Phenotypic Mutation 'wanna3' (pdf version)
Allele | wanna3 |
Mutation Type |
missense
|
Chromosome | 1 |
Coordinate | 36,817,299 bp (GRCm39) |
Base Change | A ⇒ T (forward strand) |
Gene |
Zap70
|
Gene Name | zeta-chain (TCR) associated protein kinase |
Synonym(s) | ZAP-70, TZK, Srk |
Chromosomal Location |
36,800,879-36,821,899 bp (+) (GRCm39)
|
MGI Phenotype |
FUNCTION: This gene encodes a member of the protein tyrosine kinase family. The encoded protein is essential for development of T lymphocytes and thymocytes, and functions in the initial step of T lymphocyte receptor-mediated signal transduction. A mutation in this gene causes chronic autoimmune arthritis, similar to rheumatoid arthritis in humans. Mice lacking this gene are deficient in alpha-beta T lymphocytes in the thymus. In humans, mutations in this gene cause selective T-cell defect, a severe combined immunodeficiency disease characterized by a selective absence of CD8-positive T lymphocytes. Alternative splicing results in multiple transcript variants. [provided by RefSeq, Jan 2014] PHENOTYPE: Mutant mice show T cell defects. Null mutants lack alpha-beta T cells in the thymus and have fewer T cells in dendritic and intestinal epithelium. Spontaneous and knock-in missense mutations affect T cell receptor signaling, one of the former resulting in severe chronic arthritis. [provided by MGI curators]
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Accession Number | NCBI RefSeq: NM_009539 (variant 1), NM_001289612 (variant 2), NM_001289765 (variant 3), NM_001289766 (variant 4); MGI: 99613 |
Mapped | Yes |
Amino Acid Change |
Histidine changed to Leucine
|
Institutional Source | Beutler Lab |
Gene Model |
predicted gene model for protein(s):
[ENSMUSP00000027291]
|
AlphaFold |
P43404 |
SMART Domains |
Protein: ENSMUSP00000027291 Gene: ENSMUSG00000026117 AA Change: H210L
Domain | Start | End | E-Value | Type |
SH2
|
8 |
93 |
6.73e-25 |
SMART |
SH2
|
161 |
245 |
1.59e-26 |
SMART |
low complexity region
|
257 |
265 |
N/A |
INTRINSIC |
TyrKc
|
337 |
592 |
1e-128 |
SMART |
|
Predicted Effect |
probably damaging
PolyPhen 2
Score 0.994 (Sensitivity: 0.69; Specificity: 0.97)
(Using ENSMUST00000027291)
|
Meta Mutation Damage Score |
0.9624 |
Is this an essential gene? |
Probably nonessential (E-score: 0.246) |
Phenotypic Category |
Autosomal Recessive |
Candidate Explorer Status |
loading ... |
Single pedigree Linkage Analysis Data
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|
Penetrance | |
Alleles Listed at MGI | All Mutations and Alleles(27) : Chemically induced (ENU)(7) Chemically induced (other)(1) Gene trapped(1) Spontaneous (2) Targeted(11) Transgenic(5)
|
Lab Alleles |
Allele | Source | Chr | Coord | Type | Predicted Effect | PPH Score |
mrtless
|
APN |
1 |
36820230 |
missense |
probably damaging |
1.00 |
murdock
|
APN |
1 |
36818785 |
missense |
probably damaging |
0.99 |
IGL00763:Zap70
|
APN |
1 |
36818333 |
missense |
possibly damaging |
0.81 |
IGL01635:Zap70
|
APN |
1 |
36810238 |
missense |
probably damaging |
0.99 |
IGL01918:Zap70
|
APN |
1 |
36817868 |
missense |
possibly damaging |
0.64 |
IGL02164:Zap70
|
APN |
1 |
36810267 |
missense |
probably damaging |
0.99 |
IGL02502:Zap70
|
APN |
1 |
36817887 |
splice site |
probably benign |
|
IGL02597:Zap70
|
APN |
1 |
36811001 |
nonsense |
probably null |
|
IGL03026:Zap70
|
APN |
1 |
36818798 |
missense |
possibly damaging |
0.94 |
biscayne
|
UTSW |
1 |
36820493 |
missense |
probably damaging |
1.00 |
mesa_verde
|
UTSW |
1 |
36818254 |
