key: cord-273910-fna7s9te
authors: Bochud, Pierre-Yves; Bochud, Murielle; Telenti, Amalio; Calandra, Thierry
title: Innate immunogenetics: a tool for exploring new frontiers of host defence
date: 2007-07-20
journal: Lancet Infect Dis
DOI: 10.1016/s1473-3099(07)70185-8
sha: 
doc_id: 273910
cord_uid: fna7s9te

The discovery of innate immune genes, such as those encoding Toll-like receptors (TLRs), nucleotide-binding oligomerisation domain-like receptors (NLRs), and related signal-transducing molecules, has led to a substantial improvement of our understanding of innate immunity. Recent immunogenetic studies have associated polymorphisms of the genes encoding TLRs, NLRs, and key signal-transducing molecules, such as interleukin-1 receptor-associated kinase 4 (IRAK4), with increased susceptibility to, or outcome of, infectious diseases. With the availability of high-throughput genotyping techniques, it is becoming increasingly evident that analyses of genetic polymorphisms of innate immune genes will further improve our knowledge of the host antimicrobial defence response and help in identifying individuals who are at increased risk of life-threatening infections. This is likely to open new perspectives for the development of diagnostic, predictive, and preventive management strategies to combat infectious diseases.

Environmental and host factors are important determinants of susceptibility to infection. In recent years, a rapidly growing body of evidence has underscored the importance of host genetic factors. The eff ect of genetic and environmental factors on the risk of death was assessed in a study of 960 adoptees. 1 Death of a biological parent (but not of an adoptive parent) from infection before the age of 50 years resulted in a six times increase in the relative risk of dying from infection in the adoptee, strongly suggesting that susceptibility to infection aggregates in families. Individuals who are heterozygous for haemoglobin S are known to be protected against malaria, whereas homozygous individuals have sickle-cell anaemia. 2 The high frequency of sickle-cell anaemia and other red blood cell disorders in regions where malaria is highly prevalent suggests that infectious agents (eg, Plasmodium falciparum) can exert quite substantial selective pressure on human populations. 3 Although natural immunity ensures survival of the species as a whole, individuals themselves are not likely to be immunocompetent to all pathogens, and individual diff erences in susceptibility to specifi c pathogens are quite common. 4 The development of the Human Genome Project in 1990 propelled the scientifi c community into a new era, allowing genetic mapping and the development of large-scale gene identifi cation that has greatly facilitated the study of gene polymorphisms.

We review recent advances in the fi eld of innate immunogenetics of host defences and show how an interdisciplinary approach of combining genetic epidemiology, genetics, genomics, and molecular and cellular biology will improve our understanding of the pathogenetic basis of infectious diseases, and help the development of new preventive and therapeutic treatment strategies.

Little inter-individual variation exists within the human genome. In fact, all genetic diff erences between individuals are estimated to be caused by variability in 3 million bp, which represent about 0·01% of the human genome. 5 Since the mutation rate in mammalian genomes is low (10 -⁹ per bp per year), most interindividual variations are inherited. The most frequent variation is the single nucleotide polymorphism (SNP), which occurs on average every 1300 bp. Another type of genetic mutation is the variable number of tandem repeat (VNTR); VNTRs consist of repeats of sequences ranging from a single basepair to thousands of basepairs. 6 The term microsatellite is used for repeats of one to six nucleotides, whereas repeats of longer units are called minisatellites (seven to 100 nucleotides) or, in the extreme case, satellite DNA (more than 100 nucleotides). Since the number of repeats varies among individuals, VNTRs have been widely used as genetic markers. 

Within a coding region of a gene, an SNP can either induce an aminoacid change (non-synonymous SNPs) or not (synonymous SNPs). SNPs may be located in the promoter region of a gene and therefore infl uence gene expression or splicing. Similarly, diff erent lengths of VNTR regions have been associated with diff erential gene expression. 7 Certain SNPs or VNTR alleles, or both, may be linked together so that non-functional polymorphisms can be used as genetic markers of functionally important mutations. Only 1·5% of SNPs are thought to be located in a coding region of a gene. The functions of nearly all SNPs that are located outside gene-coding or regulatory regions are unknown.

In recent years, SNP genotyping technologies with high throughput and aff ordable costs have become available. These technologies are based on a few basic biochemical reactions (hybridisation, PCR with diff erential primer extension, specifi c ligation, and diff erential cleavage), which are used on diff erent support media and can be detected by diff erent methods (fi gure 1). 8 Recent highthroughput technologies allow genotyping at low cost (ie, a few cents per SNP per sample). 9

Once markers have been typed, two main approaches can be used to analyse them: single marker analysis or haplotype analysis. A haplotype refers to the arrangement of two or more alleles on the same chromosome. Currently, there is much debate about which approach is the most appropriate. Studies have proposed that the underlying structure of the human genome can be described by use of a relatively simple framework in which the data are parsed into a series of discrete haplotype blocks. 10, 11 This observation has led to the development of haplotype tagging methods that aim to capture the haplotype structure in a candidate region. 11 Haplotype tagging refers to the concept that most of the haplotypic structure in a particular chromosomal region can be captured by genotyping a smaller number of markers than all of those that constitute the haplotypes. The crucial markers to type would be the minimum set of markers that unambiguously identify each possible haplotype.

