HLA-B alleles of the Cayapa of Ecuador: new B39 and B15 alleles

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1 Immunogenetics (1995) 42:19-27 Spfinger-Veflag 1995 Theodore L. Garber Lesley M. Butler Elizabeth A. Traehtenberg Henry A. Erlich Olga Rickards Gianfranco De Stefano David I. Watkins HLA-B alleles of the Cayapa of Ecuador: new B39 and B15 alleles Received: 3 November 1994 / Revised: 23 February 1995 Abstract Recent data suggest that HLA-B locus alleles can evolve quickly in native South American populations. To investigate further this phenomenon of new HLA-B variants among Amerindians, we studied samples from another South American tribe, the Cayapa from Ecuador. We selected individuals for HLA-B molecular typing based upon their HLA class II typing results. Three new variants of HLA-B39 and one new variant of HLA-B15 were found in the Cayapa: HLA-B*3905, HLA-B*3906, HLA-B*3907, and HLA-B*1522. A total of thirteen new HLA-B alleles have now been found in the four South American tribes studied. Each of these four tribes studied, including the Cayapa, had novel alleles that were not found in any of the other tribes, suggesting that many of these new HLA-B alleles may have evolved since the Paleo-Indians originally populated South America. Each of these 13 new alleles contained predicted amino acid replacements that were located in the peptide binding site. These amino acid The names listed for these sequences were officially assigned by the WHO nomenclature Committee in September 1994, B'3905, and November 1994, B*1522, B'3906, and B'3907. This follows the agreed policy that, subject to the conditions stated in the most recent Nomenclature Report (Bodmer et al. 1994), names will be assigned to the new sequences as they are identified. Lists of such new names will be published in the following WHO Nomenclature Report. The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers U14756 (HLA-B*1522), U15683 (HLA-B*3905), U15639 (HLA-B*3906), and U15640 (HLA-B*3907) Theodore L. Garber Lesley M. Butler David I. Watkins (ES~) Wisconsin Regional Primate Research Center and Department of Pathology, University of Wisconsin, 1220 Capitol Court, Madison, WI 53715, USA Elizabeth A. Trachtenberg Henry A. Erlich Department of Human Genetics, Roche Molecular Systems, 1145 Atlantic Ave., Alameda, CA 94501, USA Olga Rickards Gianfranco De Stefano Dipartimento di Biologia, Universit~t degli di Roma "Tor Vergata", Via della Ricerca Scientifica, Rome 00133, Italy replacements may affect the sequence motif of the bound peptides, suggesting that these new alleles have been maintained by selection, New allelic variants have been found for all common HLA-B locus antigenic groups present in South American tribes with the exception of B48. In spite of its high frequency in South American tribes, no evidence for variants of B48 has been found in all the Amerindians studied, suggesting that B48 may have unique characteristics among the B locus alleles. Introduction The major histocompatibility complex (MHC) class I and class II loci are unusually polymorphic genes that encode membrane-bound glycoproteins that play an important role in the acquired immune response (Klein 1986; Zinkernagel and Doherty 1974; Unanue 1984;, Schwartz 1985; Townsend et al. 1986; Hill et al. 1992). The excess of nonsynonymous over synonymous nucleotide substitutions in sites encoding the peptide binding site suggests that the extensive allelic diversity at these MHC loci may have been maintained by positive selection pressure (Hughes and Nei 1988, 1989; Hughes et al. 1993). Using nucleotide sequence data from the MHC class I and class II loci of primates and rodents, Takahata and Nei (1990) calculated that the large number of MHC alleles and the high average heterozygosity observed in most species could be explained by either overdominant selection or frequency-dependent selection. While allelic lineages at certain MHC loci have been conserved during evolution (Klein 1987; Fan et al. 1989; Slierendregt et al_ 1992), alleles at some of the MHC loci appear to have evolved rapidly (Belich et al. 1992; Watkins et al. 1992; McAdam et al. 1994). The slowly evolving HLA-DQA locus (Gyllensten and Erlich 1989; Kenter et al_ 1992a) represents one end of the spectrum of evolutionary rates, whereas the HLA-B locus is at the other more rapidly evolving extreme. Homologues of several of the human DRB and DQB allelic lineages are found in nonhuman primate species. Alleles of these lineages, however, tend to

