Phenotypic Mutation 'Poseidon2' (pdf version)
AllelePoseidon2
Mutation Type missense
Chromosome8
Coordinate118,304,613 bp (GRCm39)
Base Change A ⇒ T (forward strand)
Gene Plcg2
Gene Name phospholipase C, gamma 2
Synonym(s) Plcg-2, PLCgamma2
Chromosomal Location 118,225,030-118,361,881 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 transmembrane signaling enzyme that catalyzes the conversion of 1-phosphatidyl-1D-myo-inositol 4,5-bisphosphate to 1D-myo-inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) using calcium as a cofactor. IP3 and DAG are second messenger molecules important for transmitting signals from growth factor receptors and immune system receptors across the cell membrane. Mutations in this gene have been found in autoinflammation, antibody deficiency, and immune dysregulation syndrome and familial cold autoinflammatory syndrome 3. [provided by RefSeq, Mar 2014]
PHENOTYPE: Homozygotes for some null alleles show decreased B cell and impaired NK cell function. Other homozygous null alleles show aberrant separation of blood and lymphatic vessels. [provided by MGI curators]
Accession Number

NCBI RefSeq: NM_172285; MGI:97616

MappedYes 
Limits of the Critical Region 117498291 - 117635140 bp
Amino Acid Change Isoleucine changed to Phenylalanine
Institutional SourceBeutler Lab
Gene Model predicted gene model for protein(s): [ENSMUSP00000079991]
AlphaFold Q8CIH5
PDB Structure Crystal structure of the N-terminal SH2 domain of mouse phospholipase C-gamma 2 [X-RAY DIFFRACTION]
Solution structure of the SH3 domain from Phospholipase C, gamma 2 [SOLUTION NMR]
SMART Domains Protein: ENSMUSP00000079991
Gene: ENSMUSG00000034330
AA Change: I273F

DomainStartEndE-ValueType
PH 21 133 1.87e-4 SMART
PLCXc 312 456 2.29e-96 SMART
low complexity region 461 476 N/A INTRINSIC
PDB:2K2J|A 478 516 6e-17 PDB
SH2 530 623 2.24e-30 SMART
SH2 644 726 1.16e-28 SMART
SH3 772 828 3.12e-18 SMART
PH 789 910 4.31e0 SMART
PLCYc 930 1044 1.18e-66 SMART
C2 1062 1167 1.41e-15 SMART
Predicted Effect possibly damaging

PolyPhen 2 Score 0.800 (Sensitivity: 0.84; Specificity: 0.93)
(Using ENSMUST00000081232)
Meta Mutation Damage Score 0.2605 question?
Is this an essential gene? Non Essential (E-score: 0.000) question?
Phenotypic Category Autosomal Dominant
Candidate Explorer Status loading ...
Single pedigree
Linkage Analysis Data
Penetrance  
Alleles Listed at MGI

All mutations/alleles(20) : Chemically induced (ENU)(3) Gene trapped(10) Spontaneous(1) Targeted(6)

