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Human Molecular Genetics Advance Access originally published online on April 4, 2007
Human Molecular Genetics 2007 16(10):1225-1232; doi:10.1093/hmg/ddm070
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© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Characterization and investigation of single nucleotide polymorphisms and a novel TLR2 mutation in the human TLR2 gene

Sabine Merx1, Michael Neumaier1, Hermann Wagner2, Carsten J Kirschning2 and Parviz Ahmad-Nejad1,*

1 Institute for Clinical Chemistry, Medical Faculty Mannheim, University of Heidelberg, Theodor-Kutzer-Ufer 1-3, D-68167 Mannheim, Germany and 2 Institute of Medical Microbiology, Immunology and Hygiene, Technical University of Munich, 81675 Munich, Germany

* To whom correspondence should be addressed. Tel: +49 6213832222; Fax: +49 6213833819; Email: parviz.ahmad-nejad{at}ikc.ma.uni-heidelberg.de

Received January 24, 2007; Accepted March 16, 2007


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In the innate immune system, TLR2 plays a central role for the response to a wide variety of microbial and endogenous danger signals. A considerable number of genetic polymorphisms within the human TLR2 gene have been reported in non-coding and coding sequences. Except for the Arg753Gln variant, however, their clinical relevance is unclear and the assessment of the effects of amino acid substitutions on receptor function is lacking. In the present study, we have characterized all known single nucleotide polymorphisms (SNPs) of TLR2 for their functional relevance in transiently transfected HEK293 cells subsequently exposed to a specific stimulus. Among the known non-synonymous SNPs in the TLR2 coding sequence, four SNPs (Thr411Ile, Tyr715stop, Tyr715Lys and Arg753Gln) were found to be functionally relevant in our experimental setting. In addition, we identified a new mutation Arg447stop leading to a premature stop codon in the extracellular portion of the receptor. TLR2-specific stimulation of whole blood from two heterozygote donors of this mutation resulted in a reduced secretion of pro-inflammatory cytokines. Finally, we tested the prevalence of these functional genetic variants in 169 healthy individuals of Caucasian origin for the mutations in the extracellular domain and 106 individuals for the mutations in the intracellular domain of the receptor. Except for 10 heterozygote donors of the Arg753Gln variant determined to be prevalent in 9.4% of the tested individuals, none of the other SNPs was found in this population.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Toll-like receptors (TLRs) are primary key sensors of invading pathogens recognizing conserved pathogen-associated molecular patterns (PAMPs) of bacteria, viruses, fungi and protozoa and inducing host's antimicrobial defence (1–4). The susceptibility to PAMPs varies between individuals presumably due to sequence variations in genes involved in the innate immune responses. To date, 10 members of the TLR family have been described in humans. TLRs consist of extracellular leucine-rich repeat (LRR) domains, followed by a short transmembrane region and an intracellular Toll/IL-1 receptor (TIR) domain. TLR2 (GenBank accession number NM_003264.3; MIM no. 603028 [OMIM] ) mediates cellular responses to various microbial danger signals including lipoproteins, peptidoglycans from Gram-positive bacteria, mycobacterial lipoarabinomannans, zymosan from yeast and endogenous ligands, such as heat-shock-proteins HSP60 and HSP70. The ability of the TLR2 to discriminate between diacyl and triacyl lipopeptides is achieved by homodimerization (5) or heterodimerization with TLR1 and TLR6 (6,7), respectively.

Genetic polymorphisms in members of the TLR family have been demonstrated to be clinically relevant. For instance, a genetic variation in the human TLR5 gene is associated with susceptibility to Legionnaires' disease (8). Furthermore, it is reported that variations in the human TLR4 gene contribute to inflammatory diseases, such as Crohn's disease and ulcerative colitis, as well as to Gram-negative septic shock (9–11). For the Arg753Gln single nucleotide polymorphism (SNP) in the human TLR2 gene, an increased risk for the development of tuberculosis was shown (12). This variant is also strongly associated with acute rheumatic fever in children (13), and it was reported to be a risk factor for developing septic shock in the course of Gram-positive bacterial infection (14). On the other hand, genotyping studies revealed a protective effect for the Arg753Gln variant for coronary restenosis (15) and also protects from the development of late stage Lyme disease because of reduced signalling via the TLR2/TLR1-complex (16).

