The Case for Selection at CCR5-D32

Transcription

1 Open access, freely avalable onlne The Case for Selecton at CCR5-D32 PLoS BIOLOGY Pards C. Sabet 1,2*, Emly Walsh 1, Steve F. Schaffner 1, Patrck Varlly 1, Ben Fry 1, Holl B. Hutcheson 3, Mke Cullen 3, Tarje S. Mkkelsen 1, Jessca Roy 1, Nck Patterson 1, Rchard Cooper 4, Davd Rech 1,5, Davd Altshuler 1,5,6, Stephen O Bren 3, Erc S. Lander 1,7,8,9 1 Broad Insttute of MIT and Harvard, Cambrdge, Massachusetts, Unted States of Amerca, 2 Harvard Medcal School, Boston, Massachusetts, Unted States of Amerca, 3 Laboratory of Genomc Dversty, Natonal Cancer Insttute, Frederck, Maryland, Unted States of Amerca, 4 Department of Preventve Medcne and Epdemology, Loyola Unversty Medcal School, Maywood, Illnos, Unted States of Amerca, 5 Departments of Genetcs and Medcne, Harvard Medcal School, Boston, Massachusetts, Unted States of Amerca, 6 Department of Molecular Bology and Center for Human Genetc Research, Dabetes Unt, Department of Medcne, Massachusetts General Hosptal, Boston, Massachusetts, Unted States of Amerca, 7 Department of Bology, Massachusetts Insttute of Technology, Cambrdge, Massachusetts, Unted States of Amerca, 8 Whtehead Insttute for Bomedcal Research, Cambrdge, Massachusetts, Unted States of Amerca, 9 Department of Systems Bology, Harvard Medcal School, Boston, Massachusetts, Unted States of Amerca The C-C chemokne receptor 5, 32 base-par deleton (CCR5-D32) allele confers strong resstance to nfecton by the AIDS vrus HIV. Prevous studes have suggested that CCR5-D32 arose wthn the past 1,000 y and rose to ts present hgh frequency (5% 14%) n Europe as a result of strong postve selecton, perhaps by such selectve agents as the bubonc plague or smallpox durng the Mddle Ages. Ths hypothess was based on several lnes of evdence, ncludng the absence of the allele outsde of Europe and long-range lnkage dsequlbrum at the locus. We reevaluated ths evdence wth the beneft of much denser genetc maps and extensve control data. We fnd that the pattern of genetc varaton at CCR5-D32 does not stand out as exceptonal relatve to other loc across the genome. Moreover usng newer genetc maps, we estmated that the CCR5-D32 allele s lkely to have arsen more than 5,000 y ago. Whle such results can not rule out the possblty that some selecton may have occurred at C-C chemokne receptor 5 (CCR5), they mply that the pattern of genetc varaton seen at CCR5-D32 s consstent wth neutral evoluton. More broadly, the results have general mplcatons for the desgn of future studes to detect the sgns of postve selecton n the human genome. Ctaton: Sabet PC, Walsh E, Schaffner SF, Varlly P, Fry B, et al. (2005) The case for selecton at CCR5-D32. PLoS Bol 3(11): e378. Introducton The mpact of evolutonary selecton on the human populaton s of central nterest and, wth ncreasng nformaton about genetc varaton, has become a subject of ntense examnaton [1 6]. Knowledge of selectve events and selected loc provde nsght nto the genetc etology of human dsease, past and present, and nto the events that have shaped our speces. As nfectous dseases pose a major selectve force, selected varants may gve nsght nto mmunologcal defense mechansms hghlghtng mportant pathways n pathogen resstance. Evolutonary pressure generates a number of potentally detectable sgnals at a locus under selecton as compared to the neutrally evolvng genome. Because dfferent populatons are subject to dstnct selectve envronments, selecton may produce populaton-specfc alleles and greater populaton dfferentaton at an affected gene, whch can be measured wth the F ST statstc [7]. Postve selecton may also cause a rapd rse n an allele s frequency, creatng a dsparty n the age of an allele estmated from ts hgh frequency n the populaton (characterstc of an old allele) and ts long-range lnkage dsequlbrum (LD, characterstc of a young allele). LD-based methods such as the Long-Range Haplotype test have been developed to detect ths sgnal [3,8 10]. C-C chemokne receptor 5 (CCR5) s one of the most promnent reported cases of recent natural selecton n the human genome. Frst dentfed as encodng a prncpal entry receptor for HIV-1 nfecton of CD4-bearng T lymphocytes, CCR5 has been the subject of ntense focus by genetcsts [8,11 14]. A well-establshed assocaton exsts between a 32 base-par deleton varant n CCR5 (CCR5-D32) and protecton from HIV nfecton, demonstratng that CCR5 plays an mportant bologcal role n HIV entry nto cells. The frst suggeston that CCR5 may have been subject to postve selecton was a hgh proporton of nonsynonymous mutatons at CCR5, suggestng selectve pressure for amno acd dvergence [12]. More compellng evdence for selecton on CCR5-D32 came from work by