2 20 cluster by species, suggesting that diversification of these lineages has taken place after speciation (Gyllensten et at. 1990, 1991; Kenter et al_ 1992b; Geluk et al. 1993). Although an A locus lineage is shared between chimpanzees and humans, less similarity is seen between the B loci of these two species (Mayer et al. 1988; Lawlor et al. 1988; Parham et al. 1989; Chen et al. 1992; McAdam et al. 1994, 1995, and data not shown). It is more difficult, however, to detect the presence of homologues of HLA-A or B locus allelic lineages in the gibbon, orangutan, or rhesus monkey, suggesting that the MHC class I loci may have evolved more rapidly than their MHC class II counterparts (Miller et al. 1991; Chen et al. 1992). Recent evidence from isolated tribes of native South Americans, chimpanzees, and bonobos suggests that the B locus may be evolving more rapidly than other MHC class I or class II loci. The Kaingang, Guarani, and Waorani are isolated populations that express a limited number of HLA serotypes. However, DNA sequence analysis of the HLA class I alleles from members of these tribes demonstrated that some of these individuals express new HLA-B allelic variants. Furthermore, these variants apparently have arisen as a result of recombination between exon 3 of alleles extant in the population (Belich et al. 1992; Watkins et al. 1992). Similarly, chimpanzees and bonobos also appear to have evolved new B locus alleles by recombination in exon 3 (McAdam et al. 1994). To test whether rapid evolution of B locus alleles is a general phenomenon that has occurred in native South Americans, we sequenced HLA-B alleles from a fourth Amerindian tribe, the Cayapa of Ecuador. The Cayapa, or Chachi, primarily live in the tropical forest in northwest Ecuador and possibly represent the first inhabitants of Ecuador (Barriga L6pez 1987). The Cayapa belong to the Chibchan-Paezan branch of the Amerind family, one of the three major linguistic families of Native Americans (Greenberg 1987). Genetic and additional anthropological evidence supports this threefold linguistic division, suggesting that the New World was inhabited by three migrations that gave rise to the current Amerind, Na-Dene, and Aleutspeaking people (Williams et al. 1985; Greenberg et al. 1986). Other genetic evidence suggests that there may have been two distinct migrations that gave rise to the current Amerinds (Schanfield et al. 1992; Horai et al. 1993). The Kaingang, Guarani, and Waorani of earlier studies are also included in the Amerind linguistic family. There are approximately 3600 living Cayapa; other studies have not yielded genetic evidence of admixture of the Cayapa with other Ecuadorian populations (Erickson et al. 1966; Stinson et al. 1989; Rickards et at. 1994). The Cayapa DNA samples used in the current study were selected for sequencing based on the results of polymerase chain reaction (PCR) sequence-specific oligonucleotide probe (SSOP) typing of their MHC class II loci. These 13 samples were chosen because they represented several different class II haplotypes. Class II analysis of the Cayapa revealed a novel DRB1 allele (DRBl*08042) and several new class II haplotypes which were unique to the Cayapa and which probably arose after T. L. Garber et al.: New HLA-B alleles of the Cayapa the Cayapa separated from other South American Amerindians (Titus-Trachtenberg et al. 1994). This finding is suggestive of selection pressure operating to maintain diversity of these novel haplotype arrangements, and echoes the relatively rapid rate of evolution of the HLA-B locus found in the three other South American Amerind tribes studied. The novel HLA-B locus variants found in the Waorani, Kaingang, and the Guarani tribes differ from previously described Caucasian and Oriental alleles by nonsynonymous substitutions that alter predicted amino acids located in the peptide binding site. Since these substitutions are largely found in the putative peptide binding site, these