Phenotypic Mutation 'petrified' (pdf version)
Allelepetrified
Mutation Type missense
Chromosome1
Coordinate14,954,340 bp (GRCm39)
Base Change A ⇒ G (forward strand)
Gene Trpa1
Gene Name transient receptor potential cation channel, subfamily A, member 1
Synonym(s) ANKTM1
Chromosomal Location 14,942,872-14,989,086 bp (-) (GRCm39)
MGI Phenotype FUNCTION: [Summary is not available for the mouse gene. This summary is for the human ortholog.] The structure of the protein encoded by this gene is highly related to both the protein ankyrin and transmembrane proteins. The specific function of this protein has not yet been determined; however, studies indicate the function may involve a role in signal transduction and growth control. [provided by RefSeq, Jul 2008]
PHENOTYPE: Mutations in this gene result in altered nociception and neuron responses to isothiocyanate or thiosulfinate compounds like those found in mustard oil and garlic. [provided by MGI curators]
Accession Number

NCBI RefSeq: NM_177781 (variant 1), NM_001348288 (variant 2); MGI:3522699

MappedYes 
Amino Acid Change Leucine changed to Proline
Institutional SourceBeutler Lab
Gene Model predicted gene model for protein(s): [ENSMUSP00000043594]
AlphaFold Q8BLA8
SMART Domains Protein: ENSMUSP00000043594
Gene: ENSMUSG00000032769
AA Change: L853P

DomainStartEndE-ValueType
ANK 63 94 1.01e2 SMART
ANK 98 127 9.7e-8 SMART
ANK 131 161 1.36e-2 SMART
ANK 165 194 5.45e-2 SMART
ANK 198 226 3.07e2 SMART
ANK 239 268 1.99e-4 SMART
ANK 272 302 1.33e2 SMART
ANK 309 338 4.19e-3 SMART
ANK 342 371 2.34e-1 SMART
ANK 413 442 3.41e-3 SMART
ANK 446 475 5.75e-1 SMART
ANK 482 511 4.1e-6 SMART
ANK 514 543 1.68e-2 SMART
ANK 548 577 4.97e-5 SMART
Blast:ANK 580 609 2e-11 BLAST
Pfam:Ion_trans 736 975 1.8e-11 PFAM
Predicted Effect probably damaging

PolyPhen 2 Score 1.000 (Sensitivity: 0.00; Specificity: 1.00)
(Using ENSMUST00000041447)
Meta Mutation Damage Score 0.6344 question?
Is this an essential gene? Probably nonessential (E-score: 0.133) question?
Phenotypic Category Autosomal Recessive
Candidate Explorer Status loading ...
Single pedigree
Linkage Analysis Data
Penetrance  
Alleles Listed at MGI

All Mutations and Alleles(8) : Targeted(8)

