Phenotypic Mutation 'ambrosius' (pdf version)
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Alleleambrosius
Mutation Type critical splice donor site (1 bp from exon)
ChromosomeX
Coordinate36,608,577 bp (GRCm38)
Base Change C ⇒ T (forward strand)
Gene Atp11c
Gene Name ATPase, class VI, type 11C
Synonym(s) Ig, A330005H02Rik
Chromosomal Location 60,223,290-60,592,698 bp (-)
MGI Phenotype Mice homozygous or hemizugous for an ENU mutation exhibit decreased B cells associated with arrested adult B cell lymphopoiesis.
Accession Number

NCBI RefSeq: NM_001001798; MGI: 1859661 

Mapped Yes 
Amino Acid Change
Institutional SourceAustralian Phenomics Network
Ref Sequences
Ensembl: ENSMUSP00000033480 (fasta)
Gene Model not available
SMART Domains

DomainStartEndE-ValueType
low complexity region 78 91 N/A INTRINSIC
Pfam:E1-E2_ATPase 94 380 7e-17 PFAM
low complexity region 693 709 N/A INTRINSIC
Blast:PTPc_DSPc 737 842 N/A BLAST
low complexity region 853 865 N/A INTRINSIC
low complexity region 994 1013 N/A INTRINSIC
low complexity region 1068 1085 N/A INTRINSIC
Phenotypic Category anemia, decrease in B cells, hematopoietic system, homeostasis/metabolism, immune system, liver/biliary system
Penetrance 100% 
Alleles Listed at MGI

All alleles(4) : Gene trapped(4) 

Lab Alleles
AlleleSourceChrCoordTypePredicted EffectPPH Score
18nih30a APN X 36608577 critical splice donor site
IGL00578:Atp11c APN X 60240817 missense probably damaging 1.00
IGL01702:Atp11c APN X 60269903 missense possibly damaging 0.74
emptyhive UTSW X 60269987 nonsense
hit UTSW X nonsense
spelling UTSW X 60290036 missense probably damaging 1.00
R1551:Atp11c UTSW X 60236712 critical splice acceptor site probably null
R2134:Atp11c UTSW X 60276783 missense probably damaging 1.00
R3687:Atp11c UTSW X 60281644 missense probably benign 0.07
R3688:Atp11c UTSW X 60281644 missense probably benign 0.07
R4496:Atp11c UTSW X 60280744 missense probably damaging 0.97
Mode of Inheritance X-linked Recessive
Local Stock None
Repository

Australian PhenomeBank

Last Updated 05/13/2016 3:09 PM by Stephen Lyon
Record Created 02/07/2011 12:25 PM by Nora G. Smart
Record Posted 05/02/2011
Phenotypic Description
Figure 1. (a) Flow cytometric analysis of the percentage of B220+ B cells in the blood of wild type, ambrosius and 18NIH30a mice. Each white circle (wt) and black circle (amb) represents a single mouse. (b) Left panel shows primary antibody response 14 days after immunization with inactivated Bordetella pertussis (B. pert) and alum-precipitated CGG. The other panels show antibody CGG, the hapten ABA, and NP-Ficoll 6 days after booster immunization. (c) Number of lymphocytes, neutrophils, reticulocytes and erythrocytes in the blood of amb and wild type littermates. (d) Unconjugated and conjugated bilirubin in the plasma of amb and control mice. (e) Typical dysplastic focus on H & E stained liver section from amb mice at 6 months (x400). (f) Multiple mitotic figures (indicated by arrowheads) seen in a representative H & E stained liver tumor section from a mutant mouse (x200). (g) Abnormal mitotic figures with condensed, asymmetric chromatin at higher magnification (x400). Figure reproduced from reference (2).