The detection and estimation of familial aggregation is usually the fi rst step in the genetic analysis of a trait. Once familial aggregation has been documented, the traditional approach has been to narrow down the genetic region of interest by use of linkage analysis, followed by fi ne mapping and association studies (table 1) . Linkage and association studies are based on the same underlying principle: once a mutation occurs on a particular chromosome, it is subsequently transmitted to off spring together with nearby loci. This association is broken down at each successive generation by recombination (ie, homologous chromosomes pair during the meiotic cell division and exchange genetic material). When two loci are close enough on the same chromosome that their alleles cosegregate when passed on to the next generation, we say that the two loci are linked. 12 Linkage disequilibrium refers to allelic association that is caused by linkage, or in other words, that has not yet been broken up by recombination. 12 An association between two loci, such as the non-independence of alleles at these loci, may be caused not only by linkage, but also to factors such as population stratifi cation or chance. Population stratifi cation refers to the situation in which study participants are selected from genetically diff erent subpopulations. Population stratifi cation will only lead to a spurious association (and hence be a confounder) if both the allele and disease frequencies diff er across subpopulations. 13 Some researchers have argued that too much emphasis has been put on this issue and surprisingly few examples can be found that unequivocally show that population stratifi cation has led to a spurious association. 14, 15 Whereas linkage and association studies can be done in families, only association studies can be done in unrelated cases and controls (table 1). The main diff erence between related and unrelated cases is the number of meiotic events that separate them, so that unrelated cases share a much shorter chromosomal segment around a particular causative mutation than related cases. Linkage and association can be obscured by incomplete penetrance (ie, there is no one-to-one correspondence between genotype and phenotype), misdiagnoses, genetic heterogeneity (several genes can produce a similar phenotype), phenocopies (ie, environmental factors mimicking the eff ect of certain genes), and disease An important advance toward enabling effi cient wholegenome-scan association studies is the determination of linkage disequilibrium patterns on a genome-wide scale through the HapMap project. 16 Because most diseases are likely to be infl uenced by several genes and environmental factors, the analysis of gene-gene interactions (epistasis) and gene-environment interactions will represent an important task in the future, but this is, and will remain, a challenging issue for the years to come.

The innate immune system assumes an essential role in the natural host defences against microbes. The recognition of microbial pathogens, either in tissue in contact with the host's environment or in the systemic circulation after invasion of the bloodstream, is done by macrophages, dendritic cells, natural killer cells, granulocytes, and monocytes, which act as sentinels of the innate immune system (fi gure 2). This process involves coordinated action of several families of proteins, such as Toll-like receptors (TLRs), 17 nucleotide-binding oligomerisation domain (NOD)-like receptors (NLRs), 18, 19 RNA helicase-containing proteins, 20 and the C-type lectins. 21

TLRs are essential components of the innate immune system. 17, 22, 23 TLRs are type I transmembrane proteins that function as homodimers or heterodimers. The extracellular domain comprises multiple leucine-rich repeat structures that vary among diff erent TLRs and are implicated in the selective recognition of a vast range of microbial-associated molecular patterns (MAMPs). 24 So far, 12 members of the TLR family have been identifi ed in mammals. Several molecules, including CD14, 25 CD36, 26 and MD2, 25 have also been shown to participate in the sensing of microbial products and are therefore integral components of these receptor complexes. Binding of microbial products to microbial-recognition molecules activates signal transduction pathways and the transcription of immune genes that code for costimulatory molecules expressed at the cell surface or for immunoregulatory eff ector molecules (including cytokines and chemokines) released in the extracellular milieu that orchestrate the host innate immune defence response. 23, 27 In addition to lipopolysaccharide of Gram-negative bacteria, 28, 29 TLR4 detects other MAMPs that are structurally unrelated to lipopolysaccharide, such as mannan (Candida albicans) or the fusion protein of respiratory syncytial virus (fi gure 3). Other endogenous ligands, including fi brinogen, fi bronectin, hyaluronic acid, heparin sulphate, beta-defensins, or heat-shock proteins, have been reported to activate TLR4. 17 However, endotoxin contamination has been argued to account for TLR5  TL  R2 TL R1

Microbial-associated molecular patterns are recognised by transmembrane receptors (1: eg, Toll-like receptors [TLRs]), which trigger the activation of several signal-transducing pathways, leading to the production of cytokines and expression of costimulatory molecules. Cytokines induce and regulate the infl ammatory response and orchestrate the adaptive immune response. By contrast with other TLRs, TLR3, TLR7, TLR8, and TLR9 are expressed mainly in the endosomal compartment (2), where local acidifi cation is required for recognition of microbial products by their cognate receptors. Intracellular pathogens or microbial products released intracellularly after lysis of ingested microorganisms may also interact with intracytoplasmic receptors, such as nucleotide-binding oligomerisation domain-like (NLR) proteins (3), or the RNA helicase-containing molecules (4: RIG-I or MDA5). TCR=T-cell receptor.

