Phenotypic Mutation 'grand_tetons' (pdf version)
Allelegrand_tetons
Mutation Type missense
Chromosome4
Coordinate149,737,156 bp (GRCm39)
Base Change A ⇒ G (forward strand)
Gene Pik3cd
Gene Name phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit delta
Synonym(s) 2610208K16Rik, 2410099E07Rik, p110delta
Chromosomal Location 149,733,625-149,787,023 bp (-) (GRCm39)
MGI Phenotype FUNCTION: [Summary is not available for the mouse gene. This summary is for the human ortholog.] Phosphoinositide 3-kinases (PI3Ks) phosphorylate inositol lipids and are involved in the immune response. The protein encoded by this gene is a class I PI3K found primarily in leukocytes. Like other class I PI3Ks (p110-alpha p110-beta, and p110-gamma), the encoded protein binds p85 adapter proteins and GTP-bound RAS. However, unlike the other class I PI3Ks, this protein phosphorylates itself, not p85 protein.[provided by RefSeq, Jul 2010]
PHENOTYPE: Homozygotes for targeted null mutations exhibit impaired B and T cell antigen receptor signaling, reduced or ablated immune responses and decreased immunoglobulin levels. Mutants also develop inflammatory bowel disease. [provided by MGI curators]
Accession Number

NCBI RefSeq: NM_001164052, NM_001029837, NM_001164049, NM_008840, NM_00164050, NM_001164051; MGI:1098211

MappedYes 
Amino Acid Change Leucine changed to Proline
Institutional SourceBeutler Lab
Gene Model predicted gene model for protein(s): [ENSMUSP00000036434] [ENSMUSP00000101313] [ENSMUSP00000101314] [ENSMUSP00000101315] [ENSMUSP00000136045] [ENSMUSP00000112863] [ENSMUSP00000113844]
AlphaFold O35904
SMART Domains Protein: ENSMUSP00000036434
Gene: ENSMUSG00000039936
AA Change: L878P

DomainStartEndE-ValueType
PI3K_p85B 31 108 2.24e-26 SMART
PI3K_rbd 174 281 1.3e-13 SMART
PI3K_C2 309 412 1.87e-28 SMART
PI3Ka 496 685 8.56e-87 SMART
PI3Kc 776 1042 5.65e-128 SMART
Predicted Effect probably damaging

PolyPhen 2 Score 1.000 (Sensitivity: 0.00; Specificity: 1.00)
(Using ENSMUST00000038859)
SMART Domains Protein: ENSMUSP00000101313
Gene: ENSMUSG00000039936
AA Change: L877P

DomainStartEndE-ValueType
PI3K_p85B 31 108 2.24e-26 SMART
PI3K_rbd 174 281 1.3e-13 SMART
PI3K_C2 309 412 1.87e-28 SMART
PI3Ka 496 685 8.56e-87 SMART
PI3Kc 775 1041 5.65e-128 SMART
Predicted Effect probably damaging

PolyPhen 2 Score 1.000 (Sensitivity: 0.00; Specificity: 1.00)
(Using ENSMUST00000105688)
SMART Domains Protein: ENSMUSP00000101314
Gene: ENSMUSG00000039936
AA Change: L876P

DomainStartEndE-ValueType
PI3K_p85B 31 108 2.24e-26 SMART
PI3K_rbd 174 281 1.3e-13 SMART
PI3K_C2 309 412 1.87e-28 SMART
PI3Ka 496 684 1.35e-84 SMART
PI3Kc 774 1040 5.65e-128 SMART
Predicted Effect probably damaging

PolyPhen 2 Score 1.000 (Sensitivity: 0.00; Specificity: 1.00)
(Using ENSMUST00000105689)
SMART Domains Protein: ENSMUSP00000101315
Gene: ENSMUSG00000039936
AA Change: L880P

DomainStartEndE-ValueType
PI3K_p85B 31 108 2.24e-26 SMART
PI3K_rbd 174 281 1.3e-13 SMART
PI3K_C2 309 412 1.87e-28 SMART
PI3Ka 496 688 1.22e-82 SMART
PI3Kc 778 1044 5.65e-128 SMART
Predicted Effect probably damaging

