Relevance of Single-Nucleotide Polymorphisms in Human TLR Genes to Infectious and Inﬂammatory Diseases and Cancer

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REVIEW Relevance of single-nucleotide polymorphisms in human TLR genes to infectious and inﬂammatory diseases and cancer

A Trejo-de la O1,3, P Herna´ndez-Sance´n2,3 and C Maldonado-Bernal1

Innate and adaptive immune responses in humans have evolved as protective mechanisms against infectious microorganisms. Toll-like receptors (TLRs) have an important role in the recognition of invading microorganisms. TLRs are the first receptors to detect potential pathogens and to initiate the immune response, and they form the crucial link between the innate and adaptive immune responses. TLRs also have an important role in the pathophysiology of infectious and inflammatory diseases. Increasing data suggest that the ability of certain individuals to respond properly to TLR ligands may be impaired by single-nucleotide polymorphisms (SNPs) within TLR genes, resulting in an altered susceptibility to infectious or inflammatory disease that might contribute to the pathogenesis of complex diseases such as cancer. The associations between diseases and SNPs are in the early stage of discovery. Important clinical insights are emerging, and these polymorphisms provide new understanding of common diseases. This review summarizes and discusses the studies that shed light on the relevance of these polymorphisms in human infectious and inflammatory diseases and cancer.

Genes and Immunity (2014) 15, 199–209; doi:10.1038/gene.2014.10; published online 13 March 2014

INTRODUCTION complex1,9 and is also involved in the signaling response to other The family of Toll-like receptors (TLRs) has been identified as key exogenous stimuli. TLR5 binds flagellin, a bacterial protein 1,10 host molecules in the induction of the innate immune response to involved in motility, and TLR9 recognizes CpG, a repetitive microbial ligands.1 TLRs constitute a family of transmembrane sequence of unmethylated nucleic acids (guanine and 1,11 proteins, each of which recognizes a different spectrum of cytosine). The significance of individual TLRs in protection pathogen-associated molecular patterns that are unique to against infectious disease has been demonstrated clearly. microorganisms and are highly conserved across several species However, recent reports suggest that this family of receptors of microorganism. This ability of TLRs to recognize a broad have a substantial role in both the response to infectious diseases spectrum of microbial molecules enables the host to detect the and other types of pathology in which inflammation has a crucial 12 13 14 presence of pathogens rapidly before more widespread infection role, such as atherogenesis, diabetes and even cancer. TLRs 15 occurs.1 are also very important to homeostasis of the organism. To date, 11 related human TLRs (TLR1–11) have been identified The ability to respond properly to TLR ligands may be impaired and characterized,2–5 all of which share structural features by single-nucleotide polymorphisms (SNPs) within TLR genes, and (Figure 1). For example, the extracellular domain of TLRs is many reports have suggested that genetic variations in these characterized by leucine-rich repeats, which are associated with genes are related to susceptibility to diseases. The first report to TLR–ligand interactions, and an intracellular domain, which demonstrate a mutation in a TLR gene that had a physiological exhibits the Toll/interleukin-1 (IL-1) receptor domain. The Toll/IL-1 implication was the mutation detected in the mouse strain C3H/ 16,17 receptor binds to other Toll/IL-1 receptor domains of intracellular HeJ, which has a proline-to-histidine substitution at amino adaptor molecules and mediates a signaling cascade that includes acid (aa) 712 in the TLR4 protein. This variation causes an LPS- a complex series of intermediates that lead to deactivation of hyporesponsive phenotype, which makes this strain of mouse 16,17 nuclear factor-kB (NF-kB). The end result of TLR signaling is more susceptible to Gram-negative bacterial infections. upregulation of proinflammatory mediators, cytokines and chemo- Several SNPs have been reported recently in the human genes kines, and induction of the adaptive immune response.4,5 for TLRs (TLR1–10), and reports have also suggested that these All TLRs can initiate inflammation, and differences in the SNPs have a functional impact on and medical relevance to a inflammatory response between different TLRs have been wide variety of human diseases (Figure 2 and Table 1). It is documented. TLRs vary by their ligand specificity, which is important to mention that, to date, there are no reports regarding determined by the extracellular portion of the receptor. TLR2 polymorphisms in TLR11. recognizes a variety of microbial components, including lipoprotein from pathogens, lipoteichoic acid and peptidoglycan.1,6 TLR3 is the receptor for double-stranded RNA,1,7 and TLR7 and TLR1 POLYMORPHISMS TLR8 are receptors for single-stranded RNA.1,8 TLR4 is the signal- TLR1 is mapped to chromosome 4p14 and is encoded by one transducing element of the lipopolysaccharide (LPS) receptor exon. The protein recognizes triacyl lipopeptides and soluble