The ambrosius mutation was discovered during flow cytometry analysis of blood from ENU-mutagenized G3 mice (1)Ambrosius males exhibited B cell frequencies in blood that were decreased to 3% of controls (Figure 1a), but normal frequencies of T and natural killer (NK) cells (2).  This B cell deficiency caused a variable humoral immune deficiency.  Immunization with inactivated Bordetella pertussis and alum-precipitated chicken gamma globulin (CGG) coupled to the hapten azo-benzene-arsonate (ABA) showed a variably reduced primary antibody response to both antigens in the mutant animals (Figure 1b; left panel). Booster immunization with ABA-CGG 6 weeks later again showed variably decreased antibody response to the CGG protein carrier while antibodies to the ABA hapten, which depends upon antibody hypermutation and selection in germinal centres, was almost absent in all mutant animals (Figure 1b; middle panels). In contrast, the mutant mice made a normal immunoglobulin M (IgM) antibody response to the T-cell independent antigen, NP-Ficoll (Figure 1b; right panel).

 

Figure 2. Atp11c mutation reduces all B cell subsets except marginal zone B cells. (a) Representative flow cytometric analysis of bone marrow cells from Atp11c+/Y wild-type (wt) or Atp11camb/Y (amb) male mice. Left panels show percentage of B220+ B cells. Middle panels show the percentage of B220+ cells within the subsets of IgM+ IgD+ mature B cells, IgM+ IgD- immature (Imm.) B cells, and IgM- IgD- pro- and pre-B cells. Right panels are gated on B220+ IgM- IgD- cells, showing the percentage that are CD43- CD24hi pre-B cells, CD43+ CD24med pro-B cells, and CD43+ CD24- pre-pro-B cells. (b, c) Number of leukocytes and indicated B cell subsets in bone marrow from Atp11c+/Y (mean, black column; individual animals, white circles) or Atp11camb/Y (mean, white columns; individual animals, black circles). (d) Representative flow cytometric analysis of B cell subpopulations in the spleen. Left panel shows the percentage of B220+ B cells and CD3+ T cells. Middle panel is gated on B220+ cells, showing the percentage that are CD93- mature B cells and CD93+ immature (imm) B cells. Right panels show the percentage of B220+ CD93- cells within the CD21hi CD23-marginal zone (MZ) subset and the CD21med CD23+ follicular (Foll) B cell subset. (e and f) Number of leukocytes and lymphocyte subsets in the spleen. Means and individual values shown as in (b, c). Data in panels a-f are representative of at least 5 independent experiments with 2-5 mice per group and experiment. Figure reproduced from reference (2).

The percentage and number of B cells were decreased in ambrosius mice to 15-18% of the numbers in wild-type controls (Figure 2a-c). The number of CD43+ CD24med pro-B cells was 60% of normal, whereas the number of CD43low CD24hi pre-B cells and IgM+ IgD- immature B cells were only 6% and 1.8% of normal, respectively. IgM+ IgD+ mature recirculating B cells in the bone marrow were 11% of normal numbers, and these expressed much higher densities of IgM compared to wild type littermates. These data establish that ATP11c is required for B cells to differentiate normally past the pro-B cell stage.  In the spleen, the number of B cells in ambrosius animals was also decreased to 9% of that in wild-type mice (Figure 2d-f).  By contrast, marginal zone (MZ) B cells were present in normal numbers and normal surface phenotype (Figure 2d, f). Numbers of peritoneal B cells are also reduced. The B cell defect is cell autonomous as ambrosius B cells were unable to reconstitute B cells of irradiated recipients in mixed chimera experiments.  T and natural killer (NK) cells from ambrosius donors accumulated in equal proportions to wild type cells in these animals.    

 

Other phenotypes include anemia, hyperbilirubinemia and hepatocellular carcinoma (Figure 1c-g). Ambrosius is allelic to spelling, emptyhive and 18NIH30a (2;3).

Nature of Mutation
Figure 3. Membrane topology of P4-ATPases modeled after the structure of SERCA1 with 10 transmembrane (TM) segments and distinct actuator (A), phosphorylation (P) and nucleotide binding (N) domains. P4-ATPases are proposed to flip phospholipids to the cytosolic membrane layer. Motifs that are important for the catalytic function of P-type ATPases are indicated. Conversion of ATP to ADP by the N-domain leads to phosphorylation of the P-domain and transport of substrate. The A-domain dephosphorylates the P-domain. P4-ATPases can form complexes with CDC50 proteins (pink), which are predicted to have two transmembrane domains and a large, glycosylated ectoplasmic domain. Phosphatidylserines are indicated in dark blue, phosphatidylethanolamine in dark green, and phosphatidylcholine in orange. Although PC is typically localized to the extracellular side of the plasma membrane, it may also be a P4-ATPase substrate. The ambrosius mutation destroys the donor splice site of intron 27. This image is interactive. Click on the image to view other mutations found in ATP11C (red). Click on the mutations for more specific information.   