TLR4 detects lipopolysaccharide (LPS), mannan (Candida albicans), and the fusion protein of the respiratory syncytial virus. TLR2 forms a heterodimer with either TLR1 to detect triacyl lipopeptide or TLR6 to detect diacyl lipopeptide and zymosan. TLR2 is also involved in the recognition of lipoteichoic acid (LTA), peptidoglycan (PG), lipoarabinomannan (LAM), porins (Neisseria spp), glycosylphosphatidylinositol mucin (Trypanosoma spp; tGPI), and the haemaglutinnin protein (HA, measles virus). TLR3, TLR7, TLR8, and TLR9 are located in the endosomal compartment and detect nucleic acids and/or haemozoin (Plasmodium spp, TLR9). Through their intracellular domain, TLRs interact with specifi c adaptor proteins, including the myeloid diff erentiation primary response protein 88 (MyD88), the TIR domaincontaining adaptor protein (TIRAP), the TIR domain-containing adapter inducing interferon (TRIF), and the TRIFrelated adapter molecule (TRAM). These adaptors lead to the activation of several transcription factors such the nuclear factor κB (NFκB), the activating-protein 1 (AP1), and/or the interferon regulatory factors 3 and 7 (IRF3/7) that ultimately induce the production of pro-infl ammatory mediators. ss=single-stranded. ds=double-stranded.

the TLR4 specifi city of some of these putative TLR ligands. 30 TLR2 and TLR6 heterodimers detect diacyl lipopeptides, whereas TLR2 and TLR1 heterodimers recognise triacyl lipopeptides. 17 TLR2 has also been proposed to sense lipoteichoic acid, peptidoglygan, lipoarabinomannan, phospholipomannan (C albicans), zymosan (Saccharomyces cerevisiae), porins (Neisseria spp), glycosylphosphatidylinositol mucin (Trypanosoma spp), and the haemagluttinin protein of the measles virus. 17 TLR3, TLR7, TLR8, and TLR9, which are mainly expressed in endosomes, serve to detect viral or bacterial nucleic acids. TLR3 detects double-stranded RNA and TLR8 detects single-stranded RNA. 17 TLR9 senses DNA containing the unmethylated CpG motifs found in bacteria and viruses and the malaria pigment haemozoin. 17 Compartmentalisation of TLR3, TLR7, TLR8, and TLR9 thus allows the detection of pathogenic DNA and RNA within the endosomal compartment, while avoiding the detection of self-DNA and mRNA. 23 On binding of cognate ligands, the intracellular Tollinterleukin-1 receptor (TIR) domain of TLRs recruits and activates diff erent adaptor proteins, including myeloid diff erentiation primary response protein 88 (MyD88), TIR domain-containing adaptor protein, TIR domaincontaining adapter-inducing interferon β (TRIF; also known as TICAM), and TRIF-related adapter molecule, ultimately leading to the activation of several specifi c signal-transducing pathways and transcription factors such as nuclear factor κB (NFκB) and activating protein 1 (AP1; fi gure 3 and fi gure 4). 40 MyD88-dependent signalling pathways (NFκB and AP1) are activated by all TLRs, whereas MyD88-independent, TRIF-dependent signalling path ways (interferon regulatory factor [IRF] 3) are activated only by some TLRs (such as TLR3 and TLR4). The observation that diff erent TLRs may activate diff erent signalling pathways with diff erent biological conse quences shows that the innate immune system can produce pathogen-specifi c defensive responses.

In addition to the TLRs, the family of proteins comprising NOD proteins and the NALPs (neuronal apoptosis inhibitor [like] proteins), also known collectively as NLRs or NACHT-leucine-rich-repeat-containing proteins, have been shown to have a crucial role in the sensing of microbial products, invasive pathogens, and endogenous host proteins. NLRs are cytosolic proteins composed of three diff erent structural domains, a carboxy-terminal ligand-binding domain consisting of leucine-rich repeats, a nucleotide oligomerisation domain, and an aminoterminal eff ector domain consisting of various caspaserecruitment domains (CARD), a pyrin domain, or a baculoviral inhibitor-of-apoptosis repeat. 17, 41 NOD1 and NOD2 have been shown to recognise specifi c bacterial peptidoglycan motifs, 42 and to interact with TLR signalling pathways. 19, 42 NOD2 detects muramyldipeptide, a peptidoglycan fraction of Gram-positive and Gram-negative bacteria, 43 TRAF6=tumour necrosis factor-receptor-associated factor 6. SNP=single nucleotide polymorphism.

γ-D-glutamyl-meso-diaminopimelic acid, a peptidoglycan fraction found in Gram-negative bacteria and in a few Gram-positive bacteria (Listeria and Bacillus spp). 44, 45 On exposure to microbial products, NODs activate transcription factors, including NFκB and the mitogenactivated protein kinase, and induce the cleavage of prointerleukin 1β into active interleukin 1β. [46] [47] [48] The NALP subfamily of NLR proteins interact with several adaptor molecules, including ASC (apoptosis-associated specklike protein containing a CARD domain), caspase 1, and caspase 5, and are essential for the activation of interleukin 1β. 49 The NALP-related protein CARD12 (also known as IPAF) is involved in Salmonella typhimuriuminduced activation of caspase 1. 50 NALP3 is implicated in the detection of ATP, 51 bacterial RNA, 52 and uric acid crystals. 53 However, most NALPs are orphan recognition proteins with no known ligands.