1Laboratorio de Investigacioń en Inmunologıá y Proteo´ mica, Hospital Infantil de Me´xico Federico Go´mez, Me´xico City, Me´xico and 2Departamento de Asesorıá Molecular, Roche Diagnostics Me´xico, Me´xico City, Me´xico. Correspondence: Dr C Maldonado-Bernal, Laboratorio de Investigacioń en Inmunologıá, Hospital Infantil de Me´xico Federico Go´mez, Dr Ma´rquez No. 162 Col. Doctores, C.P, Me´xico City 06720, Me´xico. E-mail: [email protected] 3These authors contributed equally to this work. Received 9 September 2013; revised 30 January 2014; accepted 3 February 2014; published online 13 March 2014 TLR polymorphisms and human diseases A Trejo-de la O et al 200 Triacyl Diacyl LPS lipopetides lipopeptides Mannans Flagellin Profillin TLR1 TLR5 TLR2 TLR6 TLR2 TLR4 TLR11

NF-B p38 MAPK JNK IRFs Transcription factors TLR8 TLR7 TLR9 TLR3

dsRNA ssRNA DNA

Inflammatory cytokines Immune regulation Endosome Proliferation Survival

Figure 1. TLRs and their main ligands. dsRNA, double-stranded RNA; LPS, lipopolysaccharide; ssRNA, single-stranded RNA.

factors of Neisseria meningitides, and is functionally associated with mycobacteria; TLR1 has an important role in discriminating TLR2.18 In 2006, Sun et al.19 reported the presence of 11 SNPs in between different microbial components. Previous reports have this gene in the Swedish population. Three of these SNPs were shown that SNPs in this gene modify the activation of its protein, more frequent in patients with prostate cancer than in controls; implying that the genetic variation in the TLR1 gene might affect two of the SNPs were located in the promoter region, À 7202A/G the innate immune response and clinical susceptibility to a wide to À 6399C/T; and the third was located in the intron of the gene spectrum of pathogens. These SNPs may also confer a higher risk ( À 833C/T) (Table 1). The À 6399T allele had a synergistic for prostate cancer, but may confer protection against GC and effect when interacted with 7987G/C and 652T/C variants in leprosy.19,21,22 However, because the biological relevance of these IRAK4 (IL-1 receptor-associated kinase 4) and increased the risk of SNPs has yet to be proven, their clinical relevance requires developing prostate cancer. We know that the chronic or confirmation. recurrent inflammation is a causal factor in several human malignancies, including prostate cancer. Genetic predisposition is also a strong risk factor in the development of prostate TLR2 POLYMORPHISMS cancer. In particular, the TLR6-1-10 gene cluster is involved in TLR2 recognizes a variety of microbial components and was the prostate cancer development, and IRAK1 and 4 are critical first human TLR involved in host defense to be described. The components in the TLR signaling pathway. On the other TLR2 gene has two exons located in 4q32. hand, SNP À 7202A/G alters the putative core binding site of In 2000, Lorenz et al.24 described the substitution of a guanine the proto-oncogene PU.1. for an adenine at 2258 bp, a change that results in an arginine-to- Another SNP in the codifying region, T1805G, which changes an glutamine substitution at residue 753. When this SNP is isoleucine to a serine at aa 602 (I602S) in the extracellular region transfected into human embryonic kidney 293 (HEK293) cells, (Figure 3), diminishes the activation of NF-kB, and the synthesis of the minor allele affects the ability of the cells to respond to IL-6 diminished the inflammatory response.20 This SNP (I602S) bacterial peptides in vitro, and the cells are not activated in affects the function and cell surface expression of TLR1 and its response to these peptides. Mononuclear cells of individuals ability to transport TLR1 to the cell surface; this SNP seems to be a heterozygous for the SNP Arg753Gln exhibit less induction of protective factor against leprosy.21 About homozygous 602S allele, tumor necrosis factor-a (TNF-a) and interferon-g upon stimulation recently Yang et al.22 found that it is associated with a reduced risk with Borrelia lysate compared with individuals not exhibiting this of Helicobacter pylori-induced gastric diseases, such as gastric variation,25 although this seems to protect from the development cancer (GC) and peptic ulcer, probably via diminished T helper of late-stage Lyme disease rather than being a risk factor for the type 1 responses. disease. This 753Q allele is associated with a higher risk of Omueti et al.23 studied three SNPs found in the extracellular developing tuberculosis.26 In children, the SNP is a predisposing region of the protein: S248N, H305L and P315L. All three exhibited factor for urinary tract infection by Gram-positive pathogens27 and attenuated responses to synthetic lipopeptides and microbial increases the risk of recurrent febrile infections28 and rheumatic agonists. fever.29 In adults, this SNP increases the risk of developing familial Different reports confirm the importance of TLR1 along with Mediterranean fever30 and may affect the severity of this disease. TLR2 and TLR6 in the recognition of Gram-positive bacteria and This SNP is also associated with non-infectious diseases such as

Genes and Immunity (2014) 199 – 209 & 2014 Macmillan Publishers Limited TLR polymorphisms and human diseases A Trejo-de la O et al 201 Table 1. TLRs alleles associated with diseases

Allele SNP ID number Disease Population Reference

TLR1 À 7202G Rs377590742 Prostate cancer Swedish 19 À 6399T Rs574356 92 À 833T Rs5743604 239C Rs5743611 Pancolitis in UC American 93 602S Rs5743618 Leprosy German 21 Gastric cancer German 22

