Phenotypic Mutation 'citron' (pdf version)
Allelecitron
Mutation Type splice site (4 bp from exon)
Chromosome8
Coordinate72,139,620 bp (GRCm39)
Base Change C ⇒ T (forward strand)
Gene Jak3
Gene Name Janus kinase 3
Chromosomal Location 72,129,027-72,143,221 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 Janus kinase (JAK) family of tyrosine kinases involved in cytokine receptor-mediated intracellular signal transduction. It is predominantly expressed in immune cells and transduces a signal in response to its activation via tyrosine phosphorylation by interleukin receptors. Mutations in this gene are associated with autosomal SCID (severe combined immunodeficiency disease). [provided by RefSeq, Jul 2008]
PHENOTYPE: Mice homozygous for a knock-out allele exhibit impaired B cell development, small thymi and T cell proliferate. Point mutation homozygotes develop autoimmune inflammatory bowel disease, decreased susceptibility to malaria infection and/or increased susceptibility to bacterial infection. [provided by MGI curators]
Accession Number

NCBI RefSeq: NM_010589; NM_001190830; MGI:99928

MappedYes 
Amino Acid Change
Institutional SourceBeutler Lab
Gene Model predicted gene model for protein(s): [ENSMUSP00000060073] [ENSMUSP00000105639] [ENSMUSP00000105640]
AlphaFold no structure available at present
SMART Domains Protein: ENSMUSP00000060073
Gene: ENSMUSG00000031805

DomainStartEndE-ValueType
B41 20 254 2.2e-42 SMART
SH2 370 460 5.57e-8 SMART
low complexity region 488 503 N/A INTRINSIC
STYKc 517 773 3.58e-12 SMART
TyrKc 818 1091 4.59e-105 SMART
Predicted Effect probably benign
SMART Domains Protein: ENSMUSP00000105639
Gene: ENSMUSG00000031805

DomainStartEndE-ValueType
B41 20 254 2.2e-42 SMART
SH2 370 460 5.57e-8 SMART
low complexity region 488 503 N/A INTRINSIC
STYKc 517 773 3.58e-12 SMART
TyrKc 818 1091 4.59e-105 SMART
Predicted Effect probably benign
SMART Domains Protein: ENSMUSP00000105640
Gene: ENSMUSG00000031805

DomainStartEndE-ValueType
B41 20 254 2.2e-42 SMART
SH2 370 460 5.57e-8 SMART
low complexity region 488 503 N/A INTRINSIC
STYKc 517 773 3.58e-12 SMART
TyrKc 818 1091 4.59e-105 SMART
Predicted Effect probably benign
Meta Mutation Damage Score 0.0898 question?
Is this an essential gene? Possibly essential (E-score: 0.702) question?
Phenotypic Category Autosomal Recessive
Candidate Explorer Status loading ...
Single pedigree
Linkage Analysis Data
Penetrance  
Alleles Listed at MGI

All mutations/alleles(23) : Chemically induced (ENU)(3) Gene trapped(14) Spontaneous(1) Targeted(5)

