Phenotypic Mutation 'Meteor' (pdf version)
AlleleMeteor
Mutation Type missense
Chromosome10
Coordinate28,657,828 bp (GRCm39)
Base Change T ⇒ A (forward strand)
Gene Themis
Gene Name thymocyte selection associated
Synonym(s) Tsepa, Gasp, E430004N04Rik
Chromosomal Location 28,544,356-28,759,814 bp (+) (GRCm39)
MGI Phenotype FUNCTION: This gene encodes a protein that plays a regulatory role in both positive and negative T-cell selection during late thymocyte development. The protein functions through T-cell antigen receptor signaling, and is necessary for proper lineage commitment and maturation of T-cells. Alternative splicing results in multiple transcript variants. [provided by RefSeq, Mar 2015]
PHENOTYPE: Homozygous null mice have defects in T cell positive selection that leads to very few alpha-beta T cells being found in the periphery. [provided by MGI curators]
Accession Number

NCBI RefSeq: NM_178666; MGI:2443552

MappedYes 
Amino Acid Change Valine changed to Glutamic Acid
Institutional SourceBeutler Lab
Gene Model predicted gene model for protein(s): [ENSMUSP00000060129] [ENSMUSP00000055315] [ENSMUSP00000101155] [ENSMUSP00000123919 ] [ENSMUSP00000123894]   † probably from a misspliced transcript
AlphaFold Q8BGW0
SMART Domains Protein: ENSMUSP00000060129
Gene: ENSMUSG00000049109
AA Change: V285E

DomainStartEndE-ValueType
Pfam:CABIT 17 266 5.2e-59 PFAM
Pfam:CABIT 282 530 3.7e-48 PFAM
low complexity region 550 564 N/A INTRINSIC
Predicted Effect probably benign

PolyPhen 2 Score 0.122 (Sensitivity: 0.93; Specificity: 0.86)
(Using ENSMUST00000056097)
SMART Domains Protein: ENSMUSP00000055315
Gene: ENSMUSG00000049109
AA Change: V285E

DomainStartEndE-ValueType
Pfam:CABIT 17 272 9.3e-52 PFAM
Pfam:CABIT 282 532 5e-62 PFAM
low complexity region 550 564 N/A INTRINSIC
Predicted Effect probably benign

PolyPhen 2 Score 0.122 (Sensitivity: 0.93; Specificity: 0.86)
(Using ENSMUST00000060409)
SMART Domains Protein: ENSMUSP00000101155
Gene: ENSMUSG00000049109
AA Change: V285E

DomainStartEndE-ValueType
Pfam:CABIT 17 272 9e-52 PFAM
Pfam:CABIT 282 532 4.9e-62 PFAM
low complexity region 550 564 N/A INTRINSIC
Predicted Effect probably benign

PolyPhen 2 Score 0.122 (Sensitivity: 0.93; Specificity: 0.86)
(Using ENSMUST00000105516)
SMART Domains Protein: ENSMUSP00000123919
Gene: ENSMUSG00000049109

DomainStartEndE-ValueType
Pfam:CABIT 17 91 1.9e-10 PFAM
Predicted Effect probably benign
SMART Domains Protein: ENSMUSP00000123894
Gene: ENSMUSG00000049109
AA Change: V132E

DomainStartEndE-ValueType
Pfam:CABIT 17 86 1.9e-9 PFAM
Pfam:CABIT 129 203 5.1e-18 PFAM
Predicted Effect possibly damaging

PolyPhen 2 Score 0.903 (Sensitivity: 0.82; Specificity: 0.94)
(Using ENSMUST00000161345)
Meta Mutation Damage Score 0.1152 question?
Is this an essential gene? Probably nonessential (E-score: 0.169) question?
Phenotypic Category Unknown
Candidate Explorer Status loading ...
Single pedigree
Linkage Analysis Data
Penetrance  
Alleles Listed at MGI

All Mutations and Alleles(15) : Chemically induced (ENU)(6) Chemically induce (other) (1) Gene trapped(2) Targeted(6)