TLR2 2258A Rs5743708 Staphylococcal infection French 24 Tuberculosis Turkish 26 Coronary restenosis German 31 Acute rheumatic fever Turkish 29 Familial Mediterranean fever Turkish 34 Cytomegalovirus disease after liver transplantation American 28 Recurrent bacterial infections Turkish children 35 Preterm birth Netherlands 33 Liver allograft failure American 27 Urinary tract infection Turkish children 2029T Rs121917864 Lepromatous leprosy Korean 36 Tuberculosis Tunisian 39 597C Rs3804099 Tuberculosis Vietnamese 48 À 16934A Rs4696480 Asthma European 49 Preterm birth Netherlands 35 597C Rs3804099 Hepatocellular carcinoma Chinese 50 1350C Rs3804100 À 196 to Breast cancer Greece 51 À 174del Gastric cancer Chinese 52

TLR3 293248A Rs3775290 Stevens–Johnson syndrome Japanese 54 299698T Rs3775296 1234T Rs3775291 Colorectal carcinoma German 56 1377T Rs3775290 Cervical cancer Indian 57 2593C Type 1 diabetes African 55 2642A 2690G

TLR4 1196T Rs4986791 Hyporesponsiveness to inhaled LPS American 60 64 Septic shock Premature birth Finnish 896G Rs4986790 Hyporesponsiveness to inhaled LPS American 60 Septic shock American 64 Premature birth Finnish-British 65 SIRS Israelite 62 RSV bronchiolitis Belgain 63 Crohn’s disease and UC 67 Asthma Swedish children 66 Sepsis American 67 Cronh’s disease Caucasian Inﬂammatory bowel disease Meta-analysis

TLR5 1174T Rs5744168 Legionnaires’ disease German 87 1775G Rs2072493 1846C Rs5744174 Gastric cancer Chinese 52

TLR6 À 1401A Rs5743795 Prostate cancer Swedish 19 92

TLR7 À 120G Rs2302267 Liver inﬂammation and ﬁbrosis Caucasian 96 þ 1817T Viral infection Chinese 95

TLR8 À 129C Viral infection Chinese 95

& 2014 Macmillan Publishers Limited Genes and Immunity (2014) 199 – 209 TLR polymorphisms and human diseases A Trejo-de la O et al 202 Table 1. (Continued)

Allele SNP ID number Disease Population Reference

TLR9 À 1486C Rs187084 Low birth weight in malaria African 105 þ 1635G Rs352140 Rapid progression of HIV-1 Swiss 106 þ 1174A Rs187086 þ 1174G Rs352139 Systemic lupus erythematosus Chinese 100 þ 1031A Not known Asthma European 97 þ 2322G Rs4129009 Asthma American 97 À 1486C Rs187084 Endometrial cancer Australia 107 À 1237C Rs5743836 2848A Rs352140 Advanced cervical cancer stages Indian 57 1635A 720C Prostate cancer Swedish 19 1104C 92

TLR10 þ 1031A Not known Asthma European American 110 þ 2322G Rs4129009 111 C4G Rs7653908 Crohn’s disease New Zealand 112 A4G Rs7658893 C4T Rs11466653 Papillary thyroid cancer Korean 114

TLR11 To date there are no related studies with TLR11 polymorphisms and their association with some pathology. Abbreviations: HCV, hepatitis C virus; HIV, human immunodeﬁciency virus; LPS, lipopolysaccharide; RSV, respiratory syncytial virus; SIRS, systemic inﬂammatory response syndrome; UC, ulcerative colitis.

