Phenotypic Mutation 'special' (pdf version)
Allelespecial
Mutation Type missense
Chromosome1
Coordinate52,178,423 bp (GRCm39)
Base Change A ⇒ G (forward strand)
Gene Stat1
Gene Name signal transducer and activator of transcription 1
Synonym(s) 2010005J02Rik
Chromosomal Location 52,158,599-52,201,024 bp (+) (GRCm39)
MGI Phenotype FUNCTION: [Summary is not available for the mouse gene. This summary is for the human ortholog.] The protein encoded by this gene is a member of the STAT protein family. In response to cytokines and growth factors, STAT family members are phosphorylated by the receptor associated kinases, and then form homo- or heterodimers that translocate to the cell nucleus where they act as transcription activators. This protein can be activated by various ligands including interferon-alpha, interferon-gamma, EGF, PDGF and IL6. This protein mediates the expression of a variety of genes, which is thought to be important for cell viability in response to different cell stimuli and pathogens. Two alternatively spliced transcript variants encoding distinct isoforms have been described. [provided by RefSeq, Jul 2008]
PHENOTYPE: Homozygotes for targeted null mutations are largely unresponsive to interferon, fail to thrive, are susceptible to viral diseases and cutaneous leishmaniasis, and show excess osteoclastogenesis leading to increased bone mass. [provided by MGI curators]
Accession Number

NCBI RefSeq: NM_001205313 (variant 1), NM_009283 (variant 2), NM_001205314 (variant 3); MGI:103063

MappedYes 
Amino Acid Change Lysine changed to Glutamic Acid
Institutional SourceBeutler Lab
Gene Model predicted gene model for protein(s): [ENSMUSP00000066743] [ENSMUSP00000141132] [ENSMUSP00000140518] [ENSMUSP00000140875] [ENSMUSP00000141125] [ENSMUSP00000139746]
AlphaFold no structure available at present
SMART Domains Protein: ENSMUSP00000066743
Gene: ENSMUSG00000026104
AA Change: K286E

DomainStartEndE-ValueType
STAT_int 2 122 2.5e-61 SMART
Pfam:STAT_alpha 139 315 1.4e-56 PFAM
Pfam:STAT_bind 317 566 4.2e-82 PFAM
SH2 571 687 1.59e-1 SMART
Pfam:STAT1_TAZ2bind 715 739 2.4e-17 PFAM
Predicted Effect probably damaging

PolyPhen 2 Score 1.000 (Sensitivity: 0.00; Specificity: 1.00)
(Using ENSMUST00000070968)
SMART Domains Protein: ENSMUSP00000141132
Gene: ENSMUSG00000026104
AA Change: K286E

DomainStartEndE-ValueType
STAT_int 2 122 2.5e-61 SMART
Pfam:STAT_alpha 136 315 3.4e-65 PFAM
Pfam:STAT_bind 317 573 3.9e-118 PFAM
SH2 577 693 1.59e-1 SMART
Pfam:STAT1_TAZ2bind 721 745 2.3e-16 PFAM
Predicted Effect probably damaging

PolyPhen 2 Score 1.000 (Sensitivity: 0.00; Specificity: 1.00)
(Using ENSMUST00000186057)
SMART Domains Protein: ENSMUSP00000140518
Gene: ENSMUSG00000026104
AA Change: K286E

DomainStartEndE-ValueType
STAT_int 2 122 1.9e-65 SMART
Pfam:STAT_alpha 136 315 3.3e-62 PFAM
Pfam:STAT_bind 317 567 1.1e-118 PFAM
SH2 571 687 1e-3 SMART
Predicted Effect probably damaging

PolyPhen 2 Score 1.000 (Sensitivity: 0.00; Specificity: 1.00)
(Using ENSMUST00000186574)
SMART Domains Protein: ENSMUSP00000140875
Gene: ENSMUSG00000026104
AA Change: K286E

DomainStartEndE-ValueType
STAT_int 2 122 2.5e-61 SMART
Pfam:STAT_alpha 136 315 1.2e-64 PFAM
Pfam:STAT_bind 317 567 4.4e-121 PFAM
SH2 571 687 1.59e-1 SMART
Pfam:STAT1_TAZ2bind 715 739 3.1e-15 PFAM
Predicted Effect probably damaging

PolyPhen 2 Score 1.000 (Sensitivity: 0.00; Specificity: 1.00)
(Using ENSMUST00000186857)
SMART Domains Protein: ENSMUSP00000141125
Gene: ENSMUSG00000026104
AA Change: K286E

DomainStartEndE-ValueType
STAT_int 2 122 1.9e-65 SMART
Pfam:STAT_alpha 136 315 3.3e-62 PFAM
Pfam:STAT_bind 317 567 1.1e-118 PFAM
SH2 571 687 1e-3 SMART
Predicted Effect probably damaging