variations may have functional significance. HLA-B alleles from the Cayapa were therefore amplified using primers located in exons 2 and 3. This amplified region encoded most of the residues in the alpha 2 and alpha 3 domains involved in peptide binding and in T-cell receptor binding. The DNA sequences recovered from the Cayapa were then compared with previously described HLA alleles to determine whether novel alleles were present. Materials and methods DNA samples Genomic DNA extraction fi-om whole blood was carried out using an inorganic salting out procedure (Miller et al. 1988). PCR Amplification HLA-B locus-specific clones were amplified from genomic DNA using the primer GCBH3 (based upon Yoshida et al, 1992) and nua2enr1. PCR reactions included 25 pmoles of each primer, 50 mm KC1, 10 mm Tris-HC1 ph 8.3, 2.5 mm Mg Cla, 200 um each datr dttp, dgtr and dctp (Pharmacia, Piscataway, NJ), and 1.5 units Taq DNA polymerase (Perkin Elmer, Norwalk, CT) in a final volume of 50 ul. The first cycle denaturation was at 94 C for 30 s, followed by 40 cycles of 94 C 15 s, 55 C for 30 s, and 72 C for 1 rain 30 s, and a final extension at 72 C for 7 rain. Amplifications were carried out using a Perkin Elmer model 9600 thermocycler. PCR products were cloned into psp65 (Melton et al. 1984; Promega, Madison, WI) and sequenced with Sequenase (US Biochemicals, Cleveland, OH) using a slight modification of the manufacturer's method (Sanger et al. 1977; Garber et al. 1994). At least three copies of each allele were sequenced. 7-deazaguanine or ditp with or without terminal deoxynucleotide transferase were used to eliminate gel compressions and strong stops (Fawcett et al. 1990; McCrea et al. 1993). PCR and sequencing primers The sense PCR primer GCBH3 was located at an H/A-B-specific site in exon 2. The anti-sense PCR primer nua2enr1 was located in the conserved 3' end of exon 3. Each PCR primer had two mixed-base sites based upon reported HLA-B sequences (Zemmour and Parham 1992); 1187 and A are vector-specific sequencing primers_ IVSB is a mixed-base primer which binds to the 3' end of the intron separating exon 2 and exon 3. A-424 binds to exon 3 at position ; 1187 and IVSB are sense primers, A and A-424 are antisense primers.

3 T. L. Garber et al.: New HLA-B alleles of the Cayapa 21 Table 1 MHC class I and II alleles of the Cayapa. HLA-B locus alleles from 13 unrelated individuals were amplified using a B-locus specific primer and a conserved primer. Underlined alleles designate newly described B alleles. Most B locus alleles were found in more than one individual; references are given only for the first listing of each allele. * Titus-Trachtenberg and co-workers (1994) Sample HLA class I alleles HLA class II alleles* HLA class I alleles previously sequenced from: 1 B'4801 DRB1 *0802-DQA1*0401-DQB1*0402/ South American: Kaingang, Guarani, Waorani; DRB1 *08042-DQA1*0401-DQBl*0402 North American: Zuni (Belich et al. 1992; Watkins et al. 1992) B *1501/B*3507 B'3501 B "3905/B'3907 B *3906 B *1522/B *4004 B*3906/B "4801 B*1522/B*3507 B'4801 B'4002 B "3906/17 "4801 B*1522/B*4002 B "3907/B'4002 DRB1 *0407-DQA1 "0301-DQB1 *0302 DRB1 *0901-DQA1 *0301-DQBl *0303/ DRB1 *1602-DQA1 *0501-DQB1 *0302 DRB1 *0802-DQA1 "0401 -DQB1 *0402/ DRB1 *0901-DQA1 *0301-DQB1 *0302 DRB1 *0407-DQA1 *0301-DQB1 *0302/ DRB1 *0901-DQA1 *0301-DQBl *0303 DRB1 *1402-DQA1 *0301-DQB1 *0302/ DRB1 *1402-DQA1 *0501-DQB1 "0301 DRB1 *0407-DQA1 *0301-DQB1 *0302/ DRB1 *0802-DQA1 *0401-DQB1 *0402 DRB1 *0407-DQAI *0301-DQB1 *0302/ DRB l *0901-DQAl *0301-DQB1 *0303 DRB1 *0802-DQA1 "0401 -DQB1 *0402/ DRB1 *0901-DQAl *0301-DQB1 *0303 DRB1 *0404-DQA1 *0301-DQB1 *0302 DRB1 *0407-DQA1 *0301-DQB1 *0302/ DRBI "0411-DQA1 *0301-DQB1 *0302 DRB1 *0404-DQA1 *0301-DQB1 *0302/ DRB1 *0802-DQA1 *0401-DQB1 *0402 DRB1 *0407-DQA1 *0301-DQB1 *0302/ DRB1 *1402-DQA1 "0501 -DQB1 "0301 Caucasian (Pohla et al. 1989)/Caucasian (Theiler et al. 1993) Japanese (Ooba et al_ 1989) Cayapa/Cayapa Cayapa Cayapa/Guarani (Belich et al. 1992) Japanese (Ling et al. 1992), Caucasian (Domena et al. 1992), Zuni (Watkins et al. 1992) PCR primers Restriction endonuclease sites used for directional cloning are underlined. GCBH3; 5' GC AA GCTT GAC GAC AC(C/G) C(A/T)G TTC GTG A 3' nua2enr1; 5' GC GAATTC