Phenotypic Mutation 'Rocket' (pdf version)
AlleleRocket
Mutation Type missense
Chromosome13
Coordinate101,825,952 bp (GRCm39)
Base Change C ⇒ T (forward strand)
Gene Pik3r1
Gene Name phosphoinositide-3-kinase regulatory subunit 1
Synonym(s) p85alpha, p55alpha, PI3K, p50alpha
Chromosomal Location 101,817,269-101,904,725 bp (-) (GRCm39)
MGI Phenotype FUNCTION: [Summary is not available for the mouse gene. This summary is for the human ortholog.] Phosphatidylinositol 3-kinase phosphorylates the inositol ring of phosphatidylinositol at the 3-prime position. The enzyme comprises a 110 kD catalytic subunit and a regulatory subunit of either 85, 55, or 50 kD. This gene encodes the 85 kD regulatory subunit. Phosphatidylinositol 3-kinase plays an important role in the metabolic actions of insulin, and a mutation in this gene has been associated with insulin resistance. Alternative splicing of this gene results in four transcript variants encoding different isoforms. [provided by RefSeq, Jun 2011]
PHENOTYPE: Homozygotes for a targeted null mutation exhibit perinatal lethality associated with hepatic necrosis, chylous ascites, enlarged muscle fibers, calcification of cardiac tissue, and hypoglycemia. Mutants lacking only the major isoform are immunodeficient. [provided by MGI curators]
Accession Number

NCBI RefSeq: NM_001024955 (variant 1), NM_001077495 (variant 2); MGI:97583

MappedYes 
Amino Acid Change Arginine changed to Glutamine
Institutional SourceBeutler Lab
Gene Model predicted gene model for protein(s): [ENSMUSP00000047004] [ENSMUSP00000056774] [ENSMUSP00000140312] [ENSMUSP00000140256]
AlphaFold P26450
SMART Domains Protein: ENSMUSP00000047004
Gene: ENSMUSG00000041417
AA Change: R88Q

DomainStartEndE-ValueType
low complexity region 34 44 N/A INTRINSIC
SH2 61 144 9.96e-28 SMART
low complexity region 263 278 N/A INTRINSIC
SH2 352 434 7.33e-26 SMART
Predicted Effect probably damaging

PolyPhen 2 Score 1.000 (Sensitivity: 0.00; Specificity: 1.00)
(Using ENSMUST00000035532)
SMART Domains Protein: ENSMUSP00000056774
Gene: ENSMUSG00000041417
AA Change: R358Q

DomainStartEndE-ValueType
SH3 6 78 2.81e-11 SMART
low complexity region 79 99 N/A INTRINSIC
RhoGAP 126 298 1.94e-37 SMART
low complexity region 303 314 N/A INTRINSIC
SH2 331 414 9.96e-28 SMART
Pfam:PI3K_P85_iSH2 431 599 7.8e-67 PFAM
SH2 622 704 7.33e-26 SMART
Predicted Effect probably damaging

PolyPhen 2 Score 1.000 (Sensitivity: 0.00; Specificity: 1.00)
(Using ENSMUST00000055518)
SMART Domains Protein: ENSMUSP00000140312
Gene: ENSMUSG00000041417
AA Change: R58Q

DomainStartEndE-ValueType
low complexity region 8 16 N/A INTRINSIC
SH2 31 112 1.52e-23 SMART
Predicted Effect probably damaging

PolyPhen 2 Score 1.000 (Sensitivity: 0.00; Specificity: 1.00)
(Using ENSMUST00000185795)
SMART Domains Protein: ENSMUSP00000140256
Gene: ENSMUSG00000041417
AA Change: R37Q

DomainStartEndE-ValueType
SH2 10 93 6.5e-30 SMART
Predicted Effect probably damaging

PolyPhen 2 Score 1.000 (Sensitivity: 0.00; Specificity: 1.00)
(Using ENSMUST00000187009)
Meta Mutation Damage Score 0.9704 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 and Alleles(28) : Gene trapped(20) Targeted(8)

