Novel alleles at the lymphotoxin alpha (LTα) locus mark extended HLA haplotypes in native Africans

Novel Alleles at the Lymphotoxin Alpha (LT␣) Locus Mark Extended HLA Haplotypes in Native Africans Jianming Tang, Angela D. Myracle, Susan Allen, Etienne Karita, Rosemary Musonda, Patricia N. Fultz, and Richard A. Kaslow ABSTRACT: Genetic variations at the closely related tumor necrosis factor alpha (TNF␣ or TNF) and lymphotoxin alpha (LT␣, formerly TNF␤) loci have been well documented in various human populations, and several haplotypes spanning the MHC class I and class II loci are known to carry specific TNF alleles. Genotyping of the TNFc microsatellite within the first intron of LT␣ in 285 Rwandans and 319 Zambians revealed two predominant alleles, c1 at frequencies of 0.598 and 0.683 and c2 at 0.384 and 0.307, respectively. Overall, the distribution of TNFc genotypes containing the major alleles conformed well to the Hardy-Weinberg equilibrium in both cohorts. Two previously unrecognized minor TNFc alleles were also detected: the first, designated c0, was found in 10 native Africans and was the only allele present in 10 chimpanzees; the second, designated c3, was seen in 6 other African patients. Further genotyping at loci for HLA class I, class II, and for transporters associated with antigen processing, subunit 1 (TAP1) in those 16 individuals suggested a tight, stable extended haplotype involving c0 and 26Asn (LT␣)-TNF3 (TNF promoter ⫺238A and ⫺308G)-DRB1*1503-DQB1*0602-TAP1.2 ABBREVIATIONS AIDS acquired immune deficiency syndrome bp base pairs HIV-1 human immunodeficiency virus type 1 HLA human leukocyte antigen LT␣ lymphotoxin alpha MHC major histocompatibility complex nt nucleotides OR odds ratio PCR polymerase chain reaction SNPs single nucleotide polymorphisms

From the Program in Epidemiology of Infection and Immunity (J.T., A.D.M., R.A.K.), School of Public Health, the Division of Geographic Medicine (J.T.), Department of Medicine, the Department of Epidemiology and International Health (A.D.M., S.A., R.A.K.), and the Department of Microbiology (P.N.F.), University of Alabama at Birmingham, Birmingham, AL; the National AIDS Control Program (E.K.), Kigali, Rwanda; and the Tropical Disease Research Center (R.M.), Ndona, Zambia. Human Immunology 62, 269 –278 (2001) © American Society for Histocompatibility and Immunogenetics, 2001 Published by Elsevier Science Inc.

(333Val)-TAP1.4 (637Gly). The c3 allele was observed on another extended haplotype with 26Thr (LT␣)-TNF1 (TNF promoter ⫺238G and ⫺308G)-DQB1*0102DQB1*0501-TAP1*0101 (333Ile and 637Asp). The c3tagged haplotype further extended to Cw*15 at the HLA class I C locus, but no specific A or B alleles could be unambiguously assigned. Positive associations between c2 homozygosity and HIV-1 seronegative status in both Rwandans and Zambians (odds ratio ⫽ 2.03 and 2.00, p ⫽ 0.04 and 0.07, respectively) had little effect on the haplotype assignments. These findings suggest a preferential expansion of the human TNFc dinucleotide (CT/ AG) repeat sequence and further imply the existence of two extended MHC lineages that have not been disrupted by recombinations. Human Immunology 62, 269 –278 (2001). © American Society for Histocompatibility and Immunogenetics, 2001. Published by Elsevier Science Inc. KEYWORDS: Africans; HLA; polymorphism; LT␣; TNFc

SSCP SSP STR TAP1 TAP2 TNF TNFc

single-strand conformation polymorphism sequence-specific primers short tandem repeat (DNA sequences) transporter associated with antigen processing, subunit 1 transporter associated with antigen processing, subunit 2 tumor necrosis factor TNFc microsatellite

Address reprint requests to: Dr. Jianming Tang, Division of Geographic Medicine, BBRB Box 7, 1530 3rd Ave South, University of Alabama at Birmingham, Birmingham, AL 39294; Tel: (205) 975 8608; Fax: (205) 934 8665; E-mail: [email protected]. Received June 27, 2000; revised November 27, 2000; accepted November 30, 2000.