PolyPhen 2 Score 1.000 (Sensitivity: 0.00; Specificity: 1.00)
(Using ENSMUST00000189347)
SMART Domains Protein: ENSMUSP00000139746
Gene: ENSMUSG00000026104
AA Change: K286E

DomainStartEndE-ValueType
STAT_int 2 122 1.9e-65 SMART
Pfam:STAT_alpha 136 315 3.3e-62 PFAM
Pfam:STAT_bind 317 567 1.1e-118 PFAM
SH2 571 687 1e-3 SMART
Predicted Effect probably damaging

PolyPhen 2 Score 1.000 (Sensitivity: 0.00; Specificity: 1.00)
(Using ENSMUST00000191435)
Meta Mutation Damage Score 0.4118 question?
Is this an essential gene? Non Essential (E-score: 0.000) question?
Phenotypic Category Unknown
Candidate Explorer Status loading ...
Single pedigree
Linkage Analysis Data
Penetrance  
Alleles Listed at MGI

All Mutations and Alleles(23) : Chemically induced (ENU)(4) Chemically induced (other)(1) Radiation induced(1) Targeted(17)

Lab Alleles
AlleleSourceChrCoordTypePredicted EffectPPH Score
IGL00092:Stat1 APN 1 52161754 start codon destroyed probably null 0.50
IGL01111:Stat1 APN 1 52182120 critical splice donor site probably null
IGL01451:Stat1 APN 1 52178502 missense probably damaging 1.00
IGL01469:Stat1 APN 1 52186529 missense possibly damaging 0.87
IGL01758:Stat1 APN 1 52176080 missense probably damaging 1.00
IGL01818:Stat1 APN 1 52190437 missense probably damaging 1.00
IGL01913:Stat1 APN 1 52165716 missense probably benign 0.08
IGL01914:Stat1 APN 1 52165716 missense probably benign 0.08
IGL02304:Stat1 APN 1 52171703 missense probably benign
IGL02428:Stat1 APN 1 52182125 splice site probably benign
Accretion UTSW 1 52174780 missense possibly damaging 0.65
Aspect UTSW 1 52190408 missense probably benign 0.01
baroque UTSW 1 52183368 missense probably damaging 1.00
Compounding UTSW 1 52190440 missense probably benign 0.17
domino UTSW 1 52179747 missense probably damaging 1.00
h_moll UTSW 1 52178353 nonsense probably null
kun_ming UTSW 1 52176575 missense possibly damaging 0.52
kuomintang UTSW 1 52190404 missense possibly damaging 0.51
poison UTSW 1 52190384 splice site probably benign
roccoco UTSW 1 52162368 missense probably damaging 1.00
rollo UTSW 1 52193082 nonsense probably null
Sedimentary UTSW 1 52178388 missense probably damaging 1.00
vandegraff UTSW 1 52194178 missense probably benign 0.01
R0022:Stat1 UTSW 1 52179789 missense probably damaging 1.00
R0022:Stat1 UTSW 1 52179789 missense probably damaging 1.00
R0039:Stat1 UTSW 1 52179819 missense probably damaging 0.99
R0458:Stat1 UTSW 1 52188211 splice site probably benign
R1313:Stat1 UTSW 1 52195165 missense probably damaging 0.98
R1313:Stat1 UTSW 1 52195165 missense probably damaging 0.98
R2998:Stat1 UTSW 1 52190408 missense probably benign 0.01
R4464:Stat1 UTSW 1 52176575 missense possibly damaging 0.52
R4709:Stat1 UTSW 1 52165680 missense probably damaging 0.97
R4934:Stat1 UTSW 1 52193082 nonsense probably null
R5038:Stat1 UTSW 1 52162368 missense probably damaging 1.00
R5075:Stat1 UTSW 1 52161871 missense possibly damaging 0.73
R5223:Stat1 UTSW 1 52183401 missense probably damaging 1.00
R5600:Stat1 UTSW 1 52188101 missense probably benign 0.06
R5866:Stat1 UTSW 1 52178423 missense probably damaging 1.00
R7105:Stat1 UTSW 1 52190408 missense probably benign 0.01
R7192:Stat1 UTSW 1 52174780 missense possibly damaging 0.65
R7284:Stat1 UTSW 1 52188081 missense probably benign 0.01
R7309:Stat1 UTSW 1 52165780 splice site probably null
R7491:Stat1 UTSW 1 52191530 missense probably benign 0.31
R7680:Stat1 UTSW 1 52183368 missense probably damaging 1.00
R7825:Stat1 UTSW 1 52190467 missense probably damaging 0.98
R7915:Stat1 UTSW 1 52190440 missense probably benign 0.17
R8245:Stat1 UTSW 1 52194178 missense probably benign 0.01
R8309:Stat1 UTSW 1 52190404 missense possibly damaging 0.51
R8728:Stat1 UTSW 1 52178353 nonsense probably null
R8952:Stat1 UTSW 1 52187042 missense probably benign 0.01
R9054:Stat1 UTSW 1 52182086 missense probably damaging 1.00
R9156:Stat1 UTSW 1 52178388 missense probably damaging 1.00
R9209:Stat1 UTSW 1 52184337 missense probably benign
R9252:Stat1 UTSW 1 52174831 missense probably benign 0.03
R9337:Stat1 UTSW 1 52191429 missense probably benign 0.00
R9388:Stat1 UTSW 1 52193037 missense possibly damaging 0.81
R9530:Stat1 UTSW 1 52187160 critical splice donor site probably null
R9648:Stat1 UTSW 1 52165695 missense probably damaging 0.98
