Phenotypic Mutation 'Guardiola' (pdf version)
Allele | Guardiola |
Mutation Type |
missense
|
Chromosome | 4 |
Coordinate | 66,757,540 bp (GRCm39) |
Base Change | A ⇒ T (forward strand) |
Gene |
Tlr4
|
Gene Name | toll-like receptor 4 |
Synonym(s) | Lps, lipopolysaccharide response, Rasl2-8 |
Chromosomal Location |
66,745,788-66,765,338 bp (+) (GRCm39)
|
MGI Phenotype |
FUNCTION: This gene belongs to the evolutionarily-conserved Toll-like receptor family, whose members are type-1 transmembrane proteins that are involved in innate immunity. Toll-like receptors are characterized by an extracellular leucine-rich repeat domain that functions in ligand recognition and an intracellular toll/interleukin-1 receptor-like domain that is crucial for signal transduction. The receptor encoded by this gene mediates the innate immune response to bacterial lipopolysaccharide, a major component of the outer membrane of Gram-negative bacteria, through synthesis of pro-inflammatory cytokines and chemokines. In addition, this protein can recognize other pathogens from Gram-negative and Gram-positive bacteria as well as viral components. Mice deficient in this gene display a number of immune response-related phenotypes including hyporesponsiveness to bacterial lipopolysaccharide and increased levels of respiratory syncytial virus compared to controls. [provided by RefSeq, Sep 2015] PHENOTYPE: Homozygotes for spontaneous or targeted mutations are hyporesponsive to bacterial lipopolysaccharide and more susceptible to infection by gram negative bacteria. [provided by MGI curators]
|
Accession Number | NCBI RefSeq: NM_021297.2; MGI:96824
|
Mapped | Yes |
Amino Acid Change |
Asparagine changed to Isoleucine
|
Institutional Source | Beutler Lab |
Gene Model |
predicted gene model for protein(s):
[ENSMUSP00000045770]
[ENSMUSP00000102988]
|
AlphaFold |
Q9QUK6 |
PDB Structure |
Crystal structure of mouse TLR4 and mouse MD-2 complex [X-RAY DIFFRACTION]
Crystal structure of mouse TLR4/MD-2/lipid IVa complex [X-RAY DIFFRACTION]
Crystal structure of mouse TLR4/MD-2/LPS complex [X-RAY DIFFRACTION]
|
SMART Domains |
Protein: ENSMUSP00000045770 Gene: ENSMUSG00000039005 AA Change: N111I
Domain | Start | End | E-Value | Type |
LRR
|
76 |
99 |
7.36e0 |
SMART |
LRR
|
100 |
123 |
1.86e0 |
SMART |
LRR
|
173 |
196 |
8.24e0 |
SMART |
LRR
|
370 |
401 |
4.33e1 |
SMART |
LRR
|
468 |
492 |
2.54e2 |
SMART |
LRR
|
493 |
516 |
1.86e2 |
SMART |
LRR
|
517 |
540 |
1.67e2 |
SMART |
LRR
|
541 |
563 |
1.92e2 |
SMART |
LRRCT
|
576 |
626 |
4.74e-3 |
SMART |
transmembrane domain
|
636 |
658 |
N/A |
INTRINSIC |
TIR
|
671 |
816 |
7.3e-39 |
SMART |
low complexity region
|
822 |
833 |
N/A |
INTRINSIC |
|
Predicted Effect |
probably damaging
PolyPhen 2
Score 1.000 (Sensitivity: 0.00; Specificity: 1.00)
(Using ENSMUST00000048096)
|
SMART Domains |
Protein: ENSMUSP00000102988 Gene: ENSMUSG00000039005
Domain | Start | End | E-Value | Type |
PDB:3VQ2|B
|
22 |
86 |
2e-38 |
PDB |
SCOP:d1m0za_
|
27 |
86 |
4e-6 |
SMART |
|
Predicted Effect |
probably benign
|
Meta Mutation Damage Score |
0.9569 |
Is this an essential gene? |
Non Essential (E-score: 0.000) |
Phenotypic Category |
Autosomal Semidominant |
Candidate Explorer Status |
loading ... |
Single pedigree Linkage Analysis Data
|
|
Penetrance | |