missense |
probably damaging |
1.00 |
shazzam
|
UTSW |
1 |
36820218 |
missense |
probably damaging |
1.00 |
trebia
|
UTSW |
1 |
36820106 |
missense |
probably damaging |
1.00 |
wanna
|
UTSW |
1 |
36810064 |
missense |
probably damaging |
1.00 |
wanna2
|
UTSW |
1 |
36820493 |
missense |
probably damaging |
1.00 |
wanna4
|
UTSW |
1 |
36820446 |
missense |
probably damaging |
1.00 |
want_to
|
UTSW |
1 |
36821598 |
missense |
probably damaging |
1.00 |
waterfowl
|
UTSW |
1 |
36809892 |
start codon destroyed |
probably null |
0.03 |
zapatos
|
UTSW |
1 |
36810262 |
missense |
possibly damaging |
0.89 |
zipper
|
UTSW |
1 |
36809983 |
missense |
probably benign |
0.09 |
PIT1430001:Zap70
|
UTSW |
1 |
36818250 |
missense |
possibly damaging |
0.95 |
R0487:Zap70
|
UTSW |
1 |
36818365 |
missense |
probably damaging |
1.00 |
R0701:Zap70
|
UTSW |
1 |
36820258 |
missense |
probably damaging |
1.00 |
R0960:Zap70
|
UTSW |
1 |
36818254 |
missense |
probably damaging |
1.00 |
R1520:Zap70
|
UTSW |
1 |
36810036 |
missense |
probably damaging |
1.00 |
R2064:Zap70
|
UTSW |
1 |
36818215 |
missense |
probably benign |
|
R3623:Zap70
|
UTSW |
1 |
36818216 |
missense |
probably benign |
0.03 |
R3689:Zap70
|
UTSW |
1 |
36820493 |
missense |
probably damaging |
1.00 |
R3690:Zap70
|
UTSW |
1 |
36820493 |
missense |
probably damaging |
1.00 |
R3804:Zap70
|
UTSW |
1 |
36810223 |
missense |
possibly damaging |
0.58 |
R3840:Zap70
|
UTSW |
1 |
36817498 |
missense |
probably damaging |
1.00 |
R4260:Zap70
|
UTSW |
1 |
36818189 |
splice site |
probably benign |
|
R4383:Zap70
|
UTSW |
1 |
36820042 |
missense |
probably damaging |
1.00 |
R4632:Zap70
|
UTSW |
1 |
36817539 |
missense |
probably benign |
|
R4783:Zap70
|
UTSW |
1 |
36818254 |
missense |
probably damaging |
1.00 |
R5051:Zap70
|
UTSW |
1 |
36820532 |
missense |
probably benign |
0.00 |
R5271:Zap70
|
UTSW |
1 |
36820446 |
missense |
probably damaging |
1.00 |
R5304:Zap70
|
UTSW |
1 |
36817299 |
missense |
probably damaging |
0.99 |
R5792:Zap70
|
UTSW |
1 |
36818090 |
intron |
probably benign |
|
R5932:Zap70
|
UTSW |
1 |
36820227 |
missense |
probably damaging |
1.00 |
R5941:Zap70
|
UTSW |
1 |
36810030 |
missense |
probably damaging |
1.00 |
R6694:Zap70
|
UTSW |
1 |
36821598 |
missense |
probably damaging |
1.00 |
R6825:Zap70
|
UTSW |
1 |
36817471 |
missense |
probably damaging |
1.00 |
R7039:Zap70
|
UTSW |
1 |
36817832 |
missense |
probably benign |
|
R7704:Zap70
|
UTSW |
1 |
36818395 |
critical splice donor site |
probably null |
|
R7769:Zap70
|
UTSW |
1 |
36809983 |
missense |
probably benign |
0.09 |
R8115:Zap70
|
UTSW |
1 |
36820287 |
missense |
probably damaging |
1.00 |
R8140:Zap70
|
UTSW |
1 |
36810262 |
missense |
possibly damaging |
0.89 |
R8289:Zap70
|
UTSW |
1 |
36820218 |
missense |
probably damaging |
1.00 |
R9186:Zap70
|
UTSW |
1 |
36818832 |
missense |
possibly damaging |
0.66 |
R9540:Zap70
|
UTSW |
1 |
36817869 |
missense |
possibly damaging |
0.95 |
R9654:Zap70
|
UTSW |
1 |
36818327 |
missense |
probably benign |
0.03 |
R9674:Zap70
|
UTSW |
1 |
36810150 |
missense |
probably benign |
0.10 |
S24628:Zap70
|
UTSW |
1 |
36809892 |
start codon destroyed |
probably null |
0.03 |
Z1176:Zap70
|
UTSW |
1 |
36818257 |
nonsense |
probably null |
|
|
Mode of Inheritance |
Autosomal Recessive |
Local Stock | |
Repository | |
Last Updated |
2019-09-04 9:39 PM
by Diantha La Vine
|
Record Created |
2017-07-01 8:56 AM
by Jin Huk Choi
|
Record Posted |
2017-08-09 |
Phenotypic Description |
