An Evolutionary Analysis of Antigen Processing and Presentation across Different Timescales Reveals Pervasive Selection

English version České info

Antigen-presenting cells digest intracellular and extracellular proteins and display the resulting antigenic repertoire on cell surface molecules for recognition by T cells. This process initiates cell-mediated immune responses and is essential to detect infections. The antigenic repertoire is generated by the antigen processing and presentation pathway. Because several pathogens evade immune recognition by hampering this process, genes involved in antigen processing and presentation may represent common natural selection targets. Thus, we analyzed the evolutionary history of these genes during mammalian evolution and in the more recent history of human populations. Evolutionary analyses in mammals indicated that positive selection targeted a very high proportion of genes (24%), and revealed that many selected sites affect positions of fundamental importance to the protein function. In humans, we found different signatures of natural selection acting both on regions that are expected to regulate gene expression levels or timing and on coding variants; two human selected polymorphisms may modulate the susceptibility to Crohn's disease and to HIV-1 infection. Therefore, we provide a comprehensive evolutionary analysis of antigen processing and we show that evolutionary studies can provide useful information concerning the location and nature of functional variants, ultimately helping to clarify phenotypic differences between and within species.

Vyšlo v časopise: An Evolutionary Analysis of Antigen Processing and Presentation across Different Timescales Reveals Pervasive Selection. PLoS Genet 10(3): e32767. doi:10.1371/journal.pgen.1004189
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.pgen.1004189

Souhrn

Zdroje

1. NeefjesJ, JongsmaML, PaulP, BakkeO (2011) Towards a systems understanding of MHC class I and MHC class II antigen presentation. Nat Rev Immunol 11 (12) 823–836.

2. KesslerJH, KhanS, SeifertU, Le GallS, ChowKM, et al. (2011) Antigen processing by nardilysin and thimet oligopeptidase generates cytotoxic T cell epitopes. Nat Immunol 12 (1) 45–53.

3. SavinaA, JancicC, HuguesS, GuermonprezP, VargasP, et al. (2006) NOX2 controls phagosomal pH to regulate antigen processing during crosspresentation by dendritic cells. Cell 126 (1) 205–218.

4. HughesAL, YeagerM (1998) Natural selection at major histocompatibility complex loci of vertebrates. Annu Rev Genet 32: 415–435.

5. HansenTH, BouvierM (2009) MHC class I antigen presentation: Learning from viral evasion strategies. Nat Rev Immunol 9 (7) 503–513.

6. YangZ (2007) PAML 4: Phylogenetic analysis by maximum likelihood. Mol Biol Evol 24 (8) 1586–1591.

7. AnisimovaM, NielsenR, YangZ (2003) Effect of recombination on the accuracy of the likelihood method for detecting positive selection at amino acid sites. Genetics 164 (3) 1229–1236.

8. YangZ, WongWS, NielsenR (2005) Bayes empirical bayes inference of amino acid sites under positive selection. Mol Biol Evol 22 (4) 1107–1118.

9. MurrellB, WertheimJO, MoolaS, WeighillT, SchefflerK, et al. (2012) Detecting individual sites subject to episodic diversifying selection. PLoS Genet 8 (7) e1002764.

10. Kosakovsky PondSL, MurrellB, FourmentM, FrostSD, DelportW, et al. (2011) A random effects branch-site model for detecting episodic diversifying selection. Mol Biol Evol 28 (11) 3033–3043.

11. ZhangJ, NielsenR, YangZ (2005) Evaluation of an improved branch-site likelihood method for detecting positive selection at the molecular level. Mol Biol Evol 22 (12) 2472–2479.

12. AnisimovaM, YangZ (2007) Multiple hypothesis testing to detect lineages under positive selection that affects only a few sites. Mol Biol Evol 24 (5) 1219–1228.

13. LiuJ, ShajiD, ChoS, DuW, Gervay-HagueJ, et al. (2010) A threonine-based targeting signal in the human CD1d cytoplasmic tail controls its functional expression. J Immunol 184 (9) 4973–4981.

14. FeinbergH, TaylorME, RaziN, McBrideR, KnirelYA, et al. (2011) Structural basis for langerin recognition of diverse pathogen and mammalian glycans through a single binding site. J Mol Biol 405 (4) 1027–1039.

