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|Coordinate||106,226,465 bp (GRCm38)|
|Base Change||A ⇒ T (forward strand)|
|Gene Name||toll-like receptor 9|
|Chromosomal Location||106,222,598-106,226,883 bp (+)|
|MGI Phenotype||Mice homozygous for a knock-out allele exhibit impaired immune system response to LPS, CpG, and Leishmania bazillensis infection.|
|Amino Acid Change||Glutamine changed to Leucine|
|Institutional Source||Beutler Lab|
Q985L in Ensembl: ENSMUSP00000082207 (fasta)
|Gene Model||not available|
|Predicted Effect||probably damaging
PolyPhen 2 Score 0.986 (Sensitivity: 0.72; Specificity: 0.96)
|Phenotypic Category||immune system, MCMV susceptibility, TLR signaling defect: TNF production by macrophages|
|Alleles Listed at MGI|
|Mode of Inheritance||Autosomal Semidominant|
|Local Stock||Sperm, gDNA|
|Last Updated||2016-05-13 3:09 PM by Peter Jurek|
The CpG2 phenotype was identified in 2 separate screens for ENU-induced mutants with defects in the innate immune response. In one screen, peritoneal macrophages from G3 mice were tested for the ability to produce TNF in response to treatment with various TLR ligands (TLR Signaling Screen). CpG2 macrophages produced normal amounts of TNF in response to all TLR ligands tested, except oligodeoxynucleotides containing CpG motifs (CpG ODNs). In response to CpG-B ODN treatment, homozygous CpG2 macrophages produced reduced amounts of TNF, approximately 50% of the amount produced by C57BL/6J macrophages (Figure 1A). However, type I interferon (IFN) production by homozygous CpG2 mice was normal in response to systemic injection of CpG-A ODN (Figure 1B) (In vivo CpG Screen). In addition, Flt3-induced bone marrow-derived plasmacytoid dendritic cells produced normal amounts of type I IFN upon stimulation with CpG-A ODN (20 μg/mL) for 16 hours in vitro (Figure 1C). Heterozygous CpG2 macrophages have not been tested, and the mutation is tentatively classified as semidominant.
In a parallel screen, mice were tested for susceptibility to infection with a normally sublethal inoculum (1 x 105 CFU) of mouse cytomegalovirus (MCMV) (MCMV Susceptibility and Resistance Screen). While wild type mice showed no sign of sickness, homozygous CpG2 mice died within 6 days after infection (Figure 1D).
Among five ENU-induced TLR9 mutations identified to date (see Allelism above), the CpG2 mutation is unique in its ability to differentially affect TNF versus type I IFN production. The positions of the five mutations within TLR9 differ, with the CpG1, CpG3,and CpG5 mutations located in the eleventh, sixth, and fourteenth extracellular leucine-rich repeats (LRR), respectively, and the CpG6 mutation likely located in the αE helix of the cytoplasmic Toll/IL-1R (TIR) domain. The CpG2 mutation is positioned in the αD helix of the TIR domain. In addition to CpG1, CpG2, CpG3, CpG5, and CpG6, another strain of mice, designated effete, also exhibits impaired TNF-α responses to CpG ODN treatment. The mutant has no TLR9 mutation; the causative mutation is under investigation.
|Nature of Mutation|
The CpG2 mutation was mapped to Chromosome 9, and corresponds to an A to T transversion at position 3060 of the Tlr9 transcript, in exon 2 of 2 total exons.
The mutated nucleotide is indicated in red lettering, and causes a glutamine to leucine substitution at residue 985 of the TLR9 protein.
Please see the record for CpG1 for information about Tlr9.
The CpG2 mutation replaces a glutamine located in α-helix D of TLR9 with a leucine, and results in a hypomorphic protein with reduced responsiveness to stimulation by CpG ODN. Structural analysis and modeling of the TIR domain of TLR2 suggest that α-helix D may lie at the predicted homodimer interface of TLRs (1). However, the amino acid sequence identity between any pair of TIR domains is generally about 25% (2), implicating the specific sequence elements of the domain in the particular recognition of binding partners. The crystal structures of the TIR domains of TLR1, TLR2, and IL-1RAPL (IL-1R accessory protein-like) reveal that in fact, significant conformational differences exist between these molecules (2;3). Notably, α-helix D in IL-1RAP is oriented perpendicularly to that in TLR1 or TLR2 (2).
TLR9 α-helix D may also participate in interactions with downstream signaling molecules, and in the case of TLR9, could facilitate interactions with MyD88. Site-directed mutagenesis and functional analysis of the TIR domain of IL-1RAcP (IL-1 receptor accessory protein) suggest that loop EE, which connects β-strand E and α-helix E, is important for IL-1 receptor complex signaling integrity, including MyD88 binding (4;5). α-helix D is only two amino acids away from β-strand E (4), and may affect the conformation of the interface that mediates binding to signaling partners. Thus, the CpG2 mutation may impair TLR9-TLR9 dimerization or interactions with cytoplasmic binding partners. The mutation does not abolish all TLR9 signaling, as TNF-α production is merely reduced and type I IFN production is largely normal. The molecular basis for the differential effect on TNF versus IFN responses is unknown.
|Primers||Primers cannot be located by automatic search.|
CpG2 genotyping is performed by amplifying the region containing the mutation using PCR, followed by sequencing of the amplified region to detect the single nucleotide change.