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INTRODUCTION Genetic variations at loci for tumor necrosis factor (TNF, also known as TNF␣, TNFA or TNFa) and lymphotoxin alpha (LT␣, formerly known as TNF␤, TNFB or TNFb) have been studied extensively in the context of population genetics and disease associations [1, 2]. Several allelic variants in the TNF-LT␣ region have been shown to bear functional significance either independently or as part of extended haplotypes that also involve certain HLA class I and class II alleles [3]. For example, of the two biallelic variants at the ⫺238 (G or A) and ⫺308 (G or A) positions of the TNF␣ promoter [4, 5], ⫺308A (TNF2) has been associated with elevated promoter activity [6, 7], and ⫺308A is also tightly linked to the HLA A1-B8-DR3 haplotype [8, 9]. The numbering of nucleotide positions has been corrected more recently [1, 10], and reports of population-specific variants in the TNF-LT␣ region continue to emanate [10 –12] although their frequencies, function, and relationships to variants at commonly typed HLA loci remain to be defined across populations. Polymorphisms in the LT␣ locus include a dinucleotide (CT/AG) repeat sequence (the TNFc microsatellite) in the first intron [13], with two alleles being reported in several studies. This microsatellite is positioned in a region predicted to have multiple transcription-factor binding sites similar to those found in the 5⬘ flanking sequences [14]. Irrespective of their functional relevance, the microsatellite alleles further resolve haplotypic specificity [13, 15] when neighboring variants are also typed. Higher haplotypic specificity may be particularly important for proper interpretation of associations between TNF-LT␣ polymorphisms and disease. For example, the TNFc microsatellite c2 allele has been reported as a marker for slower rate of progression to AIDS among HIV-infected European Caucasians [16]. However, both experimental data [17–19] and epidemiologic associations based on multiple cohorts [20 –24] have demonstrated more unambiguously that genetic variants in the HLA loci mediate HIV-1 disease progression and that multilocus haplotypes in the MHC may be involved. Some of the reported relationships between TNF-LT␣ polymorphisms and the widely differing profile of human TNF␣ and LT␣ production/induction in individuals as well as populations [1, 25] are clearly debatable [26, 27]. Substantial differences between ethnic groups in both frequencies and haplotypic diversities [1] imply that the apparent effects attributable to isolated TNFLT␣ alleles can often be confounded by the presence or absence of variants at additional sites. Accordingly, comparison of allele distributions and haplotype structures between ethnically distinct populations should help dissect the role of TNF-LT␣-HLA lineages. Here, in two

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groups of native Africans enrolled in cohort studies of HIV/AIDS, we summarize findings from the TNFc microsatellite typing and describe two novel alleles along with their extended HLA haplotypes. MATERIALS AND METHODS Subjects and Genotyping Materials A group of 285 females (207 HIV-1 seropositive and 78 HIV-1 seronegative) participating in an HIV/AIDS cohort study in Kigali, Rwanda [28 –30], and another group of 319 Zambians (226 HIV-1 seropositive and 93 HIV-1 seronegative) from Lusaka [31, 32] were studied. Criteria for selecting these African patients included: (a) HIV-1 infection and risk for infection to determine eligibility to the studies; and (b) availability of genotyping materials and clinical data for analyses of outcomes. In addition, 292 healthy, unrelated, and mostly Caucasian (88%) male (68%) patients from a phase I HIV-1 vaccine trial in the United States (US normals) served as a comparison group. Ten chimpanzees (Pan troglodytes; Yerkes Regional Primate Center in Atlanta, GA, USA) were further included as an outgroup. DNA from each individual was prepared principally from buffy coats or purified peripheral mononuclear cells and with either the salting out technique [33, 34] or the QIAamp blood kit (QIAGEN Inc., Chatsworth, CA, USA). All DNA samples were diluted to 150 –200 ng ␮l⫺1 and stored at 4°C in TE buffer (10 mM Tris-HCl, pH 8.0, 2 mM EDTA) before use. PCR-Based Typing of CD4 Short Tandem Repeat Sequences on Chromosome 12 The CD4 short tandem repeat (STR) alleles were typed according to the original protocols [35], except that the sense primer (Table 1) was labeled with Cy5 fluorescent moiety to facilitate automated separation and detection of PCR amplicons on the ALFexpress DNA sequencer (Amersham Pharmacia Biotech, Piscataway, NJ, USA). In various human populations, the STR alleles show apparent sizes ranging from 85 bp to 135 bp, corresponding to 5 to 15 perfect repeats of the pentanucleotide sequence TTTTC [36]. Genotyping of TNF␣, LT␣, and TNFc Variants Single nucleotide polymorphisms (SNP) in the TNF␣ promoter positions ⫺238 and ⫺308 as well as the SNP at codon position 26 of LT␣ gene were defined by PCR-SSP using a protocol almost identical to that proposed by Fanning et al. [37], although not all SNPs in TNF␣-LT␣ region were targeted. In addition, TNFc microsatellite alleles were amplified by PCR and separated on denaturing gels as reported previously [38], except for automation as outlined in the CD4 STR