15. FeinbergH, PowleslandAS, TaylorME, WeisWI (2010) Trimeric structure of langerin. J Biol Chem 285 (17) 13285–13293.

16. VerdijkP, DijkmanR, PlasmeijerEI, MulderAA, ZoutmanWH, et al. (2005) A lack of birbeck granules in langerhans cells is associated with a naturally occurring point mutation in the human langerin gene. J Invest Dermatol 124 (4) 714–717.

17. PacletMH, HendersonLM, CampionY, MorelF, DagherMC (2004) Localization of Nox2 N-terminus using polyclonal antipeptide antibodies. Biochem J 382 (Pt 3) 981–986.

18. WallachTM, SegalAW (1997) Analysis of glycosylation sites on gp91phox, the flavocytochrome of the NADPH oxidase, by site-directed mutagenesis and translation in vitro. Biochem J 321 (Pt 3) 583–585.

19. Royer-PokoraB, KunkelLM, MonacoAP, GoffSC, NewburgerPE, et al. (1986) Cloning the gene for an inherited human disorder–chronic granulomatous disease–on the basis of its chromosomal location. Nature 322 (6074) 32–38.

20. BustamanteJ, AriasAA, VogtG, PicardC, GaliciaLB, et al. (2011) Germline CYBB mutations that selectively affect macrophages in kindreds with X-linked predisposition to tuberculous mycobacterial disease. Nat Immunol 12 (3) 213–221.

21. SchrodtS, KochJ, TampeR (2006) Membrane topology of the transporter associated with antigen processing (TAP1) within an assembled functional peptide-loading complex. J Biol Chem 281 (10) 6455–6462.

22. KochJ, GuntrumR, HeintkeS, KyritsisC, TampeR (2004) Functional dissection of the transmembrane domains of the transporter associated with antigen processing (TAP). J Biol Chem 279 (11) 10142–10147.

23. NijenhuisM, HammerlingGJ (1996) Multiple regions of the transporter associated with antigen processing (TAP) contribute to its peptide binding site. J Immunol 157 (12) 5467–5477.

24. DongG, WearschPA, PeaperDR, CresswellP, ReinischKM (2009) Insights into MHC class I peptide loading from the structure of the tapasin-ERp57 thiol oxidoreductase heterodimer. Immunity 30 (1) 21–32.

25. de GaravillaL, GrecoMN, SukumarN, ChenZW, PinedaAO, et al. (2005) A novel, potent dual inhibitor of the leukocyte proteases cathepsin G and chymase: Molecular mechanisms and anti-inflammatory activity in vivo. J Biol Chem 280 (18) 18001–18007.

26. AscherDB, CromerBA, MortonCJ, VolitakisI, ChernyRA, et al. (2011) Regulation of insulin-regulated membrane aminopeptidase activity by its C-terminal domain. Biochemistry 50 (13) 2611–2622.

27. EnenkelC, WolfDH (1993) BLH1 codes for a yeast thiol aminopeptidase, the equivalent of mammalian bleomycin hydrolase. J Biol Chem 268 (10) 7036–7043.

28. KimW, BennettEJ, HuttlinEL, GuoA, LiJ, et al. (2011) Systematic and quantitative assessment of the ubiquitin-modified proteome. Mol Cell 44 (2) 325–340.

29. ChoudharyC, KumarC, GnadF, NielsenML, RehmanM, et al. (2009) Lysine acetylation targets protein complexes and co-regulates major cellular functions. Science 325 (5942) 834–840.

30. WilsonDJ, HernandezRD, AndolfattoP, PrzeworskiM (2011) A population genetics-phylogenetics approach to inferring natural selection in coding sequences. PLoS Genet 7 (12) e1002395.

31. Genomes Project Consortium (2010) DurbinRM, AbecasisGR, AltshulerDL, AutonA, et al. (2010) A map of human genome variation from population-scale sequencing. Nature 467 (7319) 1061–1073.

32. DallE, BrandstetterH (2013) Mechanistic and structural studies on legumain explain its zymogenicity, distinct activation pathways, and regulation. Proc Natl Acad Sci U S A 110 (27) 10940–10945.