Primers for PCR amplification
CpG2(F): 5’- TAAAGGCCCTGACCAATGGCAC -3’
CpG2(R): 5’- GGCAGAGAATGAACTCCAGTCCTG -3’
1) 94°C 2:00
2) 94°C 0:30
3) 55°C 0:30
4) 72°C 1:00
5) repeat steps (2-4) 40X
6) 72°C 7:00
7) 4°C ∞
Primers for sequencing
CpG2_seq(F): 5’- GGAGCCGCAAGACTCTATTTG -3’
CpG2_seq(R): 5’- TCACTCTCCTGAAAGATGCATGG -3’
The following sequence of 1268 nucleotides (from Genbank genomic region NC_000075 for linear DNA sequence of Tlr9) is amplified:
3001 gcaccctgcc taatggcacc ctcctccaga aactcgatgt cagtagcaac agtatcgtct
3061 ctgtggtccc agccttcttc gctctggcgg tcgagctgaa agaggtcaac ctcagccaca
3121 acattctcaa gacggtggat cgctcctggt ttgggcccat tgtgatgaac ctgacagttc
3181 tagacgtgag aagcaaccct ctgcactgtg cctgtggggc agccttcgta gacttactgt
3241 tggaggtgca gaccaaggtg cctggcctgg ctaatggtgt gaagtgtggc agccccggcc
3301 agctgcaggg ccgtagcatc ttcgcgcagg acctgcggct gtgcctggat gaggtcctct
3361 cttgggactg ctttggcctt tcactcttgg ctgtggccgt gggcatggtg gtgcctatac
3421 tgcaccatct ctgcggctgg gacgtctggt actgttttca tctgtgcctg gcatggctac
3481 ctttgctagc ccgcagccga cgcagcgccc aaactctccc ttatgatgcc ttcgtggtgt
3541 tcgataaggc acagagcgca gttgccgact gggtgtataa cgagctgcgg gtgcggctgg
3601 aggagcggcg cggccgccga gccctacgct tgtgtctgga ggaccgagat tggctgcctg
3661 gccagacgct cttcgagaac ctctgggctt ccatctatgg gagccgcaag actctatttg
3721 tgctggccca cacggaccgc gtcagtggcc tcctgcgcac cagcttcctg ctggctcagc
3781 agcgcctgtt ggaagaccgc aaggacgtgg tggtgttggt gatcctgcgt ccggatgccc
3841 accgctcccg ctatgtgcga ctgcgccagc gtctctgccg ccagagtgtg ctcttctggc
3901 cccagcagcc caacgggcag gggggcttct gggcccagct gagtacagcc ctgactaggg
3961 acaaccgcca cttctataac cagaacttct gccggggacc tacagcagaa tagctcagag
4021 caacagctgg aaacagctgc atcttcatgt ctggttcccg agttgctctg cctgccttgc
4081 tctgtcttac tacaccgcta tttggcaagt gcgcaatata tgctaccaag ccaccaggcc
4141 cacggagcaa aggttggctg taaagggtag ttttcttccc atgcatcttt caggagagtg
4201 aagatagaca ccaaacccac acagaacagg actggagttc attctctgcc
PCR primer binding sites are underlined; sequencing primer binding sites are highlighted in gray; the mutated A is shown in red text.
1. Beutler, B., Jiang, Z., Georgel, P., Crozat, K., Croker, B., Rutschmann, S., Du, X., and Hoebe, K. (2006) Genetic analysis of host resistance: Toll-Like receptor signaling and immunity at large, Annu. Rev. Immunol. 24, 353-389.
2. Khan, J. A., Brint, E. K., O'Neill, L. A., and Tong, L. (2004) Crystal structure of the Toll/interleukin-1 receptor domain of human IL-1RAPL, J. Biol. Chem. 279, 31664-31670.
3. Xu, Y., Tao, X., Shen, B., Horng, T., Medzhitov, R., Manley, J. L., and Tong, L. (2000) Structural basis for signal transduction by the Toll/interleukin-1 receptor domains, Nature 408, 111-115.
4. Radons, J., Dove, S., Neumann, D., Altmann, R., Botzki, A., Martin, M. U., and Falk, W. (2003) The interleukin 1 (IL-1) receptor accessory protein Toll/IL-1 receptor domain: analysis of putative interaction sites in vitro mutagenesis and molecular modeling, J. Biol. Chem. 278, 49145-49153.
|Science Writers||Eva Marie Y. Moresco|
|Illustrators||Diantha La Vine|
|Authors||Nengming Xiao, Carrie N. Arnold, Amanda L. Blasius, Bruce Beutler|
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