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TABLE 1 Oligonucleotides used to amplify polymorphic CD4 STR, TNFc microsatellite, and TAP1 coding sequences Oligo name

Specificity

Oligo sequence (5⬘ 佥 3⬘)

Annealing sites

CD4T4CF5 CD4T4CR3 TNFc-5S TNFc-3A TAP1dH26 TAP1dL10 TAP1jH33 TAP1jL90

CD4 STR CD4 STR TNFc TNFc TAP1, exon 4 TAP1, exon 4 TAP1, exon 10 TAP1, exon 10

Cy5-TTg gAg TCg CAA gCT gAA CTA gCa gCC TgA gTg ACA gAg TgA gAA CCa Cy5-ggA ggT CTg TCT TCC gCC gc CgT TCA ggT ggT gTC ATg ggc CAg gTA ACA TCA TgT CTC g CCA TgA ACA TAC CTg gTA C ATg Tgg CTA TAC CgT TCT C AAg ATg ACT gCC TCA CCT g

302 佥 324b 390 佤 412b 8258 佥 8276d 8182 佤 8201d 27508 佥 27526e 27710 佤 27728e 31415 佥 31433e 31590 佤 31608e

a

As reported elsewhere [35]. According to GenBank sequence M86525. c Adapted from reported sequences [38]. d GenBank sequence Z15026. e GenBank sequence X66406. b

typing. The common c1 and c2 alleles of the TNFc microsatellite corresponded to a 93-bp (containing 13 CT/AG repeats) and a 95-bp (containing 14 CT/AG repeats) amplicon, respectively.

637 (exon 10), and 648 (exon 10). Variants at the first two positions were also assigned using the TAP1.1/1.2 and TAP1.3/1.4 nomenclature [41] as a further reference.

Molecular HLA Typing HLA class I A, B, C and class II DRB1 and DQB1 alleles were resolved to their 2- to 5-digit specificities based on a combination of molecular techniques including PCR with sequence-specific primers (PCR-SSP) and automated DNA sequencing as outlined elsewhere [39], with the exception that some of the more recent HLA class I typings were done using a commercial PCR-SSP kit (Pel-Freez Clinical Systems, Brown Deer, WI, USA).

Statistical Analyses Genotyping results were initially summarized as allele and genotype frequencies based on direct counting. For variants found in 2.5% to 97.5% of the subjects, conditional odds ratios (OR) with 95% confidence intervals (CI) were calculated whenever differences between ethnic groups or between HIV-1 seropositive and HIV-1 seronegative subjects within each cohort were detected. In addition, the Watterson test [42, 43] was applied to each population to compare the observed and expected homozygosity frequencies. “Apparent” two-locus linkage disequilibrium were estimated (in the absence of genetic data from families) by ␹2 and OR tests in a 2 ⫻ 2 table containing the numbers of individuals with both, one without the other, or neither of the two alleles in question [44]. A p value (聿0.01) documenting a significant association of two alleles with odds ratio 肁2.0 was treated as indicative of a positive linkage disequilibrium, and the delta value was calculated [45]. For variants in absolute linkage disequilibrium, it was necessary to add an arbitrary number of one to all cells to facilitate the analyses. Additionally, two-locus haplotypes were deemed fully reliable when alleles at one or both loci remained homozygous after two different typings that target separate regions of the allele-defining sequences [30]. The extended haplotypes were determined by the pairwise frequency and the relative strength (OR and delta values) of two-locus haplotypes.

