Phenotypic Mutation 'Cpg2' (pdf version)
Mutation Type missense
Coordinate106,103,664 bp (GRCm39)
Base Change A ⇒ T (forward strand)
Gene Tlr9
Gene Name toll-like receptor 9
Chromosomal Location 106,099,797-106,104,075 bp (+) (GRCm39)
MGI Phenotype FUNCTION: [Summary is not available for the mouse gene. This summary is for the human ortholog.] The protein encoded by this gene is a member of the Toll-like receptor (TLR) family which plays a fundamental role in pathogen recognition and activation of innate immunity. TLRs are highly conserved from Drosophila to humans and share structural and functional similarities. They recognize pathogen-associated molecular patterns (PAMPs) that are expressed on infectious agents, and mediate the production of cytokines necessary for the development of effective immunity. The various TLRs exhibit different patterns of expression. This gene is preferentially expressed in immune cell rich tissues, such as spleen, lymph node, bone marrow and peripheral blood leukocytes. Studies in mice and human indicate that this receptor mediates cellular response to unmethylated CpG dinucleotides in bacterial DNA to mount an innate immune response. [provided by RefSeq, Jul 2008]
PHENOTYPE: Nullizygous mice exhibit impaired immune responses to CpG DNA and altered susceptibility to EAE and parasitic infection. ENU-induced mutants may exhibit altered susceptibility to viral infection or induced colitis and impaired immune response to unmethylated CpG oligonucleotides. [provided by MGI curators]
Accession Number

NCBI RefSeq: NM_031178; MGI: 1932389

Amino Acid Change Glutamine changed to Leucine
Institutional SourceBeutler Lab
Gene Model not available
AlphaFold Q9EQU3
PDB Structure Crystal structure of mouse TLR9 (unliganded form) [X-RAY DIFFRACTION]
Crystal structure of mouse TLR9 in complex with inhibitory DNA4084 (form 1) [X-RAY DIFFRACTION]
Crystal structure of mouse TLR9 in complex with inhibitory DNA4084 (form 2) [X-RAY DIFFRACTION]
Crystal structure of mouse TLR9 in complex with inhibitory DNA_super [X-RAY DIFFRACTION]
Crystal Structure of the C-terminal Domain of Mouse TLR9 [X-RAY DIFFRACTION]
SMART Domains Protein: ENSMUSP00000082207
Gene: ENSMUSG00000045322
AA Change: Q985L

signal peptide 1 25 N/A INTRINSIC
LRR 62 85 1.49e2 SMART
LRR 122 144 1.41e1 SMART
LRR 198 221 4.98e-1 SMART
LRR 283 306 6.59e1 SMART
LRR 307 332 1.62e1 SMART
Blast:LRR 333 361 8e-6 BLAST
LRR 390 413 7.38e1 SMART
LRR 414 440 1.86e2 SMART
LRR 496 520 1.81e2 SMART
LRR 521 544 6.05e0 SMART
LRR 545 568 2.27e2 SMART
LRR 575 599 4.58e1 SMART
LRR 628 651 3.87e1 SMART
LRR_TYP 677 700 3.39e-3 SMART
LRR 702 724 2.27e2 SMART
LRR 726 748 3.09e2 SMART
Blast:LRRCT 761 810 4e-11 BLAST
Pfam:TIR 870 1029 7.4e-11 PFAM
Predicted Effect probably damaging

PolyPhen 2 Score 0.997 (Sensitivity: 0.41; Specificity: 0.98)
(Using ENSMUST00000062241)
Meta Mutation Damage Score Not available question?
Is this an essential gene? Probably nonessential (E-score: 0.078) question?
Phenotypic Category Autosomal Semidominant
Candidate Explorer Status loading ...
Single pedigree
Linkage Analysis Data
Penetrance 100% 
Alleles Listed at MGI

All alleles(9) : Targeted, knock-out(1) Gene trapped(1) Chemically induced(7)