Lab Alleles
AlleleSourceChrCoordTypePredicted EffectPPH Score
IGL01609:Themis APN 10 28544749 splice site probably benign
IGL01729:Themis APN 10 28637587 nonsense probably null
IGL01833:Themis APN 10 28658307 nonsense probably null
IGL02582:Themis APN 10 28637543 missense probably benign 0.00
IGL02835:Themis APN 10 28637616 intron probably benign
cloudies UTSW 10 28637195 nonsense probably null
currant UTSW 10 28658007 missense probably damaging 1.00
death_valley UTSW 10 28544723 missense probably damaging 1.00
six_flags UTSW 10 28657903 missense probably damaging 1.00
R0445:Themis UTSW 10 28658007 missense probably damaging 1.00
R0507:Themis UTSW 10 28657828 missense possibly damaging 0.90
R0709:Themis UTSW 10 28637570 missense probably benign 0.00
R1170:Themis UTSW 10 28544744 missense possibly damaging 0.80
R1442:Themis UTSW 10 28658131 missense probably damaging 0.96
R1844:Themis UTSW 10 28657753 missense probably damaging 1.00
R2004:Themis UTSW 10 28658720 missense probably benign 0.28
R2150:Themis UTSW 10 28544723 missense probably damaging 1.00
R2358:Themis UTSW 10 28739376 missense possibly damaging 0.57
R4529:Themis UTSW 10 28658331 missense possibly damaging 0.92
R4693:Themis UTSW 10 28658647 missense probably damaging 1.00
R4717:Themis UTSW 10 28665748 missense probably benign
R4801:Themis UTSW 10 28637507 missense probably benign 0.21
R4802:Themis UTSW 10 28637507 missense probably benign 0.21
R5249:Themis UTSW 10 28637195 nonsense probably null
R5557:Themis UTSW 10 28657882 missense possibly damaging 0.90
R5569:Themis UTSW 10 28657887 missense possibly damaging 0.95
R5640:Themis UTSW 10 28739372 missense probably damaging 0.99
R5735:Themis UTSW 10 28598530 missense probably benign 0.09
R6467:Themis UTSW 10 28657762 missense possibly damaging 0.47
R6523:Themis UTSW 10 28657894 missense possibly damaging 0.65
R6727:Themis UTSW 10 28657903 missense probably damaging 1.00
R7014:Themis UTSW 10 28665703 missense probably benign
R7101:Themis UTSW 10 28637422 nonsense probably null
R7185:Themis UTSW 10 28657873 missense probably benign 0.00
R7323:Themis UTSW 10 28609497 missense probably benign
R7386:Themis UTSW 10 28665743 missense probably benign 0.00
R7472:Themis UTSW 10 28637415 missense possibly damaging 0.69
R7555:Themis UTSW 10 28657698 missense possibly damaging 0.67
R7715:Themis UTSW 10 28739305 missense probably benign 0.02
R7825:Themis UTSW 10 28658470 missense probably benign 0.11
R7992:Themis UTSW 10 28637342 missense probably benign 0.02
R8112:Themis UTSW 10 28673502 makesense probably null
R8850:Themis UTSW 10 28673492 missense possibly damaging 0.83
R8954:Themis UTSW 10 28665709 missense probably benign 0.00
R9038:Themis UTSW 10 28657749 missense probably damaging 0.99
R9081:Themis UTSW 10 28544582 unclassified probably benign
R9168:Themis UTSW 10 28658233 missense probably benign 0.01
R9169:Themis UTSW 10 28658233 missense probably benign 0.01
R9170:Themis UTSW 10 28658233 missense probably benign 0.01
R9171:Themis UTSW 10 28658233 missense probably benign 0.01
R9269:Themis UTSW 10 28739390 missense probably benign 0.10
R9404:Themis UTSW 10 28665743 missense probably benign 0.00
R9518:Themis UTSW 10 28544748 critical splice donor site probably null
Mode of Inheritance Unknown
Local Stock
Repository
Last Updated 2019-09-04 9:30 PM by Diantha La Vine
Record Created 2019-01-23 10:40 AM by Bruce Beutler
Record Posted 2019-02-14
Phenotypic Description

