Phenotypic Mutation 'everglades' (pdf version)
Alleleeverglades
Mutation Type missense
Chromosome14
Coordinate20,581,016 bp (GRCm39)
Base Change A ⇒ T (forward strand)
Gene Ppp3cb
Gene Name protein phosphatase 3, catalytic subunit, beta isoform
Synonym(s) Cnab, CnAbeta, 1110063J16Rik, Calnb, PP2BA beta
Chromosomal Location 20,549,432-20,596,641 bp (-) (GRCm39)
MGI Phenotype PHENOTYPE: Homozygous null mice have small hearts and thymi, and reduced body weight. Cardiac function is normal, but mice lack a cardiac hypertrophic response to pressure overload, angiotensin II, or isopreteronol. Thymi are hypoplastic, with abnormal T cell development and reduced numbers of T cells. [provided by MGI curators]
Accession Number

NCBI RefSeq: NM_008914 (variant 1), NM_001310426 (variant 2), NM_001310427 (variant 3); MGI:107163

MappedYes 
Amino Acid Change Isoleucine changed to Lysine
Institutional SourceBeutler Lab
Gene Model predicted gene model for protein(s): [ENSMUSP00000022355] [ENSMUSP00000125722] [ENSMUSP00000125630] [ENSMUSP00000125582]
AlphaFold P48453
SMART Domains Protein: ENSMUSP00000022355
Gene: ENSMUSG00000021816
AA Change: I136K

DomainStartEndE-ValueType
low complexity region 2 20 N/A INTRINSIC
PP2Ac 65 356 5.03e-166 SMART
Predicted Effect probably damaging

PolyPhen 2 Score 0.975 (Sensitivity: 0.76; Specificity: 0.96)
(Using ENSMUST00000022355)
SMART Domains Protein: ENSMUSP00000125722
Gene: ENSMUSG00000021816
AA Change: I136K

DomainStartEndE-ValueType
low complexity region 2 20 N/A INTRINSIC
PP2Ac 65 356 5.03e-166 SMART
Predicted Effect probably damaging

PolyPhen 2 Score 0.986 (Sensitivity: 0.74; Specificity: 0.96)
(Using ENSMUST00000159027)
SMART Domains Protein: ENSMUSP00000125630
Gene: ENSMUSG00000021816
AA Change: I136K

DomainStartEndE-ValueType
low complexity region 2 20 N/A INTRINSIC
PP2Ac 65 356 5.03e-166 SMART
Predicted Effect probably damaging

PolyPhen 2 Score 0.997 (Sensitivity: 0.41; Specificity: 0.98)
(Using ENSMUST00000161445)
SMART Domains Protein: ENSMUSP00000125582
Gene: ENSMUSG00000021816
AA Change: I136K

DomainStartEndE-ValueType
low complexity region 2 20 N/A INTRINSIC
PP2Ac 65 356 5.03e-166 SMART
low complexity region 487 497 N/A INTRINSIC
Predicted Effect possibly damaging

PolyPhen 2 Score 0.936 (Sensitivity: 0.80; Specificity: 0.94)
(Using ENSMUST00000161989)
Meta Mutation Damage Score 0.9571 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(28) : Gene trapped(25) Targeted(3)

