Phenotypic Mutation 'waterfowl' (pdf version)
Allelewaterfowl
Mutation Type start codon destroyed
Chromosome1
Coordinate36,809,892 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 (variant 1), NM_001289612 (variant 2), NM_001289765 (variant 3), NM_001289766 (variant 4); MGI: 99613

MappedYes 
Amino Acid Change Methionine changed to Valine
Institutional SourceBeutler Lab
Gene Model not available
AlphaFold P43404
SMART Domains Protein: ENSMUSP00000027291
Gene: ENSMUSG00000026117
AA Change: M1V

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 null

PolyPhen 2 Score 0.025 (Sensitivity: 0.95; Specificity: 0.81)
(Using ENSMUST00000027291)
SMART Domains Protein: ENSMUSP00000139990
Gene: ENSMUSG00000026117
AA Change: M1V

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

PolyPhen 2 Score 0.013 (Sensitivity: 0.96; Specificity: 0.78)
(Using ENSMUST00000185871)
Meta Mutation Damage Score 0.8637 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
wanna4 UTSW 1 36820446 missense probably damaging 1.00
want_to UTSW 1 36821598 missense probably damaging 1.00
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 Live Mice
Repository
Last Updated 2019-03-05 6:45 PM by Diantha La Vine
Record Created 2013-09-05 6:57 PM by Kuan-Wen Wang
Record Posted 2017-02-13
Other Mutations in This Stock Stock #: S24628 Run Code: HSQ01102
Coding Region Coverage: 10x: 94.3% 20x: 88.0%
Validation Efficiency: 0/0

GeneSubstitutionChr/LocMutationPredicted EffectZygosity
Adgre4 G to A 17: 56,159,288 (GRCm39) V658I probably benign Het
Ccdc40 T to C 11: 119,122,944 (GRCm39) Y249H possibly damaging Het
D6Ertd527e C to G 6: 87,088,506 (GRCm39) T223S unknown Homo
Gbp4 G to A 5: 105,268,972 (GRCm39) R394C possibly damaging Het
Gpr183 C to A 14: 122,191,888 (GRCm39) C211F probably damaging Homo
Lcp1 A to T 14: 75,464,446 (GRCm39) I556F possibly damaging Het
Letm1 G to A 5: 33,904,788 (GRCm39) P513S probably benign Het
Letm1 G to A 5: 33,904,790 (GRCm39) P512L probably benign Het
Msh3 A to G 13: 92,483,294 (GRCm39) V283A possibly damaging Het
Nfkb2 G to T 19: 46,296,006 (GRCm39) E170D probably benign Het
Npr3 C to A 15: 11,848,649 (GRCm39) M439I probably benign Het
Or5m10 A to T 2: 85,717,782 (GRCm39) I213F possibly damaging Het
Or5m9 A to T 2: 85,877,399 (GRCm39) H191L probably benign Het
Pax5 G to A 4: 44,691,886 (GRCm39) A120V probably damaging Het
Plcb1 A to G 2: 135,179,419 (GRCm39) Y609C probably damaging Het
Plxna1 G to A 6: 89,334,318 (GRCm39) H104Y probably benign Homo
Rnf213 A to T 11: 119,305,295 (GRCm39) I509F probably damaging Het
Ryr2 T to C 13: 11,884,042 (GRCm39) S213G probably damaging Homo
Spint1 A to G 2: 119,076,096 (GRCm39) T231A probably damaging Het
Tbcel C to A 9: 42,355,796 (GRCm39) C139F probably benign Het
Thbs2 A to C 17: 14,900,235 (GRCm39) S573A probably benign Het
Tmem43 C to A 6: 91,459,300 (GRCm39) P257Q probably benign Homo
Tmprss13 A to G 9: 45,248,430 (GRCm39) probably null Het
Tnc C to T 4: 63,936,249 (GRCm39) G229D probably damaging Homo
Ugt1a10 TTCATCA to TTCA 1: 88,143,880 (GRCm39) probably benign Het
Vmn1r196 T to A 13: 22,478,006 (GRCm39) V215D probably damaging Homo
Vmn1r22 G to T 6: 57,877,317 (GRCm39) T220K probably benign Homo
Vmn2r116 G to A 17: 23,606,253 (GRCm39) M388I possibly damaging Het
Zap70 A to G 1: 36,809,892 (GRCm39) M1V probably null Homo
Zfp282 A to G 6: 47,874,815 (GRCm39) D340G probably damaging Homo
Zfp282 T to A 6: 47,881,987 (GRCm39) I558N possibly damaging Homo
Phenotypic Description

Figure 1. Waterfowl mice exhibit an increased B to T cell ratio. Flow cytometric analysis of the 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. Waterfowl mice exhibit a reduced frequency of T cells. Flow cytometric analysis of the 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. Waterfowl mice exhibit a reduced frequency of CD4+ T cells. Flow cytometric analysis of the 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. Waterfowl mice exhibit a reduced frequency of CD4+ T in CD3+ T cells. Flow cytometric analysis of the 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. Waterfowl mice exhibit a reduced frequency of CD8+ T cells. Flow cytometric analysis of the 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.