CAG C(G/T)T (C/G)TC CTT CCC GTT CTC 3' Sequencing primers 1187; 5' TCA CAC AGG AAA CAG CTA TG 3' A; 5' ATT TAG GTG ACA CTATA 3' IVSB; 5' GCT CGG GGG A(C/G)(G/T) GGG CTG AC 3' A-424; 5' GAT GTA ATC CTT GCC GTC GTA 3' recovered alleles in Figure 1. The site for the sense primer GCBH3 included adenine 102 of exon 2, which is unique to all HLA-B alleles except HLA-B*5401, an allele not found in Amerindians (Zemmour and Parham 1992)_ Use of GCBH3 restricted amplification to only B locus alleles, no products of other loci were found among all clones screened. Two of the 13 Cayapa in this study appeared to be B'480I homozygotes; only B'4801 was found among the 24 HLA-B clones from the two apparent homozygotes (12 clones from each individual). Both of the B'4801 homozygotes were heterozygous for their HLA class II loci (Table 1). Results Amplification of HLA-B-specific sequences Amplification, cloning, and sequencing of genomic DNA from 13 unrelated individuals from the Cayapa tribe resulted in the identification of four new HLA-B alleles. Sequence-based HLA typing assignments of all recovered alleles are shown in Table 1. Nucleotide alignments for coding regions and PCR priming sites are shown for all Description of the new HLA-B alleles The new B15 allele, HLA-B*1522, was found in three individuals (Table 1). HLA-B*1522 has a C at position 133 of exon 2 that is unique among all HLA-B15 alleles (Fig. 1). HLA-B*1522 was identical to B*1508 (Hildebrand et al_ 1994) with the exception of amino acids 45 and 46 of the alpha 1 domain. HLA-B*1522 had the sequence ACG GAG at positions of exon 2 which codes for

4 22 Fig. 1 Alignment of nucleotide sequences from the Cayapa_ Sequences are compared against exons 2 and 3 of HLA-B*39013 (Kato et al. 1993). The four new HLA-B alleles described in this paper ate HLA-B*1522, HLA-B*3905, HLA-B*3906, and HLA-B*3907. Potential donor sequences for microrecombination events that resulted in these four new alleles are boxed. The unique sequence found in exon 3 of HLA-B*3906 but not in any other allele found in the Cayapa is underlined. PCR primer sites used to amplify the B locus are underlined in the B'39013 sequence; see Materials and methods for primer sequences. The B locus-specific primer site was located at nucleotides of exon 2 HLA-B*39013 HLA-B*39013 HLA-B*3905 HLA-H*3906 HLA-B*3907 HLA B'4801 HLA-B*I522 HLA-B*4002 HLA-B*3507 HLA-B*4004 HLA-B*I501 HLA-B*39013 HLA-B*3905 HLA-B*3906 HLA-B*3907 HLA-B*4801 HLA-B*I522 HLA-B*4002 HLA-B*3507 HLA-B*4004 HLA-B*I501 exon 2 T. L. Garber et al.: New HLA-B alleles of the Cayapa 1 I C`CTCCCACTCCATGAGGTATTTCTACACCTCCGTGTCCC~GCCC(~3CCGCGGC`C~%GCCCCGCTTCATCTCAGTCCGCTACGTGGAC~ACACGCAGT~`CGTGA llo C(~ TTCGACAGCOoACGC CGCGAGT C CGAGAGAGGAGC C G CCGG CG CCGTGGA TAGAGCAC-GAG GGG CCGGAATATT(3GC, AC CGGAACACAC AGATC~ ~GACC... A... ~... A... G... G.G... C ~AC.,,. I C... A G T.... A... GA... A... G... G.G... C GAT..C...C... A... G... G.G... C... I exon ] 30 AACACACAGACTGACCGAGAGAGCCTG CGGAACCTGCGCGGCTACTACAACCAGAGCGAGGC CG 1 (]GTC TCA CAC C C TCCAGAC-GATGTA CC-G C TGCGACG TC~G... E]... i... ii!iil iiiliiil fill i... TTGG... C = HLA-B*39013 HLA-B*3905 HLA-B*3906 HLA-B*39f17 HLA B~4801 HLA-B*I522 HLA-B*4002 HLA-B*3507 HLA-B*40D4 HLA-B*I I00 ii GGC GGACGGGCGCCTCCTCCG GGG ATAACCAGTTCGCCTACGACGGCAAC`GA TACATCG CCTGAACGAGGACCTGAGCTCCTC<]ACCGCC`(3CGGACACC A C... C... G... ~... G... A... C... C... G... A... C... C... G... ~... G HLA B*3H013 GCGGCTCAGATCACCCAGCGCAAGTGGGAGGCGGCCCGTGTC~1CGGAGCAGCTC,AGAACCTACCTCGAGGGCACGTGCGTGGAGTC/~CTCCGCAGATACCTG HLA-B* HLA-B* HLA-B* HLA-B* T T G... C~ H L A - B * A... TG G CT HLA B' G C~ HLA-B* G CT HLA-B*40fl G C~ HLA-Be A... G CT "( C HLA B'39013 C=AC~AAC GG~GGAGAC G C TG CAG CG CG CGG threonine 45 and glutamic acid 46. At these same sites, B*1508 has ATG GCC encoding methionine and alanine, respectively. Position 45 is more variable among B locus alleles than it is in A or C locus alleles (Bjorkman and Parham 1990). The amino acid found at position 45 of the alpha 1 domain is located in the floor of the B pocket and is important in determining the motif of the second amino acid residue of the bound peptide (Saper et al. 1991; Madden et al. 1991). Due to the difference between HLA-B*1522 and B*1508 at position 