A series of fascinating articles have provided strong evidence implicating the innate immune system in the host defence against viruses. Two intracytoplasmic molecules have been implicated in the detection of viral RNA. Retinoic-acid-inducible protein I (RIG-I) 54 and melanoma diff erentiation-associated gene 5 (MDA5) 55-57 possess a CARD domain and RNA helicase domains that function as sensors of double-stranded RNA. 58 RIG-I and MDA5 signal through the adaptor molecule MAVS (mitochondrial antiviral signalling protein; also known as CARDIF or VISA) 18, 59, 60 and interact with several other signal-transducing molecules, including FADD (tumour necrosis factor receptor superfamily member 6 precursor [TNFRSF6, also know as FAS]-associated death domain protein), RIPK1 (receptor-interacting serine/threonineprotein kinase 1; also known as RIP or RIP1), TBK1 (TRAF family member-associated NF-kappa-B activator [TANK]-binding kinase-1), and IKKE (inhibitor of NFκB kinase subunit epsilon; also known as IKK-i). 40 These molecules are involved in the production of type I interferons (interferons α and β) in response to infection by RNA viruses. Therefore, RIG-I and MDA5 are able to detect single-stranded RNA present in the cytoplasmic compartment and thus not accessible to endosomal TLR3. Interestingly, RIG-I and MDA5 can discriminate between diff erent types of viruses. RIG-I is essential for the production of interferons in response to paramyxoviruses, infl uenza virus, and Japanese encephalitis virus, whereas MDA5 is crucial for detection of picornavirus. 20

A newly described form of innate immunity, termed intrinsic immunity, ensures protection by providing a constitutive, always-on line of defence, relying on intracellular obstacles to hinder the replication of pathogens. 61 This component of the immune system has gained much attention as a cornerstone of the resistance of mammals against several classes of retroelements and retroviruses. 61 Among the best studied proteins are the family of apolipoprotein B mRNA-editing enzyme catalytic polypeptide 3 (APOBEC3) proteins, which interfere with the viral lifecycle by incorporating themselves into viral particles, leading to viral DNA hypermutation on the next round of infection. 62, 63 A series of studies involving infection of human CD4+ T cells and macrophages with wild-type HIV-1 and HIV-1 defi cient in the vif gene showed that the antiviral eff ect of ABC3G (also known as CEM15 or APOBEC3G) is counteracted by Vif. 64 Interestingly, in non-human primates, ABC3G orthologues provide antiviral activity against wild-type HIV-1, 65 but not their cognate simian immunodefi ciency viruses, suggesting that virus permissiveness in diff erent primates results from species-specifi c diff erences within vif. 63 One human variant of ABC3G has been associated with rapid HIV-1 disease progression. 66 The tripartite motif (TRIM) family is a well-conserved family of proteins characterised by a structure comprising a ring-fi nger domain, one or two B-box motifs, and a predicted coiled-coil region. 67 Additionally, most TRIM proteins have additional carboxy-terminal domains. Members of the TRIM protein family are involved in various cellular processes, including cell proliferation, diff erentiation, development, oncogenesis, and apoptosis. 68, 69 Some TRIM proteins exert antiviral properties. TRIM5α is reported to restrict retroviral infection by specifi cally recognising the viral capsid and promoting its premature disassembly. 70 Human TRIM5α has limited effi cacy against HIV-1, whereas some primate TRIM5α orthologues can potently restrict this particular lentivirus. 68, 69 Substantial interspecies sequence diversity characterises TRIM5α and may underlie diff erences in the pattern and breadth of restriction of multiple lentiviruses. Human TRIM5α variants do not modify susceptibility to HIV-1; however, they change susceptibility to other retroviruses, such as N-tropic murine leukaemia virus. 71 Polymorphisms found in TRIM5α might conceivably have been selected in past epidemics by viruses unrelated to HIV-1.

The increasing availability of genomic data allows comparative analyses of genetic sequences involved in innate and intrinsic immunity. This approach, also described as evolutionary genomics, identifi es the role of adaptive forces on protein-encoding genes by determining signs of positive (diversifying) or negative (purifying) selection. For example, positive selection in the human genome indicates shifts in living conditions experienced by modern human populations, such as diff erent habitats, food sources, population densities, and exposure to pathogens. 72 Several families of innate immunity genes have been investigated by use of comparative genomics. Vertebrate TLRs are an example of evolutionary conservation that Review indicate the diffi culty for the microbes to mutate genes that encode MAMPs. 73, 74 The CD209 (DC-SIGN) proteins, a family of C-type lectins that participate in the recognition of various pathogens, display a complex pattern of evolution. Whereas CD209 has been under a strong selective constraint that prevents accumulation of aminoacid changes, CD209L (also known as DC-SIGN2 or DC-SIGNR) exhibits greater variation across human populations. 75 Such variations may be tolerated because of the potentially redundant functional activities of the molecules encoded by these genes. 76 The killer-cell immunoglobulin-like receptor (KIR) genes encode a family of receptors expressed by natural killer cells, which participate in early responses against infected or transformed cells through production of cytokines and direct cytotoxicity. 77, 78 By contrast with TLRs and CD209, only a small proportion of KIR alleles are conserved among primates, showing a rapid species-specifi c diversifi cation of the KIR gene family members and a plasticity of the genomic region that parallels that of the MHC loci. 79 Thus, the evolutionary forces driving the genesis of natural killer receptors and their HLA ligands represent a concerted response to pathogens. Finally, a remarkable success of evolutionary genomics in infectious diseases is the identifi cation of protein regions relevant for host-pathogen interactions in HIV-1 infection. Comparative analysis of the primate antiretroviral cellular defence genes encoding for ABC3G and TRIM5α have revealed the powerful selective pressures emerging from a long-standing battle between retroviruses and their hosts. [80] [81] [82] Singular aminoacids or regions (patches) contain key residues that confer primates the ability to combat HIV-1.

