FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2037961698> ?p ?o ?g. }

• W2037961698 endingPage "497" @default.
• W2037961698 startingPage "493" @default.
• W2037961698 abstract "Toll-like receptors (TLR) serve important roles in immunity against microbial pathogens, and TLR3 plays a critical role in the recognition of double-stranded RNA derived from viruses. Two specific single nucleotide polymorphisms (SNP) in the human TLR3 gene, A851T and C1234T, which result in amino acid substitutions, N284I and L412F, respectively, have been described to result in impaired receptor signaling and function in an in vitro model. Hence, an assay that could simultaneously detect these two functional TLR3 SNPs could facilitate studies into their role in the pathogenesis of human viral diseases and serve as a tool to screen viral disease predisposition. Therefore, we developed a real-time PCR assay based on a LightCycler format for simultaneous detection of A851T and C1234T SNPs within the TLR3 gene. This relatively low-cost PCR assay is proposed as a novel tool in the study of TLR3 and its role in viral disease. Toll-like receptors (TLR) serve important roles in immunity against microbial pathogens, and TLR3 plays a critical role in the recognition of double-stranded RNA derived from viruses. Two specific single nucleotide polymorphisms (SNP) in the human TLR3 gene, A851T and C1234T, which result in amino acid substitutions, N284I and L412F, respectively, have been described to result in impaired receptor signaling and function in an in vitro model. Hence, an assay that could simultaneously detect these two functional TLR3 SNPs could facilitate studies into their role in the pathogenesis of human viral diseases and serve as a tool to screen viral disease predisposition. Therefore, we developed a real-time PCR assay based on a LightCycler format for simultaneous detection of A851T and C1234T SNPs within the TLR3 gene. This relatively low-cost PCR assay is proposed as a novel tool in the study of TLR3 and its role in viral disease. Toll-like receptors (TLRs) belong to the group of pathogen recognition receptors that play critical roles in innate immunity by serving as front-line molecules that signal to the host of the presence of invading microbial pathogens. Ten TLRs have been described in humans, and of these, TLR3 has been demonstrated to be important in host antiviral responses.1Alexopoulou L Holt AC Medzhitov R Flavell RA Recognition of double-stranded RNA and activation of NF-kappaB by Toll-like receptor 3.Nature. 2001; 413: 732-738Crossref PubMed Scopus (4907) Google Scholar Experimental investigations have implicated TLR3 in the pathogenesis of many viral diseases because this receptor recognizes viral double-stranded RNA.1Alexopoulou L Holt AC Medzhitov R Flavell RA Recognition of double-stranded RNA and activation of NF-kappaB by Toll-like receptor 3.Nature. 2001; 413: 732-738Crossref PubMed Scopus (4907) Google Scholar However, the clinical relevance of these experimental findings are not yet defined, although TLR3 has been studied in the context of infections attributable to hepatitis C virus, human immunodeficiency virus, dengue virus, West Nile virus, measles virus, and members of the herpes viruses including herpes simplex viruses.2Dhiman N Ovsyannikova IG Vierkant RA Ryan JE Pankratz VS Jacobson RM Poland GA Associations between SNPs in toll-like receptors and related intracellular signaling molecules and immune responses to measles vaccine: preliminary results.Vaccine. 2008; 26: 1731-1736Crossref PubMed Scopus (129) Google Scholar3Kong KF Delroux K Wang X Qian F Arjona A Malawista SE Fikrig E Montgomery RR Dysregulation of TLR3 impairs the innate immune response to West Nile virus in the elderly.J Virol. 2008; 82: 7613-7623Crossref PubMed Scopus (144) Google Scholar4Tsai YT Chang SY Lee CN Kao CL Human TLR3 recognizes dengue virus and modulates viral replication in vitro.Cell Microbiol. 