Currently, 16 polymorphisms within the human TLR2 gene are known. Eight of these are non-synonymous mutations leading to amino acid exchanges, whereas the remaining eight are synonymous polymorphisms. The Arg753Gln variant in the TLR2 gene is the only coding SNP, which was correlated with clinically relevant effects. In this report, the Arg677Trp polymorphism originally reported to be associated with lepromatous leprosy in Koreans (17) is not included. Malhotra et al. (18) demonstrated that this polymorphism is not a true polymorphism but a variation present in a 93% homologous region of TLR2 exon 3, which is located 23 kb upstream of the human TLR2 gene. We characterized, for the first time, systematically all non-synonymous SNPs of human TLR2 using transiently transfected HEK293 cells as a reporter system. Furthermore, analysing the prevalence of the functionally relevant TLR2 SNPs by sequencing, we identified a new mutation leading to a premature stop codon at the amino acid position 447. This mutation appears to be functionally relevant, too, impairing responses towards different TLR2 stimuli.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Different sequence variations in hTLR2 affect the innate immune response to bacterial lipoprotein
Genetic human database survey revealed eight synonymous and eight non-synonymous polymorphisms (Table 1) for the human TLR2 coding sequence (GenBank accession number NM_003264.3; http://www.ncbi.nlm.nih.gov). Database search (http://www.ncbi.nlm.nih.gov; http://innateimmunity.net; http://snpper.chip.org) revealed three different hits concerning SNPs at amino acid position 715 (Tyr715Asn; Tyr715stop and Tyr715Lys). All variants, except the Tyr715Asn polymorphism, were investigated in our analyses. To avoid redundancy concerning the amino acid exchanges, we focused our analyses only on two potentially interesting SNPs, Tyr715stop and Tyr715Lys, leading to a premature stop codon and a basic amino acid, respectively.


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  		Table 1. Database results of investigated SNPs in the human TLR2 gene

 
So far, an association with a higher susceptibility to Staphylococcus infection (14), as well as to tuberculosis (12), for the Arg753Gln SNP was shown. Latest results indicate that there is a strong relationship between acute rheumatoid fever in children and the Arg753Gln variant (13). Other polymorphisms in the TLR2 gene may influence susceptibility to infections and/or progression towards sepsis or septic shock, too. However, no data have been reported regarding the impact of these non-synonymous polymorphisms on the TLR2 function. Therefore, we set out to clarify the functional relevance of eight known human TLR2 non-synonymous polymorphisms. Using NF-{kappa}B reporter gene assays, we tested the functional consequences of the mutations in a controlled cell culture system examining transiently transfected HEK293 fibroblasts (Fig. 1).


Figure 1
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  		Figure 1. Dual-luciferase assay of transiently transfected HEK293 cells with TLR2 and TLR2 variants, respectively. After transfection with the indicated plasmids, cells were plated on six-well plates, incubated overnight at 37°C, 5% CO2 and stimulated for 14 h with 1µg/ml Pam3CysSK4 (EMC Microcollections). Cells were lysed with 1x passive lysis buffer (Promega). Firefly and renilla luciferase activities were measured using the Dual-Glo Luciferase Assay System (Promega), according to manufacturer's instructions. The fold induction was calculated as a ratio of 6x NF-{kappa}B firefly luciferase and TK renilla luciferase. The figure represents merged data from four independent experiments.

 
We investigated all TLR2 non-synonymous variants functionally. Two TLR2 variants, coding for Tyr715Lys and Tyr715stop, were non-responsive upon stimulation with the synthetic TLR2 ligand Pam3CysSK4. The variants Thr411Ile and Arg753Gln showed hypo-responsiveness, whereas all other investigated polymorphisms were comparable with wild-type TLR2 (Fig. 1).