Given that the innate immune system is at the interface between the host and the pathogen, polymorphisms of innate immune genes are very likely to aff ect the host susceptibility to infections. Since the innate immune system senses only a limited number of highly conserved microbial-associated molecular patterns 23 via a limited number of receptors and signalling molecules, as anticipated, several polymorphisms have been found to confer an increased susceptibility to specifi c pathogens (table 2, table 3 , and fi gure 4).

A study from Turkey revealed an association between susceptibility to tuberculosis and an SNP (R753Q) in the TLR2 gene. 94 14 (9·3%) of 151 tuberculosis patients were homozygous for the minor allele compared with two (1·7%) of 116 healthy controls (odds ratio 6·0, 95% CI 1·3-3·9, p=0·009). Of note, R753Q was associated with decreased responsiveness to bacterial lipopeptides. 95 The role of a microsatellite polymorphism (GT repeat) in the exon 2 of TLR2 has been studied in 176 Korean 

patients with tuberculosis and 196 healthy controls. 7 A shorter GT repeat was found more frequently in tuberculosis patients than healthy individuals (49·4% vs 37·7%, p=0·02), and was associated with weaker promoter activities and lower TLR2 expression in CD14positive peripheral blood monocytes. Two nonsynonymous SNPs in the extracellular domain of TLR4 found to be in linkage disequilibrium (D299G and T399I) have been associated with an increased susceptibility to infections caused by Gram-negative bacteria, 103, 104 Brucella spp, 98 respiratory syncytial virus, 88 and P falciparum. 99 Individuals heterozygous for the D299G and T399I SNPs were hyporesponsive to lipopolysaccharide as measured by bronchospastic response after inhalation of endotoxin. 31 Furthermore, airway epithelial cells isolated from heterozygous individuals had defi cient response to lipopolysaccharide, suggesting that D299G and T399I acted in a dominant fashion with respect to the wild-type allele. 31 However, monocytes and whole blood isolated from heterozygous individuals did not show abnormal responses to lipopolysaccharide, 127, 128 suggesting that the eff ects of these mutations may vary between cell types. Although two studies had shown that D299G, 103 or D299G and T399I, 104 were associated with an increased risk of Gram-negative infections or septic shock, three subsequent studies did not fi nd an association in patients with meningococcal sepsis. [120] [121] [122] However, in one study, rare heterozygous missense mutations of TLR4 were linked with the development of meningococcal disease. 120 Unexpectedly, D299G and T399I were associated with decreased rather than increased susceptibility to Legionella pneumophila infection. 32 A stop codon polymorphism in TLR5 (R392stop), shown to abolish the ability of TLR5 to detect bacterial fl agellin, has been associated with increased susceptibility to pneumonia caused by L pneumophila. 123

Several studies have shown associations between mutations in genes encoding several proteins of the TLR signalling pathways (IRAK4, 33,34 IKBKG, [36] [37] [38] and IκBα 35, 129 ) and rare inherited immunodefi ciencies. Complete recessive interleukin-1 receptor-associated kinase 4 (IRAK4) defi ciency is characterised by recurrent infections with pyogenic bacteria at an early age that tend to disappear over time. By contrast, mutations aff ecting the other genes result in X-linked (IKBKG) or autosomaldominant (IκBα) anhydrotic ectodermal dysplasia, which is characterised by increased susceptibility to a broader range of pathogens, such as atypical mycobacteria or Pneumocystis jirovecii and a complex disorder involving impaired development of skin appendages, conical teeth, and hypotrichosis. [36] [37] [38] Taken together, these data clearly show that mutations in the genes encoding TLRs and downstream signal-transducing molecules infl uence innate immune responses and increase susceptibility to many infectious diseases. Similarly, polymorphism of cytokines and cytokine receptor genes, which are key eff ector molecules, have also been associated with altered susceptibility to invasive pathogens. 130

Polymorphisms in genes encoding NLRs have been shown to infl uence susceptibility to infl ammatory diseases. Polymorphisms in NOD2 have been associated with susceptibility to Crohn's disease, 131 

Common limitations of genetic association studies are shown in table 4. Genetic studies done to date often fail on the following factors: (1) to properly account for confounding factors (such as lack of information on ethnicity), and selection and information biases (insuffi cient data on the source population of cases and controls or study endpoints); (2) to present appropriate statistical analyses (such as lack of sample size calculation and correction for multiple testing); and (3) to provide convincing information about biological plausibility. As an example, among fi ve studies that assessed the eff ect of TLR4 polymorphisms on susceptibility to, and outcome of, severe infections, only two included more than 100 patients, 105 two provided information about patient's ethnicity, 105, 106 and only one limited the analysis to a specifi c ethnic group. 106 Comparison of data is often impaired by the fact that apparently similar studies used markedly diff erent controls groups and endpoints. [103] [104] [105] [106] [107] Proving causality is never trivial. Associations are likely to occur when noncausal markers are in linkage disequilibrium with the true disease locus. Although the replication of a fi nding in an independent sample decreases the risk of a falsepositive result, the functional signifi cance of the genetic variant should ultimately be shown in biological studies. However, proving biological plausibility may be diffi cult in view of the limitations of in-vitro studies used as proxy of complex in-vivo biological processes. For example, use of gene-silencing techniques often reduces the biological observation to that of an on/off system, which does not allow the detection of quantitative variations (ie, a dose-response eff ect) of gene expression or discrete functional alterations. With the increasing use of high-throughput genotyping techniques, the number of genetic associations that will be reported in the years to come will most probably exceed our capacity to do proper functional studies and hence to provide convincing evidence for biological plausibility. 139 36 Jain et al, 37 Jain et al, 38 functional studies should therefore focus on genetic polymorphisms that exert a strong eff ect, have been replicated by independent investigators, and have potential diagnostic or therapeutic implications. To limit the importance of positive publication bias, it will be crucial for investigators and journal editors to become less reluctant to publish well-conducted negative studies. 139