Alleles Listed at MGI | All Mutations and Alleles(18) : Chemically induced (ENU)(6) Spontaneous(6) Targeted(6)
|
Lab Alleles |
Allele | Source | Chr | Coord | Type | Predicted Effect | PPH Score |
IGL01120:Tlr4
|
APN |
4 |
66758662 |
missense |
probably benign |
0.01 |
IGL01343:Tlr4
|
APN |
4 |
66752124 |
splice site |
probably benign |
|
IGL01669:Tlr4
|
APN |
4 |
66759504 |
missense |
possibly damaging |
0.48 |
IGL01875:Tlr4
|
APN |
4 |
66757726 |
missense |
probably damaging |
1.00 |
IGL02138:Tlr4
|
APN |
4 |
66759202 |
missense |
probably damaging |
0.99 |
IGL02244:Tlr4
|
APN |
4 |
66752298 |
critical splice donor site |
probably null |
|
IGL02793:Tlr4
|
APN |
4 |
66757681 |
missense |
probably damaging |
1.00 |
IGL03269:Tlr4
|
APN |
4 |
66759033 |
missense |
probably damaging |
1.00 |
IGL03288:Tlr4
|
APN |
4 |
66757990 |
missense |
probably damaging |
0.99 |
bugsy
|
UTSW |
4 |
66757491 |
nonsense |
probably null |
|
Cruyff
|
UTSW |
4 |
66758563 |
missense |
probably damaging |
1.00 |
don_knotts
|
UTSW |
4 |
66759409 |
missense |
probably damaging |
1.00 |
Lops
|
UTSW |
4 |
66752117 |
splice site |
probably null |
|
lps3
|
UTSW |
4 |
66759334 |
missense |
probably damaging |
1.00 |
Lps4
|
UTSW |
4 |
66759379 |
missense |
probably damaging |
1.00 |
milquetoast
|
UTSW |
4 |
66757681 |
missense |
probably damaging |
1.00 |
salvador
|
UTSW |
4 |
66758443 |
missense |
probably damaging |
0.99 |
R0449:Tlr4
|
UTSW |
4 |
66757857 |
missense |
probably damaging |
0.99 |
R0481:Tlr4
|
UTSW |
4 |
66746153 |
missense |
probably benign |
0.05 |
R0576:Tlr4
|
UTSW |
4 |
66757732 |
missense |
probably benign |
0.00 |
R0827:Tlr4
|
UTSW |
4 |
66752117 |
splice site |
probably null |
|
R1488:Tlr4
|
UTSW |
4 |
66757786 |
missense |
probably damaging |
1.00 |
R1490:Tlr4
|
UTSW |
4 |
66757611 |
missense |
possibly damaging |
0.56 |
R1522:Tlr4
|
UTSW |
4 |
66757933 |
missense |
possibly damaging |
0.80 |
R1616:Tlr4
|
UTSW |
4 |
66757717 |
missense |
probably damaging |
1.00 |
R1681:Tlr4
|
UTSW |
4 |
66759342 |
missense |
probably damaging |
1.00 |
R1738:Tlr4
|
UTSW |
4 |
66759313 |
missense |
probably benign |
0.19 |
R1888:Tlr4
|
UTSW |
4 |
66759409 |
missense |
probably damaging |
1.00 |
R1888:Tlr4
|
UTSW |
4 |
66759409 |
missense |
probably damaging |
1.00 |
R1929:Tlr4
|
UTSW |
4 |
66757681 |
missense |
probably damaging |
1.00 |
R1982:Tlr4
|
UTSW |
4 |
66759272 |
missense |
probably benign |
0.40 |
R1998:Tlr4
|
UTSW |
4 |
66758707 |
missense |
probably damaging |
1.00 |
R2186:Tlr4
|
UTSW |
4 |
66758220 |
missense |
possibly damaging |
0.63 |
R2305:Tlr4
|
UTSW |
4 |
66758338 |
missense |
probably damaging |
1.00 |
R3011:Tlr4
|
UTSW |
4 |
66757491 |
nonsense |
probably null |
|
R3420:Tlr4
|
UTSW |
4 |
66757773 |
missense |
probably benign |
0.37 |
R3422:Tlr4
|
UTSW |
4 |
66757773 |
missense |
probably benign |
0.37 |
R3818:Tlr4
|
UTSW |
4 |
66759553 |
missense |
probably benign |
0.00 |
R4212:Tlr4
|
UTSW |
4 |
66758563 |
missense |
probably damaging |
1.00 |
R4213:Tlr4
|
UTSW |
4 |
66758563 |
missense |
probably damaging |
1.00 |
R4417:Tlr4
|
UTSW |
4 |
66757540 |
missense |
probably damaging |
1.00 |
R4630:Tlr4
|
UTSW |
4 |
66757477 |
missense |
probably benign |
0.44 |
R4735:Tlr4
|
UTSW |
4 |
66759435 |
missense |
probably damaging |
1.00 |
R5191:Tlr4
|
UTSW |
4 |
66759616 |
missense |
probably damaging |
0.96 |
R5613:Tlr4
|
UTSW |
4 |
