Phenotypic Mutation 'waterfowl'

• Allele: waterfowl
• Mutation Type: start codon destroyed
• Chromosome: 1
• Coordinate: 36,770,811 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: 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]
• Mapped: Yes
• Amino Acid Change: Methionine changed to Valine
• Institutional Source: Beutler Lab
• Gene Model: not available
• AlphaFold: P43404
• SMART Domains: Protein: ENSMUSP00000027291; Gene: ENSMUSG00000026117; AA Change: M1V

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

Domain	Start	End	E-Value	Type
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
• Is this an essential gene?: Probably nonessential (E-score: 0.184)
• Phenotypic Category: Autosomal Recessive
• Mode of Inheritance: Autosomal Recessive
• Local Stock: Live Mice
• 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
• Science Writers: Anne Murray
• Illustrators: Peter Jurek, Katherine Timer
• Authors: Kuan-Wen Wang, Jin Huk Choi, Ming Zeng, Bruce Beutler

Accession Number

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

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)

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. 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 ZAP70^waterfowl 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

  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.

  4. Palacios, E. H., and Weiss, A. (2004) Function of the Src-Family Kinases, Lck and Fyn, in T-Cell Development and Activation. Oncogene. 23, 7990-8000.

  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.

  9. Fallah-Arani, F., Schweighoffer, E., Vanes, L., and Tybulewicz, V. L. (2008) Redundant Role for Zap70 in B Cell Development and Activation. Eur J Immunol. 38, 1721-1733.