Lab Alleles
AlleleSourceChrCoordTypePredicted EffectPPH Score
IGL00801:Trpa1 APN 1 14961557 missense probably damaging 0.97
IGL00937:Trpa1 APN 1 14950501 splice site probably benign
IGL00957:Trpa1 APN 1 14951892 missense probably damaging 0.99
IGL01307:Trpa1 APN 1 14966771 missense probably benign 0.23
IGL01336:Trpa1 APN 1 14957104 splice site probably benign
IGL01408:Trpa1 APN 1 14959637 missense probably benign 0.03
IGL01504:Trpa1 APN 1 14952443 missense possibly damaging 0.79
IGL01543:Trpa1 APN 1 14970300 missense probably damaging 1.00
IGL01609:Trpa1 APN 1 14982607 missense probably damaging 0.99
IGL01895:Trpa1 APN 1 14957867 missense possibly damaging 0.87
IGL02449:Trpa1 APN 1 14968381 missense probably damaging 1.00
IGL02936:Trpa1 APN 1 14946193 splice site probably null
fear-2 UTSW 1 14961527 critical splice donor site probably null
R0008:Trpa1 UTSW 1 14973439 missense possibly damaging 0.53
R0008:Trpa1 UTSW 1 14973439 missense possibly damaging 0.53
R0317:Trpa1 UTSW 1 14951856 missense probably benign 0.03
R0454:Trpa1 UTSW 1 14955972 critical splice donor site probably null
R0828:Trpa1 UTSW 1 14946108 missense probably damaging 1.00
R0944:Trpa1 UTSW 1 14982585 splice site probably null
R0962:Trpa1 UTSW 1 14968387 missense possibly damaging 0.61
R1025:Trpa1 UTSW 1 14974407 missense probably benign 0.01
R1035:Trpa1 UTSW 1 14961527 critical splice donor site probably null
R1134:Trpa1 UTSW 1 14951972 missense possibly damaging 0.95
R1278:Trpa1 UTSW 1 14988947 critical splice donor site probably null
R1497:Trpa1 UTSW 1 14956036 missense probably benign 0.30
R1617:Trpa1 UTSW 1 14943899 missense probably damaging 1.00
R1800:Trpa1 UTSW 1 14944648 missense probably benign 0.04
R1856:Trpa1 UTSW 1 14969612 nonsense probably null
R1886:Trpa1 UTSW 1 14959649 missense probably benign 0.00
R2004:Trpa1 UTSW 1 14976207 missense possibly damaging 0.83
R2152:Trpa1 UTSW 1 14969625 missense probably damaging 1.00
R2172:Trpa1 UTSW 1 14951880 missense probably benign 0.01
R2198:Trpa1 UTSW 1 14980970 missense probably benign
R2221:Trpa1 UTSW 1 14973480 missense probably null 0.12
R2223:Trpa1 UTSW 1 14973480 missense probably null 0.12
R2307:Trpa1 UTSW 1 14982605 missense probably benign 0.00
R2338:Trpa1 UTSW 1 14954469 missense probably damaging 0.97
R2698:Trpa1 UTSW 1 14976222 missense probably damaging 1.00
R2872:Trpa1 UTSW 1 14957844 missense probably damaging 1.00
R2872:Trpa1 UTSW 1 14957844 missense probably damaging 1.00
R2873:Trpa1 UTSW 1 14957844 missense probably damaging 1.00
R2874:Trpa1 UTSW 1 14957844 missense probably damaging 1.00
R3418:Trpa1 UTSW 1 14944605 missense probably benign 0.01
R3419:Trpa1 UTSW 1 14944605 missense probably benign 0.01
R3796:Trpa1 UTSW 1 14963488 missense possibly damaging 0.74
R3799:Trpa1 UTSW 1 14963488 missense possibly damaging 0.74
R4238:Trpa1 UTSW 1 14954340 missense probably damaging 1.00
R4320:Trpa1 UTSW 1 14944676 missense probably benign 0.00
R4591:Trpa1 UTSW 1 14952332 splice site probably null
R4834:Trpa1 UTSW 1 14966747 missense possibly damaging 0.72
R4991:Trpa1 UTSW 1 14980970 missense probably benign 0.00
R4999:Trpa1 UTSW 1 14946085 missense probably benign 0.05
R5038:Trpa1 UTSW 1 14981090 missense probably damaging 1.00
R5055:Trpa1 UTSW 1 14946183 missense probably damaging 1.00
R5158:Trpa1 UTSW 1 14951885 missense probably benign 0.01
R5193:Trpa1 UTSW 1 14946141 missense possibly damaging 0.92
R5558:Trpa1 UTSW 1 14968492 missense probably damaging 1.00
R5578:Trpa1 UTSW 1 14957232 missense probably damaging 1.00
R5680:Trpa1 UTSW 1 14946078 missense probably benign 0.00
R5738:Trpa1 UTSW 1 14946174 missense probably damaging 1.00
R5801:Trpa1 UTSW 1 14968302 missense probably damaging 1.00
R5945:Trpa1 UTSW 1 14968359 missense probably benign 0.03
R6092:Trpa1 UTSW 1 14959710 missense probably damaging 1.00
R6776:Trpa1 UTSW 1 14982601 missense probably benign
R7126:Trpa1 UTSW 1 14960648 missense probably benign 0.00
R7154:Trpa1 UTSW 1 14952457 missense possibly damaging 0.46
R7175:Trpa1 UTSW 1 14963431 missense possibly damaging 0.90
R7258:Trpa1 UTSW 1 14973473 missense probably damaging 1.00
R7358:Trpa1 UTSW 1 14968334 missense probably damaging 1.00
R7412:Trpa1 UTSW 1 14954422 missense probably benign 0.43
R7639:Trpa1 UTSW 1 14957137 missense probably benign 0.00
R7740:Trpa1 UTSW 1 14982625 missense possibly damaging 0.72
R7815:Trpa1 UTSW 1 14974486 missense probably benign 0.01
R7854:Trpa1 UTSW 1 14951918 missense probably benign 0.00
R8112:Trpa1 UTSW 1 14974490 missense probably benign
R8217:Trpa1 UTSW 1 14957247 missense probably damaging 0.97
R8711:Trpa1 UTSW 1 14980998 missense probably damaging 1.00
R8834:Trpa1 UTSW 1 14963528 missense possibly damaging 0.60
R8907:Trpa1 UTSW 1 14963563 missense probably damaging 1.00
R8907:Trpa1 UTSW 1 14959664 missense probably benign 0.00
R9058:Trpa1 UTSW 1 14959618 missense probably damaging 1.00
R9135:Trpa1 UTSW 1 14952435 missense probably damaging 1.00
R9261:Trpa1 UTSW 1 14963465 missense probably damaging 1.00
R9266:Trpa1 UTSW 1 14980953 critical splice donor site probably null
R9287:Trpa1 UTSW 1 14956040 nonsense probably null
R9323:Trpa1 UTSW 1 14968564 missense probably benign 0.01
R9379:Trpa1 UTSW 1 14966739 missense possibly damaging 0.64
R9497:Trpa1 UTSW 1 14989026 missense probably benign 0.02
R9616:Trpa1 UTSW 1 14989077 start gained probably benign
R9666:Trpa1 UTSW 1 14973455 missense possibly damaging 0.67
X0028:Trpa1 UTSW 1 14960644 missense probably benign 0.16
Z1176:Trpa1 UTSW 1 14968574 missense probably damaging 1.00
Z1176:Trpa1 UTSW 1 14961530 missense possibly damaging 0.80
Z1176:Trpa1 UTSW 1 14951916 missense probably damaging 1.00
Mode of Inheritance Autosomal Recessive
Local Stock
Repository
Last Updated 2019-09-04 9:40 PM by Anne Murray
Record Created 2017-03-21 4:15 PM by Bruce Beutler
Record Posted 2018-07-19
Phenotypic Description