66759122 |
missense |
possibly damaging |
0.94 |
R5705:Tlr4
|
UTSW |
4 |
66752217 |
missense |
probably damaging |
1.00 |
R5726:Tlr4
|
UTSW |
4 |
66758652 |
missense |
probably benign |
|
R6021:Tlr4
|
UTSW |
4 |
66759103 |
missense |
probably damaging |
1.00 |
R6159:Tlr4
|
UTSW |
4 |
66758070 |
missense |
possibly damaging |
0.92 |
R6227:Tlr4
|
UTSW |
4 |
66758832 |
missense |
probably benign |
|
R7139:Tlr4
|
UTSW |
4 |
66758520 |
missense |
probably benign |
0.06 |
R7199:Tlr4
|
UTSW |
4 |
66759430 |
missense |
probably damaging |
0.99 |
R7220:Tlr4
|
UTSW |
4 |
66758188 |
missense |
probably benign |
|
R7337:Tlr4
|
UTSW |
4 |
66758191 |
missense |
possibly damaging |
0.86 |
R7487:Tlr4
|
UTSW |
4 |
66842659 |
missense |
probably benign |
0.00 |
R7638:Tlr4
|
UTSW |
4 |
66758443 |
missense |
probably damaging |
0.99 |
R7773:Tlr4
|
UTSW |
4 |
66757836 |
missense |
probably damaging |
1.00 |
R7814:Tlr4
|
UTSW |
4 |
66759316 |
missense |
probably damaging |
1.00 |
R7897:Tlr4
|
UTSW |
4 |
66758058 |
missense |
probably benign |
0.07 |
R8044:Tlr4
|
UTSW |
4 |
66746084 |
missense |
probably benign |
0.01 |
R8062:Tlr4
|
UTSW |
4 |
66758087 |
missense |
probably benign |
0.00 |
R8080:Tlr4
|
UTSW |
4 |
66757713 |
missense |
probably damaging |
1.00 |
R8446:Tlr4
|
UTSW |
4 |
66757673 |
missense |
probably damaging |
0.98 |
R8916:Tlr4
|
UTSW |
4 |
66847268 |
missense |
probably benign |
0.06 |
R9100:Tlr4
|
UTSW |
4 |
66758518 |
missense |
probably benign |
0.08 |
R9415:Tlr4
|
UTSW |
4 |
66746160 |
critical splice donor site |
probably null |
|
R9562:Tlr4
|
UTSW |
4 |
66759522 |
missense |
possibly damaging |
0.80 |
R9565:Tlr4
|
UTSW |
4 |
66759522 |
missense |
possibly damaging |
0.80 |
R9752:Tlr4
|
UTSW |
4 |
66757912 |
missense |
probably benign |
0.02 |
X0064:Tlr4
|
UTSW |
4 |
66758377 |
missense |
probably damaging |
0.99 |
Z1088:Tlr4
|
UTSW |
4 |
66847319 |
missense |
probably benign |
0.01 |
|
Mode of Inheritance |
Autosomal Semidominant |
Local Stock | |
Repository | |
Last Updated |
2019-09-04 9:43 PM
by Katherine Timer
|
Record Created |
2016-03-03 8:19 AM
|
Record Posted |
2016-05-06 |
Phenotypic Description |
The Guardiola phenotype was identified among N-ethyl-N-nitrosourea (ENU)-mutagenized G3 mice of the pedigree R4417, some of which showed reduced TNFα secretion from macrophages in response to the Toll-like receptor 4 (TLR4) ligand, lipolysaccharide (LPS) (Figure 1) and attenuated inflammatory responses related to decreased secretion of the proinflammatory cytokine interleukin (IL)-1β in response to priming with lipopolysaccharide (LPS) followed by flagellin treatment (Figure 2).
|
Nature of Mutation |
Whole exome HiSeq sequencing of the G1 grandsire identified 50 mutations. Both of the above anomalies were linked by continuous variable mapping to a mutation in Tlr4: an A to T transversion at base pair 66,839,303 (v38) on chromosome 4, or base pair 11,493 in the GenBank genomic region NC_000070 for the Tlr4 gene. The strongest association was found with an additive model of linkage to the normalized response to LPS, wherein three variant homozygotes and 10 heterozygotes departed phenotypically from four homozygous reference mice with a P value of 8.88 x 10-8 (Figure 3). The mutation corresponds to residue 613 in the mRNA sequence NM_021297.2 within exon 3 of 3 total exons.