coronary restenosis31 and type 1 diabetes,32 and confers a higher Tuberculosis meningitis. Another two SNPs, Pro632His and risk of allograft failure and mortality.33 People carrying the 753Q À 16 934, are associated with meningitis and asthma in allele have an increased susceptibility to cytomegalovirus children.49 The T597C polymorphism and T1350C located in the infection after liver transplantation.34 When this polymorphism is exon region are also significantly associated with a decreased risk found with another SNP, À 16934T/A, the risk of developing of developing hepatocellular carcinoma.50 A 22-bp nucleotide preterm birth is increased greatly.35 deletion at position À 196 to À 174 of the 50-untranslated Another SNP was reported by Kang and Chae36 in the Korean region showed that the ins/del and del/del genotypes and population in 2001. The C–T substitution at nucleotide 2029 bp the del allele are associated with breast cancer51 and also with results in replacement of a conserved arginine residue with the risk of developing intestinal-type GC but not diffuse-type tryptophan at aa 677. This polymorphism was associated with a GC.52 TLR2 is critical to the recognition of peptidoglycan and major risk of developing leprosy.36 People with the 677W minor lipoprotein and several atypical LPSs. TLR2 has a crucial role in allele have significantly lower serum levels of IL-12. Monocytes the defense against infectious disease, in autoimmune diseases from patients with this allele are less responsive to Mycobacterium and in the regulation of the immune system. Dysfunction in this leprae37 and produce significantly less IL-2, IL-12, interferon-g and receptor can have several consequences, and there is evidence TNF-a.38 In HEK293 cells transfected with the 677W allele, the NF- that SNPs in this gene modify the response of TLR2. Although kB activation in response to M. leprae is abolished. This contradictory reports have been published about the clinical polymorphism is also associated with susceptibility to relevance, it is clear that SNPs in TLR2 have important biological tuberculosis in the Tunisian population.39 effects in many inflammatory and infectious diseases and cancer, Transfection of both polymorphisms, R753Q and R677W, into perhaps due to the change in the activation of transcription Chinese hamster ovary cells disturbs the activation of these cells factors such as NF-kB and secretion of important inflammatory by well-established TLR2 ligands.40 However, neither of these mediators. polymorphisms is related to susceptibility to lymphatic filariasis,41 periodontal disease,42 rheumatic heart disease,43 bacteremia44 or other noninfectious diseases such as systemic lupus TLR3 POLYMORPHISMS erythematosus.45 Von Aulock et al.46 reported, in 2004, that TLR3 recognizes double-stranded RNA associated with viral peripheral blood mononuclear cells from people heterozygous for infections and induces the production of interferon type 1. TLR3 R753Q responded to lipoteichoic acid from Bacillus subtilis by maps to 4q35 and has five exons (Figure 4), although the producing similar quantities of IL-10, TNF-a and IL-8 as those protein is encoded by exons 2–5. Recent reports have shown that produced by cells from people with the ancestral gene. However, there are two polymorphisms in the extracellular region of the other polymorphisms beside the two mentioned above were protein: one caused by a substitution of adenine for thymine at found to be functionally relevant in transfected HEK293 cells;47 952 bp (N284I) and the other by a substitution of thymine for these include Thr411Ile, Arg447stop, Tyr715stop and Tyr715Lys. In cytosine at 1335 bp (L412F).53 Both modify the response of this addition, the mutation Arg447stop leads to a premature stop TLR and reduce the activity of NF-kB. The expression of the codon in the extracellular portion of TLR2, which is relevant since extracellular protein is also reduced by both SNPs, although TLR2-specific stimulation of whole blood from heterozygote no modifications of the expression of mRNA have been donors of this mutation resulted in a reduced secretion of reported.53 Other polymorphisms in the TLR3 gene are related proinflammatory cytokines.47 to susceptibility to certain diseases. For example, the Thuong et al.48 report a strong association between the TLR2 polymorphisms 293248 A/A and 299698 T/T are strongly SNP T597C located in the exon region with the development of associated with Stevens–Johnson syndrome.54 However, genetic

Genes and Immunity (2014) 199 – 209 & 2014 Macmillan Publishers Limited TLR polymorphisms and human diseases A Trejo-de la O et al 203

-7202G -6399T 2258A -833T -16934A 1174T 239C 597C 896G 1775G 602S 2029T 1196T 1846C -1401A

Triacyl lipopetides Triacyl LPS Diacyl lipopetides Mannans Flagellin lipopetides TLR1 TLR2 TLR4 TLR5 TLR6

Pancolitis in UC Septic shock Legionnaire’s Stapylococal Prostate Leprosy Premature birth disease infection cancer Asthma Prostate cancer Tuberculosis Crohn disease Gastric cancer Gastric cancer Coronary UC restenosis Asthma Pretermbirth Gastritis Metaplasia Hepatocellular carcinoma Breast cancer Gastric cancer TLR8 TLR7 Viral infection Inflammation -120T>G and Fibrosis

-1237C 293248A +1174G ssRNA 299698T +1635G 1234T 2848A 1377T -1486C 720C DNA Progression of HIV Systemic lupus erythematosus Asthma Stevens-Johnson dsRNA Endometrial cancer syndrome Prostate cancer Colorectal carcinoma Cervical cancer

Figure 2. Genetic association between TLRs and susceptibility to infections, inflammatory disease and cancer. dsRNA, double-stranded RNA; ssRNA, single-stranded RNA; UC, ulcerative colitis. variations of this gene may also predispose to noninfectious present at low frequencies. In 2001, Arbour et al.60 described two pathologies, for example, the 2593C/T, 2642C/A and 2690A/G of these polymorphic variations: A896G substitution that results in substitutions are related to the risk of developing type 1 diabetes the replacement of a conserved aspartic acid residue with a in African populations.55 Castro et al.56 reported that a missense glycine at aa 299 and C/T substitution at 1196 bp that replaces a polymorphism (Leu412Phe, rs3775291) in the TLR3 gene is an threonine with an isoleucine at aa 399. Both SNPs have been independent prognostic marker for disease-specific survival in shown to produce blunted responses to inhaled endotoxin in vitro patients with stage II colorectal carcinoma. The strong effect of and in vivo.60 The THP-1 cell line transfected with the minor allele this SNP on colorectal carcinoma survival was confirmed in of these polymorphisms displays a lower response to LPS and patients who did not receive adjuvant therapy. In 2011, Pandey markedly reduced expression of the protein on the surface of et al.57 observed that TLR3 (c.1377C/T) gene polymorphism epithelial cells.60 These SNPs also affect the synthesis of CT þ TT is marginally associated with cervical cancer inflammatory mediators such as fibrinogen61 and diminish the susceptibility in women from North India. TLR3 is a potent synthesis of IL-1b and other inflammatory cytokines.60–62 After the inducer of interferon type 1, which has anti-viral and first report of these two polymorphisms, a series of studies immunostimulatory activities. Further studies of the frequency investigated the associations between these SNPs on the and biological consequences of these SNPs, and their roles in incidence and course of other diseases. Both SNPs are related to other diseases, are needed. However, we can speculate that the risk of developing Gram-negative septic shock63,64 and functional polymorphisms of TLR3 can predispose individuals to premature birth.65 Individuals with the TLR4 299Gly/399Ile allele increased susceptibility to viral infections and cancer associated have a higher risk of developing bowel and Crohn’s diseases,66–68 with viral infections and also to diseases associated with and chronic osteomyelitis.69 They are also linked to the risk of inflammatory responses. developing urinary tract infection in children70 and also increase the risk of developing bronchiolitis caused by respiratory syncytial virus in infants.71 These alleles also confer a greater risk of TLR4 POLYMORPHISMS developing active tuberculosis after transplantation. The D299G The TLR4 gene has four exons that map to 9q32–33, and TLR4 is genotype may have a protective effect against the development the receptor for LPS, taxol, heat-shock proteins, fibrinogen and of gastric mucosa-associated lymphoid tissue lymphoma.72 other ligands. Smirnova et al.58,59 reported that TLR4 is a However, it seems not to be related to recurrence of gastric polymorphic protein with 12 single amino-acid changes that are ulcer in H. pylori-infected patients73 and gastric precancerous