45, these molecules may therefore bind peptides with different sequence motifs. HLA-B*1522 may be another example of the formation of a new allele by recombination in the New World. B'3507, which was found in the Cayapa (Table 1), and other B35 and B5 alleles all have amino acids identical to HLA-B*1522 at position (Theiler et al. 1993). B35 and B5 antigens are common in Amerindian populations (Imanishi et al. 1992; Petzl-Erler et al. 1993; Williams and McAuley 1992). HLA-B*1522 thus appeared to be the result of a recombination between a B*1508-1ike gene and a B35 or B5 allele. HLA-B*1522 was associated with three different DR alleles in three individuals also heterozygous for HLA-B. The HLA-B*1522 alleles were associated with DRBI*0407 or "0901, and DRBI*0404 or *0802 in two DR heterozygotes, and with DRBl*1402 in linkage with either DQB1 *0301-DQA1 *0302 or DQAI*O501-DQBI*0301 in another individual (Titus- Trachtenberg et al. 1994; Table 1). Finding HLA-B*1522 in association with at least three DR alleles indicates recombination has occurred to generate new haplotype diversity, and suggests that HLA-B*1522 was not generated in recent years. Three novel HLA-B39 alleles were also found among the Cayapa. All three of these new B39 alleles are distinguished by nonsynonymous substitutions resulting in predicted amino acid replacements at residues that are part of the peptide binding site. The new allele HLA-B*3905 was recovered from a single heterozygous individual and is associated with either DRBI*0901 or *0802 (Table 1). Relative to previously described B39 alleles, HLA-B*3905

5 T. L. Garber et al.: New HLA-B alleles of the Cayapa 23 Fig. 2 Alignment of the predicted amino acid sequence of the alpha 1 and alpha 2 domains. The sites of the predicted amino acid replacements of the novel alleles HLA-B*1522, HLA-B*3905, HLA-B*3906, and HLA-B*3907 are underlined and demonstrate that the new alleles could result in predicted amino acid replacements within the peptide binding site. * Indicates amino acad residues whose side chains project into or towards the peptide binding site that may affect the motif of the peptide bound; ^ Indicates residues whose side chains project outwards, possibly affecting interaction wath the TCR; + Indicates residues that may affect both the binding of peptides and TCR interaction (Bjorkman et al. 1987a, b; Bjorkrnan and Parham 1990; Madden et al. 1991) l alpha 1 domain II B~39013 J GSHSMRYFYTSVSRPGI~GEPRFISVGYVDDTQFVR Bt t ^+. ^+t ^t*^at ^~t A* A*~A t FDSDAASPREEPRAPWIEQEGPEYWDRNTQICKTNTQTDRESLRNLRGY~/NQSEA B' B~ B' Y... B' T...K... E...S... Y... B' T...K... E...S... Y... B*4B01... E...S... Y... I al )ha 2 domain i00 RMYGCDVGP....q.....II... L.. I... S... B~ T... F... Y... i...ii... L.. B*1522 F... Y... ]... B' MA... E...S... Y... i... ii B~39013 DGRLLRGHNQFAYDGKDYIALNEDLSSWTAADTAAQITQRKWEAARVAEQLRTYLEGTCVEWLRRYLENGKETLQRA Bt B~ B' B' Y... ;... R... A... E... B' Y... R... A... E... B' Y... R... S...L... A... E... B~ D.S... A... L... B* D.S... E...W.A... L... B~I D.S... E...W.A... L... has a single G to T substitution at position 219 of exon 2 that results in a predicted aspartic acid to tyrosine replacement at residue 74 within the helical region of the alpha 1 domain (Figs. 1, 2). Amino acid 74 is not a very variable site in any of the HLA class I loci; only two different amino acids are found at this position in all B locus alleles (aspartic acid and tyrosine), two amino acids in all A locus alleles (aspartic acid and histidine), and one amino acid found in all C locus alleles [(aspartic acid) (Zemmour and Parham 1992)]. Even though variability at this site is low, the residue present at amino acid 74 can affect access to pocket C (Garrett et al. 1989; Saper et al. 1991). With the exception of one other new B39 allele reported here, all other B locus alleles described in this study have a T at position 219 in exon 2 and thus any one of these alleles could have served as the donor sequence in a microexchange event creating this new allele (Fig. 1). HLA-B*3907 was the second new B39 allele recovered and it was found in two individuals (Table 1). It shared with HLA-B*3905 the G to T substitution at position 219 of exon 2, resulting in an amino acid replacement at position 