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Immunology in natura: clinical, epidemiological and evolutionary genetics of infectious diseases Lluis Quintana-Murci 1,5 , Alexandre Alcaïs 2,3,5 , Laurent Abel 2,3,5 & Jean-Laurent Casanova 2-5 The field of human genetics of infectious diseases defines the genes and alleles rendering individuals (clinical genetics) and populations (epidemiological genetics) vulnerable to infection, and studies those selected by previous infections (evolutionary genetics). These disciplines—clinical, epidemiological and evolutionary genetics—delineate the redundant and nonredundant functions of host defense genes for past and present survival in natura—in natural ecosystems governed by natural selection. These disciplines, in other words, assess the ecologically relevant and evolutionarily selected roles of human genes and alleles in protective immunity to diverse and evolving microorganisms. The genetic dissection of human immunity to infection in natura provides unique immunological insight, making it an indispensable complement to experimental immunology in vitro and in vivo in plants and animals. Infection is the greatest killer in human history. Life expectancy at birth did not exceed about 25 years until relatively recently, with advances in hygiene, vaccines and antimicrobial drugs building on Pasteur’s micro- bial theory of disease 1 . Confronted with the tremendous diversity of coevolving microbes, natural immunity ensured the overall survival of the species, despite premature death of most individuals. The microbial theory of disease identified the cause of infectious diseases and solved the most important question in human medicine, but did not unravel the causes of intrafamilial clinical heterogeneity (arguably the second most important question in medicine) in families exposed to the same microbial environment (Fig. 1). The considerable clinical variability between individuals and between populations was initially attributed to the variability of infection; that view changed when Charles Nicolle demonstrated that the same pathogen could cause both asymptomatic and symptomatic infections 1 . We now know that interindividual vari- ability results from the complex interplay between microbial and nonmi- crobial environmental factors and between genetic and nongenetic host factors 2 . Microbial factors in particular, such as the emergence of a more virulent strain at the outset of an epidemic, can play a central role in spe- cific contexts. Nevertheless, individuals with life-threatening infections can be correctly said to have some kind of immunodeficiency—either transiently induced or genetically determined. The most conservative definition of immunodeficiency is death from infection—or a threat to life, if rescued by the medicine available. Most individuals therefore suffer from immunodeficiency, the clini- cal expression of which depends on exposure to ad hoc environmental factors, whether through microbes or human intervention 1,3 . Since the 1930s, a large body of epidemiological evidence has been amassed suggesting that immunodeficiency is often inherited 1,4 . The field of human genetics of infectious diseases aims to identify the ‘susceptibility genes’ (or the ‘resistance genes’ for diseases caused by the most virulent microbes) underlying this inheritance; this field can be divided into three areas of research: clinical, epidemiological and evolutionary genet- ics (Fig. 2) 5,6 . Clinical genetics largely overlaps with mendelian genetics (that is, reflecting simple monogenic inheritance) and aims to identify the mutant alleles that render individuals vulnerable to infection or to disease (including the field of primary immunodeficiencies, PIDs) 3,7 ; epidemiological genetics focuses more on determining which alleles confer predisposition at the population level 8 ; evolutionary genetics evaluates the consequences of past infections (which may continue to persist) in the genetic make-up of present-day human populations 9,10 . All three approaches, clinical genetics in particular, have considerable clinical implications, as they shed new light on the pathogenesis of infec- tious diseases and provide new means of diagnosis and immunological intervention. The field of human genetics of infectious diseases is also of interest to immunologists. Considerable progress has recently been made in studies of immunity to infection, both in vitro and in vivo 11–14 . The roles of individual genes have been investigated in plants 15 and animals 16 , in mice in particular. Studies in mice have been based on reverse genetics (gene-targeted knockout and knock-in mutations) and forward genetics (natural mutation and random mutagenesis) 17–20 . The power of such experimental approaches lies in their capacity to offer rigorous control of many host and environmental variables; these approaches also benefit from decades of successful description of the three pillars of immu- nology—developmental, cellular and molecular immunology—in the mouse model. However, their strength is also their weakness: studies in mice are based on experimental conditions, rather than aiming to define natural functions of genes in a natural ecosystem 2 . Though this 1 Human Evolutionary Genetics Unit, Centre National de la Recherche Scientifique URA3012, Pasteur Institute, 75015 Paris, France. 2 Laboratory of Human Genetics of Infectious Diseases, Institut National de la Santé et de la Recherche Médicale U550, Necker Medical School, 75015 Paris, France. 3 University Paris René Descartes, Necker Medical School, 75015 Paris, France. 4 Pediatric Hematology and Immunology Unit, Necker Children’s Hospital, 75015 Paris, France. 5 These authors contributed equally to this work. Correspondence should be addressed to J.-L.C. ([email protected]). Published online 19 October 2007; doi:10.1038/ni1535 NATURE IMMUNOLOGY VOLUME 8 NUMBER 11 NOVEMBER 2007 1165 PERSPECTIVE PATHOGENESIS © 2007 Nature Publishing Group http://www.nature.com/natureimmunology

