment the DR4 variation. Conceivably, a small number of founding DR4 alleles may have diversified by mutation, recombination and/or gene conversion, and then increased in frequency by selection or drift. The predominant DR4 subtype is different in different groups. For example, DRB1*0404 and *0411 are the DR4 alleles in the Coreguaje (with very high frequencies of f=0.32 and f=0.47), DRB 1*0407 is predominant in the Kogui (f=0.60) and DRB1*0403 is the principal DR4 allele in the Tule (fd.22). DQAi and DQ5i The distributions of DQA 1 and DQB I in the Colombian Amerindian populations studied are lisred 177

5 Trachtenberg et al. Codnn Position 9 65 T -w C C -a- A first base pair first base pair Transillon Trnnrvcrsiun Hi5 Leu 7 In Colombia Figure 2. DPB1*3501 may have been generated from DPB1*1401 in some Colombian Amerindian populations by a C+A transversion at the first base position of codon 65. DPB1*1401, in turn, may have been generated from DPB1*0301 by a T+C transition at the first base position of codon 9. DPB1*3501 has not been found in any other Amerindian groups. Since DPB 1 *3501 has been reported in an African and Portuguese population (see text), the appearance of this allele in some Colombian Amerindian groups represents a possible example of convergent evolution and of independent origins for the same DPB 1 allele. in Tables 2 and 3, respectively. The reduction in DQ diversity in the Amerindian groups is consistent with available DRB 1 alleles, known to be in linkage disequilibrium with DQ. At the DQAl locus the alleles were predominantly *0301, *0401 and *0501, with allele frequencies as high as f= in some populations. At the DQBl locus the alleles were "0301 (f=-0.80), *0302 (f=o ), and *0402 (f=-0.65); each population has a predominant DQB 1 allele. DPBl The DPBl allelic distribution in the Colombian Amerindians (Table 4) was also restricted with the number of alleles per population ranging from three (DPBI*0402, *1401, *3501 in the Kogui, and DPBl *0301, *1401, *0402 in the Nukak) to six (Sikuani). Overall, DPB 1 *0402 (f= ) and DPBl*1401 (f=o ) are the primary DP alleles. DPB1*1301 was present in two groups (the Waunana and the Embera, both of the Pacific coast) at high frequencies (f=o ). DPB 1*3501 was found at a high frequency (f=0.27) in the Kogui, and at very low frequencies in three other populations. The DPB1*3501 and DPBl*1301 alleles are absent in all other North and South American populations. The alleles DPB 1 * 1401 and DPB 1 *3501 are absent or very rare alleles in almost all populations. However, DPB 1 * 1401 is a very common allele in Ecuadorian Amerindian groups (9; Trachtenberg, Watkins and Erlich, unpublished), and is also found at lower frequencies in Brazilian and Argentinian Indians (4; Trachtenberg, Satz and Erlich, unpublished). The DPB1*1401 and "3501 alleles differ by a single, first base change (C+A) at codon position 65 in exon 2. This first base pair transversion changes leucine to isoleucine at this codon. We suggest that DPB1*3501 may have been generated from DPB1*1401 by either a point mutation or an interallelic gene conversion event (changing CTC to ATC at codon position 65), with subsequent selection for the DPB 1 *3501 allele in the Kogui and three other Colombian populations. Assuming a gene conversion origin, a potential donor for the isoleucine (ATC) codon at position 65 could be the DPB 1 *0402 allele, which is found in high frequencies in these Colornbian populations. The highest frequency of the DPB 1 *3501 allele is found in the Kogui population. The Kogui have only two other DPB 1 alleles, including DPB1*0402 and *1401, which could be, by hypothesis, the parental alleles to DPB1*3501. DPB 1 *3501 has also been reported in an African and Portuguese population (24). In addition, one DPB 1 *3501 allele was identified recently in a Siberian population (Trachtenberg, Schanfield, and Erlich, unpublished). The possibility that DPB 1*3501 was transferred from Asia during the colonization of the Americas and lost everywhere except these Colombian groups cannot be formally excluded. If DPB 1 *3501 has, in fact, been generated recently in Colombia, this event would represent an independent origin and convergent evolution of this allele. Evidence for convergent evolution of the novel Table 4. DPBl in Colombian Amerindian Populations Kogui ljka Guahibo Nukak Sikuani lngano Waunana Ernbera Tuie / N a 178