& 2014 Macmillan Publishers Limited Genes and Immunity (2014) 199 – 209 TLR polymorphisms and human diseases A Trejo-de la O et al 204 N248S H305L I602S hTLR1

LRR M TIR 743 bp 914 bp 1805 bp G/A A/T T/G T411I R447stop R677W R753Q Y715stop Y715K hTLR2

LRR M TIR

1452 bp 1559 bp 2029 bp 2258 bp 2341 bp 2363/65 bp C/T C/T C/T G/A T/G TAT/AAA

D299GD299G T399I T399I hTLR4

LRR M TIR 896 bp 896 bp 1196 bp 1196 bp A/G A/G C/T C/T

R392 stop N592S hTLR5

LRR M TIR 1174 bp 1775 bp C/T A/G S249P V427A hTLR6

LRR M TIR 744 bp 1279 bp C/T C/T Figure 3. Schematic representation of the different SNPs in TLR genes 1, 2, 4, 5 and 6.

lesions in a Venezuelan population.74 We found that TLR4/D299G/ incidence of diabetic neuropathy,77 and SNP carriers have a T399I polymorphisms in Mexican patients were more frequent in lower rate of acute allograft rejection.78 The D299G SNP from duodenal ulcer and showed a trend in GC, and when it is TLR4 is also associated with a reduced risk of developing compared with non-atrophic gastritis and patients with TLR4 atherosclerosis and stenosis, and is also associated with polymorphisms, it expressed significantly lower levels of IL-1b, increased carotid artery compliance in young adults. The IL-6, IL-8 and GRO-a, and higher levels of TNF-a, IL-10, MCP-1 cosegregating 299Gly/399Ile variant confers a higher risk of and MIP-1a. SNPs in TLR4 gene had an association with severe developing myocardial infarction. However, these SNPs protect H. pylori-associated disease and with modified pattern of against the development of late-onset Alzheimer’s disease.79 Even inflammatory cytokines and chemokines in the gastric mucosa. though there is much evidence that these polymorphisms These results suggest that TLR4 SNPs contribute importantly to influence the susceptibility and progression of many diseases, the clinical outcome of H. pyori infection.75 other studies have failed to find associations between these Functional and genetic studies of TLR4 SNPs show a polymorphisms and various pathologies such as Candida possible association with susceptibility to Gram-negative bacterial bloodstream infection,80 acute pancreatitis,81 necrotizing infection and other bacterial and viral infections. However, is enterocolitis in newborns82 and ankylosing spondylitis.83 By necessary to carry out studies with higher sample size before contrast, newly identified polymorphisms in this gene have the role of TLR4 SNPs in susceptibility to infection will be clinical relevance. Eight SNPs for the TLR4 gene are associated understood. with a significantly lower risk of developing prostate cancer.84 The important role of TLR4 SNPs in inflammation-related Duan et al.85 reported another SNP, the G11367C polymorphism, diseases such as the chronic course of sarcoidosis has been which when transfected into HEK293 cells attenuated the reported.76 Although it seems that the minor alleles of these SNPs activation of these cells. Carriers of the 11367C variant allele in TLR mutations predispose to several pathologies, recent reports have lower plasma TNF-a level and TLR4 expression on the surface describing a protective role for these SNPs have also been of peripheral leukocytes.85 More recently, Apetoh et al.86 found published. For example, people with type 2 diabetes carrying the that breast cancer patients harboring the Asp299Gly minor allele of these polymorphisms have a substantially lower polymorphism of TLR4 relapse earlier under anthracycline-based

Genes and Immunity (2014) 199 – 209 & 2014 Macmillan Publishers Limited TLR polymorphisms and human diseases A Trejo-de la O et al 205 2593 bp 2690 bp hTLR3 C/T A/G