Linkage analysis in (B6xCBA)F2 offspring mapped the mutation to an X-chromosomal region distal to marker rs13483763 at 54,012,901 basepairs (Build NCBI37.1).  RefSeq annoted exons in this region were then captured using a custom Agilent Sureselect targeted DNA capture array using RNA baits.  This region was then sequenced using next-generation sequencing on an Illumina GAIIx sequencer.  93% of the nucleotides across all RefSeq exons in the mutation-containing region were covered with a read depth of 5 or greater (2).  Within this region, a G to A transition at position 34148572 in the Genbank genomic region NC_000086  for the Atp11c gene on chromosome X (GTAAGCAGCT-> ATAAGCAGCT). The mutation is located within the donor splice site of intron 27, one nucleotide from the previous exon.  Atp11c contains 30 total exons.  Multiple Atp11c transcripts are displayed on Ensembl and Vega.  The mutation was confirmed using standard Sanger sequencing.  PCR-amplification of cDNA from mutant and wild type bone marrow or spleen with primers located in exons 25 and 29 yielded a shorter product in the mutant animals, which was shown by sequencing to skip exon 27 and splice exon 26 to exon 28.  The resulting deletion of 104 base pairs introduced a frame-shift after amino acid 1010 with 35 aberrant amino acids and abolished the C-terminal residues encoding the last two transmembrane domains and cytoplasmic tail of the ATP11c protein (Figure 3).         

 

           <--exon 26  <--exon 27 intron 27--> exon 28-->  <--exon 28    

     ACTCTGAAG…………AATTATTTG GTAAGCAGC…………GCCTTTTCT…………TTTCCCTGA

1008 -T--L--K-…………-I--I--W-              -A--F--S-…………-F--P--* 1043

      correct       deleted                     aberrant

 

The donor splice site of intron 27, which is destroyed by the mutation, is indicated in blue; the mutated nucleotide is indicated in red.

 

Please see the record for emptyhive for information about Atp11c.

Putative Mechanism

The ambrosius mutation results in aberrant splicing of the Atp11c transcript resulting in loss of the last two transmembrane domains and the cytoplasmic tail of the protein.  It is unlikely that this transcript produces a functional, appropriately localized protein.  The phenotypes of ambrosius mice are essentially identical with other Atp11c mutants.

 

Figure 4. Effects of Bcl-2, IL-7 or BCR transgene on ATP11c-mutant B cell development. Representative flow cytometric analysis showing the frequency of B cell subpopulations in the bone marrow of (a) non-trangenic mice with mutant (amb) or wild-type (wt) Atp11c, (b) Atp11camb or wt mice with enforced expression of Bcl2 under the control of the Vav-promoter (Vav-Bcl2), (c) Atp11camb or wt mice over-expressing IL-7 under the control of the MHC II Eα gene promoter (H2Ea-117), (d) Atp11camb or wt mice expressing rearranged Igh and Igk transgenes from transgenes from the MD4 straining. (e) Graphs show the relative number of pro-B, pre-B, immature B cells in the bone marrow and B cells in the spleen of Atp11camb mutant mice with the indicated transgene, as a percentage of the mean number in wt control animals carrying the same transgene. Each circle represents one mouse, columns show means. Figure reproduced from reference (2).