74 of the alpha 1 domain helical region. HLA-B*3907 also had two nucleotide substitutions in exon 3 that were unique among all B39 alleles. Relative to all other B39 alleles, HLA-B*3907 had an A to G substitution at position 69, and a T to C substitution at position 76 of exon 3 (Fig. 1). This resulted in amino acid replacements of asparagine to aspartic acid and phenylalanine to serine at amino acids 114 and 116, respectively, of the second beta strand of the alpha 2 domain (Fig. 2)_ While these replacements within HLA-B*3907 are unique to B39, three alleles containing amino acid 114 and 116 that were identical to HLA-B*3907 were also recovered from the Cayapa (Fig. 2)_ Any of these three alleles could therefore have participated in a genetic exchange with an allele similar to HLA-B*3905 (Fig. 2). This suggests that HLA-B*3905 was formed first, followed by recombination events that created HLA-B*3907. The aspartic acid 114 and serine 116 are also encoded for by other B locus alleles common among Amerindians including B15 and B35, so it is possible that alleles other than those found in the Cayapa may have been involved in the formation of HLA-B*3907, The HLA-B*3907 alleles were found in association with either DRB1 *0802 or "0901 and DRBI*0407 or "1402 in two heterozygous individuals, implicating recombination in the generation of novel haplotypes which serves to augment the HLA diversity in this Amerindian population (Table 1). A third new B39 allele, HLA-B*3906, was recovered from three individuals (Table 1)_ The nucleotide sequence from position in exon 3 of HLA-B*3906 differentiated HLA-B*3906 from all other B39 alleles. HLA-B*3906 did not share this novel nucleotide sequence with the other two new B39 alleles or any other allele recovered from the Cayapa (Fig. 1). Compared with other B39 alleles, the nucleotide sequence of HI_A-B*3906 resulted in two predicted amino acid changes at positions 95 and 97 from leucine to tryptophan and arginine to threonine, respectively, of the first beta strand of the alpha 2 domain. These replacements are restricted to B locus alleles and are not found in any A or C locus alleles reported. Amino acid residues 95 and 97 are located in the beta-pleated sheet at the base of the peptide binding groove and are accessible to bound peptides (Bjorkman et al_ 1987a, b). HLA-B*3906 shared this short sequence with other B locus alleles that were not found among the Cayapa surveyed, such as: B*1302, B*1504, B'4006, B*5101, B'5102, B'5103, B'5201, B'5401, B'5501, B'5502, B'5601, and B'7801 (Zemmour and Parham 1992). B*1504 and B'52012 are

6 24 found in another Ecuadorian tribe, the Waorani (Watkins et al. 1992), and B51 is a common serotype among South Americans (Imanishi et al. 1992; Petzl-Erler et al. 1993), demonstrating the presence of potential donors of this short sequence in South America. The HLA-B*3906 allele was also found in association with at least two DR alleles (Table 1) including DRBI*0407 and "0901 in one HLA- B'3906 homozygote. Two other HLA-B*3906 heterozygotes were also heterozygous at DR, one carrying DRB1 *0407, *0802, the other carrying DRB1 *0407, "0411. Discussion Original New World inhabitants have long been noted to have limited HLA allelic diversity (Kostyu and Amos 1981). In general, New World populations contain fewer class I and class II alleles defined by DNA analysis than other populations. This lack of diversity probably reflects the limited genetic diversity of the founding population. Furthermore, there was an extremely high rate of mortality in the New World after contact by Europeans. This may have been due to reduced MHC allelic variability which might have, in turn, resulted in a limited number of antigenpresenting molecules appropriate for the imported Old World diseases (Black 1992). Apparently South American populations responded to selection pressure caused by endemic tropical pathogens by rapid accumulation of new B locus alleles that are not easily distinguished serologically from Western antigens (Belich et al. 1992; Watkins et al. 1992). These new alleles may have been formed by recombination between alleles that were already present in the founding Paleo-Indians to give unique combinations of residues that line the