Lab Alleles
AlleleSourceChrCoordTypePredicted EffectPPH Score
IGL00226:Jak3 APN 8 72134341 splice site probably benign
IGL00720:Jak3 APN 8 72136681 missense probably damaging 1.00
IGL00966:Jak3 APN 8 72131656 missense probably benign 0.24
IGL01147:Jak3 APN 8 72136047 missense probably benign
IGL01308:Jak3 APN 8 72137810 missense probably damaging 1.00
IGL01328:Jak3 APN 8 72132264 missense probably damaging 1.00
IGL01386:Jak3 APN 8 72136933 missense probably damaging 1.00
IGL01515:Jak3 APN 8 72133206 splice site probably null
IGL01870:Jak3 APN 8 72133434 missense probably damaging 1.00
IGL02132:Jak3 APN 8 72131124 missense probably damaging 0.99
IGL02413:Jak3 APN 8 72138763 splice site probably null
IGL02752:Jak3 APN 8 72135595 missense possibly damaging 0.50
IGL03089:Jak3 APN 8 72138727 missense probably benign 0.15
IGL03177:Jak3 APN 8 72135014 missense probably damaging 1.00
barbed UTSW 8 72131425 missense possibly damaging 0.88
beanstalk UTSW 8 72139932 missense probably benign 0.01
Bonis UTSW 8 72131898 missense probably benign 0.05
corrupt UTSW 8 72136696 missense probably damaging 1.00
daniels UTSW 8 72134299 missense possibly damaging 0.48
Deposuit UTSW 8 72138048 missense probably damaging 1.00
distortion UTSW 8 72136622 missense probably damaging 1.00
Downcast UTSW 8 72138155 missense probably benign 0.07
fake_news UTSW 8 72138601 missense probably damaging 1.00
Implevit UTSW 8 72131417 missense probably benign
mount_tai UTSW 8 72136021 missense probably damaging 1.00
potentes UTSW 8 72138702 missense probably damaging 0.99
Riot UTSW 8 72134960 missense probably damaging 1.00
thistle UTSW 8 72138027 critical splice acceptor site probably null
thistle2 UTSW 8 72138189 missense probably damaging 1.00
PIT4403001:Jak3 UTSW 8 72136993 missense probably benign 0.00
PIT4515001:Jak3 UTSW 8 72132286 missense probably benign 0.21
R0013:Jak3 UTSW 8 72136971 missense probably damaging 0.98
R0496:Jak3 UTSW 8 72135041 missense probably damaging 1.00
R0522:Jak3 UTSW 8 72134918 splice site probably benign
R0531:Jak3 UTSW 8 72139620 splice site probably benign
R0538:Jak3 UTSW 8 72138126 missense probably benign
R0612:Jak3 UTSW 8 72136021 missense probably damaging 1.00
R0744:Jak3 UTSW 8 72136622 missense probably damaging 1.00
R0833:Jak3 UTSW 8 72136622 missense probably damaging 1.00
R0836:Jak3 UTSW 8 72136622 missense probably damaging 1.00
R1183:Jak3 UTSW 8 72137194 missense probably damaging 1.00
R1420:Jak3 UTSW 8 72134182 missense possibly damaging 0.75
R1793:Jak3 UTSW 8 72138590 splice site probably benign
R1967:Jak3 UTSW 8 72134179 missense probably damaging 1.00
R1983:Jak3 UTSW 8 72140780 missense probably benign
R1983:Jak3 UTSW 8 72131019 missense possibly damaging 0.95
R2058:Jak3 UTSW 8 72138027 critical splice acceptor site probably null
R2060:Jak3 UTSW 8 72136059 nonsense probably null
R2060:Jak3 UTSW 8 72133358 nonsense probably null
R3705:Jak3 UTSW 8 72134166 missense probably damaging 1.00
R3734:Jak3 UTSW 8 72129225 unclassified probably benign
R4231:Jak3 UTSW 8 72138189 missense probably damaging 1.00
R4596:Jak3 UTSW 8 72137275 missense probably damaging 0.99
R4844:Jak3 UTSW 8 72134299 missense possibly damaging 0.48
R4897:Jak3 UTSW 8 72138048 missense probably damaging 1.00
R5038:Jak3 UTSW 8 72138702 missense probably damaging 0.99
R5469:Jak3 UTSW 8 72131417 missense probably benign
R5538:Jak3 UTSW 8 72131417 missense probably benign
R5718:Jak3 UTSW 8 72136998 missense probably damaging 1.00
R5799:Jak3 UTSW 8 72131344 missense probably damaging 1.00
R5909:Jak3 UTSW 8 72136875 missense possibly damaging 0.68
R5959:Jak3 UTSW 8 72134715 missense probably damaging 1.00
R6260:Jak3 UTSW 8 72131954 missense probably benign 0.00
R6798:Jak3 UTSW 8 72133615 missense probably damaging 0.99
R7013:Jak3 UTSW 8 72131425 missense possibly damaging 0.88
R7070:Jak3 UTSW 8 72137255 missense probably damaging 1.00
R7122:Jak3 UTSW 8 72138601 missense probably damaging 1.00
R7166:Jak3 UTSW 8 72134960 missense probably damaging 1.00
R7225:Jak3 UTSW 8 72138155 missense probably benign 0.07
R7440:Jak3 UTSW 8 72133362 missense probably benign 0.02
R7489:Jak3 UTSW 8 72136936 missense probably damaging 1.00
R7773:Jak3 UTSW 8 72131686 missense probably benign
R7779:Jak3 UTSW 8 72139932 missense probably benign 0.01
R8511:Jak3 UTSW 8 72138194 missense probably damaging 1.00
R8808:Jak3 UTSW 8 72138164 missense possibly damaging 0.71
R8859:Jak3 UTSW 8 72131160 missense probably benign 0.37
R9079:Jak3 UTSW 8 72131898 missense probably benign 0.05
R9320:Jak3 UTSW 8 72134265 missense probably benign 0.03
R9389:Jak3 UTSW 8 72136696 missense probably damaging 1.00
R9664:Jak3 UTSW 8 72131366 missense probably damaging 1.00
Z1176:Jak3 UTSW 8 72133327 missense possibly damaging 0.93
Mode of Inheritance Autosomal Recessive
Local Stock gDNA
MMRRC Submission 038161-MU
Last Updated 2019-09-04 9:48 PM by Diantha La Vine
Record Created 2014-06-14 10:20 PM by Ming Zeng
Record Posted 2015-02-11
Phenotypic Description

