Phenotypic Mutation 'wanna4' (pdf version)
Allelewanna4
Mutation Type missense
Chromosome1
Coordinate36,820,446 bp (GRCm39)
Base Change T ⇒ A (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]
Accession Number

NCBI RefSeq: NM_009539 (variant 1), NM_001289612 (variant 2), NM_001289765 (variant 3), NM_001289766 (variant 4); MGI: 99613

MappedYes 
Limits of the Critical Region 36761798 - 36782818 bp
Amino Acid Change Valine changed to Aspartic acid
Institutional SourceBeutler Lab
Gene Model predicted gene model for protein(s): [ENSMUSP00000027291]
AlphaFold P43404
SMART Domains Protein: ENSMUSP00000027291
Gene: ENSMUSG00000026117
AA Change: V547D

DomainStartEndE-ValueType
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 1.000 (Sensitivity: 0.00; Specificity: 1.00)
(Using ENSMUST00000027291)
Meta Mutation Damage Score 0.9460 question?
Is this an essential gene? Possibly nonessential (E-score: 0.322) question?
Phenotypic Category Autosomal Recessive
Candidate Explorer Status loading ...
Single pedigree
Linkage Analysis Data
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
AlleleSourceChrCoordTypePredicted EffectPPH 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
wanna3 UTSW 1 36817299 missense probably damaging 0.99
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 9:09 AM by Jin Huk Choi
Record Posted 2017-08-09
Phenotypic Description

Figure 1. Wanna4 mice exhibit increased B to T cell ratios. Flow cytometric analysis of peripheral blood was utilized to determine B and T cell frequency. 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. Wanna4 mice exhibit decreased frequencies of peripheral T cells. Flow cytometric analysis of peripheral blood was utilized to determine T cell frequency. 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. Wanna4 mice exhibit decreased frequencies of peripheral CD4+ T cells. Flow cytometric analysis of peripheral blood was utilized to determine T cell frequency. 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 4. Wanna4 mice exhibit decreased frequencies of peripheral CD4+ T cells in CD3+ T cells. Flow cytometric analysis of peripheral blood was utilized to determine T cell frequency. 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 5. Wanna4 mice exhibit decreased frequencies of peripheral naive CD4+ T cells in CD4 T cells. Flow cytometric analysis of peripheral blood was utilized to determine T cell frequency. 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 6. Wanna4 mice exhibit decreased frequencies of peripheral CD8+ T cells. Flow cytometric analysis of peripheral blood was utilized to determine T cell frequency. 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 7. Wanna4 mice exhibit decreased frequencies of peripheral CD8+ T cells in CD3+ T cells. Flow cytometric analysis of peripheral blood was utilized to determine T cell frequency. 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 8. Wanna4 mice exhibit decreased frequencies of peripheral naive CD8+ T cells in CD8 T cells. Flow cytometric analysis of peripheral blood was utilized to determine T cell frequency. 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 9. Wanna4 mice exhibit increased frequencies of peripheral central memory CD4+ T cells in CD4 T cells. Flow cytometric analysis of peripheral blood was utilized to determine T cell frequency. 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 10. Wanna4 mice exhibit increased frequencies of peripheral central memory CD8+ T cells in CD8 T cells. Flow cytometric analysis of peripheral blood was utilized to determine T cell frequency. 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 11. Wanna4 mice exhibit increased CD4 to CD8 T cell ratios. Flow cytometric analysis of peripheral blood was utilized to determine T cell frequency. 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 12. Wanna4 mice exhibit increased expression of CD44 on peripheral T cells. Flow cytometric analysis of peripheral blood was utilized to determine CD44 MFI. 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 13. Wanna4 mice exhibit increased expression of CD44 on peripheral CD4 T cells. Flow cytometric analysis of peripheral blood was utilized to determine CD44 MFI. 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 14. Wanna4 mice exhibit increased expression of CD44 on peripheral CD8 T cells. Flow cytometric analysis of peripheral blood was utilized to determine CD44 MFI. 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 15. Wanna4 mice exhibit diminished T-dependent IgG responses to ovalbumin administered with aluminum hydroxide. IgG 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 wanna4 phenotype was identified among N-ethyl-N-nitrosourea (ENU)-mutagenized G3 mice of the pedigree R5271, some of which showed an increased B to T cell ratio (Figure 1) due to reduced frequencies of T cells (Figure 2), CD4+ T cells (Figure 3), CD4+ T cells in CD3+ T cells (Figure 4), naïve CD4 T cells in CD4 T cells (Figure 5), CD8+ T cells (Figure 6), CD8+ T cells in CD3+ T cells (Figure 7), and naïve CD8 T cells in CD8 T cells (Figure 8) with concomitant increased frequencies of central memory CD4 T cells in CD4 T cells (Figure 9), and central memory CD8 T cells in CD8 T cells (Figure 10), all in the peripheral blood. Some mice showed an increase in the CD4 to CD8 T cell ratio (Figure 11) as well as increased expression of CD44 on T cells (Figure 12), CD4 T cells (Figure 13) and CD8 T cells (Figure 14). The T-dependent antibody response to ovalbumin administered with aluminum hydroxide was also diminished (Figure 15).

