Single-Nucleotide Polymorphismsin Genes Encoding Toll-Like Receptor -2, -3, -4, and -9

in Case–Control Study with Breast Cancer

Godfrey E. Etokebe,1,* Jelena Knezevic,2,* Branka Petricevic,1,3,4,* Jasminka Pavelic,2

Damir Vrbanec,3 and Zlatko Dembic1

Background: Genetic susceptibility to cancer is multifactorial, and it is known that impairment of the immunesystem could contribute to risk for getting cancer. Aim of the Study: Single-nucleotide polymorphisms (SNPs) ofToll-like receptor (TLR) 2, TLR3, TLR4, and TLR9 genes, which are important for innate immunity, were ana-lyzed for the association with breast cancer. Methods: The SNPs comprised TLR2 (c.597T>C), TLR2 (c.1350T>C),TLR3 (c.1377C>T), TLR4 (c.896A>G), and TLR9 (c.1635A>G). The allelic and genotypic frequencies of these TLRSNPs were compared between patients (n¼ 130) and controls (n¼ 101) in a case–control study from Croatia.Results: TLR SNPs were not significantly different. From the population genetics viewpoint, we found that ahypomorphic variant of TLR4 (p.Asp299Gly) allele has no specific allelic frequency (8.4%) in the Croatianpopulation (n¼ 496) compared to other Caucasians (6.5–10%). Conclusion: These results suggest that polymor-phisms in tested TLR genes are not likely to be associated with increased risk for developing breast cancer.

Introduction

It is well known that persons with mutations in onco-genes and tumor-suppressor genes are at increased risk of

developing cancer. In the immunosurveillance hypothesis,the immune system is regarded as a guardian of tissues pro-tecting their integrity from undesired mutant cells (Zitvogelet al., 2006). A dwindling immune system has been shownto increase the risk of getting cancer in animal models andhumans with immunodeficiencies or undergoing posttrans-plantation immunosuppressive therapy (Smyth et al., 2000;Girardi et al., 2001; Street et al., 2002; Smyth et al., 2004). Thegenetic susceptibility to cancer is multifactorial, with somefactors known to function in the cell cycle, apoptosis, orcell differentiation. However, the risk may also compriseadditional factors that are related to the activation of the im-mune system and chronic inflammation, as, recently, genepolymorphisms in Toll-like receptor 4 (TLR4) and the TLR6-TLR1-TLR10 gene cluster have been associated with prostatecancer (Zheng et al., 2004; Chen et al., 2005; Sun et al., 2005)and gastric lymphoma (Hellmig et al., 2005). Under selec-tive pressure from infectious microorganisms, multicellularorganisms have evolved immunological defense mechanisms,

generally categorized as innate or adaptive. Recent insightsinto the complex mechanisms of human innate immunitysuggest that genetic variability in genes encoding its compo-nents may play a role in the development of infectious andrelated diseases including cancer (Lazarus et al., 2002; Chenet al., 2005; Schroder and Schumann, 2005). It is not knownto what extent innate immunity can contribute to immuno-surveillance.

TLRs in mammals represent phylogenetically conservedtransmembrane proteins known to bind conserved molecularpatterns usually present in distantly related species like bac-teria, viruses, and parasites. They are therefore pattern rec-ognition receptors. TLRs can signal to T-cells via dendriticcells (DCs) that such molecular patterns are present within thedamaged body tissues. These include peptidoglycan, bacte-rial lipopolysaccharide, double-stranded RNA, and unme-thylated DNA as ligands for TLR2, 4, 3, and 9, respectively.Signals provided by DCs can then initiate the adaptive im-mune responses against microbial infections. Activation ofDCs by TLRs are essential for T-cell activation, up-regulationof costimulatory molecules, inhibition of regulatory T-cellactivity, and also for activation and maturation of B-cells(Rakoff-Nahoum and Medzhitov, 2009).

1Faculty of Dentistry, Institute for Oral Biology, University of Oslo, Oslo, Norway.2Division of Molecular Medicine, Laboratory of Molecular Oncology, Rudjer Boskovic Institute, Zagreb, Croatia.Departments of 3Medical Oncology and 4Oncology, Clinical Hospital Center ‘‘Zagreb,’’ University of Zagreb, Zagreb, Croatia.*These authors contributed equally to this work.