Figure 1. Meteor mice exhibit decreased frequencies of peripheral CD4+ T cells. Flow cytometric analysis of peripheral blood was utilized to determine T cell frequency. Raw 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 Meteor phenotype was identified among G3 mice of the pedigree R0507, some of which showed reduced frequencies of CD4+ T cells in the peripheral blood (Figure 1).

Nature of Mutation

Figure 2. Linkage mapping of the reduced CD4+ T cell frequency using a dominant model of inheritance. Manhattan plot shows -log10 P values (Y-axis) plotted against the chromosome positions of 70 mutations (X-axis) identified in the G1 male of pedigree R0507. Raw 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 70 mutations. The CD4+ T cell phenotype was linked by continuous variable mapping to a mutation in Themis:  a T to A transversion at base pair 28,781,832 (v38) on chromosome 10, or base pair 113,506 in the GenBank genomic region NC_000076 encoding Themis. Linkage was found with a dominant model of inheritance, wherein six variant homozygotes and five heterozygous mice departed phenotypically from three homozygous reference mice with a P value of 0.000183 (Figure 2).  

The mutation corresponds to residue 1,154 in the mRNA sequence NM_178666 within exon 4 of 6 total exons.

 

1138 AAAGAGTTTCCTGTAGTAGCTGAAGTTGTCGAA

280  -K--E--F--P--V--V--A--E--V--V--E-

The mutated nucleotide is indicated in red.  The mutation results in a valine to glutamic acid substitution at position 285 (V285E) in the Themis protein, and is strongly predicted by PolyPhen-2 to be benign (score = 0.122).

Illustration of Mutations in
Gene & Protein
Protein Prediction

Figure 3. Domain structure of Themis. The Meteor mutation results in a valine to glutamic acid substitution at position 285. This image is interactive. Click on each allele for additional information. Abbreviations: CABIT, cysteine-containing all beta in Themis; NLS, nuclear localization signal; PRR, proline-rich region. See text for more details. This image is interactive; click to view other mutations in Themis.

Themis encodes Thymocyte expressed molecule involved in selection [Themis; alternatively, Grb2-associating protein (Gasp)]. Themis contains two CABIT (cysteine-containing all beta in Themis) domains (amino acids 1-261 (CABIT1) and 262-521 (CABIT2) in Themis), a bipartite nuclear localization signal (NLS; amino acids 345-349 in Themis), and a proline-rich region (PRR; PPPRPPKHP; amino acids 555-563 in Themis) [Figure 3; (1-3)].

The Meteor mutation (V285E) is within the CABIT2 domain.

For more information about Themis, please see the record for currant.

Putative Mechanism

Themis functions at the positive selection checkpoint in thymocyte development; its mode of action is not known (1-5). Several Themis mutant mouse strains exhibited a block in positive selection, resulting in a block in the developmental progression from DP to SP cells (1-6). As a result, the mutant mice exhibited reduced numbers of thymic and peripheral CD4 and CD8 SP cells; the CD4 SP cells were more affected than the CD8 SP cell population (1-6). The role of Themis in negative selection is unclear. In several mutant mouse studies, minor defects in negative selection were observed along with defects in positive selection (1;4). The function of Themis in TCR signaling has not been resolved. Studies have reported a constitutive association of Themis with TCR signaling proteins including Vav1 (1), Itk, and PLCγ1 (4;6;7). In addition, several studies determined that, upon TCR stimulation, Themis associated with PLCγ1, LAT, and SLP-76 indicating that Themis may be a component of the SLP-76/LAT signalosome (1;4;6-8). Mutations in the noncoding region between THEMIS and PTPRK have been linked to susceptibility to celiac disease in humans (9). In addition, patients with celiac disease exhibit elevated THEMIS levels in the duodenal mucosa compared to unaffected individuals (10). Mice with mutations or deficiency in Themis phenocopy the Meteor mice in that all of the models exhibit reduced frequencies of peripheral CD4 SP cells compared to wild-type mice (1-4;10), suggesting that the Meteor mutation is hypomorphic. Thymocyte positive selection is likely defective in Meteor mice, as shown for other Themis mutants.

