|Coordinate||106,224,689 bp (GRCm38)|
|Base Change||T ⇒ C (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||Leucine changed to Proline|
|Institutional Source||Beutler Lab|
L393P in Ensembl: ENSMUSP00000082207 (fasta)
|Gene Model||not available|
|Predicted Effect||probably damaging
PolyPhen 2 Score 0.997 (Sensitivity: 0.40; Specificity: 0.98)
|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||05/13/2016 3:09 PM by Peter Jurek|
The CpG5 phenotype was identified in a screen of ENU-induced G3 mutants for altered response to Toll-like receptor (TLR) ligands (TLR Signaling Screen). Peritoneal macrophages from heterozygous CpG5 mice produce normal amounts of tumor necrosis factor (TNF)-α in response to all TLR ligands tested, except oligodeoxynucleotides containing CpG motifs (CpG ODNs). Upon stimulation with CpG ODN, heterozygous CpG5 macrophages produce reduced amounts of TNF-α. In addition, naïve B cells from whole blood of heterozygous CpG5 mice fail to proliferate upon stimulation with CpG ODN in vitro, a response recently demonstrated to be specific for CpG ODN among other TLR agonists (1).
Homozygous CpG5 mice will be tested in both the TNF bioassay and the B cell proliferation assay. Results should indicate whether CpG5 is dominant or semidominant; it has been tentatively classified as semidominant.
Although not all of the same phenotypes have been examined, those tested are identical between CpG1, CpG2, CpG3 and CpG5 mice. Sequence analysis revealed that all four strains contain mutations in Tlr9. However, the positions of the mutations differ, with the CpG1, CpG3 and CpG5 mutations located in the sixteenth, sixth and fourteenth extracellular leucine-rich repeats (LRR), respectively, and the CpG2 mutation located in the cytoplasmic Toll/IL-1R (TIR) domain. In addition to CpG1, CpG2, CpG3 and CpG5, 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|
A Tlr9 mutation corresponding to a T to C transition at position 1284, in exon 2 of 2 total exons, was identified for CpG5.
The mutated nucleotide is indicated in red lettering, and corresponds to the missense error L393P in the polypeptide chain.
The CpG5 mutation results in a leucine to proline change at position 393 of the TLR9 protein. L393 is the first leucine residue of the predicted fourteenth LRR module of the TLR9 ectodomain (Figure 1) (2).
Please see the record for CpG1 for information about Tlr9.
The CpG5 mutation substitutes leucine with proline at position 393 of the TLR9 protein, which is the predicted first leucine of the fourteenth LRR module in the ectodomain. No tertiary structural data presently exist for TLR9, making it difficult to hypothesize how the CpG5 mutation could affect either ligand binding or receptor dimerization. Recently, the crystal structure of the related TLR3 heterodimer bound to double-stranded (ds) RNA has been elucidated (see record for CpG1). The ligand-bound TLR3 heterodimer forms an M-shape with the dsRNA binding to the concave surfaces of the TLR3 heterodimer at two locations on each ectodomain (3). It has been hypothesized that TLR7, 8 and 9 ligands may also bind to the concave surface of the ectodomain at a site made up by insertions at LRR 2, 5, 8 and 11 (4). The CpG5 mutation might somehow disrupt ligand binding and/or receptor dimerization, or destroy proper folding or localization of the receptor. The CpG5 phenotype supports any and all of these possibilities.
|Primers||Primers cannot be located by automatic search.|
CpG5 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. The same primers are used for PCR amplification and for sequencing.
CpG1(F): 5’-AACTCTTCCTGGTTCCAAGGTCTG -3’
CpG1(R): 5’-GGGTATGAATGTCATTGTGTGCC -3’
1) 94°C 2:00
2) 94°C 0:15
3) 60°C 0:30
4) 68°C 1:00
5) repeat steps (2-4) 40X
6) 68°C 7:00
7) 4°C ∞
The following sequence of 850 nucleotides (from Genbank genomic region NC_000075 for linear DNA sequence of Tlr9) is amplified:
1812 aactcttcc tggttccaag gtctggtcaa cctctcggtg ctggacctaa
1861 gcgagaactt tctctatgaa agcatcaccc acaccaatgc ctttcagaac ctaacccgcc
1921 tgcgcaagct caacctgtcc ttcaattacc gcaagaaggt atcctttgcc cgcctccacc
1981 tggcaagttc ctttaagaac ctggtgtcac tgcaggagct gaacatgaac ggcatcttct
2041 tccgcttgct caacaagtac acgctcagat ggctggccga tctgcccaaa ctccacactc
2101 tgcatcttca aatgaacttc atcaaccagg cacagctcag catctttggt accttccgag
2161 cccttcgctt tgtggacttg tcagacaatc gcatcagtgg gccttcaacg ctgtcagaag
2221 ccacccctga agaggcagat gatgcagagc aggaggagct gttgtctgcg gatcctcacc
2281 cagctccgct gagcacccct gcttctaaga acttcatgga caggtgtaag aacttcaagt
2341 tcaccatgga cctgtctcgg aacaacctgg tgactatcaa gccagagatg tttgtcaatc
2401 tctcacgcct ccagtgtctt agcctgagcc acaactccat tgcacaggct gtcaatggct
2461 ctcagttcct gccgctgact aatctgcagg tgctggacct gtcccataac aaactggact
2521 tgtaccactg gaaatcgttc agtgagctac cacagttgca ggccctggac ctgagctaca
2581 acagccagcc ctttagcatg aagggtatag gccacaattt cagttttgtg acccatctgt
2641 ccatgctaca gagccttagc ctggcacaca atgacattca taccc
Primer binding sites are underlined; the mutated T is shown in red text.
1. Jiang, W., Lederman, M. M., Harding, C. V., Rodriguez, B., Mohner, R. J., and Sieg, S. F. (2007) TLR9 stimulation drives naive B cells to proliferate and to attain enhanced antigen presenting function, Eur. J Immunol. 37, 2205-2213.
2. Matsushima, N., Tanaka, T., Enkhbayar, P., Mikami, T., Taga, M., Yamada, K., and Kuroki, Y. (2007) Comparative sequence analysis of leucine-rich repeats (LRRs) within vertebrate toll-like receptors, BMC. Genomics 8, 124.
3. Liu, L., Botos, I., Wang, Y., Leonard, J. N., Shiloach, J., Segal, D. M., and Davies, D. R. (2008) Structural basis of toll-like receptor 3 signaling with double-stranded RNA, Science 320, 379-381.
|Science Writers||Eva Marie Y. Moresco|
|Illustrators||Diantha La Vine|
|Authors||Nengming Xiao, Bruce Beutler|