Lab Alleles
AlleleSourceChrCoordTypePredicted EffectPPH Score
IGL00594:Plcg2 APN 8 118282810 missense possibly damaging 0.89
IGL00911:Plcg2 APN 8 118313254 missense probably benign 0.17
IGL00952:Plcg2 APN 8 118333956 missense probably benign
IGL01115:Plcg2 APN 8 118284068 missense probably damaging 1.00
IGL01326:Plcg2 APN 8 118300738 splice site probably benign
IGL01357:Plcg2 APN 8 118340900 splice site probably benign
IGL01705:Plcg2 APN 8 118308401 missense probably damaging 1.00
IGL01755:Plcg2 APN 8 118347980 missense possibly damaging 0.48
IGL01828:Plcg2 APN 8 118316972 missense probably damaging 1.00
IGL02307:Plcg2 APN 8 118306635 critical splice donor site probably null
IGL02345:Plcg2 APN 8 118311919 missense probably damaging 0.99
IGL02448:Plcg2 APN 8 118333960 missense probably benign
IGL02587:Plcg2 APN 8 118284852 missense possibly damaging 0.80
IGL02646:Plcg2 APN 8 118330622 missense possibly damaging 0.88
IGL03409:Plcg2 APN 8 118310234 missense probably damaging 0.96
Ctenophore UTSW 8 118284057 missense probably damaging 0.98
Porifera UTSW 8 118306585 missense possibly damaging 0.79
Poseidon UTSW 8 118341977 missense probably damaging 1.00
queen UTSW 8 118308446 missense probably benign 0.00
Seahorse UTSW 8 118316574 splice site probably null
Teleost UTSW 8 118310288 missense probably damaging 1.00
Theseus UTSW 8 118323071 missense probably damaging 0.99
trident UTSW 8 118339717 missense probably benign 0.00
R0172:Plcg2 UTSW 8 118306521 missense probably benign 0.00
R0194:Plcg2 UTSW 8 118300136 splice site probably benign
R0410:Plcg2 UTSW 8 118342112 missense probably damaging 0.98
R0462:Plcg2 UTSW 8 118312044 missense probably benign 0.06
R0494:Plcg2 UTSW 8 118282843 missense probably damaging 1.00
R0522:Plcg2 UTSW 8 118341027 splice site probably null
R0612:Plcg2 UTSW 8 118300104 missense probably benign 0.01
R1239:Plcg2 UTSW 8 118282783 missense probably benign
R1367:Plcg2 UTSW 8 118341977 missense probably damaging 1.00
R1608:Plcg2 UTSW 8 118340974 missense possibly damaging 0.89
R1756:Plcg2 UTSW 8 118319447 missense probably benign 0.02
R2176:Plcg2 UTSW 8 118339733 missense probably damaging 1.00
R3500:Plcg2 UTSW 8 118339717 missense probably benign 0.00
R4043:Plcg2 UTSW 8 118339717 missense probably benign 0.00
R4654:Plcg2 UTSW 8 118231054 missense probably benign
R4883:Plcg2 UTSW 8 118333872 nonsense probably null
R4932:Plcg2 UTSW 8 118333822 missense probably benign 0.05
R5080:Plcg2 UTSW 8 118316742 missense probably benign 0.10
R5226:Plcg2 UTSW 8 118304613 missense possibly damaging 0.80
R5264:Plcg2 UTSW 8 118361532 missense possibly damaging 0.95
R5298:Plcg2 UTSW 8 118331988 missense probably benign
R5473:Plcg2 UTSW 8 118361140 missense probably benign
R5555:Plcg2 UTSW 8 118339734 nonsense probably null
R5557:Plcg2 UTSW 8 118313296 missense probably damaging 0.99
R5805:Plcg2 UTSW 8 118325234 critical splice donor site probably null
R5826:Plcg2 UTSW 8 118337583 missense probably benign 0.19
R5871:Plcg2 UTSW 8 118230956 missense probably damaging 1.00
R5894:Plcg2 UTSW 8 118231088 missense probably damaging 0.99
R6142:Plcg2 UTSW 8 118312010 missense probably benign
R6609:Plcg2 UTSW 8 118294909 missense probably benign 0.31
R6684:Plcg2 UTSW 8 118323071 missense probably damaging 0.99
R6710:Plcg2 UTSW 8 118284086 missense probably benign 0.05
R6931:Plcg2 UTSW 8 118284058 missense probably benign 0.24
R6946:Plcg2 UTSW 8 118230929 missense probably benign
R7036:Plcg2 UTSW 8 118323045 missense probably benign
R7070:Plcg2 UTSW 8 118323045 missense probably benign
R7072:Plcg2 UTSW 8 118316574 splice site probably null
R7214:Plcg2 UTSW 8 118310288 missense probably damaging 1.00
R7351:Plcg2 UTSW 8 118317049 missense possibly damaging 0.95
R7393:Plcg2 UTSW 8 118306564 missense possibly damaging 0.90
R7443:Plcg2 UTSW 8 118231028 missense probably benign 0.00
R7513:Plcg2 UTSW 8 118306592 missense probably damaging 0.99
R7609:Plcg2 UTSW 8 118284852 missense probably benign 0.01
R8134:Plcg2 UTSW 8 118284057 missense probably damaging 0.98
R8399:Plcg2 UTSW 8 118323101 missense probably damaging 1.00
R8701:Plcg2 UTSW 8 118308416 missense probably damaging 1.00
R8774:Plcg2 UTSW 8 118306585 missense possibly damaging 0.79
R8774-TAIL:Plcg2 UTSW 8 118306585 missense possibly damaging 0.79
R8938:Plcg2 UTSW 8 118231114 critical splice donor site probably null
R9003:Plcg2 UTSW 8 118342002 missense
R9286:Plcg2 UTSW 8 118331976 missense probably benign 0.19
R9318:Plcg2 UTSW 8 118323107 missense probably benign
RF008:Plcg2 UTSW 8 118300263 splice site probably null
X0027:Plcg2 UTSW 8 118282722 missense probably benign 0.03
Mode of Inheritance Autosomal Dominant
Local Stock
Repository
Last Updated 2019-09-04 9:40 PM by Diantha La Vine
Record Created 2017-06-04 10:51 AM by Xue Zhong
Record Posted 2018-04-11
Phenotypic Description