2009; 11: 604-615Crossref PubMed Scopus (149) Google Scholar5Villacres MC Literat O DeGiacomo M Du W Frederick T Kovacs A Defective response to Toll-like receptor 3 and 4 ligands by activated monocytes in chronic hepatitis C virus infection.J Viral Hepatol. 2008; 15: 137-144PubMed Google Scholar6West J Damania B Upregulation of the TLR3 pathway by Kaposi's sarcoma-associated herpesvirus during primary infection.J Virol. 2008; 82: 5440-5449Crossref PubMed Scopus (100) Google Scholar Single nucleotide polymorphisms (SNPs) occur commonly in the genetic makeup of TLR3, and the presence of these SNPs is hypothesized to influence the function of this pattern recognition receptor. In the clinical setting, SNPs could potentially be associated with a better or worse clinical outcome for patients with infectious disease. To date, there have been more than 136 SNPs described within the human TLR3 gene; however, only four were predicted to cause changes in the TLR3 protein molecule.7Ranjith-Kumar CT Miller W Sun J Xiong J Santos J Yarbrough I Lamb RJ Mills J Duffy KE Hoose S Cunningham M Holzenburg A Mbow ML Sarisky RT Kao CC Effects of single nucleotide polymorphisms on Toll-like receptor 3 activity and expression in cultured cells.J Biol Chem. 2007; 282: 17696-17705Crossref PubMed Scopus (117) Google Scholar Two of these four TLR3 SNPs (A851T [rs5743316] and C1234T [rs3775291]) have been shown to affect TLR3 function as a result of amino acid changes from Isoleucine to Asparagine (N284I) and from Phenylalanine to Leucine (L412F), respectively. In an experimental setting in vitro, these amino acid changes resulting from SNPs in the exon-coding region were demonstrated to impair TLR3 signaling in response to its ligand.7Ranjith-Kumar CT Miller W Sun J Xiong J Santos J Yarbrough I Lamb RJ Mills J Duffy KE Hoose S Cunningham M Holzenburg A Mbow ML Sarisky RT Kao CC Effects of single nucleotide polymorphisms on Toll-like receptor 3 activity and expression in cultured cells.J Biol Chem. 2007; 282: 17696-17705Crossref PubMed Scopus (117) Google Scholar Specifically, the ability to respond to the TLR3 ligand poly(I:C), as assessed by the degree of nuclear factor-kappa B–driven luciferase activity, was reduced by 30% in cells with the L412F mutation, whereas it was completely abrogated in cells with N284I mutation.7Ranjith-Kumar CT Miller W Sun J Xiong J Santos J Yarbrough I Lamb RJ Mills J Duffy KE Hoose S Cunningham M Holzenburg A Mbow ML Sarisky RT Kao CC Effects of single nucleotide polymorphisms on Toll-like receptor 3 activity and expression in cultured cells.J Biol Chem. 2007; 282: 17696-17705Crossref PubMed Scopus (117) Google Scholar On the other hand, the other SNPs, including the Y307D and S737T mutations that were also predicted to alter the TLR3 molecule, did not affect TLR3 function or have not yet been conclusively demonstrated to result in impaired TLR3 signaling.7Ranjith-Kumar CT Miller W Sun J Xiong J Santos J Yarbrough I Lamb RJ Mills J Duffy KE Hoose S Cunningham M Holzenburg A Mbow ML Sarisky RT Kao CC Effects of single nucleotide polymorphisms on Toll-like receptor 3 activity and expression in cultured cells.J Biol Chem. 2007; 282: 17696-17705Crossref PubMed Scopus (117) Google Scholar Because of their association with impaired TLR3 signaling in vitro, we hypothesized that these two specific SNPs (A851T [N284I] and C1234T [L412F]) could be associated with viral diseases in humans. The tedious and time-consuming nature of identifying A851T and C1234T SNPs using a home-brewed conventional PCR method and the costly genotyping of each clinical sample make this procedure less than desirable. Therefore, we aimed to develop an assay that could rapidly and simultaneously detect A851T and C1234T SNPs in the human TLR3 gene. The target gene, TLR3 (GenBank accession number NM_003265), is located in chromosome 4q35 and comprises five exons within its 15,942-bp transcript encoding a 3057-bp mRNA.8Pirie FJ Pegoraro R Motala AA Rauff S Rom L Govender T Esterhuizen TM Toll-like receptor 3 gene polymorphisms in South African Blacks with type 1 diabetes.Tissue Antigens. 