In recent years, innate immunogenetic studies of inherited genetic disorders have provided researchers and clinical investigators with crucial information that has improved our understanding of the host defences against microbial pathogens. Table 5 shows examples of the eff ect of recent discoveries in the fi eld of innate immunogenetics with foreseeable applications for the short, middle, and long term in areas such as vaccine development and predictive and preventive medicine. The persistence or emergence of potentially devastating infectious diseases, such as tuberculosis, malaria, HIV/ AIDS, and, most recently, severe acute respiratory syndrome or avian infl uenza, underscore the need to develop new vaccines and therapeutic treatment strategies. A better understanding of microbial genomics and genetics and host innate immunogenetics is likely to provide important information for the development of new vaccines. Vaccine immunogenicity is determined not only by the chemical and physical nature of microbial antigens and adjuvants, but also by the genetic make-up of vaccine recipients. Analyses of polymorphisms of innate immune genes may also help understand why some individuals exhibit suboptimum responses to vaccination. 140 Immuno suppression as a result of myeloablative chemotherapy, solid organ or haematological stem-cell transplantation, or corticosteroid therapy for autoimmune diseases represent other clinical conditions for which immune gene polymorphisms may help to predict the risk of life-threatening infectious complications. The recent discoveries of genes encoding TLRs, NLRs, and the related signal-transducing molecules has markedly improved our understanding of innate immunity. The availability of high-throughput genotyping techniques opens new perspectives to further improve our understanding of the pathogenesis of infectious diseases and for the development of new diagnostic, predictive, and preventive treatment strategies. Clinicians and researchers should be aware of the results and far-reaching implications of recent innate immunogenetic studies that have associated genetic polymorphisms with susceptibility to, or outcome of, infectious diseases. Collecting DNA should now be an integral part of epidemiological or clinical infectious disease studies. National and international consortia should be created to put together large cohort studies to promote and facilitate research in the fi eld.

We declare that we have no confl icts of interest. 

Relevant articles for this Review were identifi ed by searching Medline (1966 to November, 2006 ) by use of the terms "genetics", "single nucleotide polymorphisms", "Toll-like receptors" or "TLRs", "nucleotide-binding oligomerization domain receptors" or "NODS", "immunology", and "innate immunity", and by extracting references from these articles. The Review was limited to articles published in the English language.

Genetic and environmental infl uences on premature death in adult adoptees

Protection aff orded by sickle-cell trait against subtertian malareal infection

The immunogenetics of resistance to malaria

Genetic dissection of immunity to mycobacteria: the human model

A map of human genome sequence variation containing 1·42 million single nucleotide polymorphisms

Microsatellites: simple sequences with complex evolution

The association between microsatellite polymorphisms in intron II of the human Toll-like receptor 2 gene and tuberculosis among Koreans

Accessing genetic variation: genotyping single nucleotide polymorphisms

Single nucleotide polymorphism genotyping: biochemistry, protocol, cost and throughput

The structure of haplotype blocks in the human genome

Haplotype blocks and linkage disequilibrium in the human genome

Introduction and overview. Statistical methods in genetic epidemiology

Reporting, appraising, and integrating data on genotype prevalence and gene-disease associations

Population stratifi cation and spurious allelic association

Counterpoint: bias from population stratifi cation is not a major threat to the validity of conclusions from epidemiological studies of common polymorphisms and cancer

The International HapMap Consortium. The International HapMap Project

Pathogen recognition and innate immunity

Cardif is an adaptor protein in the RIG-I antiviral pathway and is targeted by hepatitis C virus

Signalling pathways and molecular interactions of NOD1 and NOD2

Diff erential roles of MDA5 and RIG-I helicases in the recognition of RNA viruses

Myeloid C-type lectins in innate immunity

Toll-like receptors in the induction of the innate immune response

Inferences, questions and possibilities in Toll-like receptor signalling

Innate immunity: an overview

LPS, TLR4 and infectious disease diversity

CD36 is a sensor of diacylglycerides

Innate immune recognition

Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene

MD-2, a molecule that confers lipopolysaccharide responsiveness on Toll-like receptor 4

Endotoxin contamination in recombinant human heat shock protein 70 (Hsp70) preparation is responsible for the induction of tumor necrosis factor alpha release by murine macrophages

TLR4 mutations are associated with endotoxin hyporesponsiveness in humans

Tolllike receptor 4 polymorphisms are associated with resistance to legionnaires' disease

Distinct mutations in IRAK-4 confer hyporesponsiveness to lipopolysaccharide and interleukin-1 in a patient with recurrent bacterial infections

Pyogenic bacterial infections in humans with IRAK-4 defi ciency

A hypermorphic IkappaBalpha mutation is associated with autosomal dominant anhidrotic ectodermal dysplasia and T cell immunodefi ciency