Figure 1. Poseidon2 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. Poseidon2 mice exhibit diminished T-independent IgM responses to 4-hydroxy-3-nitrophenylacetyl-Ficoll (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 Poseidon2 phenotype was identified among N-Nitroso-N-ethylurea (ENU)-mutagenized G3 mice of the pedigree R5226, some of which showed exhibited reduced frequencies of B1 cells in the peripheral blood (Figure 1) as well as diminished T-independent antibody response to 4-hydroxy-3-nitrophenylacetyl-Ficoll (NP-Ficoll) (Figure 2).

Nature of Mutation

Figure 3. Linkage mapping of the diminished T-independent IgM responses to NP-Ficoll using an additive model of inheritance. Manhattan plot shows -log10 P values (Y-axis) plotted against the chromosome positions of 62 mutations (X-axis) identified in the G1 male of pedigree R5226. 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 62 mutations. Both of the above anomalies were linked by continuous variable mapping to a mutation in Plcg2:  an A to T transversion at base pair 117,577,874 (v38) on chromosome 8, or base pair 79,600 in the GenBank genomic region NC_000074 encoding Plcg2. The strongest association was found with an additive model of inheritance to the normalized NP-Ficoll-associated phenotype, wherein nine variant homozygotes and 35 heterozygous mice departed phenotypically from 21 homozygous reference mice with a P value of 5.01 x 10-7 (Figure 3). A semidominant effect was observed in both of the assays.

The mutation corresponds to residue 1,018 in the mRNA sequence NM_172285 within exon 10 of 33 total exons.

1003 CGGATGACCAAGTTTATCGACGACACAATGAGA

268  -R--M--T--K--F--I--D--D--T--M--R-

The mutated nucleotide is indicated in red. The mutation results in an isoleucine to phenylalanine substitution at position 273 (I273F) in the phospholipase C gamma 2 (PLC-γ2) protein, and is strongly predicted by Polyphen-2 to be damaging (score = 0.800).

Illustration of Mutations in
Gene & Protein
Protein Prediction

Figure 4. Domain structure of PLC-γ2. PLC isozymes contain a conserved domain structure consisting of the catalytic X and Y domains located between EF-hand motifs and a calcium-binding C2 domain. There is a pleckstrin homology (PH) domain at the N-terminus. PLC-γ2 contains an additional PH domain split by two tandem Src homology 2 (SH2) domains and an SH3 domain. The Poseidon2 mutation results in an isoleucine to phenylalanine substitution at position 273. This image is interactive. Other mutations found in PLC-γ2 are noted in red. Click on each allele for more information.