Stephens et al. [8]. Ths study found that D32 occurs at hgh frequency n European Caucasans (5% 14%, wth north-south and east-west clnes) but s absent among Afrcan, Natve Amercan, and East Asan populatons, suggestng that the D32 mutaton occurred after the separaton of the ancestral founders of these populatons. Moreover, Stephens et al. [8] reported strong LD between CCR5-D32 and two mcrosatellte markers, suggestng an estmated age for the allele of only ;700 y (range 275 1,875 y). The apparent rapd rse n frequency mpled strong postve selecton, and the specfc age rased ntrgung possbltes for the selectve agent, such as the bubonc plague n Medeval Europe. Wth the recent avalablty of comprehensve nformaton Receved Aprl 13, 2005; Accepted September 8, 2005; Publshed November 1, 2005 DOI: /journal.pbo Copyrght: Ó 2005 Sabet et al. Ths s an open-access artcle dstrbuted under the terms of the Creatve Commons Attrbuton Lcense, whch permts unrestrcted use, dstrbuton, and reproducton n any medum, provded the orgnal author and source are credted. Abbrevatons: cm, centmorgan; DAF, derved allele frequency; EHH, extended haplotype homozygosty; EHL, extended haplotype length; LD, lnkage dsequlbrum; REHH, relatve extended haplotype homozygosty; SNP, sngle-nucleotde polymorphsm Academc Edtor: Andy Clark, Cornell Unversty, Unted States of Amerca *To whom correspondence should be addressed. E-mal: pards@broad.mt.edu 0001

2 Table 1. Genetc Dversty at CCR5 n Comparson wth Genetc Dversty for Regons from Two Large Emprcal Datasets Measure Populaton CCR5 Comparson Regons (64) HapMap Chromosome 3 Average heterozygosty European-Amercan ( ) 0.29 ( ) Chnese ( ) 0.26 ( ) Yoruba ( ) 0.3 ( ) Average F ST European-Amercan versus Chnese ( ) 0.09 ( ) European-Amercan versus Yoruba ( ) 0.14 ( ) Chnese versus Yoruba ( ) 0.16 ( ) Average DAF dstrbuton European-Amercan ( ) 0.41 ( ) Chnese ( ) 0.4 ( ) Yoruba ( ) 0.34 ( ) DOI: /journal.pbo t001 about patterns of allelc dversty n the human genome, we can now reexamne the case for selecton at CCR5 by comparson wth extensve emprcal data and more sophstcated predcted dstrbutons. We carred out hgh-densty sngle-nucleotde polymorphsm (SNP) genotypng around CCR5 n multple populatons, and analyzed the data wth the beneft of large genomc comparson datasets and revsed physcal and genetc maps. Our results show that CCR5-D32 does not clearly stand out n terms of genetc dversty or long-range haplotypes relatve to other varants at the locus or throughout the human genome. Results/Dscusson We genotyped CCR5-D32, two mcrosatelltes, and 70 SNPs (dbsnp data release 120, extendng 837 kbp centromere-dstal and 430 kbp centromereproxmal to the CCR5 locus (Table S1). We studed 340 chromosomes from three populatons: European-Amercans, Chnese, and Yoruba from Ngera. Eght of the European- Amercan chromosomes bore the D32 mutaton. In addton, we genotyped a subset of the SNPs n 12 D32/D32 ndvduals from the orgnal study. Ths provded a total of 32 chromosomes bearng the D32 allele. We carred out all analyses on both datasets (Table S2). We frst examned the allele frequences at SNPs around CCR5 n the European-Amercan, Yoruba, and Chnese populaton samples for evdence of selecton. As a genomewde emprcal comparson, we used two datasets. The frst s 2,359 SNPs genotyped n the same 340 samples n the three populatons. These SNPs are dstrbuted n 168 mmunologc genes from 64 loc across the genome; they were chosen accordng to the same methodology and have a smlar physcal dstrbuton as for CCR5 [15] (see Materals and Methods). The second s data for 63,149 SNPs on Chromosome 3 from the Internatonal Haplotype Map Project (HapMap, data release 16) genotyped n the same three populatons. CCR5 s not a sgnfcant outler relatve to the 168 genes or HapMap Chromosome 3 wth respect to heterozygosty and F ST (Table 1; Fgure S1). The heterozygosty statstc assesses the genetc dversty n a populaton; a selectve sweep can reduce genetc dversty and balancng selecton can ncrease genetc dversty. The F ST statstc [7] compares the frequency of an allele between populatons; a populaton-specfc selectve pressure may produce greater populaton dfferentaton at an affected gene. We also looked at the derved allele frequency (DAF) dstrbuton, whch can detect the genetc htchhkng of varaton lnked to an allele under postve selecton, and found no evdence for selecton [16] (Table 1; Fgure S2). All of these tests have lmted power, wth genotypng data ascertaned to favor common shared SNPs and usng the chmpanzee sequence for comparson. Therefore, whle the results provde no evdence for selecton, t can not be ruled out; ths could be further explored wth sequencng of a large number of