Phenotypic Mutation 'wenzhou' (pdf version)
Allelewenzhou
Mutation Type missense
Chromosome6
Coordinate71,350,856 bp (GRCm39)
Base Change A ⇒ T (forward strand)
Gene Cd8a
Gene Name CD8 subunit alpha
Synonym(s) Lyt-2, Ly-B, Ly-35, Ly-2
Chromosomal Location 71,350,411-71,356,155 bp (+) (GRCm39)
MGI Phenotype FUNCTION: [Summary is not available for the mouse gene. This summary is for the human ortholog.] The CD8 antigen is a cell surface glycoprotein found on most cytotoxic T lymphocytes that mediates efficient cell-cell interactions within the immune system. The CD8 antigen acts as a coreceptor with the T-cell receptor on the T lymphocyte to recognize antigens displayed by an antigen presenting cell in the context of class I MHC molecules. The coreceptor functions as either a homodimer composed of two alpha chains or as a heterodimer composed of one alpha and one beta chain. Both alpha and beta chains share significant homology to immunoglobulin variable light chains. This gene encodes the CD8 alpha chain. Multiple transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq, Nov 2011]
PHENOTYPE: Animals homozygous for a mutation in this gene lack CD8+CD4- cytotoxic T cells in the thymus and spleen and do not mount a cytotoxic response to alloantigens. [provided by MGI curators]
Accession Number

NCBI RefSeq: NM_001081110.2 (variant 1), NM_009857.1 (variant 2); MGI: 88346

MappedYes 
Limits of the Critical Region 71373427 - 71379171 bp
Amino Acid Change Aspartic acid changed to Valine
Institutional SourceBeutler Lab
Gene Model predicted gene model for protein(s): [ENSMUSP00000068123] [ENSMUSP00000131873]
AlphaFold P01731
PDB Structure MURINE CD8AA ECTODOMAIN FRAGMENT IN COMPLEX WITH H-2KB/VSV8 [X-RAY DIFFRACTION]
The Crystal Structure of a TL/CD8aa Complex at 2.1A resolution:Implications for Memory T cell Generation, Co-receptor Preference and Affinity [X-RAY DIFFRACTION]
CD8alpha-alpha in complex with YTS 105.18 Fab [X-RAY DIFFRACTION]
Crystal structure of a CD8ab heterodimer [X-RAY DIFFRACTION]
Crystal structure of CD8alpha-beta in complex with YTS 156.7 FAB [X-RAY DIFFRACTION]
Crystal structure of the CD8 alpha beta/H-2Dd complex [X-RAY DIFFRACTION]
SMART Domains Protein: ENSMUSP00000068123
Gene: ENSMUSG00000053977
AA Change: D107V

DomainStartEndE-ValueType
low complexity region 9 26 N/A INTRINSIC
IG 38 148 1.46e-5 SMART
transmembrane domain 195 217 N/A INTRINSIC
Predicted Effect probably benign

PolyPhen 2 Score 0.022 (Sensitivity: 0.95; Specificity: 0.81)
(Using ENSMUST00000066747)
SMART Domains Protein: ENSMUSP00000131873
Gene: ENSMUSG00000053977
AA Change: D107V

DomainStartEndE-ValueType
low complexity region 9 26 N/A INTRINSIC
IG 38 148 1.46e-5 SMART
transmembrane domain 195 217 N/A INTRINSIC
Predicted Effect probably benign

PolyPhen 2 Score 0.022 (Sensitivity: 0.95; Specificity: 0.81)
(Using ENSMUST00000172321)
Meta Mutation Damage Score 0.8322 question?
Is this an essential gene? Probably nonessential (E-score: 0.089) question?
Phenotypic Category Autosomal Recessive
Candidate Explorer Status loading ...
Single pedigree
Linkage Analysis Data
Penetrance  
Alleles Listed at MGI

All mutations/alleles(18) :  Chemically induced (ENU)(1) Spontaneous(1) Targeted(13) Transgenic(3)

