Phenotypic Mutation 'milquetoast' (pdf version)
Allele | milquetoast |
Mutation Type |
missense
|
Chromosome | 4 |
Coordinate | 66,757,681 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]
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Accession Number | NCBI RefSeq: NM_021297.2; MGI:96824
|
Mapped | Yes |
Amino Acid Change |
Histidine changed to Leucine
|
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: H158L
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 0.998 (Sensitivity: 0.27; Specificity: 0.99)
(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 |
0.1562 |
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 |
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 |
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 |
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 | |
MMRRC Submission |
038216-MU
|
Last Updated |
2019-09-04 9:44 PM
by Anne Murray
|
Record Created |
2015-09-14 8:30 AM
|
Record Posted |
2015-09-22 |
Phenotypic Description |
The milquetoast phenotype was identified among N-ethyl-N-nitrosourea (ENU)-mutagenized G3 mice of the pedigree R1929 by gene-based superpedigree analysis in which some mice from five pedigrees showed reduced TNFα secretion from macrophages in response to the Toll-like receptor 4 (TLR4) ligand, lipolysaccharide (LPS) (Figure 1).
|
Nature of Mutation |
Whole exome HiSeq sequencing of the R0827 G1 grandsire identified 109 mutations. The reduced TLR4-associated signaling phenotype was linked by continuous variable mapping to a mutation in Tlr4 using gene-based superpedigree analysis: an A to T transversion at base pair 66,839,444 (v38) on chromosome 4, or base pair 11,634 in the GenBank genomic region NC_000070. Linkage was found using an additive model of inheritance, wherein 12 variant homozygotes from five pedigrees exhibiting either null of missense Tlr4 alleles departed phenotypically from 45 homozygous reference mice and 67 heterozygous mice with a P value of 4.878 x 10-5 (Figure 2). The mutation corresponds to residue 754 in the mRNA sequence NM_021297 within exon 3 of 3 total exons.
738 AAACTCAATGTGGCTCACAATTTTATACATTCC
153 -K--L--N--V--A--H--N--F--I--H--S-
|
The mutated nucleotide is indicated in red. The mutation results in a histidine (H) to leucine (L) substitution at position 158 (H158L) in the TLR4 protein, and is strongly predicted by PolyPhen-2 to be damaging (score = 0.998).
|
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 3). The milquetoast mutation results in a histidine (H) to leucine (L) substitution at position 158 (H158L) in the TLR4 protein. H158 is within LRR5. 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 Milquetoast indicates that the mutation results in loss of TLR4 function.
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Primers |
PCR Primer
milquetoast_pcr_F: ATGGCATGGCTTACACCACC
milquetoast_pcr_R: AGTTCATGGAGCTTAATTCCCTG
Sequencing Primer
milquetoast_seq_F: CCTCTCAAACTTGATACTGACAGG
milquetoast_seq_R: CATGGAGCTTAATTCCCTGAAAGGC
|
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 402 nucleotides is amplified (chromosome 4, + strand):
1 atggcatggc ttacaccacc tctcaaactt gatactgaca ggaaacccta tccagagttt 61 ttccccagga agtttctctg gactaacaag tttagagaat ctggtggctg tggagacaaa 121 attggcctct ctagaaagct tccctattgg acagcttata accttaaaga aactcaatgt 181 ggctcacaat tttatacatt cctgtaagtt acctgcatat ttttccaatc tgacgaacct 241 agtacatgtg gatctttctt ataactatat tcaaactatt actgtcaacg acttacagtt 301 tctacgtgaa aatccacaag tcaatctctc tttagacatg tctttgaacc caattgactt 361 cattcaagac caagcctttc agggaattaa gctccatgaa ct
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 | Ying Wang, Bruce Beutler |