To further characterize the TLR2 mutant proteins and to control for equal expression of transiently introduced receptors, we performed western blot analysis of FLAG-tagged wild-type TLR2 and its mutant variants (Fig. 2). All four functionally relevant SNPs were expressed on the protein level, thus excluding the possibility that the lack of Pam3CysSK4 stimulation was due to unstable mRNA or insufficient protein synthesis. To date, four N-glycosylation sites are known in the extracellular LRR domain at amino acid positions 114, 199, 414 and 442 (19). Thus, the complex western blot pattern reflected most likely glycosylation variants (20).


Figure 2
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  		Figure 2. Immunoblot analysis for expression control of functional relevant TLR2 variants with anti-FLAG M2 monoclonal antibody (Sigma-Aldrich). For expression analysis, HEK293 cells were transiently transfected with FLAG-tagged wild-type phTLR2 and variants, respectively. Cell lysates were analysed by western blotting using the FLAG-specific anti-FLAG M2 antibody. Lane 1: marker; lane 2: negative control (pRK5); lane 3: Arg447stop; lane 4: Thr411Ile; lane 5: Tyr715Lys; lane 6: Tyr715stop; lane 7: Arg753Gln; lane 8: Pro631His and lane 9: wild-type TLR2.

 
Three functional relevant polymorphisms are rare in Caucasians
To date, only little information is available regarding the frequency of SNPs within the TLR2 gene. To gain some hints on the evolutionary pressure possibly operating on the non- and hypo-responsive sequence variants, we set out to compare their populational prevalence by sequencing parts of the extracellular LRR domain and the TIR domain. The results are summarized in Table 2. For this reason, we analysed 169 blood donors for their frequency of the Thr411Ile SNP. In our study group, the synonymous SNP Ser450Ser displayed a heterozygosity frequency of 11.24%. In the TLR2 TIR domain, the frequency was investigated for the following physiologically relevant variants: Tyr715Lys, Tyr715stop and Arg753Gln and a non-synonymous SNP Glu768Asp. Our results (Table 2) show that in this Caucasian population, the non-responsive polymorphisms Tyr715Lys, Tyr715stop and Glu768Asp are rare, as neither heterozygote nor homozygote carriers were found. In contrast, heterozygosity for the hypo-responsive Arg753Gln polymorphism was observed in 9.4% (10/106) in this study population, thus showing a correlation between the level of responsiveness upon specific stimulation and the frequency in our study population as we found only heterozygote carriers for the hypo-responsive Arg753Gln variant.


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  		Table 2. Genotyping of healthy human donors for TLR2 SNPs and a novel mutation

 
Detection of a novel TLR2 mutation
Genotyping of 169 healthy donors for common TLR2 SNPs revealed a novel, so far unknown, stop mutation in the extracellular LRR domain leading to an amino acid exchange at position 447. One donor showed heterozygosity for this new mutation (Fig. 3A). Blood donations were taken independently for two times to verify the newly discovered mutation by resequencing in an ABI PRISM 310 Genetic Analyzer. Additional genotyping via pyrosequencing showed only one female heterozygote carrier corresponding to 0.38% for the Arg447stop mutation (1/262) (Fig. 3B). All other donors had wild-type TLR2 alleles. Homozygote Arg447stop donors were not detected.


Figure 3
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  		Figure 3. Genotyping analysis of the novel Arg447stop mutation via sequencing in an ABI PRISM 310 Genetic Analyzer (Applied Biosystems) (A) and pyrosequencing in a PSQ96 MA (Biotage) (B). Wild-type TLR2 donors and heterozygote Arg447stop donors were shown.