2005; 66: 125-130Crossref PubMed Scopus (46) Google Scholar The gene sequence encodes a putative 904 amino acid residues. TLR3 comprises an extracellular horseshoe-shaped solenoid domain containing 23 leucine-rich repeats, a transmembrane domain consisting of a single α-helix, and a cytoplasmic tail containing the conserved Toll-interleukin-1 receptor (TIR) domain.8Pirie FJ Pegoraro R Motala AA Rauff S Rom L Govender T Esterhuizen TM Toll-like receptor 3 gene polymorphisms in South African Blacks with type 1 diabetes.Tissue Antigens. 2005; 66: 125-130Crossref PubMed Scopus (46) Google Scholar The TLR3 SNPs of interest, A851T and C1234T, are located within a close proximity of each other on Exon 4 of the TLR3 gene. This close proximity made our goal of developing a dual-detection single assay attainable. On approval from the Mayo Foundation Institutional Review Board, stored peripheral blood samples from 228 randomly selected liver transplant recipients were thawed for nucleic acid extraction and PCR analysis. TLR3 plasmids A851T (N284I) and C1234T (L412F) were kindly provided as gifts from Dr. C.T. Ranjith-Kumar and Dr. C. Kao (Texas A&M University, College Station, TX). The plasmid sequences were confirmed by in-house genetic sequencing using the ABI prism 3730XL DNA analyzer. Each plasmid was confirmed to contain only its designated SNP with no other unintended mutations. A wild-type construct from the N284I plasmid was generated by using the site mutation kit (Stratagene Cat. #200518). Wild-type oligonucleotides were made in-house and annealled over the top of the A851T SNP with the highlighted base-change (bold) to incorporate the wild-type sequence 5′-CTATGCTCGATCTTTCCTACAACAACTTAAATGTGGTTGGTAA-3′. The wild-type plasmid was confirmed by in-house sequencing to be pure wild-type TLR3 gene with no other unintended mutations. Nuclei acid was extracted from 228 whole blood samples from liver transplant recipients according to manufacturer's protocol using the IsoQuick Nucleic Acid Extraction kit (Catalog No. MXT-020–100, ORCA Research, Inc.). The extracted nucleic acid from the whole blood samples were eluted in 100 μl of RNase-free water. In randomly selected samples, the nucleic acid concentrations were measured using the Nanodrop spectrophotometer (Thermo Fisher Scientific). Forty samples were randomly selected and subjected to both home-brewed conventional and real-time PCR assay for comparison. Gene sequencing (ABI PRISM 3730 XL DNA Analyzers, Perkin-Elmer Applied Biosystems, Foster City, CA) was performed on all 40 samples at the DNA Sequencing Laboratory of the Advanced Genomics Technology Center (Mayo Clinic, Rochester, MN). The primers were designed for use in both the home-brewed conventional and real-time PCR assays. The primers were purchased and made in-house (Synthetic Core Laboratory, Advanced Genomics Technology Center, Mayo Clinic, Rochester, MN) using the ABI 3948 purification instrument. The hybridization probes, which were developed using the LightCycler probe design software (Roche), were used for the real-time PCR assay only. The probes were purchased from Idaho Technology (Salt Lake City, UT). The sequences for the primers and probes are shown in Table 1.Table 1Primer and Probe Sequences for the Detection of A851T and C1234T Single Nucleotide Polymorphism in Toll-Like Receptor 3 (TLR3) GenePrimers TLR3 sense primer5′-TCCCAGCCTTACAGAGAAGC-3′ TLR3 anti-sense primer5′-CCTGTGAGTTCTTGCCCAAT-3′Probes TLR3 F2 (A851T) Fluorescein Red-6405′-TGGTAACGATTCCTTTGCTTG-3′ TLR3 F3 (C1234T) Fluorescein Red-7055′-AGAATAAAATCTCAAAAATAGAGAGTGA-3′ codon represents the wild-type sequence for the TLR3 A851T single nucleotide polymorphism. The bolded A changes to a T, and this base change results in the amino acid substitution from asparagine to isoleucine. codon represents the wild-type sequence for the TLR3 C1234T single nucleotide polymorphisms. The bolded C changes to a T, and this base change results in the amino acid substitution from leucine to phenylalanine. Open table in a new tab codon represents the wild-type sequence for the TLR3 A851T single nucleotide polymorphism. The bolded A changes to a T, and this base change results in the amino acid substitution from asparagine to isoleucine. codon represents the wild-type sequence for the TLR3 C1234T single nucleotide polymorphisms. The bolded C changes to a T, and this base change results in the amino acid substitution from leucine to phenylalanine. A section of Exon 4 where the two SNPs are located in the TLR3 gene was amplified using the primer set in Table 1. The PCR reaction mixture for each sample consisted of 2.5 μl of 10X PCR buffer, 1.75 μl of 25 mM MgCl2, 0.5 μl of 10 mM dNTP, 1.0 μl of each primer at 5 μM, and 0.2 μl of AmpliTaq Gold for a total volume of 6.95 μl. A total of 100 ng of plasmid or clinical sample nucleic acid was added to the above reaction, and nucleic acid-free water was added to make up a final volume of 25 μl. Conventional home-brewed PCR was performed on a Perkin Elmer 2400 PCR system. The amplification conditions included an initial denaturation at 94°C for 5 minutes, followed by 45 cycles of denaturation (94°C for 30 s), annealing (55°C for 30 s), and extension (72°C for 45 s), with a final extension for 10 minutes at 72°C, after amplification samples were stored at 4°C. All real-time PCRs were performed in a total volume of 20 μl, which contained 15 μl of TLR3 Mastermix and 5 μl of nucleic acid sample (2 to 10 ng/μl). The TLR3 Mastermix includes 2 μl of ready-to-use hot start reaction mix for LightCycler (LightCycler FastStart DNA Master HybProbe, Roche Diagnostics, Cat. No. 12239272001), 2.4 μl of MgCl2 at 4 mmol/L, 8.4 μl of nucleic acid-free water, 0.5 μl of each primer at 0.25 μmol/L, 0.2 μl of each (A851T, C1234T) fluorescein probe, and 0.4 μl of each (A851T, C1234T) red probe. The sequences for the primers and probes are described in Table 1. The Mastermix and samples were added to a capillary tube, spun down, and PCR was performed using a LightCyler instrument (Roche). The amplification conditions included an initial denaturation step at 95°C for 10 minutes, followed by 45 cycles of denaturation (95°C for 10 s), annealing (55°C for 15 s), and extension (72°C for 15 s). Melting curve analysis was one cycle at 95°C for 0 s, 45°C for 60 s, followed by an increase in temperature to 85°C at a slope of 0.2°C/s. One cycle of a cooling-down step at 40°C for 0 s followed. The fluorescein probes covered the SNPs of interest and exhibited different peaks in melting temperatures, which was dependent on the binding to the wild-type or mutated TLR3 genes.9Hamann L Hamprecht A Gomma A Schumann RR Rapid and inexpensive real-time PCR for genotyping functional polymorphisms within the Toll-like receptor -2, -4, and -9 genes.J Immunol Methods. 2004; 285: 281-291Crossref PubMed Scopus (56) Google Scholar The LightCycler-based real-time PCR assay was designed for the simultaneous detection of the A851T and C1234T SNPs in the TLR3 gene in a single capillary tube reaction. The TLR3 A851T SNP was analyzed on fluorescence channel F2. The peak melting curve for the wild-type TLR3 plasmid (851 AA) was observed at 62.47°C. In contrast, the peak melting curve for the variant TLR3 A851T gene (851 TT) was shifted to 56.86°C, thereby generating a temperature shift difference of 5.6°C between the wild-type AA and variant TT TLR3 genes (Figure 1). Genetic sequencing of the two plasmid constructs confirmed the base change (A851T; Figure 2, A and B).Figure 2Representation of TLR3 gene sequencing for the A851T single nucleotide polymorphism. A: The wild-type gene containing AAC, which encodes for the amino acid aspargine. B: The A851T single nucleotide polymorphism with the codon ATC, which encodes for isoleucine.