Figure 1. Citron mice exhibit an increased B:T cell ratio in the periphery. Flow cytometric analysis of 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. Citron mice exhibit increased 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. Citron mice exhibit increased frequencies of peripheral IgM+ B cells. Flow cytometric analysis of peripheral blood was utilized to determine IgM+ 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 4. Citron mice exhibit an increased percentage of peripheral IgD+ B cells. Flow cytometric analysis of peripheral blood was utilized to determine IgD+ B cell percentage. 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 citron phenotype was identified among N-Nitroso-N-ethylurea (ENU)-mutagenized G3 mice of the pedigree R0531, some of which showed an increase in the B:T cell ratio (Figure 1) due to an increased frequency of B cells (Figure 2) as well as an increased frequency of IgM+ B cells (Figure 3) and a higher percentage of IgD+ B cells (Figure 4), all in the peripheral blood.   

Nature of Mutation

Figure 5. Linkage mapping of the reduced B cell frequency using a recessive model of inheritance. Manhattan plot shows -log10 P values (Y-axis) plotted against the chromosome positions of 74 mutations (X-axis) identified in the G1 male of pedigree R0531.  Normalized phenotype data are shown for single locus linkage analysis with 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 74 mutations. All of the above anomalies were linked by continuous variable mapping to a mutation in Jak3:  a C to T transition at base pair 71,686,976 (v38) on chromosome 8, or base pair 10,594 in the GenBank genomic region NC_000074 encoding Jak3. The strongest association was found with a recessive model of linkage to the normalized B cell frequency, wherein three variant homozygotes departed phenotypically from six homozygous reference mice and 11 heterozygous mice with a P value of 2.244 x 10-7 (Figure 5).  The mutation is located within the donor splice site of intron 22, four nucleotides from the previous exon. Two Jak3 protein-coding transcripts are displayed on Ensembl; both transcripts encode the same protein. The effect of the mutation at the cDNA and protein level is unknown.  One possibility, shown below, is that aberrant splicing would cause skipping of the 118 base pair exon 22 (out of 25 total exons; exon 22 encodes amino acids 990-1028) and a frame-shift that results in coding of two aberrant amino acids followed by a premature stop codon in exon 23.  

        <--exon 21      <--exon 22 intron 22-->     exon 23-->

9733 ……CCCATCTTTTG ………AGCCCATCCGCT gtgcgtcgcctgc……… GAGTTCCTGA 10866
986  ……-P--I--F--W ………-S--P--S--A-                  --S--S--*  991

         correct        deleted                      aberrant

Genomic numbering corresponds to NC_000074. The donor splice site of intron 22, which is destroyed by the mutation, is indicated in blue; the mutated nucleotide is indicated in red.

Illustration of Mutations in
Gene & Protein
Protein Prediction
Figure 6. Domain structure of JAK3. The citron mutation destroys the donor splice site of intron 22. Abbreviations: B41, 4.1, ezrin, radixin, moesin (FERM) homology domain; SH2, Src Homology 2-like. This image is interactive. Other mutations found in JAK3 are noted in red. Click on each mutation for more information.

