Phenotypic Mutation 'roccoco' (pdf version)
Alleleroccoco
Mutation Type missense
Chromosome1
Coordinate52,162,368 bp (GRCm39)
Base Change A ⇒ T (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 
Limits of the Critical Region 52119440 - 52161865 bp
Amino Acid Change Asparagine changed to Isoleucine
Institutional SourceBeutler Lab
Gene Model predicted gene model for protein(s): [ENSMUSP00000066743] [ENSMUSP00000141132] [ENSMUSP00000140518] [ENSMUSP00000140875] [ENSMUSP00000141144] [ENSMUSP00000141125] [ENSMUSP00000139746]
AlphaFold no structure available at present
SMART Domains Protein: ENSMUSP00000066743
Gene: ENSMUSG00000026104
AA Change: N75I

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 0.999 (Sensitivity: 0.14; Specificity: 0.99)
(Using ENSMUST00000070968)
SMART Domains Protein: ENSMUSP00000141132
Gene: ENSMUSG00000026104
AA Change: N75I

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 0.999 (Sensitivity: 0.14; Specificity: 0.99)
(Using ENSMUST00000186057)
SMART Domains Protein: ENSMUSP00000140518
Gene: ENSMUSG00000026104
AA Change: N75I

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 0.998 (Sensitivity: 0.27; Specificity: 0.99)
(Using ENSMUST00000186574)
SMART Domains Protein: ENSMUSP00000140875
Gene: ENSMUSG00000026104
AA Change: N75I

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 0.999 (Sensitivity: 0.14; Specificity: 0.99)
(Using ENSMUST00000186857)
SMART Domains Protein: ENSMUSP00000141144
Gene: ENSMUSG00000026104
AA Change: N75I

DomainStartEndE-ValueType
STAT_int 2 122 1.9e-65 SMART
Predicted Effect probably damaging

PolyPhen 2 Score 1.000 (Sensitivity: 0.00; Specificity: 1.00)
(Using ENSMUST00000188681)
SMART Domains Protein: ENSMUSP00000141125
Gene: ENSMUSG00000026104
AA Change: N75I

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 0.998 (Sensitivity: 0.27; Specificity: 0.99)
(Using ENSMUST00000189347)
SMART Domains Protein: ENSMUSP00000139746
Gene: ENSMUSG00000026104
AA Change: N75I

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 0.998 (Sensitivity: 0.27; Specificity: 0.99)
(Using ENSMUST00000191435)
Meta Mutation Damage Score 0.8967 question?
Is this an essential gene? Non Essential (E-score: 0.000) question?
Phenotypic Category Autosomal Recessive
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
rollo UTSW 1 52193082 nonsense probably null
Sedimentary UTSW 1 52178388 missense probably damaging 1.00
special UTSW 1 52178423 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 Autosomal Recessive
Local Stock
Repository
Last Updated 2019-09-04 9:40 PM by Diantha La Vine
Record Created 2017-04-14 9:31 AM by Evan Nair-Gill
Record Posted 2017-09-15
Phenotypic Description

Figure 1. Roccoco mice exhibit decreased frequencies of peripheral naïve CD4 T cells in CD4 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 2. Roccoco mice exhibit decreased frequencies of peripheral 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.
Figure 3. Roccoco mice exhibit increased frequencies of peripheral CD44+ 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. Roccoco mice exhibit increased frequencies of peripheral CD44+ CD4+ 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. Roccoco mice exhibit increased frequencies of peripheral effector memory CD4 T cells in CD4 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. Roccoco 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. Roccoco mice exhibit increased CD44 epression on peripheral 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 8. Roccoco mice exhibit increased CD44 epression on peripheral CD4 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 9. Roccoco mice exhibit increased CD44 epression on peripheral 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.
Figure 10. Roccoco mice exhibit decreased killing of CTL targets pulsed with a MHC-class I restricted peptide for beta-galactosidase after immunization with rSFV-beta-galactosidase. 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 roccoco phenotype was identified among N-ethyl-N-nitrosourea (ENU)-mutagenized G3 mice of the pedigree R5038 some of which showed reduced frequencies of naïve CD4 T cells in CD4 T cells (Figure 1) and naïve CD8 T cells in CD8 T cells (Figure 2) with concomitant increased frequencies of CD44+ T cells (Figure 3), CD44+ CD4 T cells (Figure 4), effector memory CD4 T cells in CD4 T cells (Figure 5), and effector memory CD8 T cells in CD8 T cells (Figure 6), all in the peripheral blood. Some mice also showed increased CD44 expression on peripheral blood T cells (Figure 7), including CD4 (Figure 8) and CD8 T cells (Figure 9). Homozygous mice showed decreased killing of CTL targets pulsed with a MHC-class I restricted peptide for beta-galactosidase after immunization with rSFV-beta-galactosidase (Figure 10).