Genotyping of Transporter Associated with Antigen Processing, Subunit 1 (TAP1) Variants Three nonsynonymous SNPs (Val333Ile, Gly637Asp, and Gln648Arg) in the coding sequences of TAP1 were typed according to diagnostic banding patterns generated by single-strand conformation polymorphism (SSCP). Two sets of oligonucleotides (Table 1) were used to amplify the polymorphic exon 4 and exon 10, respectively. PCR products were randomly labeled with (␣33 P)dATP (ICN Biomedicals, Inc., Costa Mesa, CA, USA), and single-strand conformants were formed in low-ionic strength (LIS, 10% sucrose, 0.01% bromophenol blue, 0.01% xylene cyanol) buffer and separated through electrophoresis on a 6% polyacrylamide gel [40]. Variants in each exon was assigned by comparing the gel mobility patterns of test samples with those of benchmark DNA samples whose identities had been established by sequencing. TAP1 allele assignment was based on three key amino acid positions at 333 (exon 4),

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TABLE 2 CD4 STR allele frequencies observed in Rwandans and Zambians Alleles by size (bp)

Rwandans (2N ⫽ 570)

Zambians (2N ⫽ 638)

Sub-Saharan Africans (2N ⫽ 806)a

US normalsb (2N ⫽ 584)

80 85 90 95 100 105 110 115 120 125 130 135 140 Heterozygosity Observed Expected

0 0.251c* 0.107 0.028 0.098 0.026 0.163c† 0.163c† 0.109 0.012 0.028 0.012 0.002

0 0.197 0.108 0.042 0.118 0.038 0.083 0.243 0.094 0.025 0.034 0.016 0.002

0.002 0.233 0.065 0.039 0.081 0.029 0.172 0.210 0.114 0.029 0.022 0.005 0

0 0.324 0.307d‡ 0.041 0.007d‡ 0.002d‡ 0.259d‡ 0.039d‡ 0.021d‡ 0 0 0.002 0

0.822 0.848

0.850 0.856

0.850 0.844

0.743 0.730

a Samples were derived from Kenya, Nigeria, Senegal, Zaire, Central African Republic, Namibia, and South Africa [36]. Numbers shown are the mean values for the observed alleles. b Mostly Caucasian unrelated participants in a phase I canary pox virus-based HIV vaccine trial [56, 57]. c Compared with Zambians: *p ⬍ 0.05; † p ⬍ 0.001 based on Mantel-Haenszel ␹2 tests. d Compared with Sub-Saharan Africans: ‡ p ⬍ 0.001 based on Mantel-Haenszel ␹2 tests.

RESULTS Population Characteristics as Defined by the CD4 STR A total of 12 different CD4 pentanucleotide (TTTTC) STR alleles were detected in the Rwandan and Zambian patients studied (Table 2). The first 11 alleles (85 bp to 135 bp) were all relatively common at frequencies higher than 0.01. The 12th allele at 140 bp was rare in both groups and had not been described anywhere else. Consistent with earlier work demonstrating clear population profiles for CD4 STR alleles [36], the observed distributions of CD4 STR alleles in Rwandans and Zambians were more comparable to sub-Saharan Africans than to Caucasians (Table 2). Indeed, only the 110 bp and 115 bp alleles showed intra- as well as inter-racial differences. Moreover, the observed heterozygosity for CD4 STR genotypes in Rwandans and Zambians resembled the expected frequencies as closely as that of the sub-Saharan Africans and US normals. Likewise, the frequencies of all homozygous CD4 STR genotypes closed matched their expected values (data not shown). Lower heterozygosity in US normals than Africans were consistent with fewer CD4 STR alleles in the former group. TNFc Genotyping Four TNFc alleles were detected in the Rwandan and Zambian samples (Figure 1). The most common alleles c1 and c2 had apparent molecular sizes of 93 bp and 95 bp, with predicted 13 and 14 CT/AG repeats, respec-

tively. Two minor alleles, c0 (91 bp, 12 CT/AG repeats) and c3 (97 bp, 15 CT/AG repeats), were also present (Figure 1). The c1 allele was most common in US normals (0.753), followed by Zambians (0.683), Caucasians (0.681), and Rwandans (0.598). Allele frequency of the FIGURE 1 Automated separation and detection of TNFc microsatellite alleles amplified by PCR. The allelic products were labeled with Cy5, denatured, and separated through 6% PAGE-plus (Amresco, Solon, OH, USA) gel prepared in 1⫻ TBE buffer (90 mM Tris-boric acid, pH 8.3, 2 mM EDTA) and 6 M urea. The marker DNA ranging from 85 nt to 115 nt is shown. Arrow and arrowhead indicate alleles c0 (91-nt, with 12 CT/AG repeats) and c3 (97-nt, with 15 CT/AG repeats), respectively. The most common TNFc alleles are c1 (93-nt, with 13 CT/AG repeats) and c2 (95-nt, with 14 CT/AG repeats).

