Phenotypic Mutation 'bugsy' (pdf version)
Allele | bugsy |
Mutation Type |
nonsense
|
Chromosome | 4 |
Coordinate | 66,757,491 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 |
Lysine changed to Stop codon
|
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: K95*
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 null
|
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 |
0.9755 |
Is this an essential gene? |
Non Essential (E-score: 0.000) |
Phenotypic Category |
Autosomal Recessive |
Candidate Explorer Status |
loading ... |
Single pedigree Linkage Analysis Data
|
|
Penetrance | |
Alleles Listed at MGI | All mutations/alleles(14) : Chemically induced (ENU)(3) Spontaneous(6) Targeted(5)
|
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 |
Cruyff
|
UTSW |
4 |
66758563 |
missense |
probably damaging |
1.00 |
don_knotts
|
UTSW |
4 |
66759409 |
missense |
probably damaging |
1.00 |
Guardiola
|
UTSW |
4 |
66757540 |
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 Recessive |
Local Stock | Sperm |
MMRRC Submission |
038214-MU
|
Last Updated |
2019-09-04 9:44 PM
by Katherine Timer
|
Record Created |
2015-09-11 4:21 PM
|
Record Posted |
2015-09-22 |
Phenotypic Description |
The bugsy phenotype was identified among N-ethyl-N-nitrosourea (ENU)-mutagenized G3 mice of the pedigree R3011, some of which showed reduced TNFα secretion from macrophages in response to the Toll-like receptor 4 (TLR4) ligand, lipolysaccharide (LPS) (Figure 1) and resistance to LPS-induced macrophage necroptosis (Figure 2).
|
Nature of Mutation |
Whole exome HiSeq sequencing of the G1 grandsire identified 33 mutations. Both of the above phenotypes were linked by continuous variable mapping to a mutation in Tlr4: an A to T transversion at base pair 66,839,254 (v38) on chromosome 4, or base pair 11,444 in the GenBank genomic region NC_000070. The strongest association was found with a recessive model of linkage to the reduced LPS-induced macrophage necroptosis, wherein one variant homozygote departed phenotypically from nine homozygous reference mice and 16 heterozygous mice with a P value of 6.016 x 10-23 (Figure 3). A substantial semidominant effect was observed in the TLR4 signaling assay using gene-based superpedigree analysis of pedigrees R3011 and R0827. The mutation corresponds to residue 564 in the mRNA sequence NM_021297 within exon 3 of 3 total exons.
549 GAAACAATTGAAGACAAGGCATGGCATGGCTTA
90 -E--T--I--E--D--K--A--W--H--G--L-
|
The mutated nucleotide is indicated in red. The mutation results in substitution of lysine (K) 95 to a premature stop codon (K95*) 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 5). The bugsy mutation results in substitution of lysine 95 to a premature stop codon (K95*), which 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 bugsy indicates that the mutation results in loss of TLR4 function.
|
Primers |
PCR Primer
bugsy_pcr_F: AAATCTGCAGAGTTCCTCTCC
bugsy_pcr_R: GTGAGCCACATTGAGTTTCTTTAAG
Sequencing Primer
bugsy_seq_F: TGCTCACACCATCATCACCTG
bugsy_seq_R: TAAGGTTATAAGCTGTCCAATAGGG
|
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 400 nucleotides is amplified (chromosome 4, + strand):
1 aaatctgcag agttcctctc ctgctcacac catcatcacc tgttttgctc tgtacagttt 61 tctctttaca ataacatggt atatcatatc tgtttgtatc atagtatggt agggactgtt 121 atgtcattag aaagggtttt tttttcagca aaaatacata attggtatct cttttgccca 181 taggtgtgaa attgaaacaa ttgaagacaa ggcatggcat ggcttacacc acctctcaaa 241 cttgatactg acaggaaacc ctatccagag tttttcccca ggaagtttct ctggactaac 301 aagtttagag aatctggtgg ctgtggagac aaaattggcc tctctagaaa gcttccctat 361 tggacagctt ataaccttaa agaaactcaa tgtggctcac
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.
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 | Ying Wang, Hexin Shi, Zhao Zhang, Lei Sun, Doan Dao, Bruce Beutler |