|Mutation Type||splice acceptor site (14 bp from exon)|
|Coordinate||66,833,880 bp (GRCm38)|
|Base Change||T ⇒ A (forward strand)|
|Gene Name||toll-like receptor 4|
|Synonym(s)||Rasl2-8, Lps, lipopolysaccharide response|
|Chromosomal Location||66,827,584-66,930,284 bp (+)|
|MGI Phenotype||Homozygotes for spontaneous or targeted mutations are hyporesponsive to bacterial lipopolysaccharide and more susceptible to infection by gram negative bacteria.|
|Amino Acid Change|
|Institutional Source||Beutler Lab|
|Gene Model||predicted gene model for protein(s): [ENSMUSP00000045770] [ENSMUSP00000102988]|
|Predicted Effect||probably null|
|Predicted Effect||probably null|
|Phenotypic Category||Autosomal Semidominant|
|Alleles Listed at MGI|
|Mode of Inheritance||Autosomal Semidominant|
|Last Updated||2016-12-09 10:25 AM by Katherine Timer|
|Record Created||2014-08-31 6:27 PM by Zhao Zhang|
The Lops phenotype was identified among G3 mice of the pedigree R0827, some of which exhibited decreased TNFα secretion in response to Toll-like receptor 4 (TLR4) ligand LPS (Figure 1) and impaired peritoneal macrophage inflammatory responses (i.e., decreased secretion of the proinflammatory cytokine interleukin (IL)-1β) following exposure to lipopolysaccharide (LPS) and aluminum adjuvants (Figure 2).
|Nature of Mutation|
Whole exome HiSeq sequencing of the G1 grandsire identified 86 mutations. Both of the above anomalies were linked by continuous variable mapping to a mutation in Tlr4: a T to A transversion at base pair 66,833,880 (v38) on chromosome 4, or base pair 6,070 in the GenBank genomic region NC_000070 encoding Tlr4. The strongest association was found with an additive model of linkage to the normalized amount of TNFα secretion in response to LPS, wherein 11 heterozygotes and 2 homozygous variant mice departed phenotypically from 6 homozygous reference mice [P = 1.355 x 10-5; Figure 3]. The mutation lies in the splice acceptor site of intron 1 of the 3 exon gene, and affects a thymine base 14 nucleotides from the next exon. One possibility, shown below, is that aberrant splicing may result in skipping of the 167 base pair exon 2, utilization of the acceptor splice site from intron 2, and splicing of exon 1 to exon 3. Deletion of exon 2 would result in a frame-shift and coding of one aberrant amino acid followed by a premature stop codon (the second abnormal codon after exon 1).
Genomic numbering corresponds to NC_000070. The acceptor splice site of intron 1, which is destroyed by the mutation, is indicated in blue and the mutated nucleotide is indicated in red.
Real-time PCR using three pairs of primers spanning exons 1 and 2 was performed to examine the expression of Tlr4 in Lops mice. Tlr4 cDNA levels in homozygous Lops mice were reduced compared to wild-type and heterozygous mice (Figure 4), suggesting that the Lops mutation negatively affects the function of the intron 1 acceptor splice site. It remains possible that a cryptic donor splice site in the 5’ UTR is used for splicing to the intron 1 donor splice site, but the resulting transcript is degraded by the nonsense mediated decay pathway. Reduced expression of TLR4 may account for the observed phenotype.
TLR4 has 22 predicted 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 Lops mutation likely results in abnormal splicing of Tlr4 and may cause an internal deletion of amino acids 31-86 in the extracellular domain of TLR4 (affecting LRR1 and LRR2), coding of one aberrant amino acid followed by a premature stop codon at position 32 of the 835 amino acid protein.
Please see the record for lps3 for information about Tlr4.
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 Lops indicates that the mutation results in loss of TLR4 function.
Lops(F):5'- GAATGGCAGGTATTTTGGGAGTCCAAT -3'
Lops(R):5'- TTCTTATCTAGTGGCCCATCCAACTGA -3'
Lops_seq(F):5'- GTATAGCTAATTTAAGGCCAGACTG -3'
Lops_seq(R):5'- CAGCAAGTTGTAGCACTACTG -3'
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|
|Authors||Zhao Zhang, Ying Wang, Hexin Shi, Bruce Beutler|