Novel TNFc Alleles Tag Extended HLA Haplotypes

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TABLE 3 Frequencies of TNFc alleles and genotypes observed in Rwandans, Zambians, and compared with Caucasians TNFc variants Alleles c1 c2 c0 c3 Genotypes c1/c1 c1/c2 c2/c2 c0/c1 c0/c2 c1/c3 Heterozygosity Observed Expected

Rwandans Zambians Caucasiansa US normalsb n ⫽ 285 n ⫽ 319 n ⫽ 144 n ⫽ 292 0.598c† 0.384c† 0.007 0.011

0.683 0.307 0.009 0

0.681 0.319 0 0

0.753d‡ 0.247d‡ 0 0

0.351c† 0.467 0.147c* 0.007 0.007 0.021

0.467 0.420 0.094 0.013 0.006 0

0.458 0.444 0.097 0 0 0

0.572d‡ 0.363 0.065 0 0 0

0.502 0.495

0.439 0.439

0.444 0.434

0.353 0.372

nized genotypes accounted for 3.5% of the total in Rwandans and 1.9% in Zambians. For Hardy-Weinberg equilibrium testing, in both Rwandans and Zambians the observed TNFc genotype frequencies closely corresponded to those expected from the distributions of individual alleles. For example, TNFc heterozygosity was observed in 50.2% of Rwandans, while the expected heterozygosity frequency was 49.5% (Table 3).

a North American Caucasian participants in study of hepatitis C virus infection as described elsewhere [58]. Their genetic profile as defined at the TNF␣-LT␣ loci closely resembled those seen in North American and European Caucasians [38, 59]. b Mostly Caucasian unrelated participants in a phase I canary pox virus-based HIV vaccine trial [56, 57]. c Compared with Zambians: *p ⬍ 0.05; † p ⬍ 0.01 based on MantelHaenszel ␹2 tests. d Compared with Caucasians: ‡ p ⬍ 0.05 based on Mantel-Haenszel ␹2 tests.

Distribution of TNFc Variants Between HIV-1 Seronegative and Seropositive Africans The TNFc allele carrier and genotype frequencies were compared between HIV-1 seronegative and HIV-1 seropositive subjects within each of the African groups (Table 4). The c2 homozygosity (c2/c2) was more frequent in both HIV-1 seronegative Rwandans and Zambians than their HIV-1 seropositive counterparts (OR ⫽ 2.03 and 2.00, p ⫽ 0.039 and 0.073, respectively). A reciprocal but less prominent effect was seen with c1 homozygosity. The distribution of allele carriers with either c1 or c2, on the other hand, did not differ between the HIV-1⫹ and HIV-1⫺ groups of Rwandans and Zambians (p ⫽ 0.076 to 0.164). Neither the rare c0 or c3 allele nor their haplotypes or other component alleles on these haplotypes were unevenly distributed in relation to HIV-1 infection or disease progression.

TNFc allele c2 was similar in Zambians (0.307) and Caucasians (0.319), who differed somewhat from Rwandans (0.384) and US normals (0.247) (Table 3). Marginally significant differences between Caucasians and US normals, as compared here, were driven by 35 nonCaucasian subjects among the US normals (Hispanics, African-Americans, Asians, and American Indians; data not shown). Three of the four possible genotypes involving the minor alleles c0 and c3 and the common alleles c1 and c2 were observed (Table 3), and these previously unrecog-

Characterization of Extended TNFc-related Haplotypes Rwandans and Zambians with novel TNFc alleles were further studied for their HLA/TNF/TAP1 profile (Figure 2). Nine out of 10 c0-positive individuals carried 26Asn at LT␣, ⫺238G, ⫺308G (not shown) at TNF promoter, DRB1*1503-DQB1*0602, and TAP1.2 (333Val) at TAP1, exon 4. In addition, all but one c0-positive individual also carried TAP1.4 (637Gly) at TAP1, exon 10. Similarly, the c3 allele appeared to form a stable haplotype with LT␣26Thr, TNF promoter ⫺238A, ⫺308G,