6 ~~ Table 5. HIA Class I1 DRB1-DQA1-DOE1 Haplotypes in Nine Amerindian Populations from Colombia HLA class I1 in nine Amerindian populations from Colombia ORBl-OQA1-DO81 Kogui ljka Sikuani Nukak Coreguaje lngano Waunana Embera Tule Haplotype (f) (1) (1) (f) (f) (f) (1) (f) (f) DR DR OR DR OR DR DR DR N.60 (1 (j t> 0.07 [] (0.071 ( N=60 [] N=54 [ ]=Caucasian or African American admixture c >=African American admixture N= [] N.60 N= [] 0.27 N= ( N= [] N=58 DRB 1 *08042 has been noted recently in an Ecuadorian Amerindian population, the Cayapa (6). DPB1*1401 differs from DPBl"0301, a fairly common DPB 1 allele in many different populations including Amerindians, by only a single, first base transition (T+C) at codon position 10 in exon 2. This transition changes tyrosine to histidine at codon position 10. It is conceivable that DPB1*1401 arose from DPB1*0301. Thus in two single base changes, a transition at codon position 10 and a transversion at codon 65, DPBl*3501 could have evolved from "0301, with DPB1*1401 as the intermediary allele (Figure 2). DR-DQ haplotypes in the Colombian Amerindians Whereas the complexity of DR-DQ haplotypes from the Colombian African populations will require further analysis, as discussed in materials and methods. the Amerindian haplotypes were easier to determine, using known linkage disequilibrium patterns or determining them unambiguously by homozygosity. The DR-DQ haplotypes and haplotypic frequencies for the nine Colombian Amerindian populations studied are listed in Table 4. There are five haplotypes found at high frequencies in the nine Colombian Amerindian populations. The DRB 1 * 1602-DQA 1 * DQB 1 *030 1 haplotype is found with frequencies at relatively high frequencies in the Sikuani (f=0.39), Waunana (f=0.33) and Embera (f=0.33) populations. The DRB 1*0407- DQAl*03-DQBl*0302 haplotype is found at a very high frequency (f=0.60) in the Kogui. DRB 1 *0411- DQA 1*03-DQB 1 *0302 is found at high frequency in the Coreguaje group. The DRB 1 * DQA 1 *0501-DQB 1*030 1 haplotype is found with a high frequency in the Sikuani (f=0.41) and in the Nukak (f=0.65), and is found at a lower frequency 179

7 Trachtenberg et al. in the Waunana (f=0.27). Finally, DRB 1 *0802- DQA 1 *0401-DQB 1 *0402 is found at very high frequencies in the Ijka (f=0.62), and is also common to the Ingano (f=0,18) and Coreguaje (f=0.18). These haplotypes are common to all Amerindian groups, however the very high frequencies found for some haplotypes were unusual. Some DR-DQ haplotypes in these Colombian Amerindian populations exhibited new DRB 1-DQA1- DQB 1 combinations. The novel haplotypes included DRB 1 * 1602-DQAI *03-DQB 1 *0301 in the Coreguaje, DRE31*0404-DQAI*0501-DQB 1*0301 in the Ingano, DRB 1 *0404-DQAI*050I-DQBI*O302 found in three individuals from the Coreguaje group, and the haplotype DRB1*1402-DQAI*03- DQB 1 *0301 found in the Embera and Tule populations. Another unusual finding in the Amerindian data was the presence of a novel DR8 haplotype, DRB1*08041-DQA1*0401-DQB1*0402 (f=0.04), in the Sikuani Indians (Central Plains). Virtually all Amerindian DR8's are DRB1*0802, with the exception of *08042 in the Cayapa, *0807 in the Ticuna and *0811 in the Navaho (28). The presence of a DRB1*08041 allele could reflect admixture with the Colombian Africans, however the African haplotype for this allele is DRB1* DQAl*0401-DQB 1 *0302 (1). Moreover, DR8 alleles were found only at low frequencies (f= ) in two of the Colombian African populations studied (see accompanying paper). The Amerindian allele DRB 1 *0802, which is a common Amerindian allele, is most frequently found with DQA 1 "0401-DQB 1 *0402, and therefore the *08041 allele in the Sikuani may have been generated anew from *0802, similar to the generation of DRB 1*08042 from *0802 in the Cayapa Indians of Ecuador (22). Summary Analysis of HLA class I1 alleles among 9 Amerindian populations (total N=227) from six geographically distinct regions of Colombia reveals a restriction in the class 11 diversity in the Amerindians, in striking contrast to a profusion of alleles, including