Lab Alleles
AlleleSourceChrCoordTypePredicted EffectPPH Score
IGL00737:Cd8a APN 6 71350691 missense probably benign 0.04
IGL02342:Cd8a APN 6 71350723 missense probably damaging 1.00
Alfalfa UTSW 6 71350712 missense probably damaging 0.99
Sprouts UTSW 6 71350913 missense probably damaging 0.97
PIT4618001:Cd8a UTSW 6 71350661 missense possibly damaging 0.94
R0212:Cd8a UTSW 6 71350633 missense probably benign 0.01
R1158:Cd8a UTSW 6 71350712 missense probably damaging 0.99
R1813:Cd8a UTSW 6 71350947 missense possibly damaging 0.47
R4541:Cd8a UTSW 6 71350856 missense probably benign 0.02
R5836:Cd8a UTSW 6 71350775 missense possibly damaging 0.48
R6390:Cd8a UTSW 6 71350913 missense probably damaging 0.97
R6889:Cd8a UTSW 6 71351546 missense probably damaging 1.00
R7773:Cd8a UTSW 6 71350799 missense probably benign 0.01
Z1088:Cd8a UTSW 6 71350670 missense possibly damaging 0.85
Z1177:Cd8a UTSW 6 71351577 missense possibly damaging 0.92
Mode of Inheritance Autosomal Recessive
Local Stock
Repository
Last Updated 2016-09-22 10:20 AM by Anne Murray
Record Created 2016-06-16 8:12 PM by Xue Zhong
Record Posted 2016-09-22
Phenotypic Description

Figure 1. Wenzhou mice exhibit an increase in the CD4+ to CD8+ T cell ratio. Flow cytometric analysis of peripheral blood was utilized to determine T cell frequency. Normalized log 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. Wenzhou mice exhibit a reduced frequency of peripheral blood 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 3. Wenzhou mice exhibit a reduced frequency of peripheral blood 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 4. Wenzhou mice exhibit a reduced frequency of peripheral blood effector 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 5. Wenzhou mice exhibit a reduced frequency of peripheral blood naïve 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.

The wenzhou phenotype was identified among G3 mice of the pedigree R4541, some of which showed an increase in the CD4+ to CD8+ T cell ratio (Figure 1) caused by reduced frequencies of CD8+ T cells (Figure 2), CD8+ T cells in CD3+ T cells (Figure 3), effector memory CD8+ T cells in CD8+ T cells (Figure 4), and naïve CD8+ T cells in CD8+ T cells (Figure 5).

Nature of Mutation

Figure 6. Linkage mapping of the diminished frequency of CD8+ T cells in CD3+ T cells using a recessive model of inheritance. Manhattan plot shows -log10 P values (Y-axis) plotted against the chromosome positions of 48 mutations (X-axis) identified in the G1 male of pedigree R4541. 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 48 mutations. All of the above anomalies were linked by continuous variable mapping to a mutation in Cd8a: an A to T transversion at base pair 71,373,872 (v38) on chromosome 6, or base pair 446 in the GenBank genomic region NC_000072 encoding Cd8a. The strongest association was found with a recessive model of linkage to the normalized frequency of CD8+ T cells in CD3+ T cells, wherein three variant homozygotes departed phenotypically from five homozygous reference mice and 11 heterozygous mice with a P value of 2.17 x 10-13 (Figure 6). A substantial semidominant effect was observed in most of the assays but the mutation is preponderantly recessive, and in no assay was a purely dominant effect observed. 

The mutation affects nucleotide 446 of Cd8a mRNA variants 1 (NM_001081110.2) and 2 (NM_009857.1) within exon 1 of 5 total exons and exon 1 of 4 total exons, respectively:

430 TTTTCTGCCATGAGGGACACGAATAATAAGTAC

102 -F--S--A--M--R--D--T--N--N--K--Y-

The mutated nucleotide is indicated in red. The mutation results in an aspartic acid (D) to valine (V) substitution at position 107 (D107V) of both variants of the CD8 antigen, α chain (CD8α) and is strongly predicted by PolyPhen-2 to be benign (score = 0.022).

Illustration of Mutations in
Gene & Protein
Protein Prediction
Figure 7. Domain structure of CD8α. Three complementarity-determining regions (CDR) mediate binding to peptide-MHCI complexes. SP, signal peptide; TMD, transmembrane domain; CYT, cytoplasmic tail. The wenzhou mutation results in an aspartic acid (D) to valine (V) substitution at position 107 (D107V). This image is interactive; click to view additional mutations in CD8α.

The CD8α chain (also known as Lyt-2) is a 34-37 kDa single pass type I transmembrane glycoprotein of the immunoglobulin (Ig) superfamily. The CD8αβ heterodimer functions as a coreceptor for the T cell receptor (TCR) [reviewed in (1)].  In contrast, the function of CD8αα is unknown; it has been speculated to function as a TCR corepressor that negatively regulates cellular activation (2).  Both CD8 isoforms bind to MHC class I molecules with equivalent affinity (3;4).