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Using this assay, a total of 228 blood samples from randomly selected liver transplant recipients were tested for the presence of the A851T SNP on two separate occasions. On both occasions, all samples demonstrated a peak in melting curve at 62.47°C, whereas none of the 228 samples demonstrated a shift in the melting curve characteristics. Hence, all 228 patients were assumed to carry the wild-type AA at position 851 of the TLR3 gene. To confirm this, 40 randomly selected samples were subjected for gene sequencing. Using this standard approach, all 40 samples exhibited the wild-type AA at position 851. Accordingly, the prevalence of the TLR3 A851T SNP in our selected population of 228 white liver transplant recipients was 0%. The presence of variant TLR3 gene containing the C1234T SNP was assessed on the same PCR assay using fluorescence channel F3. The wild-type TLR3 plasmid (1234 CC) had a peak melting curve at 61.25°C. In contrast, the variant TLR3 C1234T gene (1234 TT) shifted the melting curve to 57.98°C; hence, a temperature shift difference of 3.3°C (Figure 3). In the analysis of the clinical samples from 228 patients, there were 107 samples with peak melting curve at 61.25°C, suggesting wild-type TLR3 gene (1234 CC). In contrast, 19 (8%) samples had peak melting curve at 57.98°C, suggesting the presence of a variant gene (1234 TT). Notably, there were 102 (45%) patients with simultaneous double-peak in melting curve analysis, with peaks at 61.25°C and 57.98°C. Forty randomly selected clinical samples were subjected for gene sequencing, and the results confirmed the presence of the 1234 CC for the wild-type gene (61.25°C) and 1234 TT for the variant C1234T SNP (57.98°C). The clinical samples that demonstrated a double-peak in the melting curve analysis were sequenced to contain C and T at base position 1234, suggesting heterozygosity (1234 CT) for this SNP of interest (Figure 4, A–C). The ability of humans to respond to pathogen-associated molecular patterns may be impaired by SNPs within TLR genes, and this could result in an altered predisposition to, and clinical course of, various infectious diseases. In this context, the current study reports on a novel methodology that can rapidly detect and distinguish two functional A851T and C1234T SNPs in the human TLR3 gene. This assay was highly reproducible and accurate, and the results are highly congruent with the more laborious technique of genetic sequencing. Using this new assay, we demonstrate that TLR3 A851T SNP (N284I) occurs very rarely in this predominantly white population, whereas the C1234T SNP (L412F) is much more prevalent. Because these SNPs have been demonstrated in vitro to impair TLR3 signaling in response to double-stranded RNA, the availability of this assay is anticipated to facilitate further studies into the role of TLR3 A851T and C1234T SNPs in the pathogenesis of viral diseases in humans. TLR3 recognizes double-stranded RNA, which are products of viral replication. Hence, TLR3 has been postulated to be involved in the pathogenesis of various viral diseases, including but not limited to respiratory viruses such as the influenza virus,10Lau YF Tang LH Ooi EE A TLR3 ligand that exhibits potent inhibition of influenza virus replication and has strong adjuvant activity has the potential for dual applications in an influenza pandemic.Vaccine. 