Immunology in natura: clinical, epidemiological and evolutionary genetics of infectious diseases

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Immunology in natura: clinical, epidemiological and evolutionary genetics of infectious diseasesLluis Quintana-Murci1,5, Alexandre Alcaïs2,3,5, Laurent Abel2,3,5 & Jean-Laurent Casanova2-5

The field of human genetics of infectious diseases defines the genes and alleles rendering individuals (clinical genetics) and populations (epidemiological genetics) vulnerable to infection, and studies those selected by previous infections (evolutionary genetics). These disciplines—clinical, epidemiological and evolutionary genetics—delineate the redundant and nonredundant functions of host defense genes for past and present survival in natura—in natural ecosystems governed by natural selection. These disciplines, in other words, assess the ecologically relevant and evolutionarily selected roles of human genes and alleles in protective immunity to diverse and evolving microorganisms. The genetic dissection of human immunity to infection in natura provides unique immunological insight, making it an indispensable complement to experimental immunology in vitro and in vivo in plants and animals.

Infection is the greatest killer in human history. Life expectancy at birth did not exceed about 25 years until relatively recently, with advances in hygiene, vaccines and antimicrobial drugs building on Pasteur’s micro-bial theory of disease1. Confronted with the tremendous diversity of coevolving microbes, natural immunity ensured the overall survival of the species, despite premature death of most individuals. The microbial theory of disease identified the cause of infectious diseases and solved the most important question in human medicine, but did not unravel the causes of intrafamilial clinical heterogeneity (arguably the second most important question in medicine) in families exposed to the same microbial environment (Fig. 1). The considerable clinical variability between individuals and between populations was initially attributed to the variability of infection; that view changed when Charles Nicolle demonstrated that the same pathogen could cause both asymptomatic and symptomatic infections1. We now know that interindividual vari-ability results from the complex interplay between microbial and nonmi-

crobial environmental factors and between genetic and nongenetic host factors2. Microbial factors in particular, such as the emergence of a more virulent strain at the outset of an epidemic, can play a central role in spe-cific contexts. Nevertheless, individuals with life-threatening infections can be correctly said to have some kind of immunodeficiency—either transiently induced or genetically determined. The most conservative definition of immunodeficiency is death from infection—or a threat to life, if rescued by the medicine available.

Most individuals therefore suffer from immunodeficiency, the clini-cal expression of which depends on exposure to ad hoc environmental factors, whether through microbes or human intervention1,3. Since the 1930s, a large body of epidemiological evidence has been amassed suggesting that immunodeficiency is often inherited1,4. The field of human genetics of infectious diseases aims to identify the ‘susceptibility genes’ (or the ‘resistance genes’ for diseases caused by the most virulent microbes) underlying this inheritance; this field can be divided into three areas of research: clinical, epidemiological and evolutionary genet-ics (Fig. 2)5,6. Clinical genetics largely overlaps with mendelian genetics (that is, reflecting simple monogenic inheritance) and aims to identify the mutant alleles that render individuals vulnerable to infection or to disease (including the field of primary immunodeficiencies, PIDs)3,7; epidemiological genetics focuses more on determining which alleles confer predisposition at the population level8; evolutionary genetics evaluates the consequences of past infections (which may continue to persist) in the genetic make-up of present-day human populations9,10. All three approaches, clinical genetics in particular, have considerable clinical implications, as they shed new light on the pathogenesis of infec-tious diseases and provide new means of diagnosis and immunological intervention.