Lab Alleles
AlleleSourceChrCoordTypePredicted EffectPPH Score
IGL00702:Ppp3cb APN 14 20578318 missense probably benign 0.00
IGL00844:Ppp3cb APN 14 20581754 missense possibly damaging 0.95
IGL01859:Ppp3cb APN 14 20559517 missense probably damaging 0.99
IGL02490:Ppp3cb APN 14 20581726 critical splice donor site probably null
IGL02546:Ppp3cb APN 14 20551622 missense probably benign 0.00
IGL02555:Ppp3cb APN 14 20581021 missense probably damaging 1.00
IGL02724:Ppp3cb APN 14 20573645 splice site probably null
IGL02944:Ppp3cb APN 14 20578303 missense probably damaging 1.00
IGL03072:Ppp3cb APN 14 20581793 missense probably damaging 1.00
IGL03301:Ppp3cb APN 14 20574052 missense probably damaging 0.99
Copacabana UTSW 14 20581010 critical splice donor site probably null
eden_express UTSW 14 20578263 nonsense probably null
Havana UTSW 14 20581820 missense possibly damaging 0.85
justinian UTSW 14 20558611 missense possibly damaging 0.73
Prokopios UTSW 14 20570720 missense probably benign 0.05
Redwood UTSW 14 20559508 missense probably damaging 1.00
R0026:Ppp3cb UTSW 14 20581836 missense probably benign 0.00
R0050:Ppp3cb UTSW 14 20581820 missense possibly damaging 0.85
R0050:Ppp3cb UTSW 14 20581820 missense possibly damaging 0.85
R0218:Ppp3cb UTSW 14 20574044 missense probably damaging 0.99
R0479:Ppp3cb UTSW 14 20553309 splice site probably null
R1013:Ppp3cb UTSW 14 20574072 missense probably benign
R1061:Ppp3cb UTSW 14 20558682 splice site probably null
R1498:Ppp3cb UTSW 14 20559567 critical splice acceptor site probably null
R1508:Ppp3cb UTSW 14 20574492 missense probably damaging 0.99
R1719:Ppp3cb UTSW 14 20574131 missense probably benign 0.05
R1799:Ppp3cb UTSW 14 20574540 missense possibly damaging 0.81
R1883:Ppp3cb UTSW 14 20573913 missense possibly damaging 0.66
R2082:Ppp3cb UTSW 14 20558746 missense possibly damaging 0.66
R2176:Ppp3cb UTSW 14 20570720 missense probably benign 0.05
R3021:Ppp3cb UTSW 14 20573921 nonsense probably null
R3726:Ppp3cb UTSW 14 20581010 critical splice donor site probably null
R4085:Ppp3cb UTSW 14 20558611 missense possibly damaging 0.73
R4328:Ppp3cb UTSW 14 20581016 missense probably damaging 1.00
R4509:Ppp3cb UTSW 14 20565569 intron probably benign
R4600:Ppp3cb UTSW 14 20570714 missense possibly damaging 0.60
R4601:Ppp3cb UTSW 14 20570714 missense possibly damaging 0.60
R4603:Ppp3cb UTSW 14 20570714 missense possibly damaging 0.60
R4610:Ppp3cb UTSW 14 20570714 missense possibly damaging 0.60
R4611:Ppp3cb UTSW 14 20570714 missense possibly damaging 0.60
R4694:Ppp3cb UTSW 14 20551583 missense probably benign 0.00
R4749:Ppp3cb UTSW 14 20574130 missense probably damaging 1.00
R4866:Ppp3cb UTSW 14 20573911 missense probably damaging 1.00
R4911:Ppp3cb UTSW 14 20559508 missense probably damaging 1.00
R5105:Ppp3cb UTSW 14 20559490 missense possibly damaging 0.84
R5219:Ppp3cb UTSW 14 20578263 nonsense probably null
R5586:Ppp3cb UTSW 14 20570758 splice site probably benign
R5740:Ppp3cb UTSW 14 20551664 missense possibly damaging 0.76
R6649:Ppp3cb UTSW 14 20581094 missense probably damaging 1.00
R7362:Ppp3cb UTSW 14 20573719 missense probably benign 0.00
R7493:Ppp3cb UTSW 14 20558619 missense probably benign 0.01
R8291:Ppp3cb UTSW 14 20573662 missense possibly damaging 0.89
R8438:Ppp3cb UTSW 14 20565658 missense probably damaging 0.99
R8515:Ppp3cb UTSW 14 20581844 missense probably benign 0.21
R8867:Ppp3cb UTSW 14 20596517 unclassified probably benign
R9136:Ppp3cb UTSW 14 20581867 missense probably benign 0.33
R9254:Ppp3cb UTSW 14 20581874 missense probably benign
R9379:Ppp3cb UTSW 14 20581874 missense probably benign
R9516:Ppp3cb UTSW 14 20573868 missense probably damaging 1.00
R9670:Ppp3cb UTSW 14 20578314 missense probably damaging 1.00
Z1177:Ppp3cb UTSW 14 20558586 missense unknown
Mode of Inheritance Autosomal Recessive
Local Stock
Repository
Last Updated 2019-09-04 9:43 PM by Diantha La Vine
Record Created 2016-05-05 2:34 PM
Record Posted 2016-11-01
Phenotypic Description