2009; 27: 1354-1364Crossref PubMed Scopus (44) Google Scholar coxsackie virus,11Richer MJ Lavallee DJ Shanina I Horwitz MS Toll-like receptor 3 signaling on macrophages is required for survival following coxsackievirus B4 infection.PLoS ONE. 2009; 4: e4127Crossref PubMed Scopus (114) Google Scholar and rhinovirus,12Zhu L Lee PK Lee WM Zhao Y Yu D Chen Y Rhinovirus-induced major airway mucin production involves a novel TLR3-EGFR-dependent pathway.Am J Respir Cell Mol Biol. 2009; 40: 610-619Crossref PubMed Scopus (121) Google Scholar to neurotrophic viruses such as West Nile virus13Daffis S Samuel MA Suthar MS Gale Jr, M Diamond MS Toll-like receptor 3 has a protective role against West Nile virus infection.J Virol. 2008; 82: 10349-10358Crossref PubMed Scopus (267) Google Scholar and herpes simplex virus.14Zhang SY Jouanguy E Ugolini S Smahi A Elain G Romero P Segal D Sancho-Shimizu V Lorenzo L Puel A Picard C Chapgier A Plancoulaine S Titeux M Cognet C von Bernuth H Ku CL Casrouge A Zhang XX Barreiro L Leonard J Hamilton C Lebon P Heron B Vallee L Quintana-Murci L Hovnanian A Rozenberg F Vivier E Geissmann F Tardieu M Abel L Casanova JL TLR3 deficiency in patients with herpes simplex encephalitis.Science. 2007; 317: 1522-1527Crossref PubMed Scopus (875) Google Scholar The majority of these studies are based on animal and experimental models, and the translation of these important findings to the bedside is limited. One study however reported that a dominant-negative TLR3 allele was demonstrated in otherwise healthy children who developed herpes simplex encephalitis,14Zhang SY Jouanguy E Ugolini S Smahi A Elain G Romero P Segal D Sancho-Shimizu V Lorenzo L Puel A Picard C Chapgier A Plancoulaine S Titeux M Cognet C von Bernuth H Ku CL Casrouge A Zhang XX Barreiro L Leonard J Hamilton C Lebon P Heron B Vallee L Quintana-Murci L Hovnanian A Rozenberg F Vivier E Geissmann F Tardieu M Abel L Casanova JL TLR3 deficiency in patients with herpes simplex encephalitis.Science. 2007; 317: 1522-1527Crossref PubMed Scopus (875) Google Scholar thereby suggesting the clinical relevance of TLR3 in herpes simplex virus pathogenesis. Whether a particular SNP in the TLR3 gene would result in such a clinical disease predisposition has not been investigated. Single nucleotide gene polymorphisms occur commonly in the human population and may result in amino acid substitution (nonsynonymous), may affect promoter characteristics (i.e., influences the transcription of the receptor), or may be completely “null.” The TLR3 A851T and C1234T SNPs are considered nonsynonymous because the nucleotide changes result in amino acid substitutions from asparagine to isoleucine (at position 284) and leucine to phenylalamine (at position 412), respectively.7Ranjith-Kumar CT Miller W Sun J Xiong J Santos J Yarbrough I Lamb RJ Mills J Duffy KE Hoose S Cunningham M Holzenburg A Mbow ML Sarisky RT Kao CC Effects of single nucleotide polymorphisms on Toll-like receptor 3 activity and expression in cultured cells.J Biol Chem. 2007; 282: 17696-17705Crossref PubMed Scopus (117) Google Scholar In vitro experiments demonstrated a complete functional impairment in TLR3 signaling in the presence of A851T SNP (N284I), whereas a markedly diminished function was observed in the presence of the C1234T SNP (L412F).7Ranjith-Kumar CT Miller W Sun J Xiong J Santos J Yarbrough I Lamb RJ Mills J Duffy KE Hoose S Cunningham M Holzenburg A Mbow ML Sarisky RT Kao CC Effects of single nucleotide polymorphisms on Toll-like receptor 3 activity and expression in cultured cells.J Biol Chem. 2007; 282: 17696-17705Crossref PubMed Scopus (117) Google Scholar Hence, these in vitro findings would support their potential role in the clinical setting. The availability of this rapid assay would facilitate clinical studies to assess the role of these functional TLR3 A851T and C1234T SNPs in the predisposition to herpes virus infection or to various human viral diseases. Using