Phenotypic Mutation 'Cpg11' (pdf version)
AlleleCpg11
Mutation Type missense
Chromosome9
Coordinate106,101,785 bp (GRCm39)
Base Change T ⇒ C (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; UniProt: Q9EQU3

MappedYes 
Amino Acid Change Serine changed to Proline
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: S359P

DomainStartEndE-ValueType
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 0.999 (Sensitivity: 0.14; Specificity: 0.99)
(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 unknown 
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
Cpg2 UTSW 9 106103664 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 034329-JAX
Last Updated 2021-10-28 10:55 AM by Stephen Lyon
Record Created 2010-04-20 11:48 AM by Hua Huang
Record Posted 2010-04-29
Phenotypic Description

Figure 2. CpG-A-induced activation marker expression is normal in pDC harboring the CpG11 allele. pDC (CD11c+ B220+ CD11b-) were sorted from bone marrow cells cultured in the presence of Flt3L for seven days and then stimulated overnight with CpG-A (1 μM). Cell surface marker expression was analyzed by flow cytometry. Treatments were prepared in duplicate wells and shown as overlays of red and blue in each flow cytometry plot. Activation marker expression was not reduced in CpG11 homozygous mutants after CpG-A stimulation. The positive (B6: C57BL/6J) and negative controls (Unc93: Unc93b13d/3d) responded as expected.
Figure 3. CpG-A-induced IFNβ production in pDC harboring the CpG11 allele. pDC (CD11c+ B220+ CD11b-) were sorted from bone marrow cells cultured in the presence of Flt3L for seven days and then stimulated overnight with CpG-A (1 μM). IFN-β was measured in the culture supernatants by ELISA. Treatments were prepared in duplicate wells. Tlr9CpG11/CpG11 pDC exhibited an intact IFN-β response to CpG-A stimulation although the quantity of IFN-β produced was reduced compared to WT pDC. The IFN-β response to polyU (TLR7 agonist) was normal in Tlr9CpG11/CpG11 pDC. The positive (B6: C57BL/6J) and negative controls (Unc93b1 3d: Unc93b13d/3d) responded as expected.

The CpG11 phenotype was identified in a screen for ENU-induced homozygous mutants with impaired responses to Toll-like receptor (TLR) ligands (TLR Signaling Screen). Peritoneal macrophages from the index CpG11 mouse (G4562) produced normal amounts of tumor necrosis factor (TNF)-α in response to all TLR ligands tested, except oligodeoxynucleotides containing CpG motifs (CpG). CpG11 heterozygous macrophages produced intermediate levels of TNF-α relative to wild type and CpG11 homozygous macrophages in response to type B CpG (CpG-B), demonstrating that the mutation is semidominant in this assay (Figure 1). In response to CpG-A stimulation, bone marrow-derived plasmacytoid dendritic cells (pDC) from CpG11 homozygous mice upregulated the activation markers MHC-II, CD69, and CD86 similarly to pDC from WT mice (Figure 2)  Moreover, interferon-β (IFN-β) production by Tlr9CpG11/CpG11 pDC in response to CpG-A was only slightly reduced compared to that of WT pDC (Figure 3). B cells from CpG11 homozygous mice also responded normally to CpG-B stimulation.

Nature of Mutation
The candidate gene Tlr9 was sequenced directly, and a T to C transition was identified at position 1181, in exon 2 of 2 total exons.
1166 CTCCACCTGGCAAGTTCCTTTAAGAACCTGGTG
353  -L--H--L--A--S--S--F--K--N--L--V-
The mutated nucleotide is indicated in red lettering, and results in a serine to proline substitution at amino acid 359 of the TLR9 protein.
 
Please see the record for CpG1 for more information about Tlr9.
Illustration of Mutations in
Gene & Protein
Protein Prediction
Figure 4. 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 CpG11 mutation is shown in red. 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 domain and an cytoplasmic Toll/Interleukin-1 receptor (TIR) domain. The CpG11 mutation (red asterisk) results in a serine to proline substitution at amino acid 359 of the TLR9 protein in the predicted eleventh leucine rich repeat (LRR). This image is interactive. Click on the image to view other mutations found in TLR9 (red). Click on the mutations for more specific information.

The extracellular domains of TLRs contain multiple leucine rich repeats (LRRs) that mediate ligand recognition by TLRs. TLR9 has 25 LRRs in its ectodomain. The CpG11 mutation is located in the predicted eleventh leucine rich repeat (LRR) of the TLR9 ectodomain (Figure 4).

Putative Mechanism

Based on the 3D structure of TLR9 (1), the serine mutated in CpG11 lies in the surface-exposed α-helical sequence of the affected LRR, at a position thought to permit occupancy by any amino acid, but adjacent to an invariant phenylalanine. The CpG11 phenotype suggests that TLR9S359P is hypomorphic, and cell type-dependent dissimilarities in cellular responses may reflect differences in the endosomal compartments where TLR9 encounters its ligand.

Primers Primers cannot be located by automatic search.
Genotyping
CpG11 genotyping is performed by amplifying the region containing the mutation using PCR, followed by sequencing of the amplified region to detect the single nucleotide insertion.
 
Primers
CpG11 (F): 5’- CATGGACGGGAACTGCTACTACAAG -3’
CpG11(R):  5’- ATGAAGTTCTTAGAAGCAGGGGTGC -3’
 
PCR program
1) 95°C             2:00
2) 95°C             0:30
3) 56°C             0:30
4) 72°C             1:00
5) repeat steps (2-4) 29X
6) 72°C             7:00
7) 4°C              ∞
 
Primers for sequencing
CpG11_seq(F): 5’- TGAGCAATCTCACCCATCTG -3’
CpG11_seq(R): 5’- GACAACAGCTCCTCCTGCTC -3’
 
The following sequence of 885 nucleotides (from Genbank genomic region NC_­­­­­000075 for linear genomic sequence of Tlr9, sense strand) is amplified:
1433                                                          catggacg
1441 ggaactgcta ctacaagaac ccctgcacag gagcggtgaa ggtgacccca ggcgccctcc
1501 tgggcctgag caatctcacc catctgtctc tgaagtataa caacctcaca aaggtgcccc
1561 gccaactgcc ccccagcctg gagtacctcc tggtgtccta taacctcatt gtcaagctgg
1621 ggcctgaaga cctggccaat ctgacctccc ttcgagtact tgatgtgggt gggaattgcc
1681 gtcgctgtga ccatgccccc aatccctgta tagaatgtgg ccaaaagtcc ctccacctgc
1741 accctgagac cttccatcac ctgagccatc tggaaggcct ggtgctgaag gacagctctc
1801 tccatacact gaactcttcc 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 acttcat
Primer binding sites are underlined; sequencing primer binding sites are highlighted in gray; the mutated T is indicated in red.
References
Science Writers Eva Marie Y. Moresco
Illustrators Diantha La Vine
AuthorsHua Huang, Ivo Rimann, Parker Mace, Argyrios N. Theofilopoulos, Dwight Kono, Bruce Beutler
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