The 247-amino acid mouse CD8α chain consists of an N-terminal hydrophobic signal sequence (amino acids (aa) 1-27), an extracellular Ig-like domain (aa 28-139) followed by a hinge or stalk region containing three O-linked glycosylation sites (aa 140-196), a transmembrane segment (aa 197-217), and a cytoplasmic tail (aa 218-247) [Figure 7; reviewed in (5)].  In mouse CD8α, cysteines 53 and 129 form an intramolecular disulfide bond that is characteristic of an Ig fold.  Disulfide linkage with the CD8β protein is mediated by cysteines 178 and 193. The extracellular Ig-like domain of both CD8 dimers contact the α3 domain of the MHCI heavy chain, whereas CD8αα additionally contacts the α2 domain and β2-microglobulin. Three complementarity-determining regions (CDRs), consisting of the BC loop (CDR1), the C’C” loop (CDR2), and the FG loop (CDR3), are involved in binding to the α3 domain of pMHCI, and in the case of CD8αα also to β2m.  Connecting the Ig-like domain to the transmembrane domain, the stalk region of CD8α contains numerous threonine, serine, and proline residues and is heavily O-glycosylated (6-8). The cytoplasmic domain of CD8α binds to the Src family kinase Lck (see iconoclast) (9;10).  Two cysteines in the CD8α cytoplasmic domain (C227 and C229 in mouse) are necessary for this interaction (11;12).

The wenzhou mutation lies within the extracellular Ig-like domain of CD8α, in the region between the CDR2 and CDR3 regions.

For more information about Cd8a, please see the record for alfalfa.

Putative Mechanism

T cells become activated when the TCR engages a peptide antigen in complex with an MHC molecule on the surface of a target or antigen-presenting cell. TCR signaling is necessary for both adaptive immune responses and for thymocyte differentiation. The major function of CD8αβ is to recruit the kinase Lck to the TCR-MHC interaction site where it can phosphorylate immunoreceptor tyrosine-based activation motifs (ITAMs) on the cytoplasmic tails of associated CD3 proteins (see tumormouse, a mutation of Cd3e, and allia, a mutation of Cd247) (13;14).  Their phosphorylation recruits and activates other proteins including ZAP-70 (ζ-chain-associated protein of 70 kDa; see murdock), SLP-76 (SH2 domain-containing leukocyte protein of 76 kDa), and LAT (linker for activation of T cells) necessary for signaling leading to T cell activation, proliferation, and differentiation. CD8α serves as a Lck docking site (9;10) through a ‘zinc clasp’ structure that tethers Lck to the CD8α cytoplasmic tail (15). Mice deficient in CD8α and/or CD8β displayed diminished or absent CD8+ T cells in the periphery, although thymocyte differentiation to the DP stage was intact.  Consequently, cytotoxic responses against alloantigens and viral antigens were reduced in T cells from these mice. 

The CDRs are involved in binding to the α3 domain of pMHCI. It is likely that the wenzhou mutation impairs the binding of CD8αβ to pMHC1, leading to diminished numbers and frequencies of CD8 T cells in the blood as a result of defective positive selection.

Primers PCR Primer
wenzhou_pcr_F: AAAATGGACGCCGAACTTGG
wenzhou_pcr_R: GAGGGAATCTGCGTGAAACTTG

Sequencing Primer
wenzhou_seq_F: CGCCGAACTTGGTCAGAAG
wenzhou_seq_R: GAAACTTGTTCAGATGTGACCCC
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

R45410014_PCR_F: 5’- AAAATGGACGCCGAACTTGG-3’

R45410014_PCR_R: 5’- GAGGGAATCTGCGTGAAACTTG-3’

Sequencing Primers

R45410014_SEQ_F: 5’- CGCCGAACTTGGTCAGAAG-3’
 

R45410014_SEQ_R: 5’- GAAACTTGTTCAGATGTGACCCC-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 400 nucleotides is amplified (NCBI RefSeq: NC_000072, chromosome 6. Chr6:71373670-71374069):

aaaatggacg ccgaacttgg tcagaaggtg gacctggtat gtgaagtgtt ggggtccgtt       

tcgcaaggat gctcttggct cttccagaac tccagctcca aactccccca gcccaccttc      

gttgtctata tggcttcatc ccacaacaag ataacgtggg acgagaagct gaattcgtcg      

aaactgtttt ctgccatgag ggacacgaat aataagtacg ttctcaccct gaacaagttc      

agcaaggaaa acgaaggcta ctatttctgc tcagtcatca gcaactcggt gatgtacttc      

agttctgtcg tgccagtcct tcagaaaggt ttggaagtgg aggttcccgt ctcctgggtt      

ttaggggtca catctgaaca agtttcacgc agattccctc

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

References
Science Writers Anne Murray
Illustrators Katherine Timer
AuthorsMing Zeng, Xue Zhong, Bruce Beutler