Figure 2. Everglades mice exhibit decreased frequencies of peripheral blood 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. Everglades mice exhibit decreased frequencies of peripheral blood CD4+ T cells. Flow cytometric analysis of peripheral blood was utilized to determine CD4+ 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. Everglades mice exhibit decreased frequencies of peripheral blood CD8+ T cells. Flow cytometric analysis of peripheral blood was utilized to determine CD8+ 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. Everglades mice exhibit increased frequencies of peripheral blood central memory CD8+ T cells. Flow cytometric analysis of peripheral blood was utilized to determine CD8+ 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. Everglades mice exhibit increased frequencies of peripheral blood effector memory CD8+ T cells. Flow cytometric analysis of peripheral blood was utilized to determine CD8+ 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. Everglades mice exhibit reduced frequencies of peripheral blood naïve CD8+ T cells. Flow cytometric analysis of peripheral blood was utilized to determine CD8+ 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. Everglades mice exhibit increased CD44 mean fluorescence intensity (MFI) on peripheral blood T cells. Flow cytometric analysis of peripheral blood was utilized to determine CD44 MFI on T cells. 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. Everglades mice exhibit increased CD44 MFI on peripheral blood CD8+ T cells. Flow cytometric analysis of peripheral blood was utilized to determine CD44 MFI on T cells. 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. Everglades mice exhibit increased frequencies of peripheral blood natural killer (NK) cells. Flow cytometric analysis of peripheral blood was utilized to determine the frequency of NK cells. 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. Everglades mice exhibited increased total IgE after ovalbumin administration. IgE levels were determined by ELISA. 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. Everglades mice exhibited increased OVA-specific IgE after ovalbumin administration. IgE levels were determined by ELISA. 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 everglades phenotype was identified among G3 mice of the pedigree R4328, some of which showed an increase in the B:T cell ratio (Figure 1) due to a reduced frequency of T cells (Figure 2) including CD4+ T cells (Figure 3) and CD8+ T cells (Figure 4), an increased frequency of central memory CD8+ T cells (Figure 5), an increased frequency of effector memory CD8+ T cells (Figure 6), and a reduced frequency of naïve CD8+ T cells (Figure 7), all in the peripheral blood. Some mice also exhibited increased expression of CD44 on T cells (Figure 8) and on CD8+ T cells (Figure 9) as well as an increased frequency of NK cells (Figure 10), all in the peripheral blood. Some mice showed increased levels of total IgE (Figure 11) and OVA-specific IgE (Figure 12) after ovalbumin administration.

Nature of Mutation

Figure 13. Linkage mapping of the increased expression of CD44 on peripheral blood CD8+ T cells using a recessive model of inheritance. Manhattan plot shows -log10 P values (Y-axis) plotted against the chromosome positions of 54 mutations (X-axis) identified in the G1 male of pedigree R4328. 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 54 mutations. All of the above anomalies were linked by continuous variable mapping to a mutation in Ppp3cb: an A to T transversion at base pair 20,530,948 (v38) on chromosome 14, or base pair 18,303 in the GenBank genomic region NC_000080 encoding Ppp3cb. The strongest association was found with a recessive model of linkage to the normalized expression level of CD44 on CD8+ T cells, wherein four variant homozygotes departed phenotypically from 11 heterozygotes and 10 homozygous reference mice with a P value of 9.085 x 10-9 (Figure 13). A substantial semidominant effect was observed in most of the assays.

The mutation corresponds to residue 539 in the mRNA sequence NM_008914 within exon 3 of 14 total exons.

524 AGAGGTTATTTTAGTATAGAGTGTGTCTTATAT

131 -R--G--Y--F--S--I--E--C--V--L--Y-

The mutated nucleotide is indicated in red.  The mutation results in an isoleucine (I) to lysine (I) substitution at position 136 (I136K) in the encoded protein, and is strongly predicted by PolyPhen-2 to be damaging (score = 0.936).

Illustration of Mutations in
Gene & Protein
Protein Prediction
Figure 14. Domain structure of CnAβ. The everglades mutation results in an isoleucine to lysine substitution at position 136. Abbreviations: PP, poly-pro; the CnB-binding domains are indicated by a "1" and "2"; CaM, calmodulin-binding domain; Inh, inhbitory. This image is interactive. Other mutations found in CnAα are noted in red. Click on each mutation for more information.

Ppp3cb encodes calcineurin Aβ (CnAβ), a calcineurin catalytic subunit isoform. All of the CnA proteins have a catalytic domain that is highly homologous to other serine/threonine protein phosphatases (amino acids 2-310 in CnAβ) (Figure 14). Within the catalytic domain is a poly-proline motif (amino acids 11-20 in CnAβ) that is specific for the CnAβ catalytic subunit (1). The CnA proteins have three C-terminal regulatory domains that include a CnB binding domain (amino acids 256-262 and 305-310 in CnAβ), a CaM-binding domain (amino acids 401-423 in CnAβ), and an autoinhibitory domain (amino acids 474-496 in CnAβ) (1;2). Ppp3cb can be alternatively spliced upon insulin-like growth factor 1 induction to generate a CnAβ1 isoform (3). The CnAβ1 isoform does not have an autoinhibitory domain, and contains a unique C-terminal domain to CnAβ (4). The Ppp3b mutation in everglades results in isoleucine (I) to lysine (I) substitution at position 136 (I136K). Amino acid 136 is within the catalytic domain.