The waterfowl phenotype was identified among G3 mice of the pedigree R0415, some of which showed an increase in the B:T cell ratio (Figure 1), a decrease in the frequency of T cells (Figure 2), a decrease in the frequency of CD4+ T cells (Figure 3), a decrease in the frequency of CD4+ T cells in CD3+ T cells (Figure 4), and a decrease in the frequency of CD8+ T cells (Figure 5), all in the peripheral blood.

Nature of Mutation
Figure 6. Zap70 mutant mice in pedigree R0415 exhibited diminished frequencies of CD4+ T cells. Zap70 mutant homozygous mice in pedigree R0415 exhibited reduced frequencies of CD4+ T cells in the peripheral blood compared to wild-type and heterozygous littermates. Statistical analysis showed that the CD4+ T cell phenotype was linked to the R0415 mutation in Zap70 by a recessive model of inheritance.

Sequencing of a homozygous variant G3 mouse identified a mutation in Zap70:  an A to G transition at base pair 36,770,811 (v38) on chromosome 1, or base pair 9,014 in the GenBank genomic region NC_000067 encoding Zap70. The mutation in Zap70 was presumed causative because the waterfowl immune phenotypes mimic that of other alleles of Zap70 (see MGI for a list of Zap70 alleles as well as the entry for murdoch, biscayne, trebia, wanna, and wanna2). Expansion of the R0415 pedigree and assessment of peripheral blood CD4+ T cell frequency by flow cytometry confirmed that the mutation in Zap70 was the causative mutation for the phenotypes observed in waterfowl (recessive linkage, P = 1.30 x 10-8; Figure 6).


The mutation corresponds to residue 155 in the mRNA sequence NM_009539 within exon 2 of 13 total exons, residue 198 in the mRNA sequence NM_001289765 within exon 2 of 13, and residue 163 in the mRNA sequence NM_001289766 within exon 2 of 13 total exons. The mutation is not predicted to affect transcript variant 2 (NM_001289612), because this transcript encodes an isoform, TZK (alternatively, truncated ZAP kinase) with a shorter N-terminus compared to the other isoforms.

 
9005 ...GTCCAGGTCCAGCATGCCCGATCCCGCGGCG...

1       …………………………………-M--P--D--P--A--A-...

Genomic numbering corresponds to NC_000067. The mutated nucleotide is indicated in red. The mutation results in a methionine (M) to valine (V) substitution at position 1 (M1V) in the ZAP70 protein, and is strongly predicted by PolyPhen-2 to be damaging (score = 0.999).

Illustration of Mutations in
Gene & Protein
Protein Prediction
Figure 7. 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 waterfowl mutation causes a methionine to valine change at amino acid 1. 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. 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 waterfowl mutation results in a methionine (M) to valine (V) substitution at the initiation codon (Figure 7). The next available methionine occurs at residue 29. Expression of ZAP70waterfowl has not been examined.

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 waterfowl mice exhibit a similar phenotype to the wanna mice, which exhibit an ENU-induced mutation in Zap70.

Primers Primers cannot be located by automatic search.
Genotyping

Genotyping is performed by amplifying the region containing the mutation using PCR, followed by sequencing of the amplified region to detect the mutation.
 

PCR Primers

S246280001_PCR_F: 5’- GTCACGCCTATCACACTATTGACC-3’

S246280001_PCR_R: 5’- AAATGGTGGAAGCGCACGTC-3’

Sequencing Primers

S246280001_SEQ_F: 5’- GACATATCTTTGGAAACTGAAGGGTC-3’
 

S246280001_SEQ_R: 5’- GTCGTGCACCAACGACAG-3’
 

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 446 nucleotides is amplified (NCBI RefSeq: NC_000077, chromosome 1:36770541-36770986 encoding Zap70):

gtcacgccta tcacactatt gaccgccacc tcccgtgtgt gtgtggcctt cgaaagggaa       

gggaatgaca tatctttgga aactgaaggg tcagttcaga cctggggacc ttcggggtgt      

ctggatgttg tgcacaggtc cccaaaaagt cagaagtggg tttcagaagt gggtgccagg      

acagggtagc tcctctctgg aactgactcc cgtgcaaaga tgaggcacca tgatggccct      

gaaacgcctt tgcacccaca gggtccagcg atgcccgatc ccgcggcgca cctgccattc      

ttctatggca gcatctcgcg ggctgaggcc gaggagcacc tgaagctggc aggcatggcc      

gacgggctgt tcctcctgcg ccagtgtttg cgctccctgg gcggctacgt gctgtcgttg     

gtgcacgacg tgcgcttcca ccattt

Primer binding sites are underlined and the sequencing primer is highlighted; the mutated nucleotide is shown in red text (Chr. + = A>G)

References
Science Writers Anne Murray
Illustrators Peter Jurek, Katherine Timer
AuthorsKuan-Wen Wang, Jin Huk Choi, Ming Zeng, Bruce Beutler