chromosomes. We also assessed the sgnfcance of the observaton that D32 s at moderately hgh frequency (8%) n the European- Amercans but absent n the Chnese and Yoruba populatons sampled. The observaton s not exceptonal n our avalable polymorphc data: of SNPs present at smlar frequency (7% 9%) n European-Amercans, ;7% are not found n the Chnese and Yoruba populatons for the 168 genes, and 6% are not found for the same populatons for the HapMap data. These estmates are lkely to be conservatve consderng that the ascertanment of these studes favors shared polymorphsms. As more data become avalable, ths analyss should be extended by larger sample szes, more populatons, and more closely matched data (ncludng nserton/deleton polymorphsms and functonal polymorphsms). We next tested for sgnatures of selecton by examnng the extent of LD around CCR5-D32. For ths purpose, we used the Long-Range Haplotype test for selecton [3] (see Materals and Methods). Specfcally, we calculated the relatve extended haplotype homozygosty (REHH), whch s senstve to recent drectonal postve selecton, and extended haplotype homozygosty (EHH), whch s more senstve to multple selectve sweeps at a locus. To estmate the recombnaton rate, we used two measures: the genetc dstance from a famly-based lnkage study [17] and the number of observed hstorcal recombnaton events [3] (Materal and Methods). We ntally examned the centromere-dstal sde of CCR5 usng the approach of Stephens et al. [8] (Fgure 1A). Specfcally, we sorted the chromosomes nto two groups: D32-bearng and non-d32-bearng chromosomes. Consstent wth the prevous study [8], we found that the D32-bearng chromosomes have much longer LD than non-d32-bearng chromosomes: the EHH s 5.96 tmes greater than the average EHH of other varants at ths locus (REHH ¼ 5.96 at a dstance of 500 kbp or 0.25 centmorgans [cm]) (Fgure 1B). We reasoned, however, that the apparent long-range LD mght be a result of sortng the chromosome nto only two 0002

3 Fgure 1. EHH Breakdown of EHH over Dstance between the CCR5-D32 Mutaton and 63 SNPs at Increasng Dstances from the Mutaton. (A) Map of SNPs typed. (B) Comparson between D32 and a sngle non-d32 class of haplotypes. It should be noted that the D32-bearng chromosomes appear (red) to have greatly extended LD compared to the non-d32-bearng chromosomes (black). (C) Breakdown usng the eght-marker haplotype contanng the D32 mutaton. There are fve haplotypes n European-Amercans (frequences: 42%, 31%, 10%, 8%, and 8%, respectvely). Full haplotype sequences and frequences n other populatons are gven n Table S3. Notce that two of the non-d32-bearng chromosomes (black) appear to have the smlar extended LD when compared to the D32-bearng chromosomes centromere-dstal to CCR5 (red). Centromere-proxmal D32-bearng chromosomes stll have the most extended LD, ndcated wth an arrow. DOI: /journal.pbo g001 classes based on ther genotype at CCR5-D32, rather than dvdng them accordng to the full varaton seen at CCR5. Fgure 2 shows how an apparent sgnal of long-range LD can readly arse n ths fashon. Brefly, one class (for example, the non-d32) may contan multple dstnct haplotypes whose ndvdual sgnals of long-range LD may be obscured when grouped together, wth the result that the other class (for example, the D32) appears to have much longer relatve LD. In fact, ths s precsely the case for CCR5. We fully delneated the varaton at CCR5 by genotypng seven addtonal SNPs wthn the gene and defned haplotypes as prevously descrbed [18] (Fgure S3). There are fve dstnct haplotypes, ncludng the D32-bearng haplotype wth frequency 8% (Table S3). The relatve LD of the D32-bearng haplotype s sgnfcantly lower than for two other haplotypes (REHH ¼ 1.92 versus 6.77 and 3.29 at dstance 500 kbp or 0.25 cm; see Fgure 1C), ndcatng that there s no sgnfcant evdence of long-range LD on the centromere-dstal sde of CCR5. We next analyzed LD on the centromere-proxmal sde of CCR5. We frst employed the approach used n the orgnal study and agan found the D32-bearng chromosomes had much longer LD than non-d32-bearng chromosomes (REHH ¼ at a dstance of 250 kbp or 0.25 cm; see Fgure 1B). We then reanalyzed the data by dsaggregatng the chromosomes nto the fve haplotypes descrbed above. The relatve longrange LD for D32-bearng chromosomes s much lower (REHH ¼ 7.26), although t s stll the hghest among the fve haplotypes. We sought to assess whether the extent of LD n the centromere-proxmal drecton on D32-bearng chromosomes s unusual relatve to that seen across the human genome. We frst compared the results to the genome-wde dstrbuton of REHH scores for the HapMap (Release 16, and found that D32-bearng chromosomes do not clearly stand out from