33. CaglianiR, RivaS, BiasinM, FumagalliM, PozzoliU, et al. (2010) Genetic diversity at endoplasmic reticulum aminopeptidases is maintained by balancing selection and is associated with natural resistance to HIV-1 infection. Hum Mol Genet 19: 4705–14 doi: 10.1093/hmg/ddq401

34. KirkR, LamanH, KnowlesPP, Murray-RustJ, LomonosovM, et al. (2008) Structure of a conserved dimerization domain within the F-box protein Fbxo7 and the PI31 proteasome inhibitor. J Biol Chem 283 (32) 22325–22335.

35. RayK, HinesCS, Coll-RodriguezJ, RodgersDW (2004) Crystal structure of human thimet oligopeptidase provides insight into substrate recognition, regulation, and localization. J Biol Chem 279 (19) 20480–20489.

36. WattersonGA (1975) On the number of segregating sites in genetical models without recombination. Theor Popul Biol 7 (2) 256–276.

37. NeiM, LiWH (1979) Mathematical model for studying genetic variation in terms of restriction endonucleases. Proc Natl Acad Sci U S A 76 (10) 5269–5273.

38. TajimaF (1989) Statistical method for testing the neutral mutation hypothesis by DNA polymorphism. Genetics 123 (3) 585–595.

39. ZengK, FuYX, ShiS, WuCI (2006) Statistical tests for detecting positive selection by utilizing high-frequency variants. Genetics 174 (3) 1431–1439.

40. FuYX, LiWH (1993) Statistical tests of neutrality of mutations. Genetics 133 (3) 693–709.

41. WrightS (1950) Genetical structure of populations. Nature 166 (4215) 247–249.

42. BarreiroLB, Ben-AliM, QuachH, LavalG, PatinE, et al. (2009) Evolutionary dynamics of human toll-like receptors and their different contributions to host defense. PLoS Genet 5 (7) e1000562.

43. CaglianiR, RivaS, PozzoliU, FumagalliM, ComiGP, et al. (2011) Balancing selection is common in the extended MHC region but most alleles with opposite risk profile for autoimmune diseases are neutrally evolving. BMC Evol Biol 11: 171–2148-11-171.

44. AndresAM, DennisMY, KretzschmarWW, CannonsJL, Lee-LinSQ, et al. (2010) Balancing selection maintains a form of ERAP2 that undergoes nonsense-mediated decay and affects antigen presentation. PLoS Genet 6 (10) e1001157.

45. TangK, ThorntonKR, StonekingM (2007) A new approach for using genome scans to detect recent positive selection in the human genome. PLoS Biol 5 (7) e171.

46. VoightBF, KudaravalliS, WenX, PritchardJK (2006) A map of recent positive selection in the human genome. PLoS Biol 4 (3) e72.

47. KennyEE, Pe'erI, KarbanA, OzeliusL, MitchellAA, et al. (2012) A genome-wide scan of ashkenazi jewish crohn's disease suggests novel susceptibility loci. PLoS Genet 8 (3) e1002559.

48. GargiuloG, LevyS, BucciG, RomanenghiM, FornasariL, et al. (2009) NA-seq: A discovery tool for the analysis of chromatin structure and dynamics during differentiation. Dev Cell 16 (3) 466–481.

49. LiangL, MorarN, DixonAL, LathropGM, AbecasisGR, et al. (2013) A cross-platform analysis of 14,177 expression quantitative trait loci derived from lymphoblastoid cell lines. Genome Res 23 (4) 716–726.

50. WilliamsonSH, HubiszMJ, ClarkAG, PayseurBA, BustamanteCD, et al. (2007) Localizing recent adaptive evolution in the human genome. PLoS Genet 3 (6) e90.

51. GrossmanSR, ShylakhterI, KarlssonEK, ByrneEH, MoralesS, et al. (2010) A composite of multiple signals distinguishes causal variants in regions of positive selection. Science 327 (5967) 883–886.

52. SabetiPC, VarillyP, FryB, LohmuellerJ, HostetterE, et al. (2007) Genome-wide detection and characterization of positive selection in human populations. Nature 449 (7164) 913–918.

53. KelleyJL, MadeoyJ, CalhounJC, SwansonW, AkeyJM (2006) Genomic signatures of positive selection in humans and the limits of outlier approaches. Genome Res 16 (8) 980–989.

54. KimuraR, FujimotoA, TokunagaK, OhashiJ (2007) A practical genome scan for population-specific strong selective sweeps that have reached fixation. PLoS One 2 (3) e286.