The Plcg2 gene encodes the 1265 amino acid PLC-γ2 that is a member of the PLC family (Figure 4). PLC enzymes act as effector molecules in the signal transduction process by hydrolyzing phosphatidylinositol 4,5 bisphosphate (PIP2) to generate two second messengers, diacylglycerol (DAG) and inositol 1,4,5-triphosphate (IP3). PLC isozymes contain a conserved domain structure consisting of the catalytic X and Y domains located between EF-hand motifs and a calcium-binding C2 domain. Most PLC proteins also contain a pleckstrin homology (PH) domain at their N-terminus. Additional domains are present in some PLC isozymes including the γ subtypes (PLC-γ1 and -γ2), which contain an additional PH domain split by two tandem Src homology 2 (SH2) domains and an SH3 domain (1). The Poseidon2 mutation alters I273 within the EF domain. The effect of the mutation on protein expression and localization has not been determined.  

Please see the record for queen for more information about Plcg2.

Putative Mechanism

The hydrolysis of PIP2 by PLC enzymes to produce DAG and IP3 is a critical step in the signal transduction process of many pathways.  DAG is responsible for activating protein kinase C and possibly the TRP calcium influx channels, while IP3 modulates calcium responses within the cell by binding to receptors on the intracellular membrane to allow the mobilization of intracellular calcium (2)Plcg2 -/- mice display internal bleeding, osteopetrosis, impaired lymph node organogenesis and defects in the functioning of B cells, platelets, neutrophils, mast cells, dendritic cells (DCs), macrophages and natural killer (NK) cells (3-5).  A nonsense mutation in Plcg2, resulting in a truncation in the N-terminal PH domain, causes aberrant separation of blood and lymphatic vessels (6).  In addition, two ENU-generated Plcg2 point mutations located in the catalytic and split PH domains have been linked to inflammatory and autoimmune responses through PLC-γ2 hyperactivation in cells of both the innate and acquired immune system (7;8). Plcg2 -/- mice display decreased mature B cells, a block in pro-B cell differentiation, B-1 B cell deficiency, and an absence of T cell-independent (T-I) antibody production; the T cell-dependent responses are relatively normal (3;4).  These phenotypes can be attributed to a defect in BCR and pre-BCR signaling with a concomitant deficiency in B-1 cells; the persistence of some peripheral B-2 cells allows the development of a T-D response. Calcium mobilization via PLC activation can be activated by Toll-like receptor (TLR) signaling (9;10).  TLR signaling pathway culminates in MAPK and NF-κB activation resulting in the production of proinflammatory cytokines (11).  Macrophages and DCs from PLC-γ2 deficient animals display abnormal calcium mobilization and reduced production of proinflammatory cytokines in response to peptidoglycan (PGN), a Gram-positive bacterial cell wall component recognized by TLR2 (see the record for languid), and lipopoysaccharide (LPS), a Gram-negative bacterial component recognized by TLR4 (see the record for lps3) (5). The phenotypes observed in Poseidon2 mice are consistent with those found in Plcg2 -/- mice as well as in mice with deficiencies in other BCR signaling molecules.

Primers PCR Primer
Poseidon2_pcr_F: TATGTGGCTCTGAATGACATAGG
Poseidon2_pcr_R: TGGCGAAACAGGTAATCCCAC

Sequencing Primer
Poseidon2_seq_F: GCTCTGAATGACATAGGTTTGAC
Poseidon2_seq_R: GGTAATCCCACCCAGGTAAGAATCAG
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 401 nucleotides is amplified (chromosome 8, + strand):


1   tatgtggctc tgaatgacat aggtttgaca ttgggtccag tctttgatca ccttgactag
61  gtcctgagga ggaccttgcc tgtcctttgg tgactttgtg ttatcttcga catctttaag
121 gagctatggg ctcaggatct gaacaaagtc cgggagcgga tgaccaagtt tatcgacgac
181 acaatgagag agaccgcaga gcccttctta tttgtggatg aggtgagtgg gctggggcat
241 gtcacgtgag caggacttcc tttatccctt cccctcggaa ggtgtcactg tcaggtgtcc
301 gggggactga ctccgtgtaa ggagtggttt tcccagggtg gggattaatc ctgcttttca
361 ttttcctgat tcttacctgg gtgggattac ctgtttcgcc 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 Katherine Timer
AuthorsXue Zhong, Aijie Liu, Jin Huk Choi, and Bruce Beutler