other haplotypes of smlar frequency (6% 10%) (Fgures 3A and S4). Because the 120 European-Amercan chromosomes genotyped n the HapMap project have lmted power for studyng low-frequency haplotypes (P. V., B. F., E. S. L., and P. C. S., unpublshed data), we augmented the analyss by comparng all 32 D32- bearng chromosomes to smulatons wth larger sample sze [19] (see Materals and Methods). We smulated 1,000 1-mbp regons n 400 European-Amercan chromosomes under a neutral model, generatng 5,915 haplotypes matched wth a frequency smlar to the D32-bearng haplotype (6% 10%). The level of EHH for the D32-bearng haplotype was not unusually hgh on ether the centromere-dstal (p ¼ 0.49) or centromere-proxmal (p ¼ 0.15) sde of CCR5 when compared to the level seen at an equvalent recombnaton dstance for the smulated regons. The REHH (we used the EHH of the two common haplotypes for a relatve value) was also not unusually hgh (Fgure 3B). We further examned the extent of the D32-bearng haplotype n comparson to other haplotypes of smlar frequency. For ths purpose, we defned the extended haplotype length (EHL) on each sde of a haplotype to be the genetc dstance at whch the EHH score falls to 0.5. The EHL for the D32-bearng haplotype s cm on the centromere-dstal sde and cm on the centromereproxmal sde, correspondng to a total of cm (Fgures 3 and S5). We then determned the EHL for haplotypes of comparable frequency (6% 10%) for both the HapMap data (average EHL s 0.354; CCR5-D32 s the 88th percentle) and for the smulated data (average EHL s 0.453; CCR5-D32 s the 64th percentle). The dstrbuton s presented n Fgure 3. Long-range LD around rare alleles s a prevalent feature n the genome, and the EHL for CCR5-D32 therefore does not stand out n comparson to ether the HapMap or smulaton 0003

4 Fgure 2. Model of Haplotype-Based Selecton Approach The mage compares ths approach, where the varants at the gene beng studed are fully elaborated, to a model where the varants are not fully elaborated. At the top, multple SNPs are genotyped to fully defne the varants that exst n the gene. The resultant observed haplotype structure s shown n both bfurcaton dagram and EHH plot formats (see Materals and Methods). At the bottom, only one SNP s genotyped, collapsng all other varants nto a seemngly dverse super-haplotype and creatng an mpresson of extenson for the remanng haplotype. DOI: /journal.pbo g002 dataset (Table S4). The EHL for CCR5-D32 would only be sgnfcant f the recombnaton rate n ths regon were several-fold hgher than that measured by the current recombnatonal maps or by countng of hstorcal recombnaton events (Protocol S1). Gven that long-range LD s a common feature of rare alleles n European-Amercans, we wanted to test f our method would have the power to detect selecton of an 8% allele over the tme scale prevously proposed [8]. We smulated 500 regons of 1 mbp length n 400 and 120 European-Amercan chromosomes that had undergone a partal selectve sweep begnnng ether 700 or 2,000 y ago for both groups of chromosomes, carryng the selected allele to a frequency of 8%. We were able to detect recent selecton n the 400 chromosomes; 69% of selected alleles orgnatng 700 y ago and 39% of selected alleles orgnatng 2,000 y ago have EHL values above the 95th percentle when compared to the neutral dstrbuton. There s far less power n the 120 chromosomes (30% and 10% of selected alleles orgnatng 700 or 2,000 y ago, respectvely), suggestng that the HapMap dataset wll be nsuffcent to scan for rare selected alleles n European-Amercans. Fnally, we revsted the estmated date of orgn for the CCR5-D32 mutaton. The orgnal estmate [8] was based on the analyss of two mcrosatelltes that were n strong LD despte apparently beng at a consderable genetc dstance away (0.91-cM nterval and both centromere-dstal, accordng to the genetc maps that were current at the tme). Wth mprovements n the genetc map over the past 7 y [17], the mcrosatelltes were shown to be on opposte sdes of CCR5 and at a much shorter genetc dstance (0.18 cm, Fgure S6). Usng the methodology and data employed by Stephens et al. [20] (Table S5), but wth the revsed genetc map, the estmated age rses from 688 y (275 1,875 y, 95% confdence nterval) to 7,000 y (2,900 15,750 y, 95% confdence nterval ). When we expanded the analyss to nclude 32 genetc markers that have been genotyped n the D32-bearng chromosomes, the estmated age also rses, to a smlar value of 5,075 y (3,150 7,800 y, 95% confdence nterval). The SNP-based estmate of the age dffers and has tghter error bars because the denser map holds more nformaton about hstorcal recombnaton events than the two mcrosatelltes, whose genetc dversty s roughly equvalent to two SNPs (Fgure S7). The older age estmate s consstent wth unpublshed work on DNA extracted from 3,000-y-old bural stes n central Germany showng that the CCR5-D32 was present at an apprecable frequency several mllenna ago, at least n central Germany [21]. The revsed age estmate suggests the hgh frequency of the CCR5-D32 allele cannot be attrbuted solely to a strong 0004