this assay, we have embarked on an investigation to define the prevalence of these SNPs in liver transplant recipients. In this study, we report that the C1234T SNP is very prevalent genetic variation in humans, whereas the A851T is uncommon in the white population.7Ranjith-Kumar CT Miller W Sun J Xiong J Santos J Yarbrough I Lamb RJ Mills J Duffy KE Hoose S Cunningham M Holzenburg A Mbow ML Sarisky RT Kao CC Effects of single nucleotide polymorphisms on Toll-like receptor 3 activity and expression in cultured cells.J Biol Chem. 2007; 282: 17696-17705Crossref PubMed Scopus (117) Google Scholar The high prevalence rate of C1234T variant concurs with the previous report, which describes the rate to be more than 40% in asthmatic population.7Ranjith-Kumar CT Miller W Sun J Xiong J Santos J Yarbrough I Lamb RJ Mills J Duffy KE Hoose S Cunningham M Holzenburg A Mbow ML Sarisky RT Kao CC Effects of single nucleotide polymorphisms on Toll-like receptor 3 activity and expression in cultured cells.J Biol Chem. 2007; 282: 17696-17705Crossref PubMed Scopus (117) Google Scholar Moreover, the LightCycler assay was able to identify the 8% of patients who possessed the SNP in both alleles (homozygous variant), and a further 45% of patients having one allele with the C1234T variant (heterozygous). As confirmed by genetic sequencing, the LightCycler assay was able to distinguish these two variants from the wild-type gene by shifts in the melting curve characteristics. The homozygous TT variant have a completely different peak melting curve, whereas the heterozygous CT variant demonstrated double peaks, with each peak corresponding to the wild-type CC and the homozygous TT peaks. A similar pattern would have been observed with the TLR3 A851T SNP; however, all 228 liver transplant recipients demonstrated the AA wild-type peak melting curve, suggesting that this specific SNP occurs very rarely. In conclusion, we report a novel single-run PCR assay, based on the LightCycler format, which could rapidly detect and differentiate the A851T and C1234T SNPs in the TLR3 gene. We anticipate that this PCR assay can be easily adapted by other investigators because the instrument is available to many research and clinical laboratories. Hence, this assay could facilitate multiple investigations into the role of TLR3 in virus disease pathogenesis, especially because these SNPs result in impaired TLR3 function at least in vitro. In this study, we embarked on a pilot investigation of defining the prevalence of TLR3 SNPs in liver transplant recipients and observed the frequent nature of the C1234T SNP. We now plan on using this validated assay to define the clinical relevance of TLR3 in human viral diseases. Likewise, we encourage other investigators to adapt this assay in their studies and to also further determine the biological function and clinical relevance of these and other TLR3 SNPs, including the Y307D and S737T mutations, in humans. If an association with clinical disease is eventually demonstrated for these specific SNPs, this assay may eventually be useful in determining viral disease predisposition in humans. We thank Dr. C.T. Ranjith-Kumar and Dr. C. Cheng Kao for the kind gift of the plasmids, Teresa Hoff for the manuscript preparation, and the Mayo Clinic Advanced Genomics Technology Center for the genotyping of the samples and the construction of the primer sets." @default.
• W2037961698 created "2016-06-24" @default.
• W2037961698 creator A5006752272 @default.
• W2037961698 creator A5043876015 @default.
• W2037961698 date "2010-07-01" @default.
• W2037961698 modified "2023-09-29" @default.
• W2037961698 title "A Real-Time PCR Assay for the Simultaneous Detection of Functional N284I and L412F Polymorphisms in the Human Toll-Like Receptor 3 Gene" @default.
• W2037961698 cites W1981498390 @default.
• W2037961698 cites W1992151583 @default.