Exon 1 Exon 2 Exon 3 Exon 4 Exon 5

5’ 299698 bp 293248 bp 3’ A/A A/A 1377 bp 2642 bp C/T C/A hTLR7 Q11L

Exon1 Exon2 Exon3

5’ -1206 bp +1817 bp 3’ T/G G/T

hTLR8 M1V

Exon1 Exon2 Promotor Region

5’ -129 bp 3’ G/C hTLR9

Exon 1 Exon 2 -1237 bp -1237 bp +1635 bp +2848 bp T/C T/C A/G A/G 5’ 3’ Figure 4. Graphical representation of the different SNPs in TLR genes 3, 7, 8 and 9. chemotherapy compared with patients carrying the ancestral modify the responses of the protein and that this could TLR4, which determine the efficacy of anticancer therapies. compromise the immune response; however, the clinical relevance of these SNPs require further study. TLR5 POLYMORPHISMS TLR5 is the receptor of bacterial flagellin and mediates recognition TLR6 POLYMORPHISMS of many medically important pathogens. TLR5 maps to 1q33.3. The TLR6 cooperates with TLR2 to recognize diacyl lipopeptides, gene has five exons. In 2003, Hawn et al.87 reported four SNPs in lipoteichoic acid and zymosan. These TLRs belong to the same the Caucasian population. The cytosine–thymidine transition at subfamily along with TLR10 and TLR1, and their genes are located in 1174 bp changes arginine at aa 392 to a stop codon that blunts tandem in the same chromosome (4p14), although TLR6 is encoded the response to flagellin stimulation and prevents activation of the in only one exon. In 2005, Sun et al.92 reported 32 SNPs in the TLR6- NF-kB in CHO and HEK293 cells. This polymeric variation and 1-10 gene cluster. Nine SNPs are in the TLR6 gene, two are another nonsynonymous SNP (A1775G N592S) were found to be nonsynonymous changes (744C/T (S249P) and 1279C/T (V427A)) associated with susceptibility to Legionnaire’s disease.87 However, and another SNP is located in the promoter region of the gene the 392STOP polymorphism does not seem to be related to the ( À 1401A/G). Only the latter has been found to be associated with susceptibility to typhoid fever88 or allergy, and nor is it related to the risk of developing prostate cancer, although the 249P allele the risk of developing systemic lupus erythematosus in the influences the bowel disease phenotype.93 Pierik et al.93 reported a American population.89 This mutation appears to protect against negative association between 249P allele and ulcerative colitis with systemic lupus erythematosus in the same population, and proctitis, and this minor allele is also a risk factor for the individuals with the 392STOP allele produce low levels of IL-1b, development of inflammatory bowel diseases. In 2007, Cerhan IL-6 and TNF-a in response to flagellin stimulation.90 These et al.94 reported a strong association between the 427A allele and apparent contradictions in the results can be explained by the risk of developing non-Hodgkin lymphoma in the Caucasian type of study and by the population analyzed. One study was a population. Non-Hodgkin lymphoma is a malignancy of case–control study that included only women.89 The study by lymphocytes, and there is growing evidence for a role of germline Hawn et al.90 was a family study that included both men and genetic variation in immune response genes and its etiology.94 women. In 2006, Gewirtz et al.91 showed that the TLR5 392STOP Given the major role of TLR6 in diseases and the increasing allele can also protect persons of Jewish ethnicity against Crohn’s number of reports on how variations in this gene predispose to disease and that persons carrying TLR5STOP exhibit significantly several pathologies, it is important to continue to study the SNPs in lower levels of flagellin-specific IgG and IgA. In 2011, Zeng et al.52 this gene and their possible clinical consequences. We think that reported an association between the rs5744174 TC þ CC genotype the clinical outcome may be given by genetic variation in TLR6 by and an elevated risk of developing GC and a significant interaction its changes in ligand recognition and signal-transduction pathways. with H. pylori infection. The rs5744174 T4C change, which causes a substitution of leucine to phenylalanine at aa 616, may affect the response of ligand recognition and the subsequent signaling TLR7 AND TLR8 POLYMORPHISMS associated with inflammation, and thus ultimately contribute to an TLR7 and TLR8 recognize single-stranded RNA and belong to the increased risk of developing GC.52 It is clear that the SNPs in TLR5 same subfamily of TLRs as TLR9. Genes of TLR7 and TLR8 are