RF036:Stat1 UTSW 1 52191419 missense probably benign
RF060:Stat1 UTSW 1 52191419 missense probably benign
X0027:Stat1 UTSW 1 52178430 missense probably damaging 1.00
Mode of Inheritance Unknown
Local Stock
Repository
Last Updated 2019-09-04 9:38 PM by Anne Murray
Record Created 2018-02-23 2:45 PM by Bruce Beutler
Record Posted 2018-04-04
Phenotypic Description
Figure 1. Special mice exhibit decreased B to T cell ratios. Flow cytometric analysis of peripheral blood was utilized to determine B and T cell frequencies. 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. Special mice exhibit decreased frequencies of peripheral B cells. Flow cytometric analysis of peripheral blood was utilized to determine B 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. Special mice exhibit decreased frequencies of peripheral B1 cells. Flow cytometric analysis of peripheral blood was utilized to determine B1 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. Special mice exhibit increased CD4 to CD8 T cell ratios. Flow cytometric analysis of peripheral blood was utilized to determine T cell frequencies. 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. Special mice exhibit increased frequencies of peripheral CD4+ 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 6. Special mice exhibit increased frequencies of peripheral 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 7. Special mice exhibit reduced frequencies of peripheral 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 8. Special mice exhibit reduced frequencies of peripheral 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 9. Special mice exhibit reduced frequencies of peripheral naive 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 10. Special mice exhibit reduced frequencies of peripheral NK cells. Flow cytometric analysis of peripheral blood was utilized to determine NK 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 11. Special mice exhibit increased frequencies of peripheral NK T cells. Flow cytometric analysis of peripheral blood was utilized to determine NK 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 12. Special mice exhibit increased expression of CD44 on peripheral blood T cells. Flow cytometric analysis of peripheral blood was utilized to determine CD44 MFI. 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 13. Special mice exhibit increased expression of CD44 on peripheral blood CD8+ T cells. Flow cytometric analysis of peripheral blood was utilized to determine CD44 MFI. 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 special phenotype was identified among N-ethyl-N-nitrosourea (ENU)-mutagenized G3 mice of the pedigree R5866, some of which showed reduced B to T cell ratios (Figure 1) due to reduced frequencies of B cells (Figure 2) and B1 cells (Figure 3). Some mice showed increased CD4 to CD8 T cell ratios (Figure 4) due to increased frequencies of CD4+ T cells in CD3+ T cells (Figure 5) and effector memory CD8 T cells in CD8 T cells (Figure 6) with concomitant reduced frequencies of CD8+ T cells (Figure 7), CD8+ T cells in CD3+ T cells (Figure 8), and naïve CD8 T cells in CD8 T cells (Figure 9), all in the peripheral blood. Some mice also showed reduced frequencies of NK cells (Figure 10) and increased frequencies of NK T cells (Figure 11) in the peripheral blood. Expression of CD44 on peripheral blood T cells (Figure 12) and CD8+ T cells (Figure 13) was increased.

Nature of Mutation

Figure 14. Linkage mapping of the increased NK T cell frequency phenotype  using a recessive model of inheritance. Manhattan plot shows -log10 P values (Y-axis) plotted against the chromosome positions of 52 mutations (X-axis) identified in the G1 male of pedigree R5866. 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 52 mutations. All of the above anomalies were linked by continuous variable mapping to a mutation in Stat1: an A to G transition at base pair 52,139,264 (v38) on chromosome 1, or base pair 19,827 in the GenBank genomic region NC_000067. The strongest association was found with a recessive model of inheritance to the increased frequency of NK T cells, wherein five variant homozygotes departed phenotypically from 34 homozygous reference mice and 33 heterozygous mice with a P value of 6.847 x 10-16 (Figure 14).  