Please see the record Copacabana for more information about Ppp3cb.

Putative Mechanism

Calcineurin (alternatively, PP2B) is a calcium- and calmodulin (CaM)-dependent serine/threonine protein phosphatase that consists of a catalytic subunit and a regulatory calcium-binding subunit, termed calcineurin A (CnA) and calcineurin B (CnB), respectively. Calcineurin has functions in T cell activation, activation-induced cell death (AICD), T cell tolerance, ion channel regulation, cardiac myocyte hypertrophy, sperm motility, synaptic endocytosis, and Alzheimer’s disease (5-8). Upon calcineurin activation, it dephosphorylates members of the nuclear factor of activated T cells (NFAT) family, promoting their translocation from the cytosol to the nucleus and subsequent induction of the transcription of NFAT target genes such as IL-2 growth factor, IFN-γ, and IL-4.  In addition to cytokine production, calcineurin-NFAT signaling mediates T cell maturation, synaptic transmission in neurons, vascular patterning in embryonic development, hypertrophic growth of the heart, and regulation of oxidative/slow fiber content in glycolytic/fast muscles (i.e., gastrocnemius, tibialis anterior, biceps, and triceps) (9;10).

CnAβ is required for the spontaneous survival of naïve T cells (11). Naïve T cells from Ppp3cb-deficient (Ppp3cb-/-) mice exhibited increased spontaneous apoptosis that could be blocked by IL-7 and IL-15. Ppp3cb-/- mice exhibit a reduction in CD3+ T cells in the peripheral blood compared to that in wild-type mice (12). In addition, the frequency of both thymic and splenic CD4+ and CD8+ cells in the Ppp3cb-/- mice was reduced compared to that in wild-type mice. The numbers of single positive T cells increased in the Ppp3cb-/- mice with age to levels comparable to wild-type mice (8). Thymus cellularity was also reduced in the Ppp3cb-/- mice. Splenic T cells from Ppp3cb-/- mice exhibited reduced proliferation induced by CD3 cross-linking or PMA/ionomycin. CnAα is able to partially compensate for the function of CnAβ in T cell activation, but both are required for efficient cell activation. After calcineurin-induced activation, NF-AT forms a complex with FOXP3 to induce regulatory T cell (Treg) cell generation through the induction of Il2ra (CD25) and Ctla4 (CD152) (13). Loss of CnAβ expression results in deficient Treg cell generation with a concomitant expansion of mature T cells with an activated phenotype (8). Calcineurin has a several functions including a role in apoptosis of T and B cells (14-16) and neuronal cells (17). In lymphocytes, calcineurin and NF-AT function in apoptosis by mediating the induction of Fas and FasL, which transduce an apoptotic signal upon T cell activation (18;19).

The phenotype of the everglades mouse indicates that CnAβeverglades exhibits loss of function.

Primers PCR Primer
everglades_pcr_F: TGCTCATTTCCACTGGGAG
everglades_pcr_R: AGGCCACTCCAGATATTTCTTG

Sequencing Primer
everglades_seq_F: TGCTCATTTCCACTGGGAGAAAAAC
everglades_seq_R: GGCCACTCCAGATATTTCTTGATAGC
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 471 nucleotides is amplified (chromosome 14, - strand):


1   aggccactcc agatatttct tgatagcatt tatactatct catgttacag tagttagagt
61  ttaagtgata tttgttaatt tagaggcttt ttttcatatc taataatctt cttgtttcag
121 tgtgtggtga catccatggc caattttttg atctgatgaa actttttgaa gtaggaggat
181 cacctgctaa tacacgatac ctttttcttg gtgattatgt ggacagaggt tattttagta
241 tagaggtaat aatcatatct gtcatttgat ttgctattgt taatatagat tattctatta
301 attcctagta tagtaagttt aaaaaaatac ttgtattggg ttctttttaa tacctctgta
361 tagtctgaaa tatgtaggaa aagctattac ttctttttca gaccatccaa ttagttttcc
421 ttgtgtcatt tcctgtagtt acaattgttt ttctcccagt ggaaatgagc a


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, Xue Zhong, and Bruce Beutler