The field of human genetics of infectious diseases is also of interest to immunologists. Considerable progress has recently been made in studies of immunity to infection, both in vitro and in vivo11–14. The roles of individual genes have been investigated in plants15 and animals16, in mice in particular. Studies in mice have been based on reverse genetics (gene-targeted knockout and knock-in mutations) and forward genetics (natural mutation and random mutagenesis)17–20. The power of such experimental approaches lies in their capacity to offer rigorous control of many host and environmental variables; these approaches also benefit from decades of successful description of the three pillars of immu-nology—developmental, cellular and molecular immunology—in the mouse model. However, their strength is also their weakness: studies in mice are based on experimental conditions, rather than aiming to define natural functions of genes in a natural ecosystem2. Though this

1Human Evolutionary Genetics Unit, Centre National de la Recherche

Scientifique URA3012, Pasteur Institute, 75015 Paris, France. 2Laboratory

of Human Genetics of Infectious Diseases, Institut National de la Santé et de

la Recherche Médicale U550, Necker Medical School, 75015 Paris, France. 3University Paris René Descartes, Necker Medical School, 75015 Paris,

France. 4Pediatric Hematology and Immunology Unit, Necker Children’s

Hospital, 75015 Paris, France. 5These authors contributed equally to this work.

Correspondence should be addressed to J.-L.C. ([email protected]).

Published online 19 October 2007; doi:10.1038/ni1535

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limitation is not a substantial problem for studies in developmental, cellular and molecular immunology per se, studies of host-environ-ment interactions are profoundly altered by the artificial nature of the experimental setup2. The use of humanized mice, experimentally mixing human and mouse tissues, does not overcome this problem21.

The field of human genetics of infectious diseases can be used to ‘dissect’ immunity to infection in natura (Fig. 3)3: that is, natural selec-tion operates, by definition, in natural conditions. Such studies, because of their emphasis on natural conditions, have the potential to define the ecologically relevant and evolutionarily selected functions of host genes, or gene variants, for past and present survival (and biology in natura obviously applies to many other fields beyond immunology). As major breakthroughs achieved in immunological studies in vitro and in vivo provide an ideal foundation for in natura studies and bio-logical validation, so too should discoveries in natura be followed by in vitro and in vivo experimental studies for the fine dissection of mecha-nisms. Although intrinsic differences between rodents and humans may account for some discrepancies between studies in mice and men, we propose that differences between natural and experimental conditions are the most important factors involved. A gene critical in vitro and in vivo may be redundant in natura; more rarely, a gene that is redundant in vitro and in vivo may be crucial in natura. Human genetics attempts to decipher the natural history and pathogenesis of infectious diseases—the most ancient and widespread life-taking experiments of Nature—at the molecular level. In doing so, it reconciles the holistic and reductionist approaches of naturalists and chemists or, to quote Hans Selye, ‘prob-lem-finders’ and ‘problem-solvers’22.

Clinical genetics of infectious diseasesThe clinical genetics of infectious diseases emerged between 1945 and 1954. This ten-year period saw the description of the first primary immunodeficiencies (PIDs), which typically consist of mendelian traits conferring predisposition to multiple infectious diseases, the nature and range of which vary according to the disorder3,7. Over 200 PIDs have since been clinically described and over 100 genetic etiologies identi-fied. A profound paradigm shift is underway at present in the field of

PIDs3. These diseases were long thought to be exclusively rare, recessive, familial, monogenic traits conferring predisposition to multiple, recur-rent, opportunistic and fatal infections in infancy. Exceptions to each of these qualifications have gradually accumulated. Newly identified PIDs predisposing otherwise healthy individuals to a single infection are of particular interest23. In most instances, the infectious phenotype of patients with conventional PIDs was consistent with the susceptibil-ity of the corresponding mutant mice, though this was not always the case24. There is at present no relevant mouse model for some of the new PIDs, including the properdin and complement defects associated with Neisseria invasive disease25; X-linked lymphoproliferative disease, which is associated with Epstein-Barr virus disease26; and epidermodysplasia verruciformis, which is associated with papillomaviral diseases27. A more recent example is provided by the association between trypanosomiasis and apolipoprotein L-1 deficiency28.