597 TTGATACTGACAGGAAACCCTATCCAGAGTTTT
106 -L--I--L--T--G--N--P--I--Q--S--F-
|
The mutated nucleotide is indicated in red. The mutation results in an asparagine (N) to isoleucine (I) substitution at position 111 (N111I) in the TLR4 protein.
|
Illustration of Mutations in
Gene & Protein |
|
---|
Protein Prediction |
TLR4 is a type I integral membrane glycoprotein containing 835 amino acids. TLR4 has 22 predicted leucine-rich repeats (LRRs) in its ectodomain at the N-terminal half of the protein (1-3), a transmembrane domain, and a cytoplasmic Toll/IL-1R (TIR) domain (Figure 4). The Guardiola mutation results in substitution of asparagine 111 to an isoleucine (N111I); amino acid 111 is located in LRR3. Please see the record for lps3 for information about Tlr4.
|
Putative Mechanism | TLR4 is the receptor for LPS (4). Stimulation of TLR4 by LPS activates two branches of signaling, one defined by early NF-κB activation (MyD88-dependent pathway, mediated by MyD88), and another distinguished by late NF-κB activation as well as interferon responsive factor (IRF)-3 activation leading to type I IFN production and costimulatory molecule upregulation (MyD88-independent pathway, mediated by Trif) (5-7). The MyD88-dependent pathway activates expression of target genes including interleukin (IL)-6, IL-1, TNF, IL-12p40 and type I interferon (IFN), cytokines required for the inflammatory response. The MyD88-independent pathway results in the production of type I IFN. The reduction in TLR4-associated responses in Guardiola indicates that the mutation results in loss of TLR4 function.
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Primers |
PCR Primer
Guardiola_pcr_F: AATCTGCAGAGTTCCTCTCCTG
Guardiola_pcr_R: CCACATGTACTAGGTTCGTCAG
Sequencing Primer
Guardiola_seq_F: ATCACCTGTTTTGCTCTGTACAG
Guardiola_seq_R: CACATGTACTAGGTTCGTCAGATTGG
|
Genotyping | 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 10:00 7) 4°C hold
The following sequence of 463 nucleotides is amplified (chromosome 4, + strand):
1 aatctgcaga gttcctctcc tgctcacacc atcatcacct gttttgctct gtacagtttt 61 ctctttacaa taacatggta tatcatatct gtttgtatca tagtatggta gggactgtta 121 tgtcattaga aagggttttt ttttcagcaa aaatacataa ttggtatctc ttttgcccat 181 aggtgtgaaa ttgaaacaat tgaagacaag gcatggcatg gcttacacca cctctcaaac 241 ttgatactga caggaaaccc tatccagagt ttttccccag gaagtttctc tggactaaca 301 agtttagaga atctggtggc tgtggagaca aaattggcct ctctagaaag cttccctatt 361 ggacagctta taaccttaaa gaaactcaat gtggctcaca attttataca ttcctgtaag 421 ttacctgcat atttttccaa tctgacgaac ctagtacatg tgg
Primer binding sites are underlined and the sequencing primers are highlighted; the mutated nucleotide is shown in red. |
References | 1. Rock, F. L., Hardiman, G., Timans, J. C., Kastelein, R. A., and Bazan, J. F. (1998) A Family of Human Receptors Structurally Related to Drosophila Toll. Proc Natl Acad Sci U S A. 95, 588-593.
2. Medzhitov, R., Preston-Hurlburt, P., and Janeway, C. A.,Jr. (1997) A Human Homologue of the Drosophila Toll Protein Signals Activation of Adaptive Immunity. Nature. 388, 394-397.
3. Bell, J. K., Mullen, G. E., Leifer, C. A., Mazzoni, A., Davies, D. R., and Segal, D. M. (2003) Leucine-Rich Repeats and Pathogen Recognition in Toll-Like Receptors. Trends Immunol. 24, 528-533.
4. Poltorak, A., He, X., Smirnova, I., Liu, M. -., Van Huffel, C., Du, X., Birdwell, D., Alejos, E., Silva, M., Galanos, C., Freudenberg, M. A., Ricciardi-Castagnoli, P., Layton, B., and Beutler, B. (1998) Defective LPS Signaling in C3H/HeJ and C57BL/10ScCr Mice: Mutations in Tlr4 gene. Science. 282, 2085-2088.
5. Kawai, T., Adachi, O., Ogawa, T., Takeda, K., and Akira, S. (1999) Unresponsiveness of MyD88-Deficient Mice to Endotoxin. Immunity. 11, 115-122.
6. Hoshino, K., Kaisho, T., Iwabe, T., Takeuchi, O., and Akira, S. (2002) Differential Involvement of IFN-Beta in Toll-Like Receptor-Stimulated Dendritic Cell Activation. Int Immunol. 14, 1225-1231.
7. Kawai, T., Takeuchi, O., Fujita, T., Inoue, J., Muhlradt, P. F., Sato, S., Hoshino, K., and Akira, S. (2001) Lipopolysaccharide Stimulates the MyD88-Independent Pathway and Results in Activation of IFN-Regulatory Factor 3 and the Expression of a Subset of Lipopolysaccharide-Inducible Genes. J Immunol. 167, 5887-5894.
|
Science Writers | Anne Murray |
Illustrators | Peter Jurek |
Authors | Cristhiaan D. Ochoa, Ying Wang, and Bruce Beutler |