• W2037961698 cites W1995003386 @default.
• W2037961698 cites W2007430725 @default.
• W2037961698 cites W2047815636 @default.
• W2037961698 cites W2056264877 @default.
• W2037961698 cites W2056423038 @default.
• W2037961698 cites W2076361525 @default.
• W2037961698 cites W2081038211 @default.
• W2037961698 cites W2084603524 @default.
• W2037961698 cites W2121656093 @default.
• W2037961698 cites W2126004312 @default.
• W2037961698 cites W2148578992 @default.
• W2037961698 doi "https://doi.org/10.2353/jmoldx.2010.090122" @default.
• W2037961698 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/2893634" @default.
• W2037961698 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/20413676" @default.
• W2037961698 hasPublicationYear "2010" @default.
• W2037961698 type Work @default.
• W2037961698 sameAs 2037961698 @default.
• W2037961698 citedByCount "6" @default.
• W2037961698 countsByYear W20379616982012 @default.
• W2037961698 countsByYear W20379616982013 @default.
• W2037961698 countsByYear W20379616982021 @default.
• W2037961698 countsByYear W20379616982023 @default.
• W2037961698 crossrefType "journal-article" @default.
• W2037961698 hasAuthorship W2037961698A5006752272 @default.
• W2037961698 hasAuthorship W2037961698A5043876015 @default.
• W2037961698 hasBestOaLocation W20379616981 @default.
• W2037961698 hasConcept C104317684 @default.
• W2037961698 hasConcept C136449434 @default.
• W2037961698 hasConcept C153911025 @default.
• W2037961698 hasConcept C170493617 @default.
• W2037961698 hasConcept C2776709828 @default.
• W2037961698 hasConcept C2778025104 @default.
• W2037961698 hasConcept C48023723 @default.
• W2037961698 hasConcept C54355233 @default.
• W2037961698 hasConcept C70721500 @default.
• W2037961698 hasConcept C86803240 @default.
• W2037961698 hasConceptScore W2037961698C104317684 @default.
• W2037961698 hasConceptScore W2037961698C136449434 @default.
• W2037961698 hasConceptScore W2037961698C153911025 @default.
• W2037961698 hasConceptScore W2037961698C170493617 @default.
• W2037961698 hasConceptScore W2037961698C2776709828 @default.
• W2037961698 hasConceptScore W2037961698C2778025104 @default.
• W2037961698 hasConceptScore W2037961698C48023723 @default.
• W2037961698 hasConceptScore W2037961698C54355233 @default.
• W2037961698 hasConceptScore W2037961698C70721500 @default.
• W2037961698 hasConceptScore W2037961698C86803240 @default.
• W2037961698 hasFunder F4320337375 @default.
• W2037961698 hasIssue "4" @default.
• W2037961698 hasLocation W20379616981 @default.
• W2037961698 hasLocation W20379616982 @default.
• W2037961698 hasLocation W20379616983 @default.
• W2037961698 hasLocation W20379616984 @default.
• W2037961698 hasOpenAccess W2037961698 @default.
• W2037961698 hasPrimaryLocation W20379616981 @default.
• W2037961698 hasRelatedWork W1967636949 @default.
• W2037961698 hasRelatedWork W1982297066 @default.
• W2037961698 hasRelatedWork W1987584618 @default.
• W2037961698 hasRelatedWork W2041645124 @default.
• W2037961698 hasRelatedWork W2347913873 @default.
• W2037961698 hasRelatedWork W2366655330 @default.
• W2037961698 hasRelatedWork W2371450007 @default.
• W2037961698 hasRelatedWork W2390841997 @default.
• W2037961698 hasRelatedWork W3149127145 @default.
• W2037961698 hasRelatedWork W3161574264 @default.
• W2037961698 hasVolume "12" @default.
• W2037961698 isParatext "false" @default.
• W2037961698 isRetracted "false" @default.
• W2037961698 magId "2037961698" @default.
• W2037961698 workType "article" @default.