The wanna3 phenotype was identified by position-based superpedigree analysis of pedigrees R3689 and R5304. Homozygous mice in these pedigrees exhibited an increase in the B to T cell ratio (Figure 1) due to reduced frequencies of T cells (Figure 2), CD44+ T cells (Figure 3), CD4+ T cells (Figure 4), CD4+ T cells in CD3+ T cells (Figure 5), CD44+ CD4 T cells (Figure 6), naïve CD4 T cells in CD4 T cells (Figure 7), CD8+ T cells (Figure 8), CD8+ T cells in CD3+ T cells (Figure 9), CD44+ CD8 T cells (Figure 10), and naïve CD8 T cells in CD8 T cells (Figure 11) with concomitant increased frequencies of B cells (Figure 12), IgD+ B cells (Figure 13), IgM+ B cells (Figure 14), central memory CD4 T cells in CD4 T cells (Figure 15), effector memory CD4 T cells in CD4 T cells (Figure 16), central memory CD8 T cells in CD8 T cells (Figure 17), all in the peripheral blood. Some mice showed an increase in the CD4 to CD8 T cell ratio (Figure 18) and increased expression of CD44 on peripheral blood CD4 T cells (Figure 19) and CD8 T cells (Figure 20). The T-dependent antibody response to ovalbumin administered with aluminum hydroxide was diminished (Figure 21).
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Nature of Mutation |
Whole exome HiSeq sequencing of the G1 grandsire identified 86 mutations. All of the above anomalies were linked by continuous variable mapping to a mutation in Zap70: an A to T transversion at base pair 36,778,218 (v38) on chromosome 1, or base pair 16,421 in the GenBank genomic region NC_000067 encoding Zap70. The strongest association was found with a recessive model of linkage to the normalized B to T cell ratio phenotype, wherein seven variant homozygotes departed phenotypically from 12 homozygous reference mice and 27 heterozygous mice with a P value of 1.17 x 10-28 (Figure 22). A substantial semidominant effect was observed in some of the assays but the mutation is preponderantly recessive, and in no assay was a purely dominant effect observed. The mutation corresponds to residue 791 in the mRNA sequence NM_001289766 within exon 4 of 13 total exons.
775 GGGAAAACTGTATACCACTATCTCATCAGCCAG
205 -G--K--T--V--Y--H--Y--L--I--S--Q-
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The mutated nucleotide is indicated in red. The mutation results in a histidine to leucine substitution at position 210 (H210L) in the ZAP70 protein, and is strongly predicted by PolyPhen-2 to be damaging (score = 0.994).
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Illustration of Mutations in
Gene & Protein |
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Protein Prediction |
The ζ-associated protein of 70 kDa (ZAP-70) is a protein tyrosine kinase (PTK) that binds to the doubly phosphorylated immunoreceptor tyrosine-based activation motifs (ITAMS) of ζ and CD3ε chains of the T cell receptor (TCR; see the record for tumormouse). ZAP70 consists of two N-terminal Src-homology 2 (SH2) domains at amino acids and a C-terminal kinase domain (Figure 23). The SH2 domains are connected by a linker known as interdomain A, while the region between the second SH2 and catalytic domains is known as interdomain B (2). The two SH2 domains of mouse ZAP-70 occur at amino acids 10-102 and 163-254, and work cooperatively to bind to the phosphorylated tyrosines of an ITAM sequence [(D/E)xxYxxI/Lx(6-8)YxxI/L]. The wanna3 mutation results in a histidine to leucine substitution at position 210 (H210L); residue 210 is within the second SH2 domain domain. Please see the record for murdock for more information about Zap70.