55. CarlsonCS, ThomasDJ, EberleMA, SwansonJE, LivingstonRJ, et al. (2005) Genomic regions exhibiting positive selection identified from dense genotype data. Genome Res 15 (11) 1553–1565.

56. BarreiroLB, LavalG, QuachH, PatinE, Quintana-MurciL (2008) Natural selection has driven population differentiation in modern humans. Nat Genet 40 (3) 340–345.

57. CharlesworthD (2006) Balancing selection and its effects on sequences in nearby genome regions. PLoS Genet 2 (4) e64.

58. WrightSI, CharlesworthB (2004) The HKA test revisited: A maximum-likelihood-ratio test of the standard neutral model. Genetics 168 (2) 1071–1076.

59. GarriganD, HammerMF (2006) Reconstructing human origins in the genomic era. Nat Rev Genet 7 (9) 669–680.

60. WardEM, StambachNS, DrickamerK, TaylorME (2006) Polymorphisms in human langerin affect stability and sugar binding activity. J Biol Chem 281 (22) 15450–15456.

61. TatenoH, OhnishiK, YabeR, HayatsuN, SatoT, et al. (2010) Dual specificity of langerin to sulfated and mannosylated glycans via a single C-type carbohydrate recognition domain. J Biol Chem 285 (9) 6390–6400.

62. de WitteL, NabatovA, PionM, FluitsmaD, de JongMA, et al. (2007) Langerin is a natural barrier to HIV-1 transmission by langerhans cells. Nat Med 13 (3) 367–371.

63. MiyazawaM, LopalcoL, MazzottaF, Lo CaputoS, VeasF, et al. (2009) The ‘immunologic advantage’ of HIV-exposed seronegative individuals. Aids 23 (2) 161–175.

64. Wittke-ThompsonJK, PluzhnikovA, CoxNJ (2005) Rational inferences about departures from hardy-weinberg equilibrium. Am J Hum Genet 76 (6) 967–986.

65. MitchellPS, PatzinaC, EmermanM, HallerO, MalikHS, et al. (2012) Evolution-guided identification of antiviral specificity determinants in the broadly acting interferon-induced innate immunity factor MxA. Cell Host Microbe 12 (4) 598–604.

66. KosiolC, VinarT, da FonsecaRR, HubiszMJ, BustamanteCD, et al. (2008) Patterns of positive selection in six mammalian genomes. PLoS Genet 4 (8) e1000144.

67. NielsenR, BustamanteC, ClarkAG, GlanowskiS, SacktonTB, et al. (2005) A scan for positively selected genes in the genomes of humans and chimpanzees. PLoS Biol 3 (6) e170.

68. GrossmanSR, AndersenKG, ShlyakhterI, TabriziS, WinnickiS, et al. (2013) Identifying recent adaptations in large-scale genomic data. Cell 152 (4) 703–713.

69. FuW, AkeyJM (2013) Selection and adaptation in the human genome. Annu Rev Genomics Hum Genet 14: 467–489.

70. SabetiPC, SchaffnerSF, FryB, LohmuellerJ, VarillyP, et al. (2006) Positive natural selection in the human lineage. Science 312 (5780) 1614–1620.

71. AndresAM, HubiszMJ, IndapA, TorgersonDG, DegenhardtJD, et al. (2009) Targets of balancing selection in the human genome. Mol Biol Evol 26 (12) 2755–2764.

72. ManryJ, LavalG, PatinE, FornarinoS, ItanY, et al. (2011) Evolutionary genetic dissection of human interferons. J Exp Med 208 (13) 2747–2759.

73. ForniD, CaglianiR, PozzoliU, ColleoniM, RivaS, et al. (2013) A 175 million year history of T cell regulatory molecules reveals widespread selection, with adaptive evolution of disease alleles. Immunity 38 (6) 1129–1141.

74. VasseurE, BoniottoM, PatinE, LavalG, QuachH, et al. (2012) The evolutionary landscape of cytosolic microbial sensors in humans. Am J Hum Genet 91 (1) 27–37.

75. HorstD, GeerdinkRJ, GramAM, StoppelenburgAJ, RessingME (2012) Hiding lipid presentation: Viral interference with CD1d-restricted invariant natural killer T (iNKT) cell activation. Viruses 4 (10) 2379–2399.