 
Further, the TLR2 locus of the parents of the heterozygote donor was analysed by sequencing. The mother displayed heterozygosity for this mutation, too, indicating the inheritance of the mutation. Interestingly, the heterozygote carriers did not report recurrent bacterial infections. Of note, allergic responses were indicated by the two heterozygote carriers of the Arg447stop mutation. Patients' history revealed infantile eczema, an early symptom of atopic dermatitis, in childhood, and both carriers reported a contact allergy to nickel. Interestingly, the mother developed allergic responses to cat hair, specific creams and food, such as citrus fruits, apples and honey. In the last years, hay fever occurred as yet further immune system dysfunction. Additionally, the second heterozygote donor indicated an allergy to cow milk in the childhood.

Functional characterization of the stop codon mutation Arg447stop
We were interested in the functional relevance of the c.1339C > T substitution (Arg447stop) in our standardized cell culture system. HEK293 fibroblasts transfected with the respective expression vector were non-responsive upon stimulation with the synthetic ligand Pam3CysSK4 in a reporter gene assay (Fig. 1).

Cotransfection of wild-type and mutagenized plasmids (hTLR2 and hTLR2-R447x) resulted in a dominant-negative effect when the mutant receptor was expressed in increasing amounts. Cell number and mortality rate had no significant effect on our reporter gene assay, as controlled by TNF-alpha stimulation (Fig. 4).


Figure 4
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  		Figure 4. Dual-luciferase assay of transiently transfected HEK293 cells for dominant-negative effect of the Arg447stop mutation. After transfection with the indicated plasmids, cells were stimulated for 14 h with 1µg/ml Pam3CysSK4 (EMC Microcollections) (A) and 50 ng/ml human TNF-alpha (PeproTech) (B), respectively. Cells were lysed with 1x passive lysis buffer (Promega). Firefly and renilla luciferase activities were measured using the Dual-Glo Luciferase Assay System (Promega), according to manufacturer's instructions. The fold induction was calculated as a ratio of 6x NF-{kappa}B firefly luciferase and TK renilla luciferase. The figure represents merged data from three independent experiments.

 
To verify expression of the TLR2 Arg447stop mutation at mRNA level in vivo, total RNA was isolated from peripheral blood of both heterozygote carriers and controls. Expression at the mRNA level was determined by means of RT–PCR using mRNA-specific primers followed by sequencing of the PCR product (Fig. 5). Sequencing analysis of controls and heterozygote donors verified the transcription of the mutated allele.


Figure 5
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  		Figure 5. Expression control at the mRNA level of a wild-type TLR2 donor and two related heterozygote Arg447stop carriers. The length of the resultant PCR product is drawn in the graph of the gene structure (A). Total RNA was reverse-transcribed into cDNA using SuperScript II Reverse Transcriptase (Invitrogen) and oligo(dT)12–18 (Invitrogen) primer. Specific exon I and exon III spanning primers were designed using the longest published cDNA sequence for human TLR2 (AF051152.1) resulting in a PCR product of 1.7 kb. The PCR was performed with Herculase Hotstart DNA polymerase (Stratagene) followed by gel extraction (Gel Extraction Kit) and sequencing in an ABI PRISM 310 Genetic Analyzer (Applied Biosystems). Lane 1: marker; lane 2: wild-type TLR2 donor; lane 3: heterozygote Arg447stop donor 1; lane 4: heterozygote Arg447stop donor 2 and lane 5: no-template control (B).

 
To investigate the in vivo relevance of the mutation, we performed whole blood stimulations of two wild-type TLR2 carriers and the two heterozygote Arg447stop mutation individuals by subsequent measurement of pro-inflammatory cytokines. The duplex cytokine assays of human IL-6 and human TNF-alpha documented hypo-responsiveness of the heterozygote donors in comparison with wild-type individuals after stimulation with Pam3CysSK4 (Fig. 6). Similar results were obtained upon stimulation with Staphylococcus aureus-derived peptidoglycan (Fig. 6).