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Putative Mechanism | Signaling through the T cell receptor (TCR) plays a critical role at multiple stages of thymocyte differentiation, T-cell activation, and homeostasis [reviewed in (3;4)]. Syk and ZAP-70 function as critical mediators of pre-TCR and TCR signaling, with ZAP-70 having a predominant role in mature T cells (4;5). Once activated, ZAP-70 and Syk interact with and phosphorylate a number of substrates important for TCR signaling including the adaptor proteins the linker for activation of T cells (LAT) and SH2 domain-containing leukocyte protein of 76 kDa (SLP-76) (6;7). Once phosphorylated, these two adaptors serve as docking sites and organize a number of effector molecules into the correct spatiotemporal manner to allow the activation of multiple signaling pathways. Zap70 knockout mice display an arrest of T cell development at the DP stage, the second critical checkpoint important during αβ T cell development due to defective TCR-mediated selection and signaling at this stage (5;8). Although ZAP-70 has a critical role in T cell development and function, it also plays a role downstream of the BCR and in NK cells. Zap70 knockout mice display normal B cell development, mount normal antibody responses and also proliferate appropriately to various stimuli (9). The phenotype of the wanna3 mice is similar to loss-of-function alleles of Zap70.
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Primers |
PCR Primer
wanna3_pcr_F: AGGAGATTCTGGGTATGGAGCC
wanna3_pcr_R: TGGGACAGACCTCCTTCAAG
Sequencing Primer
wanna3_seq_F: GCCCATAGCATGTGTGTTAGTACC
wanna3_seq_R: GCGGTAAATTAGTCCATCCGC
|
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 1, + strand):
1 aggagattct gggtatggag cccatagcat gtgtgttagt acctgaggca caggaagtcc 61 cctccatgtt gctgtagcaa catgtgacac ccttcccccc acccccttca ggctgaggcc 121 ccggaaggag cagggcacat atgcactgtc cctggtctat gggaaaactg tataccacta 181 tctcatcagc caggacaagg ctggcaagta ctgcatcccc gaaggcacca agtttgacac 241 gctctggcag gtaggctgcc cacattctgg ggagaacccc gtgatagtgt gctcagtttg 301 gggtgggggg aggcctcacc tgacacccct tcccacagct ggtggagtac ctgaagctga 361 aggcggatgg actaatttac cgcttgaagg aggtctgtcc ca
Primer binding sites are underlined and the sequencing primers are highlighted; the mutated nucleotide is shown in red. |
References | 1. Adzhubei, I. A., Schmidt, S., Peshkin, L., Ramensky, V. E., Gerasimova, A., Bork, P., Kondrashov, A. S., and Sunyaev, S. R. (2010) A Method and Server for Predicting Damaging Missense Mutations. Nat Methods. 7, 248-249.
2. Au-Yeung, B. B., Deindl, S., Hsu, L. Y., Palacios, E. H., Levin, S. E., Kuriyan, J., and Weiss, A. (2009) The Structure, Regulation, and Function of ZAP-70. Immunol Rev. 228, 41-57.
3. Zamoyska, R., Basson, A., Filby, A., Legname, G., Lovatt, M., and Seddon, B. (2003) The Influence of the Src-Family Kinases, Lck and Fyn, on T Cell Differentiation, Survival and Activation. Immunol Rev. 191, 107-118.
5. Kadlecek, T. A., van Oers, N. S., Lefrancois, L., Olson, S., Finlay, D., Chu, D. H., Connolly, K., Killeen, N., and Weiss, A. (1998) Differential Requirements for ZAP-70 in TCR Signaling and T Cell Development. J Immunol. 161, 4688-4694.
6. Bubeck Wardenburg, J., Fu, C., Jackman, J. K., Flotow, H., Wilkinson, S. E., Williams, D. H., Johnson, R., Kong, G., Chan, A. C., and Findell, P. R. (1996) Phosphorylation of SLP-76 by the ZAP-70 Protein-Tyrosine Kinase is Required for T-Cell Receptor Function. J Biol Chem. 271, 19641-19644.
7. Zhang, W., Sloan-Lancaster, J., Kitchen, J., Trible, R. P., and Samelson, L. E. (1998) LAT: The ZAP-70 Tyrosine Kinase Substrate that Links T Cell Receptor to Cellular Activation. Cell. 92, 83-92.
8. Negishi, I., Motoyama, N., Nakayama, K., Nakayama, K., Senju, S., Hatakeyama, S., Zhang, Q., Chan, A. C., and Loh, D. Y. (1995) Essential Role for ZAP-70 in both Positive and Negative Selection of Thymocytes. Nature. 376, 435-438.
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Science Writers | Anne Murray |
Illustrators | Diantha La Vine |
Authors | Xue Zhong, Jin Huk Choi, Bruce Beutler |