GENETIC TESTING AND MOLECULAR BIOMARKERSVolume 13, Number 6, 2009ª Mary Ann Liebert, Inc.Pp. 729–734DOI: 10.1089=gtmb.2009.0045

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Malignant cancers can express tumor-specific or tumor-associated markers (antigens) that could be used as targetsfor immune attack during immunosurveillance (Bogen et al.,2000). By having the immune system recognize such tumorantigens, the repeated onset of exactly the same cancer couldbe prevented. However, malignant tumor cells, possibly byhaving an inherent ability to constantly change genotypicand phenotypic characteristics, are able to escape immunityand=or inhibit the specific (adaptive) immune system de-fenses by establishing peripheral and sometimes also centraltolerance to such antigens (de Visser et al., 2006).

As a part of systematic investigation of susceptibilityto cancer within its relationship to the immune system, wefirst chose candidate genes from those that are importantfor the maintenance of the innate immunity. Likewise, wefirst selected the most prevalent form of malignancy inwomen—breast cancer. We designed a case–control studywith individuals from Croatia and analyzed associations ofsome single-nucleotide polymorphisms (SNPs) of the TLR2,TLR3, TLR4, and TLR9 genes with the incidence of breastcancer.

Materials and Methods

Patients and controls

Blood samples were collected from 130 breast cancer pa-tients at the Department of Medical Oncology, Department ofOncology, Clinical Hospital Center ‘‘Zagreb,’’ Zagreb, and101 healthy women (Department of Transfusiology, Rijeka,Croatia).

Diagnosis was made by medical, radiological, and bio-chemical assessment of patients. Most patients presented atearly stages of breast cancer and the initial treatment wasstarted at the Department. Patients’ ages ranged from 30 to 85years at the time of taking a sample with a mean of 62.8� 11.0(�standard deviation). Controls were with a mean age of42.5� 11.1 (�standard deviation) years.

All study participants provided oral and written informedconsent. The ethics committee of the Medical ResearchCouncil at the School of Medicine, Universities of Zagreb andRijeka approved the studies.

The DNA analysis

DNA was isolated from frozen blood by salting outmethod. The polymorphisms of TLR2 (NM_003264) c.597C>T(rs3804099) and c.1350C>T (rs3804100), TLR3 (NM_003265)c.1377C>T (rs3775290), TLR4 (NM_138554) c.896A>G(rs4986790), and TLR9 (NM_017442) c.1635A>G (rs352140)(Noguchi et al., 2004) were done by TaqMan allele-specificpolymerase chain reaction assay using two different poly-merase chain reaction machines with their own correspond-ing software: MX4000 and MX3005 (Stratagene, San Diego,CA) and Applied Biosystems (ABI) 7000 (ABI, Foster City,CA). TLR3 and TLR9 SNPs were additionally mapped by re-striction enzyme digestions, which turn out to be identical.The primers and probes for TLR2, 3, 4, and 9 were purchasedfrom ABI. TLR4 (c.896A>G) primers and 6-carboxyfluore-scein (FAM) and VICTK (proprietary)-labeled probes were aspreviously published (Schmitt et al., 2002). TLR2, TLR3, andTLR9 gene primers and probes for reporter dyes (FAM=VIC)had the following sequences:

The primers and reporter probes for TLR2 (c.597T>C)were as follows (50-30):Forward: 50-CAGATCTACAGAGCTATGAGCCAAAAReverse: 50-TCTCCAGCAGTAAAATATGCTGCTTReporter 1: VIC-TCAGATGACTTACATTCTGAReporter 2: FAM-CAGATGACTTACGTTCTGA

The primers and reporter probes for TLR2 (c.1350T>C)were as follows (50-30):Forward: GAAATATTTGAACTTATCCAGCACACGAAReverse: GTTGCTAACATCTAAAATTTCCAGTGTCTTReporter 1: VIC-CAGCCTGTTACACTGTGTReporter 2: FAM-AGCCTGTTACGCTGTGT

The primers and reporter probes for TLR3 (c.1377C>T)were as follows (50-30):Forward: GCCAGGAATGGAGAGGTCTAGAAAReverse: TCAGCTGCAGGTACTTGTTGTAGReporter 1: VIC-AAAGATAGATTTCGAAAATAReporter 2: FAM-AAAGATAGATTTCAAAAATA

The primers and reporter probes for TLR9 (c.1635A>G)were as follows (50-30):Forward: TCTACCACGAGCACTCATTCACReverse: GGGCTGGCTGTTGTAGCTReporter 1: VIC-TCCAGTCGTGGTAGCTReporter 2: FAM-CAGTCGCGGTAGCT

Statistical analysis

For statistical significance, analyses were done with the useof a chi-square test using the computer software programStatcalc (2�2 or 3�3 tables) (Acastat software). Hardy–Weinberg and linkage disequilibrium analyses were per-formed using Arlequin versus 2.0 software (Genetics andBiometry Laboratory, University of Geneva, Geneva, Swit-zerland). All loci were in Hardy–Weinberg equilibrium.