5 A 25 B REHH REHH C Proporton of haplotypes Frequency Frequency Extended Haplotype Length (cm) Fgure 3. Comparsons wth Emprcal and Smulaton Data (A) and (B) Plots of relatve EHH versus frequency for CCR5 n comparson to HapMap data (release 16) for Chromosome 3 n European-Amercans (A) and 1,000 smulatons of 400 chromosomes n European-Amercans (B). Green dots represent the comparson haplotypes and the lnes represent, from bottom to top, the 50th, 75th, 95th, and 99th percentles. The red dots represent the results for eght CCR5-D32-bearng chromosomes n (A) and 32 CCR5-D32-bearng chromosomes n (B) for the centromere-proxmal sde, and the blue dots represent results for the centromere-dstal sde. (C) EHL of the haplotypes of frequency 6% 10% from the HapMap (sold green lne) and from smulatons (dotted green lne) n comparson to CCR5- D32 (red lne). DOI: /journal.pbo g003 selectve event wthn the past mllennum. If selecton dd play a role n the hgh frequency of the allele, the ntal selecton pressure must have occurred before the perod calculated n the prevous estmate [8]. It should be noted that the data do not rule out some addtonal selecton occurrng wthn the past mllenna, but none that would be detected by the methodology used n Stephens et al. or n the current paper. Our reanalyss of CCR5 shows that CCR5-D32 does not clearly stand out from the rest of the genome n terms of allele frequency dstrbuton, populaton dfferentaton, or long-range LD (Fgure S8). The hgh populaton dfferentaton and long-range LD found for CCR5-D32 are, n fact, far more common n the genome than prevously beleved, and therefore do not provde support for the hypothess of strong selecton for CCR5-D32. Usng methods descrbed both n the prevous study [8] and n the current study, and by examnng currently avalable data, there s no detectable evdence for recent selecton for CCR5-D32. Of course, the lack of support does not exclude the possblty of selecton for the allele or the locus. Gven the bology of the gene, t s certanly possble that t has been subject to some selecton despte the lack of clear evdence. We note that small-scale studes of the dstrbuton of mutatons [12 14,22] have provded suggestve evdence for selecton, but these results may be less convncng n comparson to recently avalable genome-wde dstrbutons [23]. Beyond the specfc results for CCR5, our results have mportant mplcatons for studes of selecton n the human genome. Frst, accurate assessment of LD benefts from fully delneatng the core haplotypes at a locus; t may not be suffcent to compare a haplotype of nterest to the set of all other haplotypes. Second, long-range LD around specfc alleles s a prevalent feature n the genome; the sgnfcance of LD results should therefore be assessed relatve to emprcal dstrbutons observed n genome-wde studes wth larger numbers of samples. Thrd, accurate estmates of an allele s age requre accurate genetc maps. Wth the growng avalablty of genome-wde datasets, t should soon be possble to search the genome for sgns of strong selectve events [3] by studyng the pattern of varaton at every gene relatve to a comprehensve genome-wde dstrbuton. The results should shed lght on mportant factors that have shaped our speces and may provde valuable nformaton about natural mechansms of dsease resstance. Materals and Methods Samples. DNA samples for 93 ndvduals from 12 multgeneratonal pedgrees of European-Amercan ancestry were obtaned from Corell Repostores ( DNA samples from 93 healthy ndvduals (31 mother father chld clusters) from the Yoruba n Ngera were obtaned as part of the Internatonal Collaboratve Study of Hypertenson n Blacks. DNA samples from 30 Han Chnese tros from Guanch were ncluded. DNA samples 0005