Other new examples of PIDs have enabled comparison and revealed how host defense genes that are critical in experiments conducted in ani-mal models in vivo may turn out to be redundant in the human model in natura. It has been suggested, for example, that TH1 cells, defined in terms of the cytokine signatures interleukin (IL)-12 (TH1 inducer molecule) and interferon (IFN)-γ (TH1 effector molecule), are essential for immunity to intracellular pathogens29–31. Unexpectedly, the data obtained from over 300 patients bearing mutations in the TH1 circuit have shown that these cytokines are critical for protective immunity to mycobacteria and, to a lesser extent, salmonella, but generally redun-dant for immunity to most other microbes32,33. Moreover, for some of the disorders, such as deficiency of the IL-12 receptor IL-12R-β1, clinical penetrance is relatively low34. However, one IL-12R-β1–defi-cient patient with listeriosis35 and one with leishmaniasis36 have been reported, although these numbers of patients are clearly too small for definitive conclusions to be drawn. Another example is provided by TLRs. These receptors were thought, on the basis of in vitro and in vivo studies, to be the principal ‘sensors’ of microbial products and, indeed, are also known as ‘pathogen-associated molecular pattern receptors’19,37. However, IRAK-4-deficient patients suffer mostly from pneumococcal disease and, to a lesser extent, from staphylococcal disease38,39. With the exception of a few TLR3 responses and fewer TLR4 responses, cells from these patients do not respond to any TLRs and are limited to the production of IFN in response to viral infections40. Moreover, TLR3- and UNC-93B–deficient patients, with impaired TLR3, TLR7, TLR8 and TLR9 responses, show a selective predisposition to herpes simplex encephalitis41,42. Patients bearing mutations in either of the two prin-cipal TLR pathways (the IRAK-4, MyD88-dependent pathway and the TLR3, MyD88-independent pathway) thus show a much narrower infec-tious phenotype than predicted by the mouse model.

The TH1-TH2 and TLR paradigms have been particularly prominent in immunology over the last 20 and 10 years, respectively. However, neither patients bearing mutations in TH1 signature cytokines nor patients bearing mutations in TLR responses have been found to show the predicted broad vulnerability to infections: to intracel-lular pathogens (TH1), to pathogens in general (TLR–IRAK4), or to viruses (TLR3–UNC-93B). These observations highlight the consid-erable redundancy of the immune system in natura. Intriguingly, the narrow and almost specific vulnerability of patients bearing muta-tions in the IL-12–IFN-γ circuit (mycobacteria), TLR–IRAK4 path-way (pneumococcus) and TLR3 or UNC-93B (herpes simplex virus) implies that some genes and pathways are pathogen specific, in the ecological sense of the term. Put another way, for any given pathogen, there seem to be ‘private genes’ (for example, TLR3)—those specific for responses to that pathogen—and ‘public genes’ (for example, RAG1)—those specific to many (or all) pathogens. Another central

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Louis Pasteur1822–1895

Marie Pasteur1826–1910

Jean-Baptiste1851–1908

Jeanne1850–1859

Cécile1853–1866

Marie-Louise1858–1934

Camille1863–1865

Figure 1 Pasteur’s pedigree and the genetic theory of infectious diseases. Louis Pasteur lost three young daughters to ‘fever’100. Shortly thereafter, between 1866 and 1870, he discovered that microbes caused disease in silk worms, thus demonstrating the microbial theory of disease1. Retrospectively, it is clear that they died of infectious diseases. This illustrious family is representative of most families worldwide and throughout most of human history: it was not uncommon for at least half the siblings in a family to die from infection1. But which half? In Pasteur’s own pedigree, one son and one daughter survived until adulthood, despite probable exposure to at least one of the microbes that killed their siblings. We show here Pasteur’s pedigree as it would have been drawn at around 1866, if the genetic theory of infectious diseases had emerged at that time. It is possible that the three children who died carried a mendelian trait (green), or at least some form of genetic vulnerability, predisposing them to infectious diseases.