Figure 6
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  		Figure 6. Heparin blood from two wild-type TLR2 donors and two heterozygote Arg447stop donors were stimulated for 6 h with 1 µg/ml Pam3CysSK4, 10 µg/ml S. aureus peptidoglycan and 50 ng/ml E. coli lipopolysaccharide. Subsequently, cytokine assays for human IL-6 and human TNF-alpha (R&D Systems) were performed on the Luminex100 instrument. White bars: heterozygote Arg447stop donor 1; light grey bars: heterozygote Arg447stop donor 2; dark grey bars: wild-type TLR2 donor 1 and black bars: wild-type TLR2 donor 2.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
In this study, we systematically investigated for the first time all known non-synonymous SNPs in the coding region of the human TLR2 gene for their functional relevance in a controlled cell culture system. Four non-synonymous SNPs impairing the function of the receptor were identified. Sequencing analysis revealed a novel, previously unknown mutation at amino acid position 447 (Arg447stop; c.1339C > T), which leads to a premature stop codon. Receptor function in a reporter gene assay was abolished as evidenced upon stimulation with Pam3CysSK4 as a receptor ligand. The TLR2 bearing Arg447stop mutation displayed a dominant-negative effect in reporter gene assays when it was added in increasing amounts. Two related heterozygote carriers for the Arg447stop mutation were identified and further characterized. Expression at the mRNA level was confirmed by sequencing analysis to exclude premature degradation. The heterozygote donors for the Arg447stop mutation displayed reduced secretion of the pro-inflammatory cytokines IL-6 and TNF-alpha.

Until now, reports on clinically relevant non-synonymous SNPs in TLRs are rare. Thus, we focused our attention on further characterizing non-synonymous SNPs in the human TLR2 gene. From a medical view point, it is significant that of all eight non-synonymous polymorphisms, only four showed a significant impact on receptor function.

Genotyping analysis displayed a frequency of 9.43% (10/106) heterozygote donors for the Arg753Gln variant. These data are in agreement with previously published reports investigating a large study population of Caucasian donors (21), but disagree with available database information on the NCBI homepage stating 2.8% heterozygosity frequency for Caucasians, Africans, African Americans and Hispanics. This difference can be possibly attributed to the composition of the control collective analysed. In our study population, only Caucasians were included, suggesting that this SNP may be more prevalent in this ethnic group. In contrast, none of the other non-synonymous mutations was present in the tested study populations of 169 and 106 healthy individuals. This implies that a considerable evolutionary pressure might be operating as these genetic variants are rare variants or may be private mutations. Our results possess impact for the rational design of genetic tests of the TLR2 inflammation receptor, e.g. for the design of gene arrays.

Furthermore, we identified a novel mutation at amino acid position 447 leading to a premature stop codon located in the extracellular domain in LRR16 (22). As expected, the Arg447stop variant expressed in HEK293 cells failed to trigger a response to Pam3CysSK4 stimulation.

In addition, we analysed the interaction of the Arg447stop mutant and wild-type TLR2. It has been recently shown that deletion of the first seven LRRs did not drastically affect the ability of the receptor to recognize diacylated and triacylated microbial polypetides (23), whereas the deletion of Ser424–Asn533 abolished signalling, resembling the effect of our newly discovered TLR2 mutation. The mutant protein might possibly interact with TLR1 and/or TLR2 at the extracellular domain, thereby forming non-functional heterodimers or homodimers that abolish signalling activity because of the lack of the transmembrane and intracellular TIR domains. In our experiments, a complete dominant-negative effect could not be achieved possibly due to an excessive proteasomal degradation of the truncated protein. As a consequence, the amount of available receptor on the surface may result in the measured residual activity of the TLR2 protein after premature proteasomal degradation of the truncated TLR2 Arg447stop mutant.