Jak3 encodes Janus kinase 3 (JAK3). JAK3 has seven different highly conserved JAK homology (JH) regions (JH1-JH7) [Figure 6; reviewed in (1)]. The JH1 region is the kinase domain (amino acids 818-1091), the JH2 domain corresponds to the pseudokinase domain (amino acids 517-773), the JH3 and JH4 regions comprise an Src Homology 2 (SH2)-like domain (amino acids 370-460), and the JH6 and JH7 regions consist of a 4.1, ezrin, radixin, moesin (FERM) homology domain (alternatively, B41 domain; amino acids 20-254). The citron mutation is predicted to result in deletion of exon 22 which encodes amino acids 990-1023 within the JAK3 kinase domain, a frame-shift, and coding of a premature stop codon within the JAK3 kinase domain. Expression and localization of JAK3­citron have not been examined.

Please see the record mount_tai for more information about Jak3.

Putative Mechanism

JAK3 binding is restricted to hematopoietic-specific cytokine receptors that have a γc receptor subunit (i.e., the IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21 receptors) [reviewed in (1)]. The γc receptor-associated cytokines have known functions. For example, IL-7 is necessary for T and B cell development, IL-2 functions in peripheral T cell homeostasis and antigen-driven T-cell expansion, IL-15 functions in natural killer (NK) cell differentiation, IL-4 functions in B-cell maturation and isotype switching (2). JAK3 mutations result in defective γc receptor-associated signaling and subsequent defects in lymphocyte development (2;3). Mutations in JAK3 are linked to autosomal recessive T- and NK-cell negative/B-cell positive type of severe combined immunodeficiency [TB+NK- SCID; OMIM: #600802; (4-6); reviewed in (2)]. Patients with TB+NK- SCID do not have T or NK cells, but have normal to elevated numbers of immature nonfunctional B lymphocytes (5;6). Patients with SCID have persistent, recurring infections due to loss of T cell-associated immunity.  Jak3 knockout (Jak3-/-) mice have reduced numbers of T, B, γδ T, and NK cells (7-11). B cell maturation in the Jak3-/- mice is blocked at the pre-B stage, leading to a reduced frequency of IgM+ B cells (10). In contrast to Jak3-/- mice, citron mice exhibit increased frequencies of IgM+ B cells. Similar to patients with TB+NK- SCID, the citron mice have increased frequencies of peripheral B cells, including the IgM+ and IgD+ B cells; however the frequency of peripheral T and NK cells were comparable between the citron and wild-type mice, indicating that JAK3­citron may retain some function.

Primers PCR Primer
citron_pcr_F: GCCATACTTCACATTGACGACTCCC
citron_pcr_R: AAAGTATTCCCAGTCAGGCACCGC

Sequencing Primer
citron_seq_F: AGGCCACTGTTTCACACC
citron_seq_R: ATCATGCTCAGGAACTCCTGTG
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 665 nucleotides is amplified (chromosome 8, + strand):


1   gccatacttc acattgacga ctccctcccc aggccactgt ttcacaccct gcccactctc
61  ctcactggcc acaccacacc tctgtccggc tctcaggtat gccccggagt ccctatctga
121 caacatcttc tcccgccaat ctgacgtgtg gagcttcgga gtggtgttgt acgagctctt
181 cacctactgc gacaagagct gcagcccatc cgctgtgcgt cgcctgcccc atccctcggt
241 cctccctctt gatctccaaa tcccctcctg acctctagcc cctatcctga ccccagccct
301 tcctcctgac ctccagatca tctcctgacc tcagtcctcc ctccggaatc ccagcccttc
361 ctcctcatct ccagatccct tcttgacccc agcccttccc ctgaccccca gcccttcctc
421 atgacgtcag cccccttcct gaccctaaca gtcctgccct aacttgctcc tctcccacag
481 gagttcctga gcatgatggg gcctgagcgt gaaggacccc cgctctgccg cctcctggag
541 ctgctggcag agggccgacg cctcccacca cctcccacct gccccaccga ggtgaggaag
601 gaggactcaa gtttctcagt ttcaattctt gattgacagc agcggtgcct gactgggaat
661 acttt


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

References
Science Writers Anne Murray
Illustrators Peter Jurek
AuthorsMing Zeng, Kuan-Wen Wang, Jin Huk Choi, Bruce Beutler