Phenotypic Mutation 'wanna' (pdf version)
Allelewanna
Mutation Type missense
Chromosome1
Coordinate36,810,064 bp (GRCm39)
Base Change A ⇒ G (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; Ensembl: ENSMUST00000027291, ENSMUST00000070299

MappedYes 
Amino Acid Change Histidine changed to Arginine
Institutional SourceBeutler Lab
Gene Model not available
AlphaFold P43404
SMART Domains Protein: ENSMUSP00000027291
Gene: ENSMUSG00000026117
AA Change: H58R

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)
SMART Domains Protein: ENSMUSP00000139990
Gene: ENSMUSG00000026117
AA Change: H58R

DomainStartEndE-ValueType
SH2 8 85 1.9e-16 SMART
Predicted Effect probably damaging

PolyPhen 2 Score 1.000 (Sensitivity: 0.00; Specificity: 1.00)
(Using ENSMUST00000185871)
Meta Mutation Damage Score Not available 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 100% 
Alleles Listed at MGI

All alleles(15) : Targeted, knock-out(2) Targeted, other(7) Gene trapped(1) Spontaneous(2) Chemically induced(3)

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
wanna2 UTSW 1 36820493 missense probably damaging 1.00
wanna3 UTSW 1 36817299 missense probably damaging 0.99
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 Sperm
Repository

none

Last Updated 2019-03-05 6:51 PM by Diantha La Vine
Record Created 2010-06-02 4:59 PM by Carrie N. Arnold
Record Posted 2011-01-05
Phenotypic Description

The wanna mutation was discovered while screening N-ethyl-N-nitrosourea (ENU)-mutagenized G3 mice for aberrant T-dependent and T-independent B cell responses. Wanna mice lack a T-dependent immuoglobin G (IgG) response to model antigens encoded by a recombinant suicide vector based on the Semliki Forest Virus (rSFV), but display normal T-independent IgM responses to haptenated ficoll and serum levels of IgA. Flow cytometric analysis of peripheral blood lymphocytes from the four original index mice showed a lack of peripheral CD4+ and CD8+ T cells (lower panels in CD4 vs CD8 plots), but had normal frequencies of natural killer (NK) cells (lower panels in CD8 vs NK1.1 plots) and mature B cells (not shown).

Nature of Mutation

The wanna mutation was mapped to Chromosome 1 by outcrossing homozygous animals to the closely related C57BL/10J strain and backcrossing F1 animals back to wanna mice. The DNA samples from phenotypically wild type and mutant animals were pooled separately and analyzed by bulk segregation analysis (BSA) using a panel of 124 single nucleotide polymorphisms (SNPs) (1).  Sequencing of the candidate gene Zap70 on Chromosome 1 found an A to G transition at position 264 of the Zap70 transcript using Genbank record NM_009539, in exon 2 of 13 total exons.

248 GACGTGCGCTTCCACCATTTCCCCATCGAGCGC
53  -D--V--R--F--H--H--F--P--I--E--R-

The mutated nucleotide is indicated in red lettering and causes a histidine to arginine change at amino acid 58 of the encoded protein.

Illustration of Mutations in
Gene & Protein
Protein Prediction
Figure 5. 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 wanna mutation causes a histidine to arginine change at amino acid 58. 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 wanna mutation results in a histidine to arginine substitution of a conserved residue in the N-terminal SH2 domain (Figure 5).  Along with the C-terminal SH2 domain, this domain is required to bind to the phosphorylated tyrosines of immunoreceptor tyrosine-based activation motifs (ITAMS) of the ζ and CD3ε chains of the T cell receptor (TCR; see the record for tumormouse).  The N-terminal SH2 domain makes van der Waals contacts with the second phosphorylated tyrosine of an ITAM [(D/E)xxYxxI/Lx(6-8)YxxI/L] according to crystal structure studies (FIgure 6) (2).  The phenotypes of wanna mice provide in vivo confirmation that His 58 is important for ZAP70 function.

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

Primers Primers cannot be located by automatic search.
Genotyping
Wanna genotyping is performed by amplifying the region containing the mutation using PCR, followed by sequencing of the amplified region to detect the single nucleotide transition.
 
Primers
Wanna(F): 5’- ACTGACTCCCGTGCAAAGATGAGG -3’
Wanna(R): 5’- TCAAGCATAGCACATGCTGAGGAC -3’
 
PCR program
1) 95°C             2:00
2) 95°C             0:30
3) 56°C             0:30
4) 72°C             1:00
5) repeat steps (2-4) 29X
6) 72°C             7:00
7) 4°C               8
 
Primers for sequencing
Wanna_seq(F): 5'- ATGATGGCCCTGAAACGC -3'
Wanna_seq(R): 5'- GAGGACTACAGCCTATAGTCTCTG -3'
 
The following sequence of 777 nucleotides (NCBI Mouse Genome Build 37.1, Chromosome 1, bases 36,827,587 to 36,828,363) is amplified:
 
 
actgactccc gtgcaaagat gaggcaccat gatggccctg aaacgccttt gcacccacag
ggtccagcga tgcccgatcc cgcggcgcac ctgccattct tctatggcag catctcgcgg
gctgaggccg aggagcacct gaagctggca ggcatggccg acgggctgtt cctcctgcgc
cagtgtttgc gctccctggg cggctacgtg ctgtcgttgg tgcacgacgt gcgcttccac
catttcccca tcgagcgcca actcaacggc acgtacgcca tcgcgggcgg gaaggcgcac
tgcggcccgg ccgagctctg ccagttctac tctcaggacc ccgacgggct gccctgcaac
ctgcgtaagc cgtgtaaccg gccgcccgga ctggagccac agcccggggt cttcgactgc
ctgcgtgatg ctatggtgcg cgactacgtg cgccagacct ggaagctgga ggtgagggcg
tggtcagagg gggagtgggc gaggcatgga ggctgatgct gtgtcttcat tggcttgcaa
gatgctctga ggcctgggat ttggaagtga cagaatgctg aggccctagt tgagcccatg
acctggaagg aggacttgca ggtccaggct tgcgcagcct gaatgatgct gtggtgcttc
accatctatg atgcgcctga aggtgccaga gaggaccatg aagggcaggc ctgtggccta
cagaaggatg gcagacagag actataggct gtagtcctca gcatgtgcta tgcttga

Primer binding sites are underlined; sequencing primer binding sites are highlighted in gray; the mutated T is indicated in red.
References
Science Writers Nora G. Smart
Illustrators Diantha La Vine
AuthorsCarrie Arnold, Elaine Pirie, and Bruce Beutler
Edit History
2011-08-17 10:18 AM (current)
2011-08-16 4:14 PM
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