PolyPhen 2 Score 1.000 (Sensitivity: 0.00; Specificity: 1.00)
(Using ENSMUST00000105690)
SMART Domains Protein: ENSMUSP00000136045
Gene: ENSMUSG00000039936
AA Change: L880P

DomainStartEndE-ValueType
PI3K_p85B 31 108 2.24e-26 SMART
PI3K_rbd 174 281 1.3e-13 SMART
PI3K_C2 309 412 1.87e-28 SMART
PI3Ka 496 688 1.22e-82 SMART
PI3Kc 778 1044 5.65e-128 SMART
Predicted Effect probably damaging

PolyPhen 2 Score 1.000 (Sensitivity: 0.00; Specificity: 1.00)
(Using ENSMUST00000177654)
SMART Domains Protein: ENSMUSP00000112863
Gene: ENSMUSG00000039936
AA Change: L879P

DomainStartEndE-ValueType
PI3K_p85B 31 108 2.24e-26 SMART
PI3K_rbd 174 281 1.3e-13 SMART
PI3K_C2 309 412 1.87e-28 SMART
PI3Ka 496 687 1.8e-80 SMART
PI3Kc 777 1043 5.65e-128 SMART
Predicted Effect probably damaging

PolyPhen 2 Score 1.000 (Sensitivity: 0.00; Specificity: 1.00)
(Using ENSMUST00000118704)
SMART Domains Protein: ENSMUSP00000113844
Gene: ENSMUSG00000039936
AA Change: L873P

DomainStartEndE-ValueType
PI3K_p85B 31 108 2.24e-26 SMART
PI3K_rbd 174 281 1.3e-13 SMART
PI3K_C2 309 408 6.47e-23 SMART
PI3Ka 492 681 8.56e-87 SMART
PI3Kc 771 1037 5.65e-128 SMART
Predicted Effect probably damaging

PolyPhen 2 Score 0.999 (Sensitivity: 0.14; Specificity: 0.99)
(Using ENSMUST00000122059)
Meta Mutation Damage Score 0.9682 question?
Is this an essential gene? Probably essential (E-score: 0.961) question?
Phenotypic Category Autosomal Recessive
Candidate Explorer Status loading ...
Single pedigree
Linkage Analysis Data
Penetrance  
Alleles Listed at MGI

All Mutations and Alleles(19) : Chemically induced (ENU)(1) Gene trapped(9) Targeted(9)

Lab Alleles
AlleleSourceChrCoordTypePredicted EffectPPH Score
IGL01308:Pik3cd APN 4 149741917 missense probably damaging 1.00
IGL01536:Pik3cd APN 4 149737123 missense probably damaging 1.00
IGL01636:Pik3cd APN 4 149738772 missense possibly damaging 0.82
IGL02794:Pik3cd APN 4 149739028 missense probably benign
Helena UTSW 4 149736277 missense probably damaging 1.00
stinger UTSW 4 149741776 missense probably damaging 1.00
F5770:Pik3cd UTSW 4 149741776 missense probably damaging 1.00
R0003:Pik3cd UTSW 4 149740836 critical splice donor site probably null
R0309:Pik3cd UTSW 4 149747677 missense probably damaging 1.00
R1246:Pik3cd UTSW 4 149744257 missense probably damaging 1.00
R1259:Pik3cd UTSW 4 149735105 nonsense probably null
R1533:Pik3cd UTSW 4 149739653 missense probably damaging 1.00
R1756:Pik3cd UTSW 4 149743207 missense probably benign 0.02
R1796:Pik3cd UTSW 4 149738576 missense possibly damaging 0.83
R1887:Pik3cd UTSW 4 149737091 missense probably damaging 1.00
R1988:Pik3cd UTSW 4 149747660 missense probably damaging 1.00
R2089:Pik3cd UTSW 4 149737156 missense probably damaging 1.00
R2091:Pik3cd UTSW 4 149737156 missense probably damaging 1.00
R4997:Pik3cd UTSW 4 149743441 missense probably damaging 1.00
R5391:Pik3cd UTSW 4 149743588 missense probably damaging 0.98
R5603:Pik3cd UTSW 4 149743312 missense probably benign
R6282:Pik3cd UTSW 4 149744200 missense probably benign 0.00
R6453:Pik3cd UTSW 4 149736759 missense probably damaging 1.00
R7286:Pik3cd UTSW 4 149744171 missense probably benign 0.08
R7423:Pik3cd UTSW 4 149736220 critical splice donor site probably null
R7508:Pik3cd UTSW 4 149739040 missense possibly damaging 0.78
R7665:Pik3cd UTSW 4 149738507 missense possibly damaging 0.70
R7897:Pik3cd UTSW 4 149741726 missense probably benign 0.06
R8039:Pik3cd UTSW 4 149744323 missense possibly damaging 0.91
R8476:Pik3cd UTSW 4 149736277 missense probably damaging 1.00
R9015:Pik3cd UTSW 4 149740055 missense probably benign 0.06
R9252:Pik3cd UTSW 4 149740087 missense possibly damaging 0.88
R9704:Pik3cd UTSW 4 149739839 missense probably benign 0.17
V7580:Pik3cd UTSW 4 149741776 missense probably damaging 1.00
V7581:Pik3cd UTSW 4 149741776 missense probably damaging 1.00
V7582:Pik3cd UTSW 4 149741776 missense probably damaging 1.00
V7583:Pik3cd UTSW 4 149741776 missense probably damaging 1.00
X0023:Pik3cd UTSW 4 149744491 missense probably benign 0.04
Z1176:Pik3cd UTSW 4 149739304 frame shift probably null
Mode of Inheritance Autosomal Recessive
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Repository
Last Updated 2019-09-04 9:42 PM by Anne Murray
Record Created 2016-06-20 9:31 AM
Record Posted 2016-09-20
Phenotypic Description