a novel DQB 1 *02 allele, found in the neighboring Colombian African populations (see accompanying paper). There are however, very significant differences in the distribution of the class I1 alleles between these Colombian Amerindian groups. Presumably, the observed differences between the Colombian Indians could reflect either drift, selection or both. The two most similar populations are the Waunana and the Embera, both on the Pacific coast. Both of these groups have a high frequency of DPB 1 * 130 I, an allele absent from other Amerindian groups. The Waunana and the Embera also have similar frequencies for DRB1*0407 and *1402 and their related DR-DQ haplotypes. The Kogui and the Ijka, who live in nearby environs in the Sierra Nevada, also have similar DR-DQ distributions; all DR-DQ haplotypes found in the Kogui are also found in the Ijka. The Ijka, however, also exhibit many other DR-DQ haplotypes. Both populations also carry the DPB1*3501, found at low frequencies in three groups and at a high frequency (f=0.27) in the Kogui. In addition, the Kogui show no class I1 admixture with either CaucasianMestizo or African American admixture, and the Ijka, who trade with the neighboring Mestizo communities, have more admixture than any other, Colombian Amerindian group. While the Kogui and the Ijka populations have very different cultural structures and are not known to intermarry, the class I1 allele distribution indicates that they may have had some interpersonal communication in the past. Studies of other Amerindian groups, including many North American populations (1,5), and South American Amerindians from Ecuador (9, 21; Trachtenberg, Watkins and Erlich, unpublished) Brazil and Argentina (4; Trachtenberg, Satz and Erlich, unpublished) indicate a reduction in diversity of class I1 alleles similar to that found in the Colombian Amerindian populations. Comparisons of Colombian Amerindian alleles with those of AsiadPolynesian-Pacific origin, ( 1 ; Trachtenberg, unpublished), do not support a Polynesian origin of Colombian Amerindians. Moreover, in contrast to the Colombian Amerindians, the Polynesian populations, although isolated, show a remarkable degree of HLA diversity. The reduction in HLA class I1 allelic variation in the Colombian Amerindians may be due to a population bottleneck during the migrations that led to the colonizing of the Americas, or conceivably to genetic drift, in these small, relatively isolated South American groups. However, the extensive diversity of the nearly isolated Colombian African groups argues against the genetic drift explanation. An alternative theory of allelic restriction in the Colombian Amerindians would be a loss of alleles due to illness or to environmental stresses with subsequent positive directional selection in response to infectious pathogens, driving certain few alleles to the very high frequencies observed. None of the arguments for the reduction in HLA variability seen in the Colombian Amerindians are mutually exclusive. It is worth noting, however, that the alleles absent from the Colombian Amerindian populations are also missing from virtually all other Amerindian groups studied thus far. This is consistent with a 180

8 bottleneck during colonization rather than genetic drift in these small populations. Acknowledgments The authors would like to thank the peoples of Colombia for participating in the Expedicion Humana and C. Rodas for her assistance in sample collection and DNA extractions. Many thanks also to Kathy Levenson and Jera Wodehouse for help with manuscript preparation. This work was supported in part by a grant from the NIH to HAE (#HL ), and by the Expedicion Humana, Instituto de Genetica Humana, Pontificia Universidad Javeriana, Bogota, Colombia. References 1. Imanishi T, Akaza T. Kimura A, Tokunaga K, Gojoberi T. In: Tsuji K, Aizawa M, Sasazuki T, ed. HLA 1991, Vol I. Oxford: Oxford University Press, 1992: Belich MP, Madrigal JA, Hildebrand WH et al. Unusual HLA-B alleles in two tribes of Brazilian Indians. Nature 1992: 357: Watkins DI, McAdam SN, Liu X et al. New recombinant HLA-B alleles in a tribe of South American Amerindians indicate rapid evolution of MHC class I loci. 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