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point concerning experiments in natura is that there is no such thing as absolute specificity: for example, about half of IL-12R-β1–defi-cient patients present with clinical salmonellosis33,34 and about half of the IRAK4–deficient patients with staphylococcal disease39. In any event, otherwise healthy children with life-threatening infectious diseases may have mendelian predispositions to those diseases; one key issue here concerns whether genes and alleles conferring such mendelian predispositions to infectious diseases have a substantial impact at the population level. This question is addressed by epide-miological genetics.

Epidemiological genetics of infectious diseasesThe term ‘epidemiology’ was coined in the mid-nineteenth century to describe the scientific study of epidemics43. Studies combining genetics and epidemiology emerged in the early twentieth century, and epide-miological genetics, also referred to as ‘genetic epidemiology’, became a unified discipline in the 1980s44. This field investigates the interaction of genetic factors with environmental factors and their role in the develop-ment of common diseases in human populations2. Examples of men-delian genetics of infectious diseases in this context include autosomal recessive deficiencies of Duffy antigen receptor for chemokines (DARC), the glycosphingolipid P antigen, the chemokine receptor CCR5, and fucosyltransferase 2 (FUT2), which confer resistance to Plasmodium

vivax45, parvovirus B19 (ref. 46), HIV47 and noroviruses48, respectively. Because most individuals do not have such recessive deficiencies, they therefore show autosomal dominant predisposition to these pathogens, which illustrates the impact of mendelian genetics at the population level. The risk attributable to other, rare mendelian predispositions is unknown38,41,42,49. However genetic epidemiological studies mostly investigate the nonmendelian component of predisposition, using two main approaches8. ‘Linkage studies’ aim to identify a chromosomal region segregating nonrandomly with the phenotype of interest within families; ‘association studies’ test for a significant association between a genetic polymorphism and a phenotype within a population2,8. Common infectious diseases are generally seen as reflecting polygenic inheritance: that is, reflecting the presence of several susceptibility genes, each making at most a modest contribution to the phenotype5. However, there is no formal proof of polygenic predisposition in individual human beings or even in specific populations5, though the recent development of genome-wide association studies50 may make it possible to demon-strate bona fide polygenic predisposition to infection.

Since 1996, genome-wide screens in studies of infectious diseases have involved linkage studies only. Loci detected by such analyses that are expected to exert a substantial effect on a given phenotype are known as ‘major genes’5,23. Several major gene loci have been mapped in individu-als susceptible to certain infectious diseases: the first to be mapped, on chromosome 5q31–q33, controls levels of Schistosoma mansoni infec-tion51, and the most recently mapped, on chromosome 8q12, confers a predisposition to pulmonary tuberculosis52. The identification of such major gene loci is important, as these loci bridge the gap between men-delian and polygenic inheritance and demonstrate the existence of a continuous spectrum of genetic predisposition to infectious diseases23. This is the case particularly for tuberculosis, for which mendelian pre-disposition has been demonstrated49. Over time the concept of ‘major genes’ may evolve further, particularly with technological advances in genomics, as it has already moved from a strictly segregation-based concept to a linkage-based one. Genes identified in association studies may also be designated major genes if they have a substantial impact on