Figure 1. Grand_tetons 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 2. Grand_tetons mice exhibit decreased frequencies of peripheral B1a cells in B1 cells. Flow cytometric analysis of peripheral blood was utilized to determine B1a 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. Grand_tetons mice exhibit increased dsDNA-induced type I interferon production in macrophages. ELISA was utilized to determine IFN production. 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. Grand_tetons mice exhibit an increase in the ratio of OVA-specific IgE to the amount of total IgE in the peripheral blood. ELISA was utilized to determine IgE production. 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 5. Grand_tetons mice exhibit an increase in the amount of OVA-specific IgE in the peripheral blood. ELISA was utilized to determine IgE production. 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 6. Grand_tetons mice exhibit increased proliferation of MCMV in macrophages. PCR was utilized to determine MCMV titres. 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. Grand_tetons mice exhibit diminished T-dependent IgG responses to ovalbumin administered with aluminum hydroxide. IgG 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 8. Grand_tetons mice exhibit diminished T-independent IgM responses to NP-Ficoll. IgM 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 grand_tetons phenotype was identified among G3 mice of the pedigree R2089, some of which showed a reduction in the frequency of B1 cells (Figure 1) and a decrease in the frequency of B1a cells in B1 cells (Figure 2) in the peripheral blood. Some mice also showed an increase in dsDNA induced type I IFN production in macrophages (Figure 3), an increase in the ratio of OVA-specific IgE over the total IgE (Figure 4) due to an increase in OVA-specific IgE (Figure 5), and an increased proliferation of mouse cytomegalovirus (MCMV) in macrophages (Figure 6). The T-dependent antibody response to ovalbumin administered with aluminum hydroxide was diminished (Figure 7). In addition, the T-independent antibody response to 4-hydroxy-3-nitrophenylacetyl-Ficoll (NP-Ficoll) was reduced (Figure 8).

Nature of Mutation

Figure 9. Linkage mapping of the increased amount of OVA-specific IgE using a recessive model of inheritance. Manhattan plot shows -log10 P values (Y-axis) plotted against the chromosome positions of 58 mutations (X-axis) identified in the G1 male of pedigree R2089. 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 58 mutations. Continuous variable mapping of the above anomalies showed comparable linkage to two mutations on chromosome 4: Pik3cd and Nfx1. The mutation in Pik3cd was deemed causative as the grand_tetons immune phenotypes mimicked that of other Pik3cd alleles (see MGI for a list of Pik3cd alleles as well as the record for stinger). The mutation in Pik3cd is a T to C transition at base pair 149,652,699 (v38) on chromosome 4, or base pair 49,868 in the GenBank genomic region NC_000070 encoding Pik3cd. The strongest association was found with a recessive model of linkage to the normalized amount of OVA-specific IgE, wherein one variant homozygote departed phenotypically from four homozygous reference mice and nine heterozygous mice with a P value of 1.401 x 10-11 (Figure 9).  A dominant effect was observed in the B1 cell assay.