TABLE 4 Comparison of TNFc variants among HIV-1⫹ and HIV-1⫺ groups from Rwanda and Zambia

Rwandans (n ⫽ 285) TNFc c1/c1 TNFc c1/c2 TNFc c2/c2 Others Zambians (n ⫽ 319) TNFc c1/c1 TNFc c1/c2 TNFc c2/c2 Others a

Mantel-Haenszel ␹2 tests.

HIV-1⫺ n (%)

HIV-1⫹ n (%)

OR (95% CI)

p Valuea

21 (26.9) 36 (46.2) 17 (21.8) 4 (5.1)

79 (38.2) 97 (46.9) 25 (12.1) 6 (2.9)

0.60 (0.34–1.06) — 2.03 (1.02–4.01) —

0.08 — 0.04 —

37 (39.8) 42 (45.2) 13 (14.0) 1 (1.1)

112 (49.6) 92 (40.7) 17 (7.5) 5 (2.2)

0.67 (0.42–1.10) — 2.00 (0.93–4.30) —

0.11 — 0.07 —

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FIGURE 2 HLA profile in Rwandans (RWA) and Zambians (ZMB) carrying novel TNFc alleles. The MHC and related loci being typed are drawn to approximate scale [60]. Curved double arrows separate the evolutionarily conserved HLA blocks between which recombination often occur to generate new haplotypes [47]. Two additional recombination hot spots (indicated by arrows) are fine mapped to HLA-B [52] and TAP2 [49]. For homozygous genotypes the alleles are only listed once. Genetic variants that form stable haplotypes despite the recombination hot spots are either underlined or highlighted in bold. Partial data from 10 chimpanzees show only a single haplotype sequence. In addition, the TNFa308G/G homozygous genotype (not shown) is found in all 16 Africans as well as 10 chimpanzees.

DRB1*0102-DQB1*0501, and TAP1*0101 (333Ile ⫹ 637 Asp). Greater divergence in class I profiles in the c0- and c3-positive subjects suggested that the c0- and c3-linked haplotypes did not extend to the class I A locus (Figure 2). On the other hand, in both Rwandans and Zambians B*4501 was enriched in the TNFc*c0-carriers (OR ⫽ 7.2, delta ⫽ 0.0044, p ⫽ 0.0005). Meanwhile, all TNFc*c3-positive Rwandans had Cw*15. The rarity of both c3 (allele frequency ⫽ 0.011) (Table 2) and Cw*15 (allele frequency ⫽ 0.018) [39] in this ethnic group pointed to a highly stable TNFc*c3-Cw*15 haplotype (OR ⫽ 386.4, delta ⫽ 0.0102, p ⬍ 0.0001).

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Findings from Chimpanzees The TNFc microsatellite, TNF promoter, LT␣, and TAP1 exon 4, exon 10 were successfully typed for ten chimpanzees. In contrast to the considerable variation observed in humans, only a single haplotype was detected in the chimpanzees: c0 (TNFc)-26Thr (LT␣)TNF1 (TNF promoter ⫺238G and ⫺308G)-TAP1.2 (333Val)-TAP1.4 (637Gly) (Figure 2). This c0-linked chimpanzee haplotype differed from the c0-linked human haplotype only at the LT␣-26Asn/Thr site. With TNFc located only ⬍300 bp away from the LT␣Asn26Thr site, it seems likely that the LT␣-26Asn allele and its haplotypes are derived from the c0-related lineage. Genotyping of other MHC loci in chimpanzees did not yield reliable results. DISCUSSION Novel allelic variants continue to surface in the well studied TNF-LT␣ region, especially in non-Caucasian populations examined with favorable investigative methods [10 –12]. Our findings of new TNFc alleles in Africans parallel the detection of other new alleles in the MHC region [40] and at the unrelated CD4 STR locus (Table 2). The full spectrum and complexity of TNFLT␣ alleles and haplotypes may increase further as the search for additional variants intensifies. The significance of these more recently discovered polymorphisms re-