6 from a chmpanzee, gorlla, and orangutan were obtaned from Corell Repostores. Genotype data. We genotyped 71 SNPs n and around and the CCR5-D32 usng the mass spectrometry-based MassArray platform provded by Sequenom (San Dego, Calforna, Unted States), mplemented as prevously descrbed [18]. The names, locatons, alleles, and flanks for all SNPs used are gven n Table S1. Mcrosatellte genotypng was conducted at the McGll Unversty and Genome Quebec Innovaton Center (Quebec, Canada), by use of MultProbe and MnTrak Lqud Handlng Systems (Perkn-Elmer, Wellesley, Calforna, Unted States) and 3730 DNA sequencers (Advanced Bosystems, Foster Cty, Calforna, Unted States). PCR was performed wth fluorescently labeled markers n standard condtons (annealng temperature of 54 8C). We also used genotypes of 2,359 SNPs, dstrbuted n 168 mmunologc genes from 64 loc throughout the genome n the same three populatons [15]. SNPs were selected from publc databases n multple batches over a 1.5-y perod from July 2002 to December Preference was gven to double ht SNPs whch have been shown to be more lkely to be valdated [24]. These crtera may bas our ascertanment of haplotype structure and may reduce the representaton of rare and populaton-specfc varaton; we comment n the paper where ths bas mght affect our observatons. We used publcly avalable data from the Internatonal Haplotype Map Project as a comparatve dstrbuton of varance n the genome wth whch to compare our results ( Phasng. We prepared these fles usng Genehunter ( broad.mt.edu/ftp/dstrbuton/software/genehunter/) to uncover unambguous phasng usng famly data [25]. The chld chromosomes were then dscarded, and we kept only the ndependent parent chromosomes. We then used PHASE ( stephens/software.html [26,27]) to obtan complete phased data. Smulatons. We used a computer program that smulates gene hstory wth recombnaton based on a neutral model of evoluton descrbed elsewhere [19,28]. The program was modfed to generate data comparable wth that collected from the three populatons Chnese, European-Amercan, and Yoruba. The smulatons were calbrated to provde data consstent wth the HapMap wth respect to varous genetc measures (ncludng F ST, heterozygosty, and mnor-allele frequency dstrbuton) and used model parameters (ncludng demography and recombnaton rate) consstent wth current estmates [19]. We smulated a long regon (1 mbp n length) of DNA and then mmcked the SNP selecton strategy used by the SNP Consortum [29], whch was the source of most of the SNPs n our study. We modfed the program to generate smulatons of a partal selectve sweep n 400 European-Amercan chromosomes, where 32 chromosomes had a common ancestor 700 y ago as per Stephens et al. [8]. We also tested where the 32 chromosomes had a common ancestor 2,000 y ago. F ST. Mean parwse dstance fxaton ndex, F ST, was used to calculate genetc dfferentaton between the three populatons [30,31]. F ST parttons the total varance nto wthn- and betweenpopulaton components, quantfyng the nbreedng effect of populaton substructure. Heterozygosty. Ne s measure of heterozygosty [32], the probablty that any two randomly chosen samples from a populaton are the same, was used to calculate SNP dversty: p ¼ n Xk ð1 p ð1þ n 1 ¼1 where n s the number of chromosomes n the sample, k s the number of alleles at a locus, and p s the frequency of the th allele. DAF dstrbuton. We calculated the DAF dstrbuton for all SNPs where t was lkely that the ancestral allele could be determned by genotypng a representatve chmpanzee, gorlla, and orangutan. If there was a consensus prmate allele across all successfully genotyped prmates, t was dentfed as the ancestral allele. Otherwse, no ancestral allele was defned. EHH. EHH assesses the age of each haplotype at a gene by measurng the decay of the extended ancestral haplotype (.e., SNPs far away from the gene), whch occurs over tme wth recombnaton. For a populaton of ndvduals sharng core haplotype X, EHH s the probablty that any two randomly chosen samples of core haplotype X have the same extended haplotype [3]. It s a measure of the decay of LD across a regon of the genome that has two advantages: frst, t can be used wth mult-allelc markers so a core haplotype model can be studed f desred, and second, t measures LD across a regon wth many loc and not just between a par of loc. The EHH s calculated as: EHH t ¼ X s ð e t ð c ð2þ t where t s the core haplotype tested, c s the number of samples of a partcular core haplotype, e s the number of samples for a partcular extended haplotype, and s s the number of unque extended haplotypes. To correct for local varaton n recombnaton rates, we can compare the EHH of a tested core haplotype to that of other core haplotypes present at the locus, usng the relatve EHH measure (.e., REHH). REHH s the factor by whch EHH decays on the tested core haplotype compared to the decay of EHH on all other core haplotypes combned. One must frst calculate the EHH, the decay of EHH on all other core haplotypes combned. For ths, we use the followng equaton where n s the number of dfferent core haplotypes: X n X s ð e j¼1;j6¼t ¼1 EHH ¼ X s ð c ð3þ ¼1 ¼1;6¼t The relatve EHH (.e., REHH) s smply EHH t = EHH. EHH and REHH were calculated for all haplotypes n all haplotype blocks for CCR5, HapMap Release 16 