Phenotypic Mutation 'waterfowl' (pdf version)
Allelewaterfowl
Mutation Type start codon destroyed
Chromosome1
Coordinate36,770,811 bp (GRCm38)
Base Change A ⇒ G (forward strand)
Gene Zap70
Gene Name zeta-chain (TCR) associated protein kinase
Synonym(s) ZAP-70, TZK, Srk
Chromosomal Location 36,761,798-36,782,818 bp (+)
MGI 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.
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 Methionine changed to Valine
Institutional SourceBeutler Lab
Gene Model not available
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)
Phenotypic Category decrease in CD4+ T cells, decrease in CD4+ T cells in CD3+ T cells, decrease in CD8+ T cells, decrease in T cells, increase in B:T cells
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 36781149 missense probably damaging 1.00
murdock APN 1 36779704 missense possibly damaging 0.95
IGL00763:Zap70 APN 1 36779252 missense possibly damaging 0.81
IGL01635:Zap70 APN 1 36771157 missense possibly damaging 0.51
IGL01918:Zap70 APN 1 36778787 missense probably benign 0.00
IGL02164:Zap70 APN 1 36771186 missense probably damaging 0.98
IGL02502:Zap70 APN 1 36778806 splice site 0.00
IGL02597:Zap70 APN 1 36771920 nonsense probably null 0.00
IGL03026:Zap70 APN 1 36779717 missense probably damaging 1.00
biscayne UTSW 1 36781412 missense
mesa_verde UTSW 1 36779173 missense
trebia UTSW 1 36781025 missense probably damaging 1.00
wanna UTSW 1 36770983 missense probably damaging 1.00
wanna2 UTSW 1 36781412 missense probably damaging 1.00
wanna3 UTSW 1 36778218 missense probably damaging 0.99
wanna4 UTSW 1 36781365 missense
R0487:Zap70 UTSW 1 36779284 missense probably damaging 1.00
R0701:Zap70 UTSW 1 36781177 missense probably damaging 1.00
R0960:Zap70 UTSW 1 36779173 missense probably damaging 1.00
R1520:Zap70 UTSW 1 36770955 missense probably damaging 1.00
R2064:Zap70 UTSW 1 36779134 missense probably benign 0.00
R3023:Zap70 UTSW 1 36782631 missense noncoding transcript
R3623:Zap70 UTSW 1 36779135 missense probably benign 0.03
R3689:Zap70 UTSW 1 36781412 missense probably damaging 1.00
R3690:Zap70 UTSW 1 36781412 missense probably damaging 1.00
R3695:Zap70 UTSW 1 36782627 missense noncoding transcript
R3804:Zap70 UTSW 1 36771142 missense possibly damaging 0.58
R3840:Zap70 UTSW 1 36778417 missense probably damaging 1.00
R4260:Zap70 UTSW 1 36779108 missense noncoding transcript
R4383:Zap70 UTSW 1 36780961 missense probably damaging 1.00
R4632:Zap70 UTSW 1 36778458 missense probably benign
R4783:Zap70 UTSW 1 36779173 missense probably damaging 1.00
R5051:Zap70 UTSW 1 36781451 missense probably benign 0.00
R5271:Zap70 UTSW 1 36781365 missense probably damaging 1.00
R5291:Zap70 UTSW 1 36771962 missense noncoding transcript
R5304:Zap70 UTSW 1 36778218 missense probably damaging 0.99
R5792:Zap70 UTSW 1 36779009 missense noncoding transcript
R5932:Zap70 UTSW 1 36781146 missense probably damaging 1.00
R5941:Zap70 UTSW 1 36770949 missense probably damaging 1.00
S24628:Zap70 UTSW 1 36770811 start codon destroyed probably null 0.03
Mode of Inheritance Autosomal Recessive
Local Stock Live Mice
Repository
Last Updated 08/08/2017 7:58 PM by Diantha La Vine
Record Created 09/05/2013 6:57 PM by Kuan-Wen Wang
Record Posted 02/13/2017
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: 55,852,288 V658I probably benign Het
Ccdc40 T to C 11: 119,232,118 Y249H possibly damaging Het
Cpne5 C to T 17: 29,162,322 R192K noncoding transcript Het
D6Ertd527e C to G 6: 87,111,524 T223S unknown Homo
Gbp4 G to A 5: 105,121,106 R394C possibly damaging Het
Gm17638 A to G 15: 77,966,223 D1147G probably benign Het
Gpr183 C to A 14: 121,954,476 C211F probably damaging Homo
Lcp1 A to T 14: 75,227,006 I556F possibly damaging Het
Letm1 G to A 5: 33,747,444 P513S probably benign Het
Letm1 G to A 5: 33,747,446 P512L probably benign Het
Msh3 A to G 13: 92,346,786 V283A possibly damaging Het
Nfkb2 G to T 19: 46,307,567 E170D probably benign Het
Npr3 C to A 15: 11,848,563 M439I probably benign Het
Olfr1023 A to T 2: 85,887,438 I213F possibly damaging Het
Olfr1034 A to T 2: 86,047,055 H191L probably benign Het
Pax5 G to A 4: 44,691,886 A120V probably damaging Het
Plcb1 A to G 2: 135,337,499 Y609C probably damaging Het
Plxna1 G to A 6: 89,357,336 H104Y probably benign Homo
Pqlc3 C to A 12: 16,997,710 V123F probably benign Het
Rnf213 A to T 11: 119,414,469 I509F probably damaging Het
Ryr2 T to C 13: 11,869,156 S213G probably damaging Homo
Spint1 A to G 2: 119,245,615 T231A probably damaging Het
Tbcel C to A 9: 42,444,500 C139F probably benign Het
Thbs2 A to C 17: 14,679,973 S573A probably benign Het
Tmem43 C to A 6: 91,482,318 P257Q probably benign Homo
Tmprss13 A to G 9: 45,337,132 probably null Het
Tnc C to T 4: 64,018,012 G229D probably damaging Homo
Ugt1a6a TTCATCA to TTCA 1: 88,216,158 unknown Het
Vmn1r196 T to A 13: 22,293,836 V215D probably damaging Homo
Vmn1r22 G to T 6: 57,900,332 T220K probably benign Homo
Vmn2r116 G to A 17: 23,387,279 M388I possibly damaging Het
Zap70 A to G 1: 36,770,811 M1V probably null Homo
Zfp282 A to G 6: 47,897,881 D340G probably damaging Homo
Zfp282 T to A 6: 47,905,053 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).

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. Click on the image to view other mutations found in ZAP-70 (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