Results

TLR SNPs and breast cancer

Genotypic frequencies of TLR2–4 and TLR9 SNPs are listedin Table 1. The statistical analyses showed no significant dif-ferences in frequencies between breast cancer patients andcontrols, indicating a lack of association with the disease.

The allelic frequencies of TLR2–4 and TLR9 alleles atvarious SNPs are presented in Table 2. Allelic frequencies ofselected TLR SNPs were not significantly different betweenbreast cancer patients and healthy women controls. This tooindicated no correlation with breast cancer.

The DNA samples used as controls (n¼ 101 women),for comparisons with patients, were selected out of the 496healthy individuals who were also typed for indicated TLRSNPs. Comparisons between patients and all healthy controlssimilarly showed no significant differences in allelic and ge-notypic frequencies of TLR SNPs (data not shown). The latterresult could have been predicted, as allelic or genotypic fre-quencies of each TLR SNP in healthy women did not differmuch from the whole population (data not shown).

Discussion

Association of inherited polymorphisms of the innate im-mune gene TLR4 with the risk of prostate cancer was the first

730 ETOKEBE ET AL.

evidence that a putative weakness in innate immunity couldpredispose for cancer (Chen et al., 2005). However, it is wellknown that viruses can predispose for cancer (human papil-loma viruses), so it is reasonable to suggest that some othercancers might be linked to hereditary traits in the immunesystem. Although no known virus has ever been linked tobreast cancer, perhaps, they might in the future, because thisfield is one of the less actively investigated areas of cancerresearch. We hypothesized that predisposition to developcancer might be due to faults in innate immunity as a part of amultigenetic risk for cancer. Association studies using poly-morphic genome tags such as microsatellite repeats and SNPscould impart knowledge whether inherent weakness in theinnate immune system might influence the risk of getting thedisease in the first place, and whether this can, in addition,modulate the course of cancer.

We found that there were no associations between breastcancer and selected TLR polymorphisms. However, due toinherent complexity of a multigenetic risk, it is possible thatvarious populations have different combinations of genescomprising susceptibility for this type of cancer. Here weshowed that frequency of rare TLR4 hypomorphic allele299Gly is 8.4% (n¼ 496), suggesting that the frequency of thisvariant in Croatian population is in concordance with otherCaucasians (6.5–10%).

Polymorphisms found in TLRs have the potential to be as-sociated with complex human disease (for a review see Laz-arus et al., 2002; Schroder and Schumann, 2005; Garantziotiset al., 2008), and especially chronic inflammatory states that

can probably predispose to development of cancer. To get aclearer picture of the associations reported in literature thusfar, it is important to review the current state of disease as-sociations and polymorphisms in TLR family of genes usedin this study. The TLR2, TLR3, and TLR9 SNPs used in thepresent study were first identified in a healthy Japanesepopulation (Noguchi et al., 2004). The TLR4 p.Asp299Gly SNPis the best studied SNP thus far, which has numerous diseaseassociations. Others were not associated with any disease,except for TLR2 (c.1350T>C).

The TLR2 (c.1350T>C) rare variant (C) was associated withprotection for developing type I diabetes mellitus (Park et al.,2004). Other TLR2 SNPs were correlated with several dis-eases. The hypomorphic variants TLR2 p.Arg753Gln andp.Arg677Trp correlated with the incidence of sepsis in a whitepopulation and with the incidence of lepromatous leprosy inan Asian population, respectively (Schroder et al., 2003). TLR2p.Arg753Gln was also associated with coronary restenosis(Hamerman et al., 2005) and increased incidence of tubercu-losis in Turkish population (Ogus et al., 2004). The latter wasnot associated with susceptibility to tuberculosis in Tunisianpopulation, but p.Arg677Trp was (Ben-Ali et al., 2004). In-terestingly, TLR2 p.Arg753Gln heterozygosity seems to im-part protection to Lyme disease (Schroder and Schumann,2005).