& 2014 Macmillan Publishers Limited Genes and Immunity (2014) 199 – 209 TLR polymorphisms and human diseases A Trejo-de la O et al 206 located in tandem in Xp22 and are encoded by two exons. In intracellular and extracellular CpG motifs, by modified TLR9, could 2007, genetic screening in the Chinese population report four change the signal-transduction pathways and the activation of polymorphisms in both genes: TLR7 Gln11Leu, þ 1817G4T, TLR8 transcriptions factors such as interferon-regulatory factors and Met1Val and À 129G4C.95 Schott et al.96 reported five different NF-kB. TLR7 SNPs in Caucasian patients with chronic hepatitis C infection. The substitution at À 120T4G appears to offer protection from the development of inflammation and fibrosis in male patients TLR10 POLYMORPHISMS because those with the À 120G allele had little or no TLR10 is the only orphan member among the human TLRs. One inflammation and fibrosis compared with those lacking the major hindrance to studies of TLR10 is that it lacks a rodent À 120G allele, who exhibited grades 2–4 inflammation and higher homolog. This has hampered the assignation of a natural or stages of fibrosis.96 The hepatitis C infection is caused by an RNA synthetic ligand to TLR10 because most TLR ligands have been virus, and then a role for TLR7 in the immune response against defined using mutant or genetically deficient mouse models. As in virus is likely. Polymorphisms in TLR7 can change immune the case of TLR7 and TLR8, which have a common locus on response and severity of inflammatory response, decreasing chromosome X, TLR1, TLR6 and TLR10 also share a common locus inflammation and fibrosis in the liver. on chromosome 4p14 and are structurally similar to one another (Human Genome Resources; National Center for Biotechnology Information, ncbi.nlm.nih.gov).3 In 2005, Hasan et al.108 found that TLR9 POLYMORPHISMS TLR10 had no mouse homolog because of the interruption of the TLR9 is essential for recognition of CpG DNA. The TLR9 gene maps mouse TLR10 gene by a retroviral sequence. They observed for the to 3p21.3 and is encoded by two exons, the second of which is the first time that the TLR10 protein complex exists as a homodimer major coding region. In 2003, Lazarus et al.97 reported 20 SNPs and associates with TLR1, TLR2, TLR7 and TLR9. The expression within the two exons in different ethnic groups in the American profile of TLR10 has been reported to be restricted to B cells and population,97 but only four SNPs were identified in all samples: plasmacytoid dendritic cells.108,109 In 2004, Lazarus et al.110 À 1486T/C, À 1237T/C, þ 1174G/A and þ 2848A/G, and there are resequenced 78 SNPs (1 SNP per 106 bp) in the European- a lot of publications that have linked to the predisposition of a American population and found a significant association between wide variety inflammatory diseases. The first two variations may two SNPs (c. þ 1031G4A, c. þ 2322A4G) and physician- act as potential binding sites for transcription factors. The change diagnosed asthma. By contrast, the polymorphism rs4129009 of a cytosine for a thymine at À 1237 was associated with an showed a highly significant inverse association with atopic asthma increased risk for asthma among the European-American cohort. in children.111 In independent studies, Abad et al.112 and Morgan Another report found that the À 1237C allele was significantly et al.113 found an association between rs7658893 and rs7653908 more frequent in patients with Crohn’s disease.98 This SNP is also and Crohn’s disease in the New Zealand population. More associated with a higher risk of developing a chronic refractory recently, an SNP in TLR10 was associated with tumor size in type of pouchitis, and this association is strongest when the patients with papillary thyroid cancer; a missense SNP in TLR10 À 260T allele of CD14 is also present.99 The À 1237C allele is (rs11466653 Met326Thr) showed a significant association with associated with the risk of developing systemic lupus small tumor size.114 erythematosus in Asian populations, but the other three polymorphisms are not.100,101 However, the À 1237C allele is not related to allergy susceptibility in German and Japanese TLR11 populations102,103 or with primary biliary cirrhosis, although the To date, there are no related studies with TLR11 polymorphisms change of a guanine for an adenine at 2848 bp was more frequent and their association with some pathology. in patients with primary biliary cirrhosis and was associated with a significantly greater expression of TLR9 and with a higher intracellular IgM concentration in B cells from patients and CONCLUSION healthy individuals.104 In a study unpublished from our group, in a In summary, TLRs are germline-encoded innate immune sensors Mexican population, we found association of À 1237T/C that recognize conserved microbial structures and induces polymorphism with GC in patients infected with H. pylori signaling cascades that increase the production of inflammatory (manuscript in preparation). The À 1486C polymorphism mediators by macrophages, neutrophils, dendritic cells and other exacerbates the manifestations of Plasmodium falciparum cell types. These processes activate immediately mechanisms of infection and increases the risk of developing adverse fetal innate host defense, as well as initiate and orchestrate the outcomes in women with placental malaria.105 adaptive immune response involved in the processes of cell Other polymorphisms of TLR9, þ 1635A/G and þ 1174G/A, are proliferation, survival, apoptosis, angiogenesis, and tissue remo- in linkage disequilibrium in the Caucasian population and increase deling and repair. Polymorphisms in TLR genes may shift balance the risk for rapid progression of HIV-1 infection.106 Haplotype between pro- and anti-inflammatory cytokines, modulating the analysis revealed that the combination of the variant alleles for risk of infection, chronic inflammation and cancer. Other TLR9, rs187084 and rs5743836, was associated with a significantly important roles for TLRs are to sense tissue damage in response decreased risk of developing endometrial cancer in individuals to endogenous ligands released from damaged tissues, necrotic with CC genotype compared with those with TT.107 The AA cells or cancer cells. Also, the TLRs provide tolerance to bacterial genotype of the G2848A polymorphism showed borderline products from normal flora and conserve homeostasis. Suscept- significance in conferring a marginally increased risk for ibility to or protection against human infectious diseases has been advanced cancer stages compared with the early stages.57 TLR9 shown to be associated directly with SNPs in TLR genes, which has been shown to be important in infectious diseases and has a may induce inappropriate TLR functions. Many studies support the role in modulating the immune response. The relationship association between polymorphisms of genes encoding TLR between mutations in this gene and the broad spectrum of proteins and susceptibility to infectious and inflammatory diseases described is unusual, and there are no other TLR diseases, but few researches have analyzed any associations with polymorphisms that are associated with viral, bacterial and cancer. Although TLRs are evolutionarily conserved receptors, it is parasitic infections, and with inflammatory diseases and cancer. clear that important modifications in these genes have occurred Although the biological effects of these SNPs have not been during evolution and that genetic variations impair TLR function. studied in detail, we can speculate that the recognition of These modifications may influence the frequency and course of