peptide binding site but do not usually affect the supertypic serological specificity. Each of the South American tribes studied, including the Cayapa from this study, had unique HLA-B variants not found in other tribes, suggesting that these new variants arose de novo within each population; the restricted distribution of these alleles suggests that evolution of these new alleles must have occurred within the last 15,000-50,000 years since the New World was first populated by the Paleo-Indians. In the Cayapa, each of the new HLA-B alleles is found on more than a single class II haplotype; this observation indicates that although these alleles may have a relatively recent origin, there has been sufficient time for recombination to occur between the class 1 and class II regions. The rapid accumulation of new HLA-B alleles within these small populations also suggests that these novel variants conferred an immunological advantage to individuals and that these alleles have been maintained in the population by selection. Most of the new HLA-B alleles found in native South Americans appear to have been formed by recombination resulting in nonsynonymous substitutions at the predicted peptide binding site. New alleles formed by recombination can have large effects upon the specificity of the peptide bound. For example, HLA-B*5301 is a common allele T. L. Garber et al.: New HLA-B alleles of the Cayapa found in West Africa that is identical to B'3501 except for codons This results in five amino acid differences between B35 and B53 including the Bw4/Bw6 epitope (Allsopp et al. 1991)_ When peptides are eluted from purified B35 and B53 molecules, there is a difference in the dominant amino acid motif bound by each HLA molecule. B35-bound peptides have two dominant residues at position two (proline) bound by the B pocket and position nine (tyrosine) bound in the F pocket, whereas peptides eluted from B53 molecules only have a single dominant residue at position two (proline). B53 is associated with resistance to severe malaria in the Gambia and can bind a conserved nonameric peptide derived from a liver stage malarial protein (Hill et al. 1991, 1992). The B35 molecule binds and presents peptides which includes polymorphic epitopes from the circumsporozoite protein; however, B35 is not associated with immunity to severe malaria. The differences between the B53 molecule and the B35 molecule includes three positions in the peptide binding site which demonstrates that small numbers of amino acid differences localized to the peptide binding site may play an important role in protection from pathogens. Similar to the proposed origin of the B'5301 allele (Allsopp et al. 1991), all of the new alleles found in South American populations, including those found in the Cayapa, were apparently formed by recombinationmediated replacements at the predicted peptide binding site, suggesting that these new alleles may also have altered peptide specificity. Among all the New World populations studied, one B locus allele, HLA-B*4801, has been found that lacks any sequence variants (Table 2)_ Serologically detected B48 is also present in some Asiatic populations (Imanishi et al. 1992), which implies that the origin of the B48 preceded the peopling of the New World and that B48 was present in members of the founding populations (Williams et al. 1985; Horai et al. 1993). The lack of molecularly defined variants of B'4801 is uncharacteristic of class I serogroups with such wide distribution and high frequency among South American Natives. B15, B35, B39, B40 and B5 are serologically defined antigens that are also widely distributed among tribes in South America; unlike B48, new DNA sequence variants based upon these widespread serotypes were found in each of four tribes studied including the Cayapa (Belich et al. 1992; Watkins et al_ 1992). B'4801 encodes a predicted amino acid replacement at position 245 of the alpha 3 domain in the same position that may reduce the affinity of A'6801 for CD8 (Salter et al. 1989)_ The lack of variants in the predicted peptide binding site of B'4801 in any North and or South Amerindian tribe taken together with possibly reduced affinity of B'4801 for CD8 suggests that the B'4801 allele may not serve an important role in an immune response. It seems unlikely, however, that B48 has been strongly selected against because B48 homozygotes do apparently occur (Table I). An alternative hypothesis is that B48 has been maintained in Asiatic and New World populations, at high frequencies in some groups (Imanishi et al. 1992; Williams and McAuley 1992; Markow et al. 1993), because of an as yet undescribed selective advantage