Chromosome 3, and the 1,000 smulated regons (120-chromosome and 500-chromosome sample sets). Haplotypes were placed nto 20 bns based on ther frequency. p-values were obtaned by log-transformng the EHH and REHH n the bns to acheve normalty, and calculatng the mean and standard devaton. All analyss was carred out usng the Sweep software program (P. V., B. F., E. S. L., and P. C. S., unpublshed data). Observed hstorcal recombnaton (marker breakdown, all EHH). When comparng EHH/REHH values across regons, t s mportant to make sure that the value s beng calculated at a smlar genetc dstance. Ths wll soon be replaced wth better cm values, but, where they are not known, ths can be matched by the marker breakdown, that s the degree to whch each added marker at a further dstance causes the extended haplotypes to decay for all core haplotypes [3]. Ths gves an evaluaton of how much hstorcal recombnaton (observed recombnants) has occurred over a dstance from the core, and therefore what genetc dstance s beng looked at. Ths can be calculated as all EHH. X n X s ð e j¼1 ¼1 allehh ¼ X s ð c ð4þ where n s the number of dfferent core haplotypes, c s the number of samples of a partcular core haplotype, e s the number of samples of a partcular extended haplotype, and s s the number of unque extended haplotypes. Bfurcaton dagram. To vsualze the breakdown of LD on core haplotypes, we used bfurcaton dagrams [3]. The root of each dagram s a core haplotype, dentfed by a dark-blue crcle. The dagram s bdrectonal, portrayng both proxmal and dstal LD. Movng n one drecton, each marker s an opportunty for a node; the dagram ether dvdes or not, dependng on whether both or only one allele s present. Thus, the breakdown of LD on the core haplotype background s portrayed at progressvely longer dstances. The thckness of the lnes corresponds to the number of samples wth the ndcated long-dstance haplotype. Supportng Informaton Fgure S1. F ST and Heterozygosty for SNPs wthn 100 kbp of CCR5 Compared to 100-kbp Sldng Wndows for HapMap Release 16 for European-Amercans Found at DOI: /journal.pbo sg001 (54 KB DOC). Fgure S2. The DAF Dstrbuton of CCR5 Compared to 100-kbp Sldng Wndows for HapMap Release 16 for European-Amercans Found at DOI: /journal.pbo sg002 (62 KB DOC). ¼1 0006

7 Fgure S3. Haplotype Bfurcaton Dagrams n European-Amercans Found at DOI: /journal.pbo sg003 (231 KB DOC). Fgure S4. The REHH versus Frequency Dstrbuton at Matched Genetc Dstance [17] Found at DOI: /journal.pbo sg004 (99 KB DOC). Fgure S5. Estmatng the Rate of Degradaton of EHH Found at DOI: /journal.pbo sg005 (40 KB DOC). Fgure S6. Remappng of Mcrosatellte Markers from Frst Study Gven the Improved Genomc Maps Found at DOI: /journal.pbo sg006 (45 KB DOC). Fgure S7. Mcrosatellte Genotypng Found at DOI: /journal.pbo sg007 (71 KB DOC). Fgure S8. Comparson of Overall Genetc Dversty and Specfc Haplotype EHH n Dfferent Populatons Found at DOI: /journal.pbo sg008 (38 KB DOC). Protocol S1. Recombnaton-Rate Estmates for CCR5 from Famly- Based Lnkage Studes (decode and Marshfeld Maps), from Prelmnary Sperm-Typng, and from Populaton Genetcs (LDhat) Found at DOI: /journal.pbo sd001 (32 KB DOC). Table S1. Informaton for D32 (rs333), 70 SNPs, and Two Mcrosatelltes Used n the Study Found at DOI: /journal.pbo st001 (30 KB XLS). Table S2. CCR5-D32 EHH Values for Eght European-Amercan Chromosomes versus the 32 Total Genotyped Chromosomes Found at DOI: /journal.pbo st002 (23 KB DOC). Table S3. Haplotype Frequences for Sx Varants n Strong LD at CCR5, Genotyped n the Three Populaton Samples Found at DOI: /journal.pbo st003 (23 KB DOC). Table S4. Extended Haplotype Length for Haplotypes of Dfferent Frequency on HapMap Chromosome 3 n European-Amercans Found at DOI: /journal.pbo st004 (23 KB DOC). Table S5. Detals of CCR5-D32 Date Estmates Found at DOI: /journal.pbo st005 (26 KB DOC). Accesson Number The LocusLnk ( accesson number for the C-C chemokne receptor 5 s Acknowledgments PCS s funded by the Damon Runyon Cancer Research Foundaton and by a L Oreal award for Women n Scence. EW was funded by the Cancer Research Insttute. We thank Andre Verner and hs colleagues at McGll Unversty and Genome Quebec Innovaton Center for ther work on mcrosatellte genotypng. We thank Mary Carrngton, Dan Rchter, Parsa Sabet, and three anonymous revewers for ther suggestons and revews of our manuscrpt. Competng nterests. The authors have declared that no competng nterests exst. Author contrbutons. PCS, EW, MC, DA, SO, and ESL conceved and desgned the experments. PCS, EW, MC, and JR performed the experments. PCS, SFS, PV, BF, TSM, NP, and DR analyzed the data. SFS, PV, BF, RC, HH, and ESL contrbuted reagents/materals/analyss