The mutation corresponds to residue 1,155 in the mRNA sequence NM_001205313 within exon 10 of 25 total exons.

1140 GAGGAGTTGGAACAGAAATTCACCTATGAGCCC

281  -E--E--L--E--Q--K--F--T--Y--E--P-

The mutated nucleotide is indicated in red. The mutation results in a lysine to glutamic acid substitution at amino acid 286 (K286E) in the STAT1 protein, and is strongly predicted by PolyPhen-2 to be damaging (score = 1.000).

Illustration of Mutations in
Gene & Protein
Protein Prediction

Figure 15. 3D and domain structure of the STAT1 protein. A) 3D representation of STAT1 based on crystalized structures of human STAT1 residues 1-683 (PDB 1YVL). The residue affected by the domino mutation is shown in red. 3D image was created using UCSF Chimera. B) Domain structure of STAT1. CC=Coiled Coil domain; DBD = DNA binding domain; LD = Linker domain; SH2=Src Homology 2 domain; TAD = Transcriptional activation domain. The critical tyrosine phosphorylation site is found at amino acid 701. The special mutation results in a lysine to glutamic acid substitution at amino acid 286 (K286E) in the STAT1 protein). Other mutations found in STAT1 are noted in red. Click on the mutations for more specific information. Click on the 3D structure to view it rotate.

Signal transducer and activator of transcription (STAT)-1 is one of seven STAT family members identified in mammals. The STAT proteins serve the dual functions of signal transduction and activation of transcription. STAT1 is a 755 amino acid protein, and like all STATs, contains an N-terminal helical domain (N-domain), a four-helix bundle (coiled coil), a central Ig-like DNA binding domain, a helical linker domain, an SH2 domain, and a C-terminal transactivation domain (TAD) (Figure 15).

The special mutation results in a lysine to glutamic acid substitution at amino acid 286 (K286E) in the STAT1 protein; amino acid 286 is within the coiled coil domain.

Please see the record for domino for more information about Stat1.

Putative Mechanism

The STAT proteins are transcription factors found latent in the cytoplasm until they are activated by extracellular signaling proteins such as cytokines, growth factors and peptides. Stimulation by these extracellular signaling proteins leads to activation of intracellular tyrosine kinases that in turn phosphorylate STATs, causing them to move into the nucleus and activate transcription of target genes. STAT1 is required for IFN signaling.  NK cell function is enhanced by IFNs, and Stat1-/- NK cells have impaired cytotoxicity relative to wild type NK cells (1)

Stat1-/- mice have no gross developmental abnormalities, but are highly sensitive to bacterial and viral infections such as Listeria monocytogenes and VSV infection (2;3). Cells from these mice are unresponsive to IFN-α and IFN-γ, although they respond normally to several other stimuli including EGF and interleukin 10 (2;3). In humans, rare STAT1 deficiency and several STAT1 point mutations have been identified in patients with recurrent bacterial and/or viral infections (4-6). Cells from these patients fail to respond to IFN-α or IFN-γ. Interestingly, one patient with complete STAT1 deficiency was able to clear at least some viruses including polio virus type III (from vaccination) and parainfluenza type II (6).

The phenotypes observed in the special mice indicate loss of STAT1special function.

Primers PCR Primer
special_pcr_F: TCCCATGTGATGCCCATAGC
special_pcr_R: TGAAAACCGATTAGCCCGTC

Sequencing Primer
special_seq_F: GCCCATAGCATGTGGAGATTC
special_seq_R: ATCCAAAGAGTTCCTGGGC
Genotyping

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 361 nucleotides is amplified (chromosome 1, + strand):


1   tcccatgtga tgcccatagc atgtggagat tctgtgactc tctcatctag tgtgttttgg
61  gactgttgtt catggagacc ccatgtcctg taccctccca ggttcaccat tgttgcagag
121 accctgcagc agatccgtca gcagcttaaa aagctggagg agttggaaca gaaattcacc
181 tatgagcccg accctattac aaaaaacaag caggtgttgt cagatcgaac cttcctcctc
241 ttccagcagc tcattcagag gtaaggctag gcagactgac tatccagaag gccatctatg
301 gtcatttgga cagtttggaa gggtttgatg gtggagcgtg gagtttggct actctgtttt
361 g


Primer binding sites are underlined and the sequencing primers are highlighted; the mutated nucleotide is shown in red.

References

Science Writers Anne Murray
Illustrators Diantha La Vine
AuthorsXue Zhong, Jin Huk Choi, Evan Nair-Gill, Jianhui Wang, and Bruce Beutler