Lab Alleles
AlleleSourceChrCoordTypePredicted EffectPPH Score
IGL00323:Pik3r1 APN 13 101827044 start codon destroyed probably benign
IGL00484:Pik3r1 APN 13 101838255 missense probably benign 0.08
IGL00911:Pik3r1 APN 13 101894169 utr 5 prime probably benign
IGL01620:Pik3r1 APN 13 101822728 missense probably damaging 1.00
IGL01872:Pik3r1 APN 13 101825625 missense probably benign 0.01
IGL02544:Pik3r1 APN 13 101823784 missense probably damaging 1.00
IGL02959:Pik3r1 APN 13 101894037 missense probably benign 0.02
anubis UTSW 13 101839284 nonsense probably null
Astro_boy UTSW 13 101838240 missense probably damaging 1.00
Pennywhistle UTSW 13 101825914 missense probably damaging 0.96
Starburst UTSW 13 101894166 start codon destroyed probably null 0.99
R0635:Pik3r1 UTSW 13 101893926 missense probably benign
R0751:Pik3r1 UTSW 13 101822866 splice site probably null
R0787:Pik3r1 UTSW 13 101827031 missense probably benign 0.30
R0845:Pik3r1 UTSW 13 101822772 missense probably benign 0.45
R0891:Pik3r1 UTSW 13 101837974 missense probably benign
R1066:Pik3r1 UTSW 13 101825171 missense probably damaging 1.00
R1184:Pik3r1 UTSW 13 101822866 splice site probably null
R1735:Pik3r1 UTSW 13 101822882 missense probably damaging 1.00
R2474:Pik3r1 UTSW 13 101839284 nonsense probably null
R3015:Pik3r1 UTSW 13 101823771 missense probably damaging 1.00
R3419:Pik3r1 UTSW 13 101828723 missense probably benign 0.17
R3876:Pik3r1 UTSW 13 101821465 missense probably benign 0.01
R3964:Pik3r1 UTSW 13 101825193 missense possibly damaging 0.75
R4175:Pik3r1 UTSW 13 101838241 missense probably benign 0.25
R4175:Pik3r1 UTSW 13 101838240 missense probably damaging 1.00
R4422:Pik3r1 UTSW 13 101830892 missense probably benign
R4890:Pik3r1 UTSW 13 101894118 missense probably damaging 1.00
R5038:Pik3r1 UTSW 13 101825952 missense probably damaging 1.00
R5117:Pik3r1 UTSW 13 101828744 missense probably benign
R6066:Pik3r1 UTSW 13 101822828 missense possibly damaging 0.72
R6254:Pik3r1 UTSW 13 101825914 missense possibly damaging 0.89
R7421:Pik3r1 UTSW 13 101825644 missense probably damaging 1.00
R7507:Pik3r1 UTSW 13 101845490 missense probably benign 0.00
R7538:Pik3r1 UTSW 13 101825914 missense probably damaging 0.96
R7605:Pik3r1 UTSW 13 101839346 missense probably benign
R7739:Pik3r1 UTSW 13 101846205 missense probably benign 0.01
R8695:Pik3r1 UTSW 13 101894062 missense probably benign 0.40
R9146:Pik3r1 UTSW 13 101825136 splice site probably benign
R9315:Pik3r1 UTSW 13 101894166 start codon destroyed probably null 0.99
R9678:Pik3r1 UTSW 13 101839289 missense probably damaging 1.00
Mode of Inheritance Autosomal Dominant
Local Stock
Repository
Last Updated 2019-09-04 9:40 PM by Diantha La Vine
Record Created 2017-05-17 1:36 PM
Record Posted 2019-02-05
Phenotypic Description

Figure 1. Rocket 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. Rocket mice exhibit decreased frequencies of peripheral B1a 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. Rocket mice exhibit decreased frequencies of peripheral B1a 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 4. Rocket mice exhibit decreased frequencies of peripheral B1b cells. Flow cytometric analysis of peripheral blood was utilized to determine B1b 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. Rocket mice exhibit decreased frequencies of peripheral NK T cells. Flow cytometric analysis of peripheral blood was utilized to determine NK 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. Rocket mice exhibit increased frequencies of peripheral B1b cells in B1 cells. Flow cytometric analysis of peripheral blood was utilized to determine B1b 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. Rocket mice exhibit increased frequencies of peripheral B2 cells. Flow cytometric analysis of peripheral blood was utilized to determine B2 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. Rocket mice exhibit decreased expression of B220 on peripheral B cells. Flow cytometric analysis of peripheral blood was utilized to determine B220 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.

The Rocket phenotype was identified among G3 mice of the pedigree R5038, some of which showed reduced frequencies of B1 cells (Figure 1), B1a cells (Figure 2), B1a cells in B1 cells (Figure 3), B1b cells (Figure 4), and NK T cells (Figure 5) with concomitant increased frequencies of B1b cells in B1 cells (Figure 6) and B2 cells (Figure 7), all in the peripheral blood. Some mice showed reduced expression of B220 on peripheral B cells (Figure 8).

Nature of Mutation

Figure 9. Linkage mapping of the B1a cells in B1 cells phenotype using a dominant 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 Pik3r1: a G to A transition at base pair 101,689,444 (v38) on chromosome 13, or base pair 78,774 in the GenBank genomic region NC_000079 encoding Pik3r1. The strongest association was found with an additive/dominant model of inheritance to the normalized frequency of B1a cells in B1 cells in the peripheral blood, wherein 27 heterozygous mice departed phenotypically from 14 homozygous reference mice with a P value of 1.185 x 10-9 (Figure 9); no homozygous variant mice were present in pedigree R5038. 

The mutation corresponds to residue 1,694 in the mRNA sequence NM_001077495 within exon 9 of 16 total exons and to residue 605 in the mRNA sequence NM_001024955 within exon 3 of 10 total exons.

78758 GGGACCTTTTTGGTACGAGACGCATCTACTAAA

353 -G--T--F--L--V--R--D--A--S--T--K- (p85α)

83  -G--T--F--L--V--R--D--A--S--T--K- (p55α/p50α)

Genomic numbering corresponds to NC_000079. The mutated nucleotide is indicated in red. The mutation results in an arginine to glutamine substitution at residue 358 (R358Q) in the p85α protein as well as an R88Q substitution in p55α and p50α. The mutation is strongly predicted by PolyPhen-2 to be damaging in both transcripts (score = 1.000).