tools. PCS, EW, DA, and ESL wrote the paper. & References 1. Olson S (2002) Populaton genetcs. Seekng the sgns of selecton. Scence 298: Bamshad M, Woodng SP (2003) Sgnatures of natural selecton n the human genome. Nat Rev Genet 4: Sabet PC, Rech DE, Hggns JM, Levne HZ, Rchter DJ, et al. (2002) Detectng recent postve selecton n the human genome from haplotype structure. Nature 419: Akey JM, Eberle MA, Reder MJ, Carlson CS, Shrver MD, et al. (2004) Populaton hstory and natural selecton shape patterns of genetc varaton n 132 genes. PLoS Bol 2: e286. DOI: /journal.pbo Walsh EC, Mather KA, Schaffner SF, Farwell L, Daly MJ, et al. (2003) An ntegrated haplotype map of the human major hstocompatblty complex. Am J Hum Genet 73: Rockman MV, Hahn MW, Soranzo N, Losel DA, Goldsten DB, et al. (2004) Postve selecton on MMP3 regulaton has shaped heart dsease rsk. Curr Bol 14: Taylor MF, Shen Y, Kretman ME (1995) A populaton genetc test of selecton at the molecular level. Scence 270: Stephens JC, Rech DE, Goldsten DB, Shn HD, Smth MW, et al. (1998) Datng the orgn of the CCR5-D32 AIDS-resstance allele by the coalescence of haplotypes. Am J Hum Genet 62: Tshkoff SA, Varkony R, Cahnhnan N, Abbes S, Argyropoulos G, et al. (2001) Haplotype dversty and lnkage dsequlbrum at human G6PD: Recent orgn of alleles that confer malaral resstance. Scence 293: Toomajan C, Ajoka RS, Jorde LB, Kushner JP, Kretman M (2003) A method for detectng recent selecton n the human genome from allele age estmates. Genetcs 165: Dean M, Carrngton M, Wnkler C, Huttley GA, Smth MW, et al. (1996) Genetc restrcton of HIV-1 nfecton and progresson to AIDS by a deleton allele of the CKR5 structural gene. Hemophla Growth and Development Study, Multcenter AIDS Cohort Study, Multcenter Hemophla Cohort Study, San Francsco Cty Cohort, ALIVE Study. Scence 273: Carrngton M, Kssner T, Gerrard B, Ivanov S, O Bren SJ, et al. (1997) Novel alleles of the chemokne-receptor gene CCR5. Am J Hum Genet 61: Carrngton M, Dean M, Martn MP, O Bren SJ (1999) Genetcs of HIV-1 nfecton: Chemokne receptor CCR5 polymorphsm and ts consequences. Hum Mol Genet 8: Bamshad MJ, Mummd S, Gonzalez E, Ahuja SS, Dunn DM, et al. (2002) A strong sgnature of balancng selecton n the 59 cs-regulatory regon of CCR5. Proc Natl Acad Sc U S A 99: Walsh EC, Sabet P, Hutcheson HB, Fry B, Schaffner SE, et al. (2005) Searchng for sgnals of evolutonary selecton n 168 genes related to mmune functon. Hum Genet. In press. 16. Fay JC, Wu CI (2000) Htchhkng under postve Darwnan selecton. Genetcs 155: Kong A, Gudbjartsson DF, Sanz J, Jonsdottr GM, Gudjonsson SA, et al. (2002) A hgh-resoluton recombnaton map of the human genome. Nat Genet 31: Gabrel SB, Schaffner SF, Nguyen H, Moore JM, Roy J, et al. (2002) The structure of haplotype blocks n the human genome. Scence 23: Schaffner S, Foo C, Gabrel SB, Rech D, Daly MJ, et al. (2005) Calbratng a coalescent smulaton of human genome sequence varaton. Genome Res. In press. 20. Rech DE, Goldsten DB (1999) Estmatng the age of mutatons usng varaton at lnked markers. In: Goldsten DB, Schlotter C, edtors. Mcrosatelltes: Evoluton and applcatons. Oxford: Oxford Unversty Press. pp Duncan SR, Scott S, Duncan CJ (2005) Reapprasal of the hstorcal selectve pressures for the CCR5-D32 mutaton. J Med Genet 42: Woodng S, Stone AC, Dunn DM, Mummd S, Jorde LB, et al. (2005) Contrastng effects of natural selecton on human and chmpanzee C-C chemokne receptor 5. Am J Hum Genet 76: Nelsen R, Bustamante C, Clark AG, Glanowsk S, Sackton TB, et al. (2005) A scan for postvely selected genes n the genomes of humans and chmpanzees. PLoS Bol 3: e170. DOI: /journal.pbo Rech DE, Gabrel SB, Altshuler D (2003) Qualty and completeness of SNP databases. Nat Genet 33: Kruglyak L, Daly MJ, Reeve-Daly MP, Lander ES (1996) Parametrc and nonparametrc lnkage analyss: A unfed multpont approach. Am J Hum Genet 58: Stephens M, Donnelly P (2003) A comparson of bayesan methods for haplotype reconstructon from populaton genotype data. Am J Hum Genet 73: Stephens M, Smth NJ, Donnelly P (2001) A new statstcal method for haplotype reconstructon from populaton data. Am J Hum Genet 68: Hudson RR (1990) Gene genealoges and the coalescent process. Oxf Surv Evol Bol. 7: Sachdanandam R, Wessman D, Schmdt SC, Kakol JM, Sten LD, et al. (2001) A map of human genome sequence varaton contanng 1.42 mllon sngle nucleotde polymorphsms. Nature 409: Ne M, Chesser RK (1983) Estmaton of fxaton ndces and gene dverstes. Ann Hum Genet 47: Akey JM, Zhang G, Zhang K, Jn L, Shrver MD (2002) Interrogatng a hghdensty SNP map for sgnatures of natural selecton. Genome Res 12: Ne M (1987) Molecular evolutonary genetcs. New York: Columba Unversty Press. 512 p. 0007