Primers PCR Primer
Meteor_pcr_F: GCATTTAAGTCAGGAGAGTGCTTACCA
Meteor_pcr_R: GACAGCTCCTTGTGAAGTGTATGGAAA

Sequencing Primer
Meteor_seq_F: TTGGAAGATTATCTCTACCCAGACAC
Meteor_seq_R: TCATAGGCCGTGGGGAAC
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 750 nucleotides is amplified (chromosome 10, + strand):


1   gcatttaagt caggagagtg cttaccattt gtagtagtaa gaaagaagtg atatttgctg
61  agttttacat aaatataagg gttgggtaac ctctgtttct tgagttgagc atgttggtat
121 atatatcatt ttgttttcat caaaaagttt caaaactgaa aagtctaaca taaggaaaat
181 tatattatga ttatttgaat tcttttctcc ctcctaaatg aaaattatct atggtattgg
241 aagattatct ctacccagac accatctctt gagagctctt taaacaaaat gtcagttgtc
301 ttctctttca ttttcagttc aaaaggacat agtccggatt ctcccaagtt tagacgttga
361 agtcaaagat attactgatt cttatgatgc taactggttt cttcagttgc tgtctacgga
421 cgaccttttt gaaatgacca gcaaagagtt tcctgtagta gctgaagttg tcgaaatatc
481 tcaagggaac cacctgcccc aaagtatttt gcagcgagag aaaaccattg tcatccacaa
541 aaagtaccag gcttcgagga tcttagcttc agaaattcgc agcaatttcc ctaaaagaca
601 cttcttgatc cctattagct acaaaggcaa gttcaaaaga agaccacggg agttccccac
661 ggcctatgac ctgcagatag ctaagagcag gaaagaaact ctccacgtgg tggccaccaa
721 agctttccat acacttcaca aggagctgtc 


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

References
  9. Dubois, P. C., Trynka, G., Franke, L., Hunt, K. A., Romanos, J., Curtotti, A., Zhernakova, A., Heap, G. A., Adany, R., Aromaa, A., Bardella, M. T., van den Berg, L. H., Bockett, N. A., de la Concha, E. G., Dema, B., Fehrmann, R. S., Fernandez-Arquero, M., Fiatal, S., Grandone, E., Green, P. M., Groen, H. J., Gwilliam, R., Houwen, R. H., Hunt, S. E., Kaukinen, K., Kelleher, D., Korponay-Szabo, I., Kurppa, K., MacMathuna, P., Maki, M., Mazzilli, M. C., McCann, O. T., Mearin, M. L., Mein, C. A., Mirza, M. M., Mistry, V., Mora, B., Morley, K. I., Mulder, C. J., Murray, J. A., Nunez, C., Oosterom, E., Ophoff, R. A., Polanco, I., Peltonen, L., Platteel, M., Rybak, A., Salomaa, V., Schweizer, J. J., Sperandeo, M. P., Tack, G. J., Turner, G., Veldink, J. H., Verbeek, W. H., Weersma, R. K., Wolters, V. M., Urcelay, E., Cukrowska, B., Greco, L., Neuhausen, S. L., McManus, R., Barisani, D., Deloukas, P., Barrett, J. C., Saavalainen, P., Wijmenga, C., and van Heel, D. A. (2010) Multiple Common Variants for Celiac Disease Influencing Immune Gene Expression. Nat Genet. 42, 295-302.
Science Writers Anne Murray
Illustrators Diantha La Vine
AuthorsJin Huk Choi, Xue Zhong, and Bruce Beutler