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Figure 2 Human genetics of infectious diseases. The field can be divided into three areas of research. Clinical genetics aims to define the alleles rendering individuals vulnerable to severe infection and focuses on one or a few affected subjects (purple individuals). These alleles typically have a profound effect on gene function and occur at low frequency. Their impact on the fitness of the overall population is, therefore, generally negligible and these alleles probably appeared very recently in the population gene pool. Epidemiological genetics focuses on identifying the alleles conferring predisposition at the population level. It is based on large case-control and/or familial cohorts including both affected individuals (purple) and unaffected individuals (green). The alleles identified typically have a more modest effect on gene function, but are more frequent than those identified by clinical genetics. They therefore have a moderate impact on population fitness and can be transmitted through a large number of generations. Evolutionary genetics evaluates the consequences of past infections (which may persist) in the genetic make-up of current human populations. It classically makes use of large random samples of the population consisting mostly of healthy individuals of different ethnic origins. The alleles identified as being under selection (either positive, balancing or negative) have a strong impact on population fitness. Protective alleles under positive or balancing selection tend to increase in frequency over time, whereas those targeted by negative selection tend to disappear from the population. Genomic regions targeted by such selective events contain specific ‘genomic signatures’.

In vitro

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Figure 3 The human model: a genetic dissection of immunity in natura. Variation in protective immunity to infection can be studied using experimental models in vitro (at the cellular level) or in vivo (at the organismal level). Alternatively, the observation of human beings in natural ecosystems governed by natural selection provides an approach in natura. Green and purple individuals represent unaffected and affected subjects, respectively.

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disease susceptibility, even if they are not amenable to linkage mapping owing to the rarity with which the familial phenotype is observed; this is the case, for example, for the sickle cell mutation HbS in the hemoglo-bin-B gene (HBB), which confers resistance to Plasmodium falciparum malaria5. Finally, major genes may be specific to a given population, and, for a given infectious disease (for example, leprosy), may be specific to a given phenotype (for example, paucibacillary leprosy).

The first successful positional cloning of a major locus in a com-mon infectious disease was for leprosy. Both the development of leprosy per se upon exposure to Mycobacterium leprae and the clinical features of the disease (ranging from paucibacillary to multibacillary forms) depend on the genetic make-up of the human host32,53. Major genes were long thought to exist, based on the results of segregation studies54. The first genome-wide linkage study of paucibacillary leprosy in India detected a major locus on chromosome 10p13 (ref. 55), which has yet to be precisely identified. The second study, in Vietnam, mapped a major gene for susceptibility to leprosy per se to chromosome 6q25 (ref. 56). Further association studies narrowed the leprosy-susceptibility locus to the regulatory region shared by PARK2, a gene encoding an E3-ubiquitin ligase called Parkin, and PACRG (Parkin coregulated gene)57, revealing an unexpected role for ubiquitination in immunity to mycobacteria58. In a further study the gene encoding lymphotoxin alpha, LTA, was shown to be a second major susceptibility gene for leprosy per se in the same

Vietnamese sample59. LTA and PARK2-PACRG variants have indepen-dent and additive effects on the risk of leprosy, particularly in patients with early disease onset. Because in vitro and in vivo studies in mice are not possible because M. leprae shows human tropism, only genome-wide studies in natura were able to track the source of immunity to M. leprae.

Evolutionary genetics of infectious diseasesThe field of human evolutionary genetics of infectious diseases searches for the evolutionary ‘footprints’ of natural selection exerted by past infec-tions, present in the genomes of present-day healthy human populations. It investigates how natural selection by infections has shaped the vari-ability of host defense genes9,60. Genetic variants that are advantageous for the host increase in frequency in the population as a result of ‘posi-tive selection’; selection favoring allelic variability within a population (for example, heterozygote advantage) is known as ‘balancing selection’; and deleterious gene variants conferring a disadvantage for the host tend to be eliminated by ‘negative selection’6,10,60. Each type of natural selection leaves a specific molecular ‘signature’ in the human genome, different from that expected under selective neutrality6,60. A gene evolves under selective neutrality61 when the mutations accumulated do not influence host fitness or survival, for example from infection. As most genomic regions evolve under neutrality62, the specific regions ‘targeted’ by natural selection reflect the importance of genes residing there for host defense in natura6,10,63. Because infectious diseases have exerted strong selective pressures on humans, dissecting the variability and degree of selection of genes involved in immunity can provide insight into the mechanisms of host defense6,64. Evolutionary genetics thus identifies the genes and their variants involved in the natural history, current susceptibility to, and pathogenesis of, infectious diseases10. One limitation of this approach, however, is that it may be difficult to identify the pathogens involved in past events, particularly if those pathogens are now extinct, or to rule out other, noninfectious selective forces that may have affected certain genes.