The mutation corresponds to residue 2,917 in the mRNA sequence NM_001029837 within exon 20 of 23 total exons.


 

49852 ATTGAGGAATTCACCCTCTCCTGTGCTGGCTAC

875   -I--E--E--F--T--L--S--C--A--G--Y-


 

Genomic numbering corresponds to NC_000070. The mutated nucleotide is indicated in red.  The mutation results in a leucine (L) to proline (P) substitution at position 880 (L880P) in all of the p110δ isoforms, and is strongly predicted by PolyPhen-2 to be damaging (score = 1.000) (1).

Illustration of Mutations in
Gene & Protein
Protein Prediction
Figure 10. Domain structure of p110δ. The grand_tetons mutation (L880P) is shown. Abbreviations: ABD, adaptor binding domain; RBD, Ras binding domain. See the text for more details. This image is interactive; click to view other Pik3cd mutations.
Figure 11. Crystal structure of mouse p110δ. The crystal structure of p110δ is comprised of amino acids 106-1044. The helices are blue, while the beta strands are purple. Image is interactive, click to rotate. Figure was adapted from PDB:2X38.

Pik3cd encodes p110δ, one of four (with p110α, p110β, and p110γ) catalytic phosphatidylinositol 3-kinase (PI3K) class IA subunits (Figure 10 & 11). All of the p110 PI3K catalytic subunits contain an adaptor-binding domain (ABD; amino acids 1-104), a Ras-binding domain (RBD; amino acids 189-278), a C2 domain (amino acids 324-474), a helical domain (amino acids 500-674), and a kinase domain (split into N- and C-lobes; amino acids 675-829 and 830-1044, respectively) (2;3). p110δ has a basic-region, leucine-zipper (bZIP)-like domain (amino acids 400-439) that overlaps with the C2 domain and a Pro-rich region (amino acids 292-311) between the RBD and the C2 domains; the C2 and bZIP-like domains as well as the Pro-rich region are proposed to mediate protein-protein interactions with proteins that contain SH3 domains (4).

The grand_tetons mutation results in a leucine (L) to proline (P) substitution at position 880 (L880P) in all of the p110δ isoforms. Residue 880 is within the C-lobe of the kinase domain.

Please see the record stinger for more information about Pik3cd.

Putative Mechanism

PI3Ks are highly conserved lipid signaling kinases. After cell stimulation by growth factors, hormones, cytokines, or antigens, the PI3Ks are recruited to the inner face of the plasma membrane where they phosphorylate phosphatidylinositol (PtdIns), PtdIns 4-phosphate, and/or PtdIns-4,5-bisphosphate (PtdIns(4,5)P2; PIP2) at the D3 position of the inositol ring, generating their respective D3’ phosphorylated derivatives [e.g., PIP2 phosphorylation generates the second messenger phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P3; PIP3); (4;5); reviewed in (6;7)]. To form a functional class I PI3K, a p110 catalytic subunit forms a heterodimer with a p85 regulatory subunit [(3;5;8); reviewed in (6)].

