Phenotypic Mutation 'Cruyff' (pdf version)
AlleleCruyff
Mutation Type missense
Chromosome4
Coordinate66,758,563 bp (GRCm39)
Base Change T ⇒ A (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

MappedYes 
Amino Acid Change Isoleucine changed to Asparagine
Institutional SourceBeutler 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: I452N

DomainStartEndE-ValueType
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

DomainStartEndE-ValueType
PDB:3VQ2|B 22 86 2e-38 PDB
SCOP:d1m0za_ 27 86 4e-6 SMART
Predicted Effect probably benign
Meta Mutation Damage Score 0.9014 question?
Is this an essential gene? Non Essential (E-score: 0.000) question?
Phenotypic Category Autosomal Recessive
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
AlleleSourceChrCoordTypePredicted EffectPPH 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
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
Repository
Last Updated 2020-07-29 6:45 PM by External Program
Record Created 2016-02-26 6:40 AM by Cristhiaan D. Ochoa
Record Posted 2016-04-28
Phenotypic Description
Figure 1. Cruyff mice exhibited reduced TNFα secretion in response to the TLR4 ligand, LPS. TNFα levels were determined by ELISA. Normalized data are shown. Abbreviations: WT, wild-type; REF, homozygous reference mice; HET, heterozygous variant mice; VAR, homozygous variant mice. Mean (μ) and standard deviation (σ) are indicated.
Figure 2. Cruyff mice exhibited resistance to macrophage necroptosis in response to the TLR4 ligand, LPS. Normalized data are shown. Abbreviations: WT, wild-type; REF, homozygous reference mice; HET, heterozygous variant mice; VAR, homozygous variant mice. Mean (μ) and standard deviation (σ) are indicated.
Figure 3. Cruyff mice secreted decreased amounts of IL-1β in response to priming with lipopolysaccharide (LPS) followed by nigericin treatment. IL-1β levels were determined by ELISA. Normalized data are shown. Abbreviations: WT, wild-type; REF, homozygous reference mice; HET, heterozygous variant mice; VAR, homozygous variant mice. Mean (μ) and standard deviation (σ) are indicated.

The cruyff phenotype was identified among N-ethyl-N-nitrosourea (ENU)-mutagenized G3 mice of the pedigree R4213, 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). Some mice also exhibited attenuated inflammatory responses related to decreased secretion of the proinflammatory cytokine interleukin (IL)-1β in response to priming with lipopolysaccharide (LPS) followed by nigericin treatment (Figure 3).

Nature of Mutation
Figure 4. Linkage mapping of the increased LPS-induced necroptosis of macrophage cells using a recessive model of inheritance. Manhattan plot shows -log10 P values (Y-axis) plotted against the chromosome positions of 37 mutations identified in the G1 male of pedigree R4213 (X-axis). Normalized phenotype data are shown for single locus linkage analysis with consideration of G2 dam identity.  Horizontal pink and red lines represent thresholds of P = 0.05, and the threshold for P = 0.05 after applying Bonferroni correction, respectively.

Whole exome HiSeq sequencing of the G1 grandsire identified 37 mutations. All of the above phenotypes were linked by continuous variable mapping to a mutation in Tlr4:  a T to A transversion at base pair 66,840,326 (v38) on chromosome 4, or base pair 12,516 in the GenBank genomic region NC_000070. The strongest association was found with a recessive model of linkage to the resistance to LPS-induced macrophage necroptosis, wherein 9 variant homozygotes departed phenotypically from 4 homozygous reference mice and 12 heterozygous mice with a P value of 1.859 x 10-19 (Figure 4).  

The mutation corresponds to residue 1,636 in the mRNA sequence NM_021297 within exon 3 of 3 total exons.

 

1620 CTACTTTACCTTGACATCTCTTATACTAACACC

447  -L--L--Y--L--D--I--S--Y--T--N--T-

The mutated nucleotide is indicated in red.  The mutation results in an isoleucine (I) to asparagine (N) substitution at position 452 (I452N) in the TLR4 protein, and is strongly predicted by PolyPhen-2 to be damaging (score = 1.000).

Illustration of Mutations in
Gene & Protein
Protein Prediction

Figure 4. Protein and domain structure of TLR4. A) Schematic representation of TLR9 based on crystalized structures of mouse TLR3 LRR (PBD 3CIG) and human TLR2 TIR (1FYW) domains. The residue affected by the Lps3 mutation is highlighted. 3D image was created using UCSF Chimera. B) TLR4 is an 835 amino acid protein with an extracellur domain (pink) of leucine rich repeats (LRR), a short transmembrane domain and a cytoplasmic Toll/Interleukin-1 receptor (TIR) domain. The cruyff mutation (red asterisk) results in an esults in substitution of isoleucine (I) 425 to an asparagine (I452N) in the TLR4 protein. This image is interactive. Click on the image to view other mutations found in TLR4 (red). Click on the mutations for more specific information.

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 cruyff mutation results in substitution of isoleucine 452 to an asparagine (I452N); amino acid 452 is located in LRR16.

 
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 cruyff indicates that the mutation results in loss of TLR4 function.

Primers PCR Primer
Cruyff_pcr_F: GTAGAAATGCACTGAGCTTTAGTGG
Cruyff_pcr_R: CCCCAAGATATTTGTTCCAATTGAC

Sequencing Primer
Cruyff_seq_F: GCTGTTCTTATTCTGATTTGGGAAC
Cruyff_seq_R: CCAATTGACATTTAGAAAGATCCAGG
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   gtagaaatgc actgagcttt agtggttgct gttcttattc tgatttggga acaaacagcc
61  tgagacactt agacctcagc ttcaatggtg ccatcattat gagtgccaat ttcatgggtc
121 tagaagagct gcagcacctg gattttcagc actctacttt aaaaagggtc acagaattct
181 cagcgttctt atcccttgaa aagctacttt accttgacat ctcttatact aacaccaaaa
241 ttgacttcga tggtatattt cttggcttga ccagtctcaa cacattaaaa atggctggca
301 attctttcaa agacaacacc ctttcaaatg tctttgcaaa cacaacaaac ttgacattcc
361 tggatctttc taaatgtcaa ttggaacaaa tatcttgggg 


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
AuthorsCristhiaan D. Ochoa, Ying Wang, Hexin Shi, and Bruce Beutler