Genes and Immunity (2014) 199 – 209 & 2014 Macmillan Publishers Limited TLR polymorphisms and human diseases A Trejo-de la O et al 207 infectious and inflammatory diseases and cancer. Currently, there 11 Hemmi H, Takeuchi O, Kawai T, Kaisho T, Sato S, Sanjo H et al. A Toll-like receptor is not enough evidence available to understand whether specific recognizes bacterial DNA. Nature 2000; 408: 740–745. infections or diseases are more or less likely to be influenced by 12 Frantz S, Ertl G, Bauersachs J. Mechanisms of disease: Toll-like receptors in TLR polymorphisms, but initial evidence suggests that TLR cardiovascular disease. Nat Clin Pract Cardiovasc Med 2007; 4: 444–454. polymorphisms influence cellular production of cytokines and 13 Kim JK. Fat uses a TOLL—road to connect inflammation and diabetes. Cell Metab chemokines and may be other important immune functions that 2006; 4: 417–419. change the course of the inflammatory or infectious diseases and 14 Chen K, Huang J, Gong W, Iribarren P, Dunlop NM, Wang JM. Toll-like receptors cancer. in inflammation, infection and cancer. Int Immunopharmacol 2007; 7: 1271–1285. 15 Rakoff-Nahoum S, Paglino J, Eslami-Varzaneh F, Edberg S, Medzhitov R. Recog- There is a strong relationship between persistent infection, nition of commensal microflora by toll-like receptors is required for intestinal chronic inflammation and cancer. It is unclear the mechanism by homeostasis. Cell 2004; 118: 229–241. which this inflammatory response leads to cancer, but the 16 Hoshino K, Takeuchi O, Kawai T, Sanjo H, Ogawa T, Takeda Y et al. Cutting edge: involvement of TLRs in the inflammatory response has been Toll-like receptor 4 (TLR4)-deficient mice are hyporesponsive to lipopoly- clearly demonstrated and the SNPs in these genes to modify the saccharide: evidence for TLR4 as the Lps gene product. J Immunol 1999; 162: inflammatory response, and then also modify the course of the 3749–3752. cancer process, which could depend on the tissue involved and 17 Poltorak A, He X, Smirnova I, Liu MY, Van Huffel C, Du X et al. Defective LPS the change in the inflammatory response. SNPs have been signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene. Science reported in promoter regions, introns and exons. Those located in 1998; 282: 2085–2088. 18 Takeda K, Takeuchi O, Akira S. Recognition of lipopeptides by Toll-like receptors. a coding region may affect the expression of the mRNA or create a J Endotoxin Res 2002; 8: 459–463. new binding site for transcription factors. Those coding non- 19 Sun J, Wiklund F, Hsu FC, Ba¨lter K, Zheng SL, Johansson JE et al. Interactions of synonymous SNPs are thought to affect the interaction between sequence variants in interleukin-1 receptor-associated kinase4 and the toll-like the receptor and its ligand, coreceptors or other proteins essential receptor 6-1-10 gene cluster increase prostate cancer risk. Cancer Epidemiol for vesicular traffic and signaling; changes in these interactions Biomarkers Prev 2006; 15: 480–485. may modify the TLR response. Because the relevance of genetic 20 Hawn TR, Misch EA, Dunstan SJ, Thwaites GE, Lan NT, Quy HT et al. A common background to the predisposition to diseases and the importance human TLR1 polymorphism regulates the innate immune response to lipopep- of TLRs in inflammatory and infectious diseases and cancer, tides. Eur J Immunol 2007; 37: 2280–2289. further studies involving large samples would provide more 21 Johnson CM, Lyle EA, Omueti KO, Stepensky VA, Yegin O, Alpsoy E et al. Cutting edge: a common polymorphism impairs cell surface trafficking and information about the effects of SNPs in these genes. This would functional responses of TLR1 but protects against leprosy. J Immunol 2007; 178: allow us to understand better their effects and their possible roles 7520–7524. in disease prognosis and prevention based on genomic risk 22 Yang CA, Scheibenbogen C, Bauer S, Kleinle C, Wex T, Bornschein J et al. A markers for cancer and therapy, which could be very important in frequent Toll-like receptor 1 gene polymorphism affects NK- and T-cell INF-g the area of personalized medicine. production and is associated with Helicobacter pylori-induced gastric disease. Helicobacter 2013; 18: 13–21. 23 Omueti KO, Mazur DJ, Thompson KS, Lyle EA, Tapping RI. 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