Nature of Mutation

Figure 11. Linkage mapping of the increased frequency of effector memory CD8 T cells using a recessive model of inheritance. Manhattan plot shows -log10 P values (Y-axis) plotted against the chromosome positions of 64 mutations (X-axis) identified in the G1 male of pedigree R5038. 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 64 mutations. All of the above anomalies were linked by continuous variable mapping to a mutation in Stat1: an A to T transversion at base pair 52,123,209 (v38) on chromosome 1, or base pair 3,772 in the GenBank genomic region NC_000067 encoding Stat1. The strongest association was found with a recessive model of inheritance to the increased frequency of effector memory CD8 T cells in CD8 T cells, wherein three variant homozygotes departed phenotypically from 22 homozygous reference mice and 16 heterozygous mice with a P value of 5.909 x 10-24 (Figure 11).  

The mutation corresponds to residue 523 in the mRNA sequence NM_001205313 within exon 4 of 25 total exons.

507 CGCTTTTCTCTGGAGAATAATTTCTTGTTGCAG

70  -R--F--S--L--E--N--N--F--L--L--Q-

The mutated nucleotide is indicated in red. The mutation results in an asparagine to isoleucine substitution at amino acid 75 (N75I) in the STAT1 protein, and is strongly predicted by PolyPhen-2 to be damaging (score = 0.999).

Illustration of Mutations in
Gene & Protein
Protein Prediction
Figure 12. 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 roccoco mutation results in an asparagine to isoleucine substitution at amino acid 75 (N75I). This image is interactive. 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, a central Ig-like DNA binding domain, a helical linker domain, an SH2 domain, and a C-terminal transactivation domain (TAD) (Figure 12).

The roccoco mutation results in an asparagine to isoleucine substitution at amino acid 75 (N75I) in the STAT1 protein; amino acid 75 is within the N-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 roccoco mice indicates loss of STAT1roccoco function.

Primers PCR Primer
roccoco_pcr_F: CAAATGTCTGGCACTTCCTATAC
roccoco_pcr_R: CAGCCACATAAAGAGAGTGTATCTG

Sequencing Primer
roccoco_seq_F: GTCAGTTGTGTGTGATAGAAACAC
roccoco_seq_R: AGAGAGTGTATCTGTACTTCTGTAAC
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 415 nucleotides is amplified (chromosome 1, + strand):


1   caaatgtctg gcacttccta tacttttttc atgtcagttg tgtgtgatag aaacactacg
61  cgaattgcta ataaaacaag gtcaggttaa aacgcaaagc taaattcccg ctgttgtagg
121 gagcacgctg cctatgatgt ctcgtttgcg accatccgct tccatgacct cctctcacag
181 ctggacgacc agtacagccg cttttctctg gagaataatt tcttgttgca gcacaacata
241 cggaaaagca agcgtaatct ccaggtacgg actggctttg agcctcactt gaagtgtctc
301 cgctgactta aaaccttttc tttatttgga ctcagcagca gatgctactg agtgcacgct
361 cactgggcat tgaaagaagt tacagaagta cagatacact ctctttatgt ggctg


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, Peter Jurek, Katherine Timer
AuthorsEvan Nair-Gill, Xue Zhong, Bruce Beutler