The strongest evidence for selection in the human genome has been obtained for genes involved in immunity65. The HLA class I and II loci are the most polymorphic human genes, owing to both positive and balancing selection66–69. As HLA molecules present microbial antigenic peptides to T cells, the polymorphism of HLA genes probably reflects the selective pressure imposed by the diversity of pathogens66. Killer cell immunoglobulin-like receptors (KIRs) are similarly polymorphic owing to both positive and balancing selection70. And it is interactions

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Figure 4 Examples of genes characterized by clinical, epidemiological and evolutionary genetic approaches in natura. (a) HLA. Clinical genetics has identified affected individuals (red) with mendelian deficiencies in HLA class I or II genes who are vulnerable to a large number of pathogens. Epidemiological genetics has found that certain HLA alleles confer vulnerability to specific infections. In addition, heterozygosity (stripes) at the HLA class I locus is associated with delayed progression to symptomatic disease (red) in individuals infected (pink) with HIV or HTLV-1. Finally, evolutionary genetics has shown that the extreme polymorphism of HLA class I and II genes, and the consequent high heterozygosity (rainbow colors) at the population level result from a long process of balancing and positive selection, probably due to the selective pressures imposed by pathogen diversity. (b) CCR5. Clinical genetics has shown that homozygosity for the ∆32 CCR5 allele (dark green) confers almost complete mendelian resistance to R5-tropic HIV-1, with little cost to fitness. Epidemiological genetics has shown that HIV-infected individuals heterozygous for the ∆32 CCR5 allele (pink stripes) show delayed progression to AIDS (red). Finally, evolutionary genetics has found evidence that ∆32 is a recent mutational event and is very likely to have undergone positive selection in the European population, which presently consists of healthy people who are ∆32 homozygous (dark green), ∆32 heterozygous (half light green, half dark green) or wild type (light green) at CCR5.

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between KIRs and HLA class I ligands that regulate the development and response of human NK cells71: combinations of variable HLA and KIR molecules diversify T and NK cell repertoires, thereby contributing to the survival of human populations worldwide70,72. The selection of particular HLA or KIR alleles does not seem to have been guided by a particular pathogen, and the polymorphism of these alleles is universal. Other selection signatures are specific to particular populations and/or pathogens10,60. The most thoroughly documented example is provided by the selective pressures exerted by malaria on some erythrocyte gene variants73. For example, despite the death of most homozygous children with sickle cell disease, the HbS allele has reach high frequencies in Africa (up to 30%) because heterozygosity confers some resistance to the life-threatening forms of P. falciparum malaria74–76. Additionally, the high frequency of the HbC allele (up to 20%) in Western Africa attests of the strong selective pressure exerted by malaria77,78. Finally, the African DARC null allele (DARC*0, also known as FY*0), which imparts com-plete resistance to P. vivax infection in homozygotes79,80, has been driven to near-fixation by positive selection in sub-Saharan Africa81, whereas it remains rare elsewhere.

Evolutionary genetics can also unmask genes under strong negative selection. A striking example is provided by the C-type lectin DC-SIGN, which acts as a cell adhesion receptor and pathogen recognition recep-tor82: negative selection has prevented the accumulation of amino-acid changes in this protein over time83. These findings are consistent with a key role for this lectin in pathogen recognition and immune response82,84. Finally, the evolutionary genetic approach can also be used to show that gene variability is neutral owing to a lack of ecological rel-evance for protective immunity. For example, mannose-binding lectin (MBL) deficiency has been associated with an increase in susceptibility to several infectious diseases85; however, alleles conferring MBL defi-ciency are very common worldwide (up to 30%), suggesting that they may also protect against some other infectious agent(s)86. Yet the pattern of variation at MBL2, encoding MBL, is consistent with strictly neutral evolution, indicating that the high worldwide prevalence of deleteri-ous MBL2 alleles is the result of genetic drift and that MBL is largely redundant in immunity87,88. A mirror image of this situation is provided by the human caspase-12 gene (CASP12). The worldwide spread of an inactive CASP12 allele has resulted from strong positive selection, with survival of sepsis being the proposed selective force that has driven this allele to near-fixation in most human populations89. As did the associa-tion studies described above on epidemiological genetics, evolutionary genetics will benefit from high-throughput sequencing and genotyping facilities, making it possible to search for signs of selective events at much larger genomic scales (for example, focusing on a set of structurally or functionally related immunity genes).