Measurements of cytokine production in two heterozygous carriers for the Arg447stop mutation revealed reduced secretion of the pro-inflammatory cytokines IL-6 and TNF-alpha, following stimulation of heparin-treated whole blood with Pam3CysSK4 or peptidoglycan, respectively. Both heterozygote Arg447stop carriers secreted only residual amounts of IL-6 and TNF-alpha, despite the presence of one functional TLR2 wild-type allele. Similar to this Arg447stop mutation, a stop codon polymorphism at amino acid position 392 (1174C > T) has been located in the extracellular LRR domain within the human TLR5 gene. Specifically, Hawn et al. (8) found that this functionally relevant SNP is associated with Legionnaire's disease. Stimulation of peripheral blood mononuclear cell (PBMCs) of heterozygote carriers of the TLR5 Arg392stop variant with the cognate stimulus flagellin did not result in detectable IL-6 production, suggesting a dominant-negative effect of this truncated TLR5 gene product. The heterozygote Arg447stop carriers in our study showed a less pronounced dominant-negative effect on the release of pro-inflammatory cytokines. This reduced secretion is likely to be caused by a ‘dilution’ of the truncated Arg447stop mutant protein with an excess of possible functional dimerization partners. Indeed, although TLR5 exclusively forms homodimers (24), TLR2 is known to build homotypic (5) and heterotypic complexes with TLR1 (6) upon specific stimulation. Furthermore, it is conceivable that the truncated TLR2 Arg447stop protein might be unstable, prematurely ubiquitinated and thus rapidly degraded by the proteasome.

Further investigations of the Arg447stop mutation should elucidate its potential role with respect to the susceptibility to inflammatory and infectious diseases of its carriers as well as the frequency of this mutation in other populations. To date, several studies regarding association of TLR2 polymorphisms with pathophysiology of specific clinical conditions have been published. Controversial data exist for the TLR2 Arg753Gln variant regarding atopic diseases (25,26). However, various allergies of both heterozygote carriers of the Arg447stop mutation point towards the need for comprehensive association studies to evaluate the clinical importance of TLR2 SNPs in allergic diseases. The presented data, especially on the Arg447stop mutation, are relevant for future clinical studies examining the significance of TLR-SNPs and help to envisage new strategies in clinical diagnosis as this mutation may play a role in various inflammatory and infectious diseases.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Material
Dulbecco's modified Eagle's medium (DMEM) was obtained from PAA (Pasching, Austria). Penicillin, streptomycin and fetal bovine serum (FBS) were obtained from Biochrom AG (Berlin, Germany). Pam3CysSK4 was purchased from EMC Microcollections (Tuebingen, Germany), human TNF-alpha from PeproTech (London, UK), S. aureus peptidoglycan from InvivoGen (Toulouse, France) and Escherichia coli LPS from Sigma-Aldrich (Taufkirchen, Germany). The monoclonal anti-FLAG M2 antibody for western blot analysis was obtained from Sigma-Aldrich. TRIzol Reagent and Superscript II RT for RNA isolation and cDNA synthesis were obtained from Invitrogen (Karlsruhe, Germany).

Methods
Human subjects
All donors gave written consent for blood donation. The healthy controls for sequencing and genotyping were recruited from the Institute for Clinical Chemistry and students from different practical courses. For characterization of the novel c.1339C > T (Arg447stop) mutation, we investigated a family with three members (two female donors and one male donor) of which both female donors were heterozygote carriers of the Arg447stop mutation.

Hundred and six donors were genotyped for SNPs in the TLR2 TIR domain, whereas a total of 169 donors were characterized by sequencing in an ABI PRISM 310 Genetic Analyzer (Applied Biosystems, Darmstadt, Germany). Pyrosequencing analysis was done for the Arg447stop mutation in additional 93 individuals. All donors investigated are of Caucasian origin.

Genotyping of human TLR2 SNPs
Genotyping of the Thr411Ile, Tyr715stop, Tyr715Lys and Arg753Gln SNPs as well as the novel mutation Arg447stop was done by sequencing in an ABI PRISM 310 Genetic Analyzer (Applied Biosystems). After PCR amplification of a 303 bp fragment encompassing the Thr411Ile SNP (primer sequences: T2_LRR_s: AAT TCA GCC TGT GAG GAT GC and T2_LRR_as: AGT TGC GGC AAA TTC AAA GA) and the Arg447stop mutation, sequencing of 169 donors was performed. For genotyping of the Tyr715stop, Tyr715Lys and Arg753Gln SNPs, a total of 106 donors were investigated after PCR amplification of a 393 bp fragment comprising these three SNPs (primer sequences: T2_TIR_s: GAT GCC TAC TGG GTG GAG AA and T2_TIR_as: CGC AGC TCT CAG ATT TAC CC). Extraction of genomic DNA for the novel Arg447stop mutation was done twice independently, as well as sequencing analysis with the sense and antisense primer.