Lab Alleles
AlleleSourceChrCoordTypePredicted EffectPPH Score
IGL00864:Tlr9 APN 9 106102206 missense probably damaging 1.00
IGL01764:Tlr9 APN 9 106103004 missense probably damaging 1.00
IGL02077:Tlr9 APN 9 106102704 missense possibly damaging 0.90
IGL02232:Tlr9 APN 9 106102136 missense probably damaging 1.00
IGL02851:Tlr9 APN 9 106101929 nonsense probably null
Asura UTSW 9 106101846 missense probably damaging 1.00
Cpg1 UTSW 9 106102206 missense probably damaging 1.00
Cpg11 UTSW 9 106101785 missense probably damaging 1.00
Cpg3 UTSW 9 106101351 missense probably damaging 1.00
Cpg5 UTSW 9 106101888 missense probably damaging 1.00
Cpg6 UTSW 9 106103792 missense probably damaging 1.00
cpg7 UTSW 9 106102548 missense probably benign 0.00
Meager UTSW 9 106101338 missense probably damaging 1.00
PIT4498001:Tlr9 UTSW 9 106100721 missense probably benign 0.00
R0058:Tlr9 UTSW 9 106102164 missense possibly damaging 0.90
R0058:Tlr9 UTSW 9 106102164 missense possibly damaging 0.90
R0071:Tlr9 UTSW 9 106100777 missense probably benign
R0071:Tlr9 UTSW 9 106100777 missense probably benign
R0126:Tlr9 UTSW 9 106102881 missense probably benign 0.01
R0165:Tlr9 UTSW 9 106103286 missense probably benign 0.10
R0534:Tlr9 UTSW 9 106102086 missense probably benign 0.01
R0585:Tlr9 UTSW 9 106102275 missense probably benign 0.01
R1527:Tlr9 UTSW 9 106100949 missense probably benign 0.09
R1712:Tlr9 UTSW 9 106101248 missense probably damaging 1.00
R1817:Tlr9 UTSW 9 106102142 missense probably benign
R1940:Tlr9 UTSW 9 106101846 missense probably damaging 1.00
R2117:Tlr9 UTSW 9 106102536 missense probably damaging 1.00
R2656:Tlr9 UTSW 9 106101140 missense probably benign 0.05
R3700:Tlr9 UTSW 9 106101278 missense probably damaging 1.00
R4600:Tlr9 UTSW 9 106101732 missense probably damaging 1.00
R4608:Tlr9 UTSW 9 106102173 missense probably damaging 0.99
R4612:Tlr9 UTSW 9 106101006 missense probably damaging 1.00
R4959:Tlr9 UTSW 9 106101876 missense probably benign
R5173:Tlr9 UTSW 9 106103151 missense possibly damaging 0.49
R5472:Tlr9 UTSW 9 106101512 missense probably damaging 1.00
R5572:Tlr9 UTSW 9 106102836 missense possibly damaging 0.47
R5618:Tlr9 UTSW 9 106101938 missense possibly damaging 0.47
R5820:Tlr9 UTSW 9 106099906 critical splice donor site probably null
R6393:Tlr9 UTSW 9 106102136 missense probably damaging 1.00
R6397:Tlr9 UTSW 9 106102305 missense probably damaging 1.00
R6455:Tlr9 UTSW 9 106101198 missense probably damaging 1.00
R7385:Tlr9 UTSW 9 106102463 missense probably damaging 1.00
R7455:Tlr9 UTSW 9 106101729 missense probably benign 0.00
R7561:Tlr9 UTSW 9 106103148 missense probably benign 0.00
R8889:Tlr9 UTSW 9 106099834 start gained probably benign
R8892:Tlr9 UTSW 9 106099834 start gained probably benign
R8926:Tlr9 UTSW 9 106103213 missense probably benign
R9221:Tlr9 UTSW 9 106101972 missense probably damaging 1.00
R9228:Tlr9 UTSW 9 106102752 missense possibly damaging 0.49
R9581:Tlr9 UTSW 9 106101510 missense probably damaging 1.00
R9689:Tlr9 UTSW 9 106100721 missense probably benign 0.00
R9697:Tlr9 UTSW 9 106100723 nonsense probably null
R9788:Tlr9 UTSW 9 106101006 missense probably damaging 1.00
Z1176:Tlr9 UTSW 9 106100862 missense probably benign 0.03
Mode of Inheritance Autosomal Semidominant
Local Stock Sperm, gDNA
MMRRC Submission 030020-UCD
Last Updated 2016-05-13 3:09 PM by Peter Jurek
Record Created unknown
Record Posted 2007-07-30
Phenotypic Description
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.
980  -Y--V--R--L--R--Q--R--L--C--R--Q-
The mutated nucleotide is indicated in red lettering, and causes a glutamine to leucine substitution at residue 985 of the TLR9 protein.
Illustration of Mutations in
Gene & Protein
Protein Prediction
Figure 3. Protein and domain structure of TLR9. (A) Schematic representation of TLR9 based on crystalized structures of mouse TLR9 LRR (PBD 3WPF) and human TLR2 TIR (1FYW) domains. The residue affected by the CpG2 mutation is highlighted. 3D image was created using UCSF Chimera. (B) TLR9 is a 1032 amino acid protein with an extracellur domain (pink) of leucine rich repeats (LRR), a short transmembrane (TM) domain (blue) and a cytoplasmic Toll/Interleukin-1 receptor (TIR) domain (green). The CpG2 mutation (red asterisk) results in a glutamine to leucine substitution at residue 985 of the TLR9 protein. This image is interactive. Click on the image to view other mutations found in TLR9. Click on each mutation for more specific information.
The CpG2 mutation results in a glutamine to leucine change at position 985 of the TLR9 protein, which lies in α-helix D of the TIR domain. The position of the mutation is shown within the crystal structure of the similar TLR2 TIR domain in Figure 2 (PDB ID 1FYW; see the record for languid). The mutation produces a hypomorphic allele, which may lose the ability to dimerize with TIR domains of other TLR9 subunits, or to bind downstream signaling partners.
Please see the record for CpG1 for information about Tlr9.
Putative Mechanism
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
PCR program
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
The following sequence of 1268 nucleotides (from Genbank genomic region NC_000075 for linear DNA sequence of Tlr9) is amplified:
2983                                               taaaggcc ctgaccaatg
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.
Science Writers Eva Marie Y. Moresco
Illustrators Diantha La Vine
AuthorsNengming Xiao, Carrie N. Arnold, Amanda L. Blasius, Bruce Beutler
Edit History
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