Concluding remarksClinical, epidemiological and evolutionary genetics use complementary approaches based on individual patients, populations of patients and healthy general populations, respectively, to define the contributions of host defense genes to survival in natura (Fig. 2). As illustrated in this perspective, these three fields have evolved in parallel—with little interaction among them—since the 1950s.

The HLA genes were the first to be studied from all three angles (Fig. 4a). Mendelian disorders affecting HLA class II gene transcription, owing to mutations in the non-DNA-binding coactivator CIITA or any sub-unit of the transcription factor RFX, are characterized by developmental and functional defects in CD4+ T cells that confer vulnerability to many pathogens90. Mendelian disorders affecting HLA class I protein expres-sion on the cell surface owing to mutations in TAP and tapasin, proteins essential for class I antigen presentation, are characterized by impairment

of the development and function of CD8+ T cells and, paradoxically, confer vulnerability to bacteria but not viruses24. At the population level, certain HLA alleles have been shown to confer predisposition to certain infections, leprosy and tuberculosis in particular8,32. In addition, hetero-zygosity at HLA class I or class II loci has been shown to be associated with a more favorable outcome of some viral infections, such as HIV-1 (ref. 91), HBV (ref. 92) and HTLV-1 (ref. 93). Finally, the extreme level of polymorphism of HLA has been shown to result from both balancing and positive selection, strongly suggesting strong selective pressure on these loci66–69. Though many questions remain unsolved, it is clear that HLA genes are indispensable for host defense at the individual and population levels, and that multiple infectious agents are responsible for the selection of their extremely high degree of polymorphism.

A more recent example of a gene studied from clinical, epidemiologi-cal and evolutionary genetics is that encoding the chemokine receptor CCR5 (Fig. 4b). Homozygosity for the ∆32 CCR5 allele confers almost complete, mendelian resistance to R5-tropic HIV-1, with little cost of fitness to the host47, though homozygous individuals were recently reported to be vulnerable to West Nile virus infection94. Association studies also showed that HIV-infected individuals heterozygous for the ∆32 CCR5 allele were delayed in progression to AIDS47. The ∆32 CCR5 allele stems from a single founder event (estimated to be about 2,000 to 3,000 years ago), and a strong signature of balancing selection in the CCR5 promoter has been documented in Europeans95. The fre-quency of the allele in European populations (about 10% in western and central Europe) and the long-range linkage disequilibrium pat-tern at the CCR5 locus are highly suggestive of positive selection96,97. However, the intensity98 and nature47,99 of the selective pressure—pos-sibly infectious—remain a matter of debate. HIV has clearly emerged too recently to have been the selective force on CCR5, but other, more ancient pathogens also using CCR5 as a receptor for entry may have been involved96,99. In any event, despite the limited known role of wild type CCR5 in host defense at individual and population levels, a single mutant allele of this gene has proven to be of great value to the host in the specific context of exposure to a given pathogen.

These complementary sets of data from studies on HLA and from CCR5, provide integrated descriptions of the roles of HLA and CCR5 genes in natura. A main goal in the human genetics of infectious diseases is now to investigate other human genes from these three standpoints.

ACKNOWLEDGMENTSWe thank the members of our laboratories for discussions and our patients and collaborators worldwide for their trust and patience. Supported by INSERM, University Paris René Descartes, Institut Pasteur, Agence Nationale de la Recherche, the BNP Paribas Foundation, the Schlumberger Foundation, the European Union, the Dana Foundation, the March of Dimes and the Howard Hughes Medical Institute.

Published online at http://www.nature.com/natureimmunologyReprints and permissions information is available online at http://npg.nature.com/reprintsandpermissions/

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