PI3Kδ has several immune functions, several of which are directly related to the phenotypes observed in the grand_tetons mice. Active p110δ is required for naïve CD4+ T cell proliferation and efficient IFNγ production in response to antigen (9-11). PI3Kδ is essential for the differentiation and expansion of T helper 1 (TH1) and TH2 cells (11) as well as for the development, differentiation, and function of CD4+CD25+FOXP3+ regulatory T (Treg)-cells (12). PI3Kδ is required for IFNγ, TNFα, and GM-CSF secretion by natural killer (NK) cells as well as for NK cell development, differentiation, and cytotoxicity (13;14). PI3Kδ is required for T cell-dependent and T cell-independent antibody production after antigen stimulation, IgM-specific antibody-induced B cell proliferation, B cell receptor (BCR)-induced DNA synthesis and proliferation, and B cell survival after LPS treatment (10;15-18). PI3Kδ regulates the IgE isotype switch from IgG1 through transcriptional regulation at the epsilon locus and regulation of activation-induced cytidine deaminase expression (19). In addition, CD5+ B1 cells were not detected among the peritoneal lymphocytes in the Pik3cd-/- mice (16). The levels B1 and B2 B cells in the peritoneal cavity and CD21hi CD23lo MZ B cells of the spleen were reduced (16).

Two Pik3cd-/- mouse models (Pik3cdtm1Jni, MGI:2446574; Pik3cdtm1Tnr; MGI:2388047) exhibited reduced levels of serum IgM and IgG1 levels compared to wild-type mice (16;17). The Pik3cdtm1Jni mice exhibited reduced TNP-specific IgM and IgG1 T-independent humoral responses compared to wild-type mice after injection with TNP-LPS; the levels of TNP-specific IgG2a, IgG2b, and IgG3 were comparable to those in wild-type mice (17). After immunization with TNP-Ficoll, the TNP-specific levels of IgM, IgG1, IgG2b, IgG3, and IgG2a were reduced in the Pik3cd-/- mice compared to wild-type mice (17). The Pik3cdtm1Tnr mice exhibited reduced T cell-independent IgM and IgG3 responses to DNP-Ficoll compared to wild-type mice (16). The Pik3cdtm1Jni mice did not exhibit a T-dependent response to TNP-KLH (17). The Pik3cdtm1Tnr mice produced significantly less T cell-dependent DNP-specific IgM, IgG1 IgG2a, IgG2b, and IgG3 antibodies seven days after DNP-KLH injection (16). NP-KLH immunization of mice with T cell-specific deletion of Pik3cd (T p110δfl/fl) resulted in reduced numbers of CD95+B220+ germinal center (GC) B cells and reduced ratio of GCs per B cell follicle compared to p110δfl/fl (CD4cre-) controls; NP-specific IgG1 in the T p110δfl/fl mice were only slightly decreased, but the high-affinity NP-specific IgG1 response was impaired (20). In a B cell-specific Pik3cd knockout (B p110δfl/fl) mouse, IgM and high affinity NP-specific IgG1 titers were similar between B p110δfl/fl and p110δfl/fl mice after NP-CGG immunization, but antigen-specific IgE levels were increased 30-fold after immunization (20).

Similar to the Pik3cd mutant mouse models described above, the grand_tetons mice are unable to mount either a T-dependent or a T cell-independent B cell response. Similarity between grand_tetons and Pik3cd-deficient phenotypes is consistent with a strongly hypomorphic or null effect of the Leu880Pro mutation in grand_tetons.

Primers PCR Primer
grand_tetons_pcr_F: TTCCATGCCCCTTGAGAGAC
grand_tetons_pcr_R: TTTTAGGCCTAAGCTCTGGGAC

Sequencing Primer
grand_tetons_seq_F: AGGGCTCACAGCGTTCTC
grand_tetons_seq_R: GGCCTAAGCTCTGGGACTATATAC
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 4, - strand):


1   ttttaggcct aagctctggg actatatact ttacattctg ttgagtaaag cacgccaaaa
61  ggctagactc agagaaagag aggcagaccc ccttctggta aagggttgca gtctggtgta
121 ccatgacctc caatcttccc cctcagggag gccctggatc gggccattga ggaattcacc
181 ctctcctgtg ctggctactg tgtggccaca tatgttctgg gcatcggtga ccggcacagc
241 gacaacatca tgatcagaga gagtgggcag gtaggggcgt gtggctgggt ggctttgcta
301 tagggctgac gtcccgtctg gcctgctggc ccctttgctc tgggtttggt ggccccgaga
361 acgctgtgag ccctgctggt gtcttcttcg cctgggtctc tcaaggggca tggaa


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, Katherine Timer
AuthorsMing Zeng, Xue Zhong, Tao Yue, Jin Huk Choi, James Butler, and Bruce Beutler