76. RessingME, LuteijnRD, HorstD, WiertzEJ (2012) Viral interference with antigen presentation: Trapping TAP. Mol Immunol 55: 139–42 doi: 10.1016/j.molimm.2012.10.009

77. MomburgF, RoelseJ, HowardJC, ButcherGW, HammerlingGJ, et al. (1994) Selectivity of MHC-encoded peptide transporters from human, mouse and rat. Nature 367 (6464) 648–651.

78. KimE, KwakH, AhnK (2009) Cytosolic aminopeptidases influence MHC class I-mediated antigen presentation in an allele-dependent manner. J Immunol 183 (11) 7379–7387.

79. EvnouchidouI, KamalRP, SereginSS, GotoY, TsujimotoM, et al. (2011) Coding single nucleotide polymorphisms of endoplasmic reticulum aminopeptidase 1 can affect antigenic peptide generation in vitro by influencing basic enzymatic properties of the enzyme. J Immunol 186 (4) 1909–1913.

80. FierabracciA, MililloA, LocatelliF, FruciD (2012) The putative role of endoplasmic reticulum aminopeptidases in autoimmunity: Insights from genomic-wide association studies. Autoimmun Rev 12 (2) 281–288.

81. ReevesEP, LuH, JacobsHL, MessinaCG, BolsoverS, et al. (2002) Killing activity of neutrophils is mediated through activation of proteases by K+ flux. Nature 416 (6878) 291–297.

82. AverhoffP, KolbeM, ZychlinskyA, WeinrauchY (2008) Single residue determines the specificity of neutrophil elastase for shigella virulence factors. J Mol Biol 377 (4) 1053–1066.

83. LiDN, MatthewsSP, AntoniouAN, MazzeoD, WattsC (2003) Multistep autoactivation of asparaginyl endopeptidase in vitro and in vivo. J Biol Chem 278 (40) 38980–38990.

84. SandersRW, VenturiM, SchiffnerL, KalyanaramanR, KatingerH, et al. (2002) The mannose-dependent epitope for neutralizing antibody 2G12 on human immunodeficiency virus type 1 glycoprotein gp120. J Virol 76 (14) 7293–7305.

85. KawashimaT, BaoYC, NomuraY, MoonY, TonozukaY, et al. (2006) Rac1 and a GTPase-activating protein, MgcRacGAP, are required for nuclear translocation of STAT transcription factors. J Cell Biol 175 (6) 937–946.

86. LambethJD (2004) NOX enzymes and the biology of reactive oxygen. Nat Rev Immunol 4 (3) 181–189.

87. VernotB, StergachisAB, MauranoMT, VierstraJ, NephS, et al. (2012) Personal and population genomics of human regulatory variation. Genome Res 22 (9) 1689–1697.

88. RohrlichPS, FazilleauN, GinhouxF, FiratH, MichelF, et al. (2005) Direct recognition by alphabeta cytolytic T cells of hfe, a MHC class ib molecule without antigen-presenting function. Proc Natl Acad Sci U S A 102 (36) 12855–12860.

89. VilellaAJ, SeverinJ, Ureta-VidalA, HengL, DurbinR, et al. (2009) EnsemblCompara GeneTrees: Complete, duplication-aware phylogenetic trees in vertebrates. Genome Res 19 (2) 327–335.

90. WernerssonR, PedersenAG (2003) RevTrans: Multiple alignment of coding DNA from aligned amino acid sequences. Nucleic Acids Res 31 (13) 3537–3539.

91. AnisimovaM, BielawskiJP, YangZ (2002) Accuracy and power of bayes prediction of amino acid sites under positive selection. Mol Biol Evol 19 (6) 950–958.

92. Kosakovsky PondSL, PosadaD, GravenorMB, WoelkCH, FrostSD (2006) Automated phylogenetic detection of recombination using a genetic algorithm. Mol Biol Evol 23 (10) 1891–1901.

93. Kosakovsky PondSL, FrostSD (2005) Not so different after all: A comparison of methods for detecting amino acid sites under selection. Mol Biol Evol 22 (5) 1208–1222.

94. DelportW, PoonAF, FrostSD, Kosakovsky PondSL (2010) Datamonkey 2010: A suite of phylogenetic analysis tools for evolutionary biology. Bioinformatics 26 (19) 2455–2457.

95. TinaKG, BhadraR, SrinivasanN (2007) PIC: Protein interactions calculator. Nucleic Acids Res 35 (Web Server issue) W473–6.