SNP information
Information about all SNPs, refSNP ID and heterozygosity frequency was obtained from the NCBI (http://www.ncbi.nlm.nih.gov) and the Innate Immunity (http://innateimmunity.net) homepage, except data about the Tyr715Lys SNP were received from the CHIP SNPper homepage (http://snpper.chip.org). Sequencing analysis revealed the novel TLR2 mutation c.1339C > T (Arg447stop).

For cDNA (GenBank accession number NM_003264.3; MIM no. 603028 [OMIM] ) numbering, nucleotide +1 corresponds to A of the ATG translation initiation codon in the reference sequence.

Plasmids and mutagenesis
The phTLR2 plasmid was generated by PCR from human cDNA (GenBank accession number NM_003264.3; GI: 68160956; MIM no. 603028 [OMIM] ) and cloned into the mammalian expression vector pcDNA3.1/Hygro (+) (Invitrogen). The coding region of the hTLR2 gene and some variants were cloned into p3xFLAG-CMVTM-14 (Sigma-Aldrich) for western blot analysis. All used TLR2 SNPs were obtained from the NCBI homepage, except the Tyr715Lys and the novel TLR2 mutation Arg447stop. Oligo information for cloning, mutagenesis and sequencing is available on request. Mutations were generated into the phTLR2 plasmid via QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA). The sequence of all hTLR2 variants and the hTLR2 wild-type sequence were confirmed by the sequence analysis (ABI PRISM 310 Genetic Analyzer, Applied Biosystems).

Transfection of HEK293 cells and dual-luciferase assay
HEK293 cells were cultured in DMEM medium (PAA) supplemented with 10% FBS, penicillin and streptomycin (all Biochrom AG). For dual-luciferase assay, 3.5x105 HEK293 cells were transfected in six-well plates by calcium phosphate method with 50 ng 6x NF-{kappa}B firefly luciferase, 5 ng thymidine kinase renilla luciferase pGL4.74 (hRLuc/TK) (Promega, Mannheim, Germany), 1 µg phTLR2 and 1 µg hTLR2 variant, respectively. After 24 h of transfection, cells were stimulated for 14 h with 1 µg/ml Pam3CysSK4 (EMC Microcollections) and 50 ng/ml human TNF-alpha (PeproTech), respectively. Cells were lysed with 1x passive lysis buffer (Promega). Firefly and renilla luciferase activities were measured on a Victor3 (PerkinElmer LAS, Rodgau-Juegesheim, Germany) using the Dual-Glo Luciferase Assay System (Promega), according to manufacturer's instructions. The fold induction was calculated as a ratio of 6x NF-{kappa}B firefly luciferase and TK renilla luciferase.

Protein analysis
Western blot analysis for expression control of functional relevant hTLR2 SNPs and the relevant Arg447stop mutation was performed with anti-FLAG M2 monoclonal antibody (Sigma-Aldrich). For expression analysis, wild-type phTLR2 and some SNPs were cloned into p3xFLAG-CMVTM-14 (Sigma-Aldrich). HEK293 cells were cultured in DMEM (PAA), supplemented with 10% FBS, penicillin and streptomycin (all Biochrom AG) and transfected with 1 µg phTLR2-FLAG and mutant hTLR2-FLAG by calcium phosphate method. Twenty-four hours after transfection, cells were lysed in radioimmunoprecipitation assay (RIPA)-buffer containing protease inhibitor cocktail (Sigma-Aldrich). Protein concentration was determined by bicinchorinic acid assay (BCA) protein assay (Uptima Interchim, Montluçon, France). Approximately 15 µg protein lysate was separated on a 10% SDS–PAGE, transferred on a nitrocellulose membrane and probed with 5 µg/ml anti-FLAG M2 monoclonal antibody (Sigma-Aldrich). Detection was performed with ImmunStar AP Chemiluminescent Kit (Bio-Rad Laboratories, Munich, Germany), according to manufacturer's instructions.