A novel X-linked disorder of immune defi ciency and hypohidrotic ectodermal dysplasia is allelic to incontinentia pigmenti and due to mutations in IKKgamma (NEMO)

Specifi c missense mutations in NEMO result in hyper-IgM syndrome with hypohydrotic ectodermal dysplasia

Specifi c NEMO mutations impair CD40-mediated c-Rel activation and B cell terminal diff erentiation

X-linked anhidrotic ectodermal dysplasia with immunodefi ciency is caused by impaired NF-kappaB signaling

NOD-LRR proteins: role in host-microbial interactions and infl ammatory disease

The role of Toll-like receptors and Nod proteins in bacterial infection

Host recognition of bacterial muramyl dipeptide mediated through NOD2. Implications for Crohn's disease

Nod1 detects a unique muropeptide from Gram-negative bacterial peptidoglycan

An essential role for NOD1 in host recognition of bacterial peptidoglycan containing diaminopimelic acid

RICK/Rip2/ CARDIAK mediates signalling for receptors of the innate and adaptive immune systems

Role of nod2 in the response of macrophages to Toll-like receptor agonists

Heterotypic interactions among NACHT domains: implications for regulation of innate immune responses

NLRs join TLRs as innate sensors of pathogens

Diff erential activation of the infl ammasome by caspase-1 adaptors ASC and Ipaf

Cryopyrin activates the infl ammasome in response to toxins and ATP

Bacterial RNA and small antiviral compounds activate caspase-1 through cryopyrin/Nalp3

Goutassociated uric acid crystals activate the NALP3 infl ammasome

The RNA helicase RIG-I has an essential function in double-stranded RNA-induced innate antiviral responses

The V proteins of paramyxoviruses bind the IFN-inducible RNA helicase, MDA-5, and inhibit its activation of the IFN-beta promoter

Expression analysis and genomic characterization of human melanoma diff erentiation associated gene-5, MDA-5: a novel type I interferon-responsive apoptosis-inducing gene

MDA-5: an interferon-inducible putative RNA helicase with double-stranded RNA-dependent ATPase activity and melanoma growth-suppressive properties

Shared and unique functions of the DExD/H-box helicases RIG-I, MDA5, and LGP2 in antiviral innate immunity

VISA is an adapter protein required for virus-triggered IFN-beta signaling

IPS-1, an adaptor triggering RIG-I-and MDA5-mediated type I interferon induction

Lentiviral vectors and antiretroviral intrinsic immunity

Role and mechanism of action of the APOBEC3 family of antiretroviral resistance factors

Innate cellular defenses of APOBEC3 cytidine deaminases and viral counter-defenses

Isolation of a human gene that inhibits HIV-1 infection and is suppressed by the viral Vif protein

A single amino acid determinant governs the species-specifi c sensitivity of APOBEC3G to Vif action

APOBEC3G genetic variants and their infl uence on the progression to AIDS

The tripartite motif family identifi es cell compartments

TRIM family proteins: retroviral restriction and antiviral defence

Control of viral infectivity by tripartite motif proteins

Specifi c recognition and accelerated uncoating of retroviral capsids by the TRIM5alpha restriction factor

Highfrequency persistence of an impaired allele of the retroviral defense gene TRIM5alpha in humans

A map of recent positive selection in the human genome

Toll-like receptor 5 recognizes a conserved site on fl agellin required for protofi lament formation and bacterial motility

The evolution of vertebrate Toll-like receptors

Novel member of the CD209 (DC-SIGN) gene family in primates

The heritage of pathogen pressures and ancient demography in the human innate-immunity CD209/CD209L region

MHC class I molecules and KIRs in human history, health and survival

The impact of variation at the KIR gene cluster on human disease

Single haplotype analysis demonstrates rapid evolution of the killer immunoglobulin-like receptor (KIR) loci in primates

Ancient adaptive evolution of the primate antiviral DNA-editing enzyme APOBEC3G

Positive selection of primate TRIM5alpha identifi es a critical species-specifi c retroviral restriction domain

Patterns of evolution of host proteins involved in retroviral pathogenesis

Polymorphisms in CD14, mannose-binding lectin, and Toll-like receptor-2 are associated with increased prevalence of infection in critically ill adults

CD14 and TLR4 gene polymorphisms in adult periodontitis

Combined carriership of TLR9 −1237C and CD14 −260T alleles enhances the risk of developing chronic relapsing pouchitis

CD14 promoter polymorphism −159C>T is associated with susceptibility to chronic Chlamydia pneumoniae infection in peripheral blood monocytes

The CD14 functional gene polymorphism −260 C>T is not involved in either the susceptibility to Chlamydia trachomatis infection or the development of tubal pathology

Association between common Toll-like receptor 4 mutations and severe respiratory syncytial virus disease

Genetic polymorphisms of CD14, Toll-like receptor 4, and caspase-recruitment domain 15 are not associated with necrotizing enterocolitis in very low birth weight infants

Gene variants of the bactericidal/permeability increasing protein and lipopolysaccharide binding protein in sepsis patients: gender-specifi c genetic predisposition to sepsis

TLR1 and TLR6 polymorphisms are associated with susceptibility to invasive aspergillosis after allogeneic stem cell transplantation

Polymorphisms in Toll-like receptor 2 are associated with increased viral shedding and lesional rate in patients with genital HSV-2 infection

A microsatellite polymorphism in intron 2 of human Toll-like receptor 2 gene: functional implications and racial diff erences