Nature of Mutation

Figure 16. Linkage mapping of the B to T cell ratio phenotype using a recessive model of inheritance. Manhattan plot shows -log10 P values (Y-axis) plotted against the chromosome positions of 61 mutations (X-axis) identified in the G1 male of pedigree R5271. 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 61 mutations. All of the above anomalies were linked by continuous variable mapping to mutations in two genes on chromosome 1: Zap70 and Tbc1d8. The mutation in Zap70 was presumed causative because the wanna4 phenotypes mimic that of other Zap70 alleles (see MGI). The Zap70 mutation is a T to A transversion at base pair 36,781,365 (v38) on chromosome 1, or base pair 19,568 in the GenBank genomic region NC_000067 encoding Zap70.  The strongest association was found with a recessive model of inheritance to the normalized B to T cell ratio phenotype, wherein two variant homozygotes departed phenotypically from 15 homozygous reference mice and 22 heterozygous mice with a P value of 5.392 x 10-54 (Figure 16).  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 1,802 in the mRNA sequence NM_001289766 within exon 12 of 13 total exons.

1786 ATGAAGGGCCCCGAGGTCCTGGACTTCATCAAG
542  -M--K--G--P--E--V--L--D--F--I--K-
 

The mutated nucleotide is indicated in red.  The mutation results in a valine to aspartic acid substitution at position 547 (V547D) in the ZAP70 protein, and is strongly predicted by PolyPhen-2 to be damaging (score = 1.000).

Illustration of Mutations in
Gene & Protein
Protein Prediction
Figure 17. Structure of ZAP-70. Mouse Zap-70 is a 618 amino acid protein tyrosine kinasen (PTK) that consists of two N-terminal Src-homology 2 (SH2) domains and a C-terminal kinase domain. The SH2 domains are connected by a linker known as interdomain A (IDA), while the region between the second SH2 and catalytic domains is known as interdomain B (IDB). The aspartic acid (D) of the residue 459 is the proton acceptor during the catalytic cycle. Several tyrosine (Y) residues located within interdomain B are phosphorylated following TCR stimulation (291, 314, and 318). Phosphorylation of Tyr 492 is required for ZAP-70 activation, while Tyr 491 phosphorylation negatively regulates ZAP-70 function. The wanna4 mutation causes a valine to aspartic acid substitution at position 547 (V547D). The 3D structure is human ZAP70. UCSF Chimera structure based on PDB 2OZO. This image is interactive. Other mutations found in ZAP-70 are noted in red. Click on the mutations for more specific information. Click on the 3D structure to view it rotate.

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 17). 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 wanna4 mutation results in a valine to aspartic acid substitution at position 547 (V547D); residue 547 is within the kinase domain.

Please see the record for murdock for more information about Zap70.

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 wanna4 mice is similar to loss-of-function alleles of Zap70.

Primers PCR Primer
wanna4_pcr_F: CAGAGTGCATCAACTTTCGGAAG
wanna4_pcr_R: CTAAGGGTAAAGGTAGCGCTGC

Sequencing Primer
wanna4_seq_F: AAGTTCTCCAGCCGCAGTGAC
wanna4_seq_R: TAAAGGTAGCGCTGCCTGGG
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 403 nucleotides is amplified (chromosome 1, + strand):


1   cagagtgcat caactttcgg aagttctcca gccgcagtga cgtctggagc tatggggtca
61  ccatgtggga ggccttctcc tatggccaga agccctacaa ggtaggctgg gcagtttggc
121 aacggtgggc tggggaggtg gaccctggct cctcacacac gaatgccttt gtccctggcc
181 tgagcagaaa atgaagggcc ccgaggtcct ggacttcatc aagcagggta agaggatgga
241 atgtccgccg gagtgtcctc ctgagatgta tgcacttatg agtgactgct ggatctacaa
301 gtgagttcca ggggggcggg gggggggggc ggggatgtgg ccgtacagcc ctgagcctag
361 cgaggatctc ctcaagccca ggcagcgcta cctttaccct tag


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
AuthorsJin Huk Choi, Xue Zhong, Bruce Beutler