Analysis of the c.1339C > T (Arg447stop) mutation
DNA sequencing
DNA was isolated from fresh blood donations of three family members. From one donor of these, DNA isolation and sequencing was done twice independently. DNA sequencing was performed as mentioned earlier.

For further frequency information of the Arg447stop mutation additionally 93 individuals were analysed via pyrosequencing in a PSQ96TM MA (Biotage, Uppsala, Sweden) using the Pyro Gold Reagents (Biotage, Uppsala, Sweden) and specific primers (T2_R447x_for: biotin-TGC CTG AAA CTT GTG AGT GG; T2_R447x_rev: GGG AAT GCA GCC TGT TAC ACT and T2_R447x_seq: CAG CCT GTT ACA CTG TGT AT). PCR was performed using GoTaq® Flexi DNA Polymerase (Promega). About 40 ng sample DNA was added to a reaction volume of 50 µl containing 10 µl 5x buffer, 3 mM MgCl2, 0.2 mM deoxyribonucleoside triphosphate mix (Eppendorf, Hamburg, Germany) and 0.6 µM of each primer. PCR conditions were 95°C for 3 min, followed by 45 cycles of 95°C for 30 s, 58°C for 45 s and 72°c for 1 min, final elongation was 72°C for 5 min.

Expression control of the mRNA expression level
Total RNA was isolated from 1 ml of citrate blood via TRIzol Reagent (Invitrogen) after erythrocyte lysis with ammonium chloride, according to manufacturer's instructions. Total RNA was reverse-transcribed using SuperScript II Reverse Transcriptase (Invitrogen) and oligo(dT)12–18 primer (Invitrogen). Specific exon I and exon III spanning primers (TLR2_mRNA_as: CCA GTG CTG TCC TGT GAC AT and TLR2_mRNA_s: GTG ACT GCT CGG AGT TCT CCC) were designed using the longest published cDNA sequence for human TLR2 (AF051152 [GenBank] .1), resulting in a PCR product of 1.7 kb. PCR was performed with Herculase® Hotstart DNA polymerase (Stratagene) followed by gel extraction (Gel Extraction Kit, Qiagen, Hilden, Germany) and sequencing in an ABI PRISM 310 Genetic Analyzer (Applied Biosystems).

Measurement of cytokines
Heparin blood was stimulated for 6 h with 50 ng/ml lipopolysaccharide (LPS), 1 µg/ml Pam3CysSK4 and 10 µg/ml S. aureus peptidoglycan. Following stimulation, plasma was collected and analysed for the presence of human IL-6 and TNF-alpha. Cytokines were measured with Fluorokine® MAP Human Base Kit A and Human IL-6 Fluorokine MAP and Human TNF-alpha Fluorokine MAP (R&D Systems, Wiesbaden, Germany), according to the manufacturer's protocol. Heparin blood was stimulated three times independently from two healthy female controls. Cytokines from both heterozygous donors were only investigated once. All analyses were performed using the Luminex 100 platform (Progen, Heidelberg, Germany).


   ACKNOWLEDGEMENTS
 
We gratefully thank C.E., R.E. and K.-P.E. for their sedulous support during the entire work. We appreciate the excellent technical assistance of K. Hanel and W. Zimmer for the Luminex100 platform. We gratefully thank R. M. Vabulas for critical reading of this manuscript. This work was funded by the Fritz-Thyssen-Stiftung; grant number: Az. 10.03.2.115 [EC] .

Conflict of Interest statement. None declared.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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