96. SchymkowitzJ, BorgJ, StricherF, NysR, RousseauF, et al. (2005) The FoldX web server: An online force field. Nucleic Acids Res 33 (Web Server issue) W382–8.

97. DehouckY, KwasigrochJM, GilisD, RoomanM (2011) PoPMuSiC 2.1: A web server for the estimation of protein stability changes upon mutation and sequence optimality. BMC Bioinformatics 12: 151-2105-12-151.

98. CapriottiE, FariselliP, CasadioR (2005) I-Mutant2.0: Predicting stability changes upon mutation from the protein sequence or structure. Nucleic Acids Res 33 (Web Server issue) W306–10.

99. CeredaM, SironiM, CavalleriM, PozzoliU (2011) GeCo++: A C++ library for genomic features computation and annotation in the presence of variants. Bioinformatics 27 (9) 1313–1315.

100. ThorntonK (2003) Libsequence: A C++ class library for evolutionary genetic analysis. Bioinformatics 19 (17) 2325–2327.

101. FayJC, WuCI (2000) Hitchhiking under positive darwinian selection. Genetics 155 (3) 1405–1413.

102. GautierM, VitalisR (2012) Rehh: An R package to detect footprints of selection in genome-wide SNP data from haplotype structure. Bioinformatics 28 (8) 1176–1177.

103. SchaffnerSF, FooC, GabrielS, ReichD, DalyMJ, et al. (2005) Calibrating a coalescent simulation of human genome sequence variation. Genome Res 15 (11) 1576–1583.

104. HudsonRR (2001) Two-locus sampling distributions and their application. Genetics 159 (4) 1805–1817.

105. FumagalliM, CaglianiR, PozzoliU, RivaS, ComiGP, et al. (2009) Widespread balancing selection and pathogen-driven selection at blood group antigen genes. Genome Res 19 (2) 199–212.

106. StephensM, SmithNJ, DonnellyP (2001) A new statistical method for haplotype reconstruction from population data. Am J Hum Genet 68 (4) 978–989.

107. StephensM, ScheetP (2005) Accounting for decay of linkage disequilibrium in haplotype inference and missing-data imputation. Am J Hum Genet 76 (3) 449–462.

108. BandeltHJ, ForsterP, RohlA (1999) Median-joining networks for inferring intraspecific phylogenies. Mol Biol Evol 16 (1) 37–48.

109. GriffithsRC, TavareS (1995) Unrooted genealogical tree probabilities in the infinitely-many-sites model. Math Biosci 127 (1) 77–98.

110. GriffithsRC, TavareS (1994) Sampling theory for neutral alleles in a varying environment. Philos Trans R Soc Lond B Biol Sci 344 (1310) 403–410.

111. EvansPD, GilbertSL, Mekel-BobrovN, VallenderEJ, AndersonJR, et al. (2005) Microcephalin, a gene regulating brain size, continues to evolve adaptively in humans. Science 309 (5741) 1717–1720.

112. ThomsonR, PritchardJK, ShenP, OefnerPJ, FeldmanMW (2000) Recent common ancestry of human Y chromosomes: Evidence from DNA sequence data. Proc Natl Acad Sci U S A 97 (13) 7360–7365.

113. GlazkoGV, NeiM (2003) Estimation of divergence times for major lineages of primate species. Mol Biol Evol 20 (3) 424–434.

114. SamsonM, LibertF, DoranzBJ, RuckerJ, LiesnardC, et al. (1996) Resistance to HIV-1 infection in caucasian individuals bearing mutant alleles of the CCR-5 chemokine receptor gene. Nature 382 (6593) 722–725.

115. PlummerFA, BallTB, KimaniJ, FowkeKR (1999) Resistance to HIV-1 infection among highly exposed sex workers in nairobi: What mediates protection and why does it develop? Immunol Lett 66 (1–3) 27–34.

116. FowkeKR, NagelkerkeNJ, KimaniJ, SimonsenJN, AnzalaAO, et al. (1996) Resistance to HIV-1 infection among persistently seronegative prostitutes in nairobi, kenya. Lancet 348 (9038) 1347–1351.

117. PurcellS, NealeB, Todd-BrownK, ThomasL, FerreiraMA, et al. (2007) PLINK: A tool set for whole-genome association and population-based linkage analyses. Am J Hum Genet 81 (3) 559–575.