Novel TNFc Alleles Tag Extended HLA Haplotypes

mains to be seen, but increased haplotype and genotype diversity could pose procedural difficulties and/or uncertainties in HLA matching for transplantation, in experimental analyses of their relative function, and in epidemiologic studies of genetic associations. Previous work on the TNFc locus has repeatedly revealed only the c1 and c2 alleles, which we were able to confirm in two separate North American cohorts. The c0 and c3 alleles described here, at allele carrier frequencies between 1.9% and 3.5%, are probably unique to Africans; these alleles and the haplotypes they mark will probably continue to circulate in the Rwandan and Zambian populations or spread into others. However, failure to uncover the c0 or c3 allele in ongoing studies of racially admixed Brazilians (J. Tang et al., unpublished) suggests that these novel TNFc alleles may occur only in native Africans. TNFc genotypes and those defined at the unrelated CD4 STR locus clearly conformed well to the HardyWeinberg equilibrium in both African populations. Consistent decrease in c2 homozygosity among HIV-1 seropositive compared with seronegative Rwandans and Zambians could have slightly distorted the distribution of the TNFc alleles as observed. However, this minimal distortion in the observed TNFc allele distribution did not account for the apparently strong haplotypes formed with c0 and c3 and seems unlikely to signal a strong association between TNFc variants and HIV-1 infection. The apparent effects of c2 homozygosity and not heterozygosity on HIV-1 infection await further assessment in light of comprehensive HLA typing along with characterization of non-genetic factors (e.g., sexually transmitted infections and multiple sexual partners) important in HIV-1 infection. Although there is no evidence that TNFc alleles themselves encode distinctive functions, the extended haplotypes are worth noting. At least two haplotypes were marked by the c0 and c3 alleles, and both were quite stable on the more centromeric MHC class II region despite the physical distance and two well-known recombination hot spots: the first located between central MHC (the delta block) and class II region (the gamma block) [46, 47], and the second within the TAP2 gene [48 –50]. Extension of the c0- and c3-tagged haplotypes well into the class II region must imply that these recombination hot spots were inactive on these haplotypes. In contrast, the haplotype marked by the c0 allele did not tightly link to specific variants in the HLA alpha block (HLA-A), supporting the notion that haplotypes are often disrupted by recombinations between the alpha and beta blocks and between HLA-B and TNF [46, 47, 51]. On the other hand, tight linkage between Cw*15, a rare allele in Rwandans [39], and the TNFc*c3 allele (all in Rwandans) appears to represent another

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exception, suggesting that recombination hot spots between TNF and HLA-B (the beta block), and between HLA-B exon 2 and exon 3 [52], became less active on the c3-Cw*15 haplotype. Because the chimpanzees all carried the c0 allele on a stable extended haplotype it is also possible that the TNFc*c3-Cw*15 haplotype is relatively new and has not been subjected to multiple recombinations. Either way, the TNFc*c3-tagged haplotype is likely to be detected in South Asian, Australian Aborigine, and East Asian populations who have the highest reported frequency of Cw*15 [53]. Genotyping of chimpanzees provided further insight into the general haplotypic evolution in the MHC. First, it appeared that the c0 (12 CT/AG repeats) is the most ancestral human TNFc sequence that is still shared by chimpanzees. Second, the c0-related haplotype has been highly conserved at the TNF and TAP1 loci in both humans and chimpanzees. Third, human TNFc sequence has expanded in size from the smallest ancestral allele c0, implying that such preferential expansion is no more than 6 million years old because humans and chimpanzees last shared an ancestor about 5.5 to 5.9 million years ago [54, 55]. ACKNOWLEDGMENTS

We thank staff and study participants in the two HIV/AIDS cohort studies in Rwanda and Zambia. We also thank Drs. Kent J. Weinhold (Duke University Medical Center), Mark Mulligan, Paul Geopfert, and the NIAID AIDS Vaccine Evaluation Group for providing samples from HIV-1 volunteers. We are indebted to C. Rivers, C. Costello, L. J. Yee, D. Munfus, Y.-T. Zhang, P. Xia, X.-C. Bai, J. Meinzen-Derr, and U. Fideli for technical assistance and data management. This work has been supported jointly by Center for AIDS Research and the Health Services Foundation, University of Alabama at Birmingham. Additional support was provided from grants AI40591, AI41951, and AI41530 from NIAID.

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