Illustration of Mutations in
Gene & Protein
Protein Prediction
Figure 10. Domain structures of p85 isoforms. Pik3r1 encodes p85α, p55α and p50α. Common to all isoforms are the presence of two C-terminal SH2 domains flanking a p110-binding domain (iSH2). The smaller isoforms differ at their N-terminus and contain a unique 35-amino-acid (p55α) or five-amino-acid (p50α) sequence. Unique to the larger isoforms are the N-terminal SH3 domain, RhoGAP domain and two proline-rich regions. The Rocket mutation results in an arginine to glutamine substitution at residue 358 (R358Q) in the p85α protein as well as an R88Q substitution in p55α and p50α. This image is interactive. Additional Pik3r1 mutations are noted in red. Click on each allele for more information.

Pik3r1 encodes p85α, a regulatory subunit of class IA phosphatidylinositol 3-kinases (PI3Ks). To form a functional class I PI3K, a p110 catalytic subunit forms a heterodimer with a p85 regulatory subunit (1;2). In activated cells, the p85 subunit recruits the p110 subunit to the plasma membrane and activates it (3-5). Conversely, the p85 subunit also inhibits the enzymatic activity of the p110 subunit in quiescent cells (6). The p85 subunits also mediate the interactions of the PI3Ks with the cytoplasmic domains of receptors as well as with adaptor proteins (7).

p85α, p55α, and p50α are splice variants of Pik3r1 (3;8;9). The p55α and p50α isoforms have two SH2 (Src homology 2) domains [nSH2 (N-terminal SH2 domain) and cSH2 (C-terminal SH2 domain)] and a p110-binding domain [iSH2 (inter SH2 domain)]. The p85α isoform has the nSH2, cSH2, and iSH2 domains, but also has a SH3 domain at the N-terminus (amino acids 6-78) and a RhoGAP domain (amino acids 126-298). Between the SH3 and RhoGAP domain and between the RhoGAP and nSH2 domain are proline-rich regions.

The Rocket mutation results in an arginine to glutamine substitution at residue 358 (R358Q) in the p85α protein as well as an R88Q substitution in p55α and p50α. Amino acid 358 in p85α and amino acid 88 within p55α and p50α is within the first nSH2 domain.

For more information about Pik3r1, please see the record for anubis.

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); (2;10); reviewed in (7;11)].  For more information on the PI3K signaling pathway, please see the record for sothe and stinger.

PIK3R1 mutations are linked to immunodeficiency-36 (IMD36; OMIM: #616005(12)), agammalobulinemia-7 (AGM7; OMIM: #615214(13)), and SHORT (Short stature, Hyperextensible joints, Ocular depression, Rieger anomaly, and Teeth delay) syndrome (OMIM: #269880(14;15)). Patients with IMD36 had decreased numbers of naïve CD4+ and CD8+ T cells; one patient had decreased numbers of memory B cells (12). A patient with AGM7 exhibited defects in early B cell development (13).

Pik3r1-/- chimeric mice (using a Rag2-deficient blastocyst complementation system) had reduced numbers of peripheral blood mature B cells and reduced serum levels of IgM, IgG1, IgG2a, IgG3, and IgA (16). The remaining B cells exhibited reduced proliferative responses after exposure to anti-IgM, anti-CD40, and lipopolysaccharide; T cell development and proliferative responses were normal. The anubis mice exhibited defects in T cell development similar to patients with IMD36 (12), but in contrast to the Pik3r1-/- chimeric mice (16). The immune phenotypes in the Rocket mice indicate that p85α/p50α/p55αRocket exhibits loss-of-function.

Primers PCR Primer
Rocket_pcr_F: TTCTGATGCAGAAGGAGGGC
Rocket_pcr_R: ATCCCCAGTGCAGAAGAAGG

Sequencing Primer
Rocket_seq_F: AGGGCTCTCAGGATGGG
Rocket_seq_R: AGAAGGGAAGCTCTGTGTGTG
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 436 nucleotides is amplified (chromosome 13, - strand):


1   atccccagtg cagaagaagg gaagctctgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt
61  gtgtgtgtgt ctttgtaagt gtgtggttac tcaaagttga aaagtaagta tggtcttata
121 ctgtgttcat tttagggaag aagtgaatga aaaactccga gacactgctg atgggacctt
181 tttggtacga gacgcatcta ctaaaatgca cggcgattac actcttacac taaggtgagc
241 caggaagtca gctgtaattg cgatgtctca gttgtcatga aaacatttct tcattagatt
301 gcctattttt cctccttaaa ttagcatata aagtaatggg tttcactata ggattttttt
361 tcaaacatct tcgattcgtc cctttctcct actgccttct cctccccatc ctgagagccc
421 tccttctgca tcagaa


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
AuthorsXue Zhong, Jin Huk Choi, and Bruce Beutler