The Arg753GLn polymorphism of the human Toll-like receptor 2 gene in tuberculosis disease

A novel polymorphism in the Toll-like receptor 2 gene and its potential association with staphylococcal infection

Lack of association between Toll-like receptor 2 polymorphisms and susceptibility to severe disease caused by Staphylococcus aureus

Heterozygous Arg753Gln polymorphism of human TLR-2 impairs immune activation by Borrelia burgdorferi and protects from late stage Lyme disease

TLR4 polymorphism in Iranian patients with brucellosis

Toll-like receptor (TLR) polymorphisms in African children: common TLR-4 variants predispose to severe malaria

Common polymorphisms of Toll-like receptors 4 and 9 are associated with the clinical manifestation of malaria during pregnancy

The Toll-like receptor 4 (Asp299Gly) polymorphism is a risk factor for gram-negative and haematogenous osteomyelitis

Role of TLR4 receptor polymorphisms in Boutonneuse fever

Human Toll-like receptor 4 mutations but not CD14 polymorphisms are associated with an increased risk of Gram-negative infections

Relevance of mutations in the TLR4 receptor in patients with Gram-negative septic shock

TLR4 and TNF-alpha polymorphisms are associated with an increased risk for severe sepsis following burn injury

Eff ects of functional Toll-like receptor-4 mutations on the immune response to human and experimental sepsis

Polymorphisms in Toll-like receptor 4 and the systemic infl ammatory response syndrome

Functional gene polymorphisms in aggressive and chronic periodontitis

Gingival epithelial cells heterozygous for Toll-like receptor 4 polymorphisms Asp299Gly and Thr399ile are hypo-responsive to Porphyromonas gingivalis

Gene polymorphisms in pro-infl ammatory cytokines are associated with systemic infl ammation in patients with severe periodontal infections

Tolllike receptor (TLR) 2 and 4 mutations in periodontal disease

Toll-like receptor 4

Thr399Ile polymorphisms are a risk factor for Candida bloodstream infection

Candida-specifi c interferon-gamma defi ciency and Toll-like receptor polymorphisms in patients with chronic mucocutaneous candidiasis

Role of the Toll-like receptor 4 Asp299Gly polymorphism in susceptibility to Candida albicans infection

Relationship between a Toll-like receptor-4 gene polymorphism, bacterial vaginosis-related fl ora and vaginal cytokine responses in pregnant women

Diff erences in infl ammatory cytokine and Toll-like receptor genes and bacterial vaginosis in pregnancy

The Toll-like receptor 4 Asp299Gly variant: no infl uence on LPS responsiveness or susceptibility to pulmonary tuberculosis in The Gambia

Innate immunity genes infl uence the severity of acute appendicitis

The role that the functional Asp299Gly polymorphism in the Toll-like receptor-4 gene plays in susceptibility to Chlamydia trachomatis-associated tubal infertility

Assay of locus-specifi c genetic load implicates rare Toll-like receptor 4 mutations in meningococcal susceptibility

Variation in Toll-like receptor 4 and susceptibility to group A meningococcal meningitis in Gambian children

A functional polymorphism of Toll-like receptor 4 is not associated with likelihood or severity of meningococcal disease

A common dominant TLR5 stop codon polymorphism abolishes fl agellin signaling and is associated with susceptibility to legionnaires' disease

Host susceptibility and clinical outcomes in Toll-like receptor 5-defi cient patients with typhoid fever in Vietnam

Polymorphisms in Toll-like receptor 9 infl uence the clinical course of HIV-1 infection

Innate immune receptor genetic polymorphisms in pouchitis: is CARD15 a susceptibility factor?

Heterozygous Tolllike receptor 4 polymorphism does not infl uence lipopolysaccharide-induced cytokine release in human whole blood

Monocytes heterozygous for the Asp299Gly and Thr399Ile mutations in the Toll-like receptor 4 gene show no defi cit in lipopolysaccharide signalling

Inherited disorders of human Toll-like receptor signaling: immunological implications

Cytokine promoter polymorphisms in severe sepsis

Host recognition of bacterial muramyl dipeptide mediated through NOD2. Implications for Crohn's disease

Nod2 is a general sensor of peptidoglycan through muramyl dipeptide (MDP) detection

CARD15 mutations in Blau syndrome

Early-onset sarcoidosis and CARD15 mutations with constitutive nuclear factor-kappaB activation: common genetic etiology with Blau syndrome

Both donor and recipient NOD2/CARD15 mutations associate with transplant-related mortality and GvHD following allogeneic stem cell transplantation

Mutation of a new gene encoding a putative pyrin-like protein causes familial cold autoinfl ammatory syndrome and Muckle-Wells syndrome

Chronic infantile neurological cutaneous and articular syndrome is caused by mutations in CIAS1, a gene highly expressed in polymorphonuclear cells and chondrocytes

The bare lymphocyte syndrome and the regulation of MHC expression

Host genetic determinants in hepatitis C virus infection

Lipopolysaccharides from distinct pathogens induce diff erent classes of immune responses in vivo

This work was supported by grants from the Swiss Foundation for Medical and Biological Grants (1121 to PYB), the Swiss National Science Foundation (81LA-65462 to PYB, 32-49129.96 and 3100-066972.01 to TC), the Bristol-Myers Squibb Foundation (to TC), the Leenaards Foundation (to PYB and TC), and the Santos-Suarez Foundation for Medical Research (to TC and AT).
