Phenotypic Mutation 'Consequential' (pdf version)
AlleleConsequential
Mutation Type missense
Chromosome18
Coordinate46,693,846 bp (GRCm39)
Base Change T ⇒ C (forward strand)
Gene Ticam2
Gene Name TIR domain containing adaptor molecule 2
Synonym(s) TRAM, Tirp
Chromosomal Location 46,691,298-46,707,600 bp (-) (GRCm39)
MGI Phenotype FUNCTION: [Summary is not available for the mouse gene. This summary is for the human ortholog.] TIRP is a Toll/interleukin-1 receptor (IL1R; MIM 147810) (TIR) domain-containing adaptor protein involved in Toll receptor signaling (see TLR4; MIM 603030).[supplied by OMIM, Apr 2004]
PHENOTYPE: Homozygous inactivation of this gene affects TLR4-mediated immune responses. [provided by MGI curators]
Accession Number

NCBI RefSeq: NM_173394; MGI:3040056

MappedYes 
Amino Acid Change Isoleucine changed to Methionine
Institutional SourceBeutler Lab
Gene Model predicted gene model for protein(s): [ENSMUSP00000066239]
AlphaFold Q8BJQ4
SMART Domains Protein: ENSMUSP00000066239
Gene: ENSMUSG00000056130
AA Change: I80M

DomainStartEndE-ValueType
Pfam:TIR_2 78 192 2e-10 PFAM
Predicted Effect probably damaging

PolyPhen 2 Score 1.000 (Sensitivity: 0.00; Specificity: 1.00)
(Using ENSMUST00000070084)
Meta Mutation Damage Score 0.3078 question?
Is this an essential gene? Non Essential (E-score: 0.000) question?
Phenotypic Category Autosomal Semidominant
Candidate Explorer Status loading ...
Single pedigree
Linkage Analysis Data
Penetrance  
Alleles Listed at MGI

All mutations/alleles(6) : Chemically induced(ENU)(1) Targeted(5)

Lab Alleles
AlleleSourceChrCoordTypePredicted EffectPPH Score
IGL00587:Ticam2 APN 18 46693880 missense probably benign 0.04
Branch UTSW 18 46693718 missense probably damaging 1.00
Messi UTSW 18 46693989 nonsense probably null
R0056:Ticam2 UTSW 18 46693401 missense possibly damaging 0.61
R0056:Ticam2 UTSW 18 46693401 missense possibly damaging 0.61
R0666:Ticam2 UTSW 18 46693718 missense probably damaging 1.00
R1676:Ticam2 UTSW 18 46693677 missense probably damaging 1.00
R2209:Ticam2 UTSW 18 46693467 missense probably damaging 1.00
R4927:Ticam2 UTSW 18 46693846 missense probably damaging 1.00
R4928:Ticam2 UTSW 18 46693989 nonsense probably null
R6841:Ticam2 UTSW 18 46693998 missense probably benign 0.02
R7489:Ticam2 UTSW 18 46693584 missense probably damaging 1.00
R8407:Ticam2 UTSW 18 46693590 missense probably damaging 1.00
R9166:Ticam2 UTSW 18 46694048 missense probably damaging 1.00
R9451:Ticam2 UTSW 18 46693766 missense probably damaging 1.00
R9467:Ticam2 UTSW 18 46693748 missense probably damaging 1.00
R9508:Ticam2 UTSW 18 46693748 missense probably damaging 1.00
R9711:Ticam2 UTSW 18 46693658 missense probably damaging 1.00
Z1177:Ticam2 UTSW 18 46693915 missense possibly damaging 0.85
Mode of Inheritance Autosomal Semidominant
Local Stock
Repository
Last Updated 2019-09-04 9:39 PM by Anne Murray
Record Created 2017-08-28 1:12 PM by Bruce Beutler
Record Posted 2017-10-20
Phenotypic Description

Figure 1. Consequential mice exhibited decreased IL-1β secretion 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 Consequential phenotype was identified among N-ethyl-N-nitrosourea (ENU)-mutagenized G3 mice of the pedigree R4927, some of which showed 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 1).