7 T. L. Garber et al.: New HLA-B alleles of the Cayapa Table 2 New variants for all Amerindian B locus alleles have been found with the sole exception of HLA-B48. New alleles first described in Kaingang, Guarani (Belich et al_ 1992), Waorani (Watkins et al. 1992), and Cayapa are listed, demonstrating the presence of molecularly defined allelic variants for all alleles analyzed with the exception of HLA-B*4801. B'4802 is not included, since it probably represents a large scale recombination between B'4801 and B'3501. The leader peptide and most of the alpha 1 domain of B'4802 is identical to B'4801, whereas the remainder of B'4802 is identical to B'3501; B'4802 also reacts with some B35-specific antisera (Watkins et al. 1992) Antigen New alleles South American population B 15 B'1504 Guarani, Waorani B*1522 Cayapa B35 B'3504 Waorani B'3505 Kaingang, Guarani B *3506 Kaingang B 39 B'3903 Waorani B'3905 Cayapa B *3906 Cayapa B'3907 Cayapa B40 B'4003 Guarani B *4004 Guarani B48 none all B51 B'5104 Kaingang, Guarani B52 B'52012 Waorani from recombination in exon 2. This situation is also true in chimpanzees and bonobos, where recombination in exon 3 was more frequent than in exon 2 (McAdam et al. 1994). The HLA-B locus, therefore, appears to have evolved by recombination in different South American populations over the last to years. This rapid evolution of new B locus alleles is also evident in comparisons of chimpanzee, bonobo, and human B locus alleles. Alleles with sequence similarity to exon 2 of HLA-B48, B57, B7, B15, and B27 are found in chimpanzees and bonobos. Comparison of exon 3 of the chimpanzee and human B alleles, however, reveals a patchwork of motifs with no sequence similarity easily detectable in this exon (McAdam et al. 1994). Thus, the new recombinant HLA-B locus alleles in the South American tribes are an example of the process of interallelic recombination generating diversity at the B locus. Selection for maintenance of these new variants may be stronger in populations like the Amerindians that were founded by a group of individuals with a relatively low number of almic lineages. Acknowledgments This work was supported by grants RR00167 and A from the National Institutes of Health (DIW). We would like to acknowledge R. Guderian and A. Guevara for their help in blood collection. 25 conferred by this allele. In one of the Cayapan B'4801 homozygotes, B'4801 is associated with the DRBI*0802 allele and on the other haplotype, with the DRBl*08042 allele. This observation is consistent with the hypothesis that DRBl*08042 was derived in the Cayapa from DRBl*0802 (Titus-Trachtenberg et al. 1994). One novel feature of the new HLA-B alleles from the Cayapa is that some of these alleles are apparently the result of genetic exchange involving short segments of exon 2. All of the previously described Amerindian HLA-B locus alleles (with the exception of B'4802) could have been the result of recombination of small regions of exon 3 encoding the beginning of the alpha 2 domain. These nonsynonymous substitutions occurred in the beta sheet in the floor of the peptide binding site and affected peptide binding pockets C, D, E, and E Unlike the previously described variants found in native South Americans, HLA-B*1522 contains a substitution in exon 2 that may affect the sequence motif of the peptide bound by changing the specificity of the B pocket. In addition, two of the three newly described B39 variants have a tyrosine at position 74 of exon 2 in the alpha helix of the peptide binding site, This replacement in the alpha 1 domain might affect the residue bound in the C pocket similar to previously described replacements at positions 95 and 97 of the alpha 2 domain which also affects the C and E pockets (Garrett et al. 1989). 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