Figure 1. Petrified mice exhibited reduced innate fear responses. 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 petrified phenotype was identified among N-ethyl-N-nitrosourea (ENU)-mutagenized G3 mice of the pedigree R4238, some of which showed reduced innate fear responses compared to wild-type littermates (Figure 1).

Nature of Mutation
Figure 2. Linkage mapping of the reduced innate fear responses using a recessive model of inheritance. Manhattan plot shows -log10 P values (Y-axis) plotted against the chromosome positions of 43 mutations (X-axis) identified in the G1 male of pedigree R4238. 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 43 mutations. The innate fear phenotype was linked by continuous variable mapping to a mutation in Trpa1: a T to C transition at base pair 14,884,116 (v38) on chromosome 1, or base pair 34,899 in the GenBank genomic region NC_000067 encoding Trpa1. Linkage was found with a recessive model of inheritance, wherein two variant homozygotes departed phenotypically from two homozygous reference mice and seven heterozygous mice with a P value of 1.535 x 10-5 (Figure 2).  

The mutation corresponds to residue 2,585 in the mRNA sequence NM_177781 within exon 21 of 27 total exons.

2659 AACTTCCTACTGTATCTTCAAAGGTTTGAGAAC
848  -N--F--L--L--Y--L--Q--R--F--E--N-

 

The mutated nucleotide is indicated in red.  The mutation results in a leucine (L) to proline (P) substitution at position 853 (L853P) in the TRPA1 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 organization and topology of TRPA1. The petrified mutation results in a leucine to proline substitution at position 853. Domain and topology information is from SMART and UniProt. Other mutations found in TRPA1 are noted. Click on each mutation for more information.

Transient receptor potential ankyrin 1 (TRPA1; alternatively, ANKTM1) is one of 28 members of the mammalian transient receptor potential (TRP) channel superfamily. The TRP channels can be subdivided into six subfamilies: TRP Canonical (TRPC), TRP Melastatin (TRPM), TRP Ankyrin (TRPA), TRP Mucolipin (TRPML), TRP Polycystin (TRPP), and TRP Vanilloid (TRPV). Mammals do not have a representative of the seventh TRP subfamily, TRPN. TRPA1 is the only member of the TRPA family.

The TRP proteins have large intracellular N- and C-terminal tail as well as six transmembrane (TM)-spanning domains, with a putative hydrophobic, pore-forming region between the fifth and sixth domains.  The TRP proteins also have variable combinations of other domains within the cytoplasmic regions of the proteins including ankyrin repeats, coiled-coil regions, a NUDIX domain, an EF hand Ca2 +-binding motif, a CIRB motif, a PDZ motif, and a TRP box [reviewed in (1)].  TRPA1 also has three EF hands within the N-terminus that mediates channel activation in response to increased levels of intracellular calcium (2). The N-terminal tail of TRPA1 is 721 amino acids in length and contains 14 ankyrin repeats. The ankyrin repeat domain (ARD) is proposed to mediate trafficking of TRPA1 to the plasma membrane or plasma membrane insertion (3). In addition, ankyrin repeats facilitate protein-protein interactions. The ARD is proposed to function in the regulation of gating and integration of multiple stimuli. TRPA1 has a “TRP-like” domain in the C-terminal tail (4). The TRP domain is a short hydrophobic segment that is necessary for phosphatidylinositol 4,5-bisphosphate (PIP2) binding (a ubiquitously expressed phospholipid that is a regulator of channel function). Within the C-terminus is a coiled-coil that mediates subunit interactions.