The TLR4 (c.896A>G) (p.Asp299Gly) mutation directlyaffects the TLR4 function, as the rare 299Gly allele has only5–10% of the common allele’s signaling activity in transfectionexperiments in vitro (Arbour et al., 2000; Schmitt et al., 2002). In

Table 1. Genotypic Frequencies of Various Toll-Like Receptor Single-Nucleotide Polymorphisms

in Patients with Breast Cancer (Mostly Ca. Mammae Ductale Invasivum and Lobulare) and Controls

Controls n (%)a Patients n (%)a

SNP wt=wt wt=var var=var wt=wt wt=var var=var pb

TLR2 (597 C=T)b 26 (29.2) 48 (53.9) 15 (16.9) 29 (32.6) 44 (49.4) 16 (18.0) 0.7TLR2 (1350 T=C) 84 (88.4) 11 (11.6) 0 (0) 76 (85.4) 13 (14.6) 0 (0) 0.6TLR3 (1377 C=T) 46 (47.9) 42 (43.8) 8 (8.3) 58 (45.3) 56 (43.8) 14 (10.9) 0.8TLR4 (896 A=G) 84 (84.8) 15 (15.2) 0 (0) 110 (84.5) 20 (15.4) 0 (0) 1TLR9 (1635 A=G) 36 (37.1) 49 (50.5) 12 (12.4) 45 (34.6) 60 (46.2) 25 (19.2) 0.23

aWild type (wt; common allele) and variant forms (var; rare allele) of listed SNPs. Total number (n) of tested patients was 130, apart fromTLR2 (n¼ 89) and TLR3 (n¼ 128). Total number (n) of tested controls was 101, but distributed differently: TLR2þ597 (n¼ 89), TLR2þ1350

(n¼ 95), TLR3 (n¼ 96), TLR4 (n¼ 99), and TLR9 (n¼ 97).b2 degrees of freedom (df)-analysis; controls versus patients.SNP, single-nucleotide polymorphism; TLR, toll-like receptor.

Table 2. Allelic Frequencies of Various Toll-Like Receptor Single-Nucleotide Polymorphisms

in Breast Cancer Patients and Controls

Controls n (%)a Patients n (%)a

SNP wt var wt var pb

TLR2 (597 T=C)b 100 (56.2) 78 (43.8) 102 (57.3) 76 (42.7) 0.9TLR2 (1350 T=C) 179 (94.2) 11 (5.8) 165 (92.7) 13 (7.3) 0.6TLR3 (1377 C=T) 134 (69.8) 58 (30.2) 172 (67.2) 84 (32.8) 0.8TLR4 (896 A=G) 183 (92.4) 15 (7.6) 240 (92.3) 20 (7.7) 1TLR9 (1635 A=G) 121 (62.4) 73 (37.6) 150 (57.7) 110 (42.3) 0.25

aWild-type alleles (wt; common alleles) and their variant forms (var). Total numbers of tested patients and controls were the same as inTable 1.

b2�2 tables (w2) statistical analysis (controls vs. patients).

TLR-GENE SNPS AND BREAST CANCER 731

contrast, no reduction in sensitivity to endotoxin (lipopoly-saccharide [LPS]), inflammatory cytokine production, or an-timicrobial effects was found ex vivo in peripheral bloodmononuclear cells (Imahara et al., 2005; van der Graaf et al.,2005). Possibly other factors that can affect the expression ofantiinflammatory cytokine genes like interleukin-10 and in-terleukin-4 need to be stratified before we could fully assessthe in vivo influence of this mutant. It was reported that299Gly mutation confers beneficial effects at the populationlevel, because TLR4 (p.Asp299Gly) is associated with hypor-esponsiveness to inhaled lipopolysaccharide in humans (Ar-bour et al., 2000), reduced atherosclerosis (Kiechl et al., 2002),reduced vascular inflammation and clinical diabetes (Koleket al., 2004), decreased susceptibility of rheumatoid arthritis(Radstake et al., 2004), lower incidence of myocardial infarc-tion (Balistreri et al., 2004; Holloway et al., 2005), diminishedseverity of the systemic inflammatory response syndrome(Child et al., 2003), reduced neuropathy in type 2 diabetes(Rudofsky et al., 2004), resistance to Legionnaire’s disease(Hawn et al., 2005), protection against late-onset Alzheimerdisease (Minoretti et al., 2006), and longevity (Balistreri et al.,2004). However, it has also detrimental effects, since it cor-relates with predisposition to developing septic shock withGram-negative bacteria (Lorenz et al., 2002b), severe sepsisafter burn injury (Barber et al., 2004), severe respiratory syn-cytial virus disease (Tal et al., 2004), susceptibility to period-ontitis (Schroder et al., 2005), increased risk of premature birth(Lorenz et al., 2002a), susceptibility to inflammatory boweldisease (Franchimont et al., 2004; Braat et al., 2005; Brand et al.,2005) especially its colonic localization (Ouburg et al., 2005),brucellosis (Rezazadeh et al., 2006), and asthma (FagerasBottcher et al., 2004). Importantly, Hellmig et al. (2005) found alower frequency of heterozygotes with the rare 299Gly allelein patients with gastric lymphomas. Recently, in Swedish(Zheng et al., 2004) and Chinese (Chen et al., 2005) studygroups another TLR4 polymorphism (c.11381G>C) was as-sociated with modest risk of developing prostate cancer. Theauthors hypothesized that increased risk of chronic infectionand chronic inflammation (provided by the variant TLR4 al-lele at 11381) in prostate might also increase the susceptibilityto developing prostate cancer. However, in the present study,allelic and genotypic frequencies of the TLR4 (p.Asp299Gly)SNP showed almost no differences between patients andcontrols (Table 1).