Nature of Mutation

Figure 2. Linkage mapping of reduced IL-1β secretion in response to priming with lipopolysaccharide (LPS) followed by nigericin treatment using an additive model of inheritance. Manhattan plot shows -log10 P values (Y-axis) plotted against the chromosome positions of 71 mutations (X-axis) identified in the G1 male of pedigree R4927. Normalized phenotype data are shown for single locus linkage analysis without 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 71 mutations. The NLRP3 inflammasome defect phenotype was linked by continuous variable mapping to mutations in two genes: Kcnn2 and Ticam2. The mutation in Ticam2 was presumed causative as immune phenotypes are attributed to mutations in Ticam2 (see MGI and the record for Branch). The Ticam2 mutation is an A to G transition at base pair 46,560,779 (v38) on chromosome 18, or base pair 13,755 in the GenBank genomic region NC_000084 encoding Ticam2. Linkage was found with an additive model of inheritance, wherein two variant homozygotes and 30 heterozygotes departed phenotypically from 12 homozygous reference mice with a P value of 8.646 x 10-6 (Figure 2). 

The mutation corresponds to residue 716 in the mRNA sequence NM_173394 within exon 3 of 3 total exons.

699 TTCCTCAAATTTGTGATACTTCATGCAGAAGAT
75  -F--L--K--F--V--I--L--H--A--E--D-

 

The mutated nucleotide is indicated in red.  The mutation results in an isoleucine to methionine substitution at position 80 (I80M) in the TRAM protein, and is strongly predicted by PolyPhen-2 to be damaging (score = 1.000).

Illustration of Mutations in
Gene & Protein
Protein Prediction
Figure 3. Domain structure of TRAM.  The Toll/IL-1 receptor (TIR) domain is shown. The Consequential mutation results in an isoleucine to methionine substitution at position 80 (I80M). This image is interactive; click to view additional Ticam2 mutations.

Ticam2 [Toll-interleukin 1 receptor (TIR) domain-containing adaptor molecule-2; also TRAM (Trif-related adaptor molecule)] is a 232 amino acid protein adaptor in TLR4 signaling (Figure 3). TRAM contains a central Toll/IL-1 receptor (TIR) domain (amino acids 78-222), a conserved region of approximately 200 amino acids which mediates homo- and heterotypic protein interactions during signal transduction (1;2). The Consequential mutation results in an isoleucine to methionine substitution at position 80 (I80M) in the TRAM protein; residue 80 is within an undefined N-terminal region of TRAM upstream of the TIR domain.

For more information about Ticam2, please see the record for Branch.

Putative Mechanism

The twelve mouse TLRs and ten human TLRs recognize a wide range of structurally distinct molecules, and all signal through only four adaptor proteins known to date: MyD88, Tirap (Mal; see the record for torpid), TICAM-1 (TRIF) and TRAM (3). TLR signaling through these adaptors initiates a cascade of signaling events involving various kinases, adaptors and ubiquitin ligases, ultimately leading to transcriptional activation of cytokine [e.g., TNF-α, interleukin (IL)-1, IL-6] and other genes through the transcription factors NF-κB, AP-1, interferon responsive factor (IRF)-3, and IRF-7. TRAM activates NF-κB, IRF-3 and IRF-7 downstream of TLR4 (2;4). The diminished response to LPS indicates loss of TRAM function.

Primers PCR Primer
Consequential_pcr_F: TCACAGAATTCATCAGGGAAGTG
Consequential_pcr_R: AAGACGGCCATGAGTCAGAC

Sequencing Primer
Consequential_seq_F: ACTGGAAGTTACACCAGGTGTCTC
Consequential_seq_R: CTCCAAGAATTCTGAGGAAGCCTG
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 18, - strand):


1   aagacggcca tgagtcagac tccaagaatt ctgaggaagc ctgcttgcgt ggttttgtgg
61  agcagagcag tggatcagag ccaccaacag gagagcagga ccaacctgag gcaaaggggg
121 cggggcctga ggagcaagat gaagaagagt tcctcaaatt tgtgatactt catgcagaag
181 atgacaccga cgaggccctc agagtccagg atctactgca aaacgacttt ggtatcaggc
241 cggggatcgt tttcgccgag atgccgtgcg gaagactgca tttgcagaat ctagacgatg
301 cggtcaatgg gtcggcctgg accatcttgt tactgactga gaacttccta agagacacct
361 ggtgtaactt ccagttctac acttccctga tgaattctgt ga


Primer binding sites are underlined and the sequencing primers are highlighted; the mutated nucleotide is shown in red.

References
Science Writers Anne Murray
Illustrators Diantha La Vine
AuthorsHexin Shi and Bruce Beuter