The petrified mutation results in a leucine (L) to proline (P) substitution at position 853 (L853P); Leu853 is within the fourth transmembrane domain.

For more information about Trpa1, please see the record for fear-2.

Putative Mechanism

The TRPV, TRPM, and TPRA subfamilies function in the sensory detection transduction of nociception and pain. The TRP channels function by facilitating the transmembrane flow of cations (i.e. Na+ and Ca2+) down electrochemical gradients to depolarize the cell as well as to mediate signal transduction (5;6). TRP channels can be activated through the activation of phospholipase C (PLC) by G protein-coupled receptors and receptor tyrosine kinases.  Activation of PLC leads to hydrolysis of PIP2, producing diacylglycerol (DAG) and inositol (1,4,5) triphosphate (IP3).  TRPA1 is activated by several stimulants, including allyl isothiocyanate (AITC; a compound in horseradish, wasabi, and mustard), allicin and diallyl disulfide (in raw garlic), cinnamaldehyde (in cinnamon), gingerol (in ginger), thymol (in thyme), eugenol (in cloves), and carvacrol (in oregano), acrolein and tear gases (environmental irritants), and endogenous stimulants including reactive oxygen, nitrogen, and carbonyl species, hydrogen peroxide, peroxynitrite, and 4-hydroxynonenal (7;8). TRPA1 is also activated by endogenous inflammatory proteins, facilitating the transduction of nociceptive signals related to tissue damage and inflammation. After injury, the cyclooxygenase pathway leads to the production of prostaglandins. The prostaglandin 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) activates dissociated dorsal root ganglion cells (9).

In humans, patients with elevated pain sensitivity exhibit differential DNA methylation in the vicinity of the TRPA1 gene, indicating that this may be a contributing factor in individual differences in pain sensitivity (10). A gain-of-function mutation in TRPA1 (N855S) is linked to familial episodic pain syndrome (FEPS; OMIM: #615040) (11). Asn855 is within the fourth transmembrane domain. FEPS is an autosomal dominant neurological disorder characterized by early-onset of episodic upper body pain triggered by fatigue, fasting, cold, illness, and/or physical exertion (11). The pain episodes also included breathing difficulties, tachycardia, sweating, generalized pallor, peribuccal cyanosis, and stiffness of the abdominal wall. The patients did not report altered pain sensitivity outside of the episodes.

Trpa1-deficient (Trpa1-/-) mice exhibit behavioral deficits in response to mechanical stimulation, AITC, and cold temperatures (12). Two independent knockout mouse models were generated and characterized. One model was reported to not have menthol-insensitive, cold-activated trigeminal neurons (13). In addition, this model was comparable to wild-type mice in the latency to lift the paw in cold plate assays, flinching in acetone-induced cooling of the paw, and in temperature preference assay (13). Bautista et al. concluded that TRPA1 does not behave as a cold sensor. In the second TRPA1 knockout model, the mice exhibited reduced paw lifting upon exposure to a cold plate as well as reduced responses to acetone application compared to wild-type mice ((12).

TRPA1 acts as a chemosensor for 2MT/TMT (14). Trpa1−/− mice displayed diminished freezing response to 2MT or TMT as compared to wild-type and heterozygous littermates (14). 2MT-evoked c-fos induction was reduced in the CeA, vPAG, and the paraventricular nucleus (PVN) of the hypothalamus in Trpa1−/− brains relative to that in Trpa1+/− brains. Furthermore, TRPA1-expressing trigeminal neurons mediate 2MT-evoked innate freezing behavior. The phenotype of the petrified mice indicates loss of TRPA1petrified function.

Primers PCR Primer
petrified_pcr_F: CAGTCTACACATTGAATTTCAGAGCC
petrified_pcr_R: TGTCAGATTTCTCCATGTGGTC

Sequencing Primer
petrified_seq_F: ACATTGAATTTCAGAGCCCTCTAC
petrified_seq_R: CCTTCCTTCTCTATCCAGAAAAGG
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 1, - strand):


1   tgtcagattt ctccatgtgg tcattctcct tccttctcta tccagaaaag gaattacttc
61  ctggattaca acaatgctct ggaatgggtt atctatacaa ctagtatcat cttcgtgttg
121 cccttgttcc tcaacatccc agcgtatatg cagtggcaat gtggagcaat agcgatattc
181 ttctactgga tgaacttcct actgtatctt caaaggtaag accagtttgg atgatgttta
241 cattctttaa gccttttaat atcaaagaaa gaactattat aggtgaggga aatttgtaac
301 aaatttctaa catgtttttc actgttccca attgttttag atatattcta atatcgtaat
361 gctgaagggt agagggctct gaaattcaat gtgtagactg 


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
AuthorsQinghua Liu and Bruce Beutler