The Chinese population has almost nonexistent TLR4299Gly allele (Hang et al., 2004). However, another TLR4polymorphism seems to play a role in disease association, asintronic (119 C=A) SNP was recently correlated with ischemicstroke (Lin et al., 2005).

The other TLR SNPs investigated in the present work werefound in 50- and 30-flanking regions of the genes (Noguchiet al., 2004), and it is not known whether they influence thetranscriptional activity of these genes. Such polymorphismsmight be helpful as markers to other more functionally rele-vant allelic variants within these loci, provided they are inlinkage disequilibrium.

The TLR3 gene polymorphisms (c.2593C>T, c.2642C>A,and c.2690A>G) were associated with diabetes mellitus type Iin South African Blacks (Pirie et al., 2005).

The TLR9 gene polymorphism was associated with hyper-immunoglobulin M in primary billiary cirrhosis (Kikuchi et al.,2005).

So far, case–control studies have indicated that variousTLR SNPs are perhaps detrimentally associated with someinfectious diseases, immune-related syndromes, and auto-immunity, while being beneficial in some other conditionsand diseases (Schroder and Schumann, 2005). By contrast,some researchers were not able to confirm some favorable andsome disadvantageous effects of TLR4 Asp299Gly polymor-phism as pointed out by a recent study (van der Graaf et al.,2005). For instance, a large study in France revealed neitherbeneficial nor detrimental influence of TLR4 299Gly SNP oncoronary heart disease (myocardial infarction) (Morange et al.,2004). Further, some studies even found the opposite, that is,increased risk for myocardial infarction in carriers of TLR4299Gly variant (Edfeldt et al., 2004). Similar conflicts werereported for the involvement of TLR4 Asp299Gly in inflam-matory bowel disease (Torok et al., 2004; Lakatos et al., 2005;Oostenbrug et al., 2005), rheumatoid arthritis (Kilding et al.,2003), preterm delivery (Hartel et al., 2004), periodontitis(Folwaczny et al., 2004), atherosclerosis (Netea et al., 2004),and asthma (Noguchi et al., 2004; Adjers et al., 2005). Theremight be several reasons why case–control studies can yieldfalse-positive or false-negative, and therefore not reproduc-ible results (Kathiresan et al., 2004). Apart from possiblevariations in diagnostic procedures, patient selection, andmethodological problems, it is well known that differences inpopulation history have produced characteristic patterns ofSNP allele frequencies when ethnic groups are compared, andperhaps such population stratification might be one of thecauses for the differences.

To conclude, there were no associations between breastcancer and selected TLR polymorphisms. However, it isstill possible that various populations may have differentcombinations of immunologically important genes com-prising susceptibility for breast cancer. This should invitemulticenter-based design and analysis of disease associa-tion studies, as the role of innate and adaptive immunity inpredisposition to breast cancer should be addressed in thefuture.

Acknowledgments

This work was financially supported by the CroatianMinistry of Science, Education and Sports, grant number 098-0982464-2394. We thank technical assistance of medical staffat the Clinical Hospital Centre ‘‘Rebro,’’ Zagreb, for help withpatients. We thank Sanja Balen, Department of Transfusiol-ogy, Medical Faculty, University of Rijeka, for collectingcontrol samples.

Disclosure Statement

No competing financial interests exist.

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Address correspondence to:Jelena Knezevic, Dr.Sc.

Division of Molecular MedicineLaboratory of Molecular Oncology

Rud-er Boskovic InstituteBijenicka cesta 54

Zagreb 10 000Croatia

E-mail: jknezev@irb.hr

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