The transition from pro- to pre-B cells is dependent on signaling through the interleukin-7 (IL-7) receptor and successful rearrangement of immunoglobulin heavy chain genes (4).  In order to determine at what point the Atp11c mutation affected B cell development, ambrosius mutants were crossed with: 1) Vav-Bcl2 transgenic mice to inhibit apoptosis(5); 2) H2Ea-Il7 transgenic mice with greatly increased IL-7 (6) or 3) MD4 transgenic mice (7) with Ig heavy and light chain genes already rearranged (Figure 4).  The Bcl2 transgene partially restored numbers of pre-B and immature B cells in ambrosius animals (Figure 4b,e).  However spleen B cells remained at 6% of the numbers in Atp11c+/Y Vav-Bcl2 controls and 31% of wild type mice, and continued to exhibit high IgM.  H2Ea-Il7 transgenic mice with a normal Atp11c gene exhibited a 5-fold increase in bone marrow pro-B cells and 10-fold increase in pre-B cell numbers relative to wild-type mice (Figure 4c,e). By contrast, there was no effect of increased IL-7 on the number of pro-B, pre-B, or immature B cells in Atp11camb/Y H2Ea-Il7 mice compared to Atp11camb/Y mice without the IL-7 transgene. Increased IL-7 was therefore unable to rescue development of ATP11c-deficient B cells, and instead the mutation abolished the effects of transgenic IL-7 on pro-B and pre-B cells in the bone marrow although IL-7Rα expression is increased in ambrosius pro-B cells (not shown).  MD4 transgenic mice carrying an already rearranged Ig heavy and light chain bypass and suppress RAG-mediated recombination of the endogenous Ig-genes, lowering the number of pro-B cells and pre-B cells to 12% and 2% of normal numbers, respectively, and replacing them with IgM+ IgD- immature B cells that are present in normal numbers in the bone marrow.  Whereas the Atp11camb mutation decreased the number of pro-B cells in non-transgenic mice, it increased the number of pro-B cells in MD4 transgenic mice to 150% of the numbers in control MD4 animals with normal ATP11c (Figure 4e).  The increased numbers of pro-B cells is likely to reflect a developmental delay in activating the rearranged Ig-transgenes within the Atp11camb/Y pro-B cell population. The number of immature B cells in the bone marrow of Atp11camb/Y MD4 animals was nevertheless only partly restored to 11% of the numbers in Atp11c+/0 MD4 mice, whereas spleen and circulating B cells were partly restored to 37% of those in MD4 transgenic mice with normal ATP11c. Bypassing the pre-BCR signaling step thus alleviated, but did not eliminate the need for ATP11c.

 

Figure 5. Decreased pro-B cell transition from Ig- to Ig+ cells and absence of response to IL-7 transgene despite increased expression of IL7Rα. (a) Representative flow cytometric analysis of the surface expression of CD24 and intracellular expression of IgM (cµ) gated on surface IgM- B220low 7AAD- B cells in bone marrow from non-transgenic or IL-7 transgenic mice with wild type (wt) or mutant (amb) Atp11c, or from Cd79-/- and Rag-/- mice. Numbers indicate percent of gated cells in each quadrant. (b) Percent of intracellular IgM+ CD24+ cells gated as in (a), and mean fluorescence intensity (MFI) of CD24 staining on these cells. (c) Analysis of bone marrow from mice expressing the rearranged MD4 Igh and Igk transgenes, showing the % of B220low IgD- cells expressing surface IgM and CD24 (upper panel) or CD19 (lower panel). Figure reproduced from reference (2).

To further characterize the developmental block in B cells in ambrosius mice, staining for cytoplasmic m(cµ) heavy chains was examined. A severely reduced number of developing B cells expressed cµ in ambrosius mice (Figure 5a,b). Null mutations eliminating the RAG1 recombinase (see the record for maladaptive) or the CD79a (Igα) subunit of the pre-BCR and BCR resulted in no or reduced percentages of B cells expressing cµ consistent with their inability to recombine the heavy chain genes or to assemble or signal through the pre-BCR, respectively. When the need for Ig-gene rearrangement and pre-BCR signaling was bypassed in MD4 transgenic mice, ATP11c-deficiency still greatly reduced the frequency of B220low B cells that expressed the Ig genes and instead there was an expanded population of pro-B cells that had not yet activated Ig transgene expression (Figure 5c). Collectively, these results indicate that the onset of heavy chain expression and the response to pre-BCR assembly are both diminished in the absence of normal ATP11c.

 

Figure 6. Atp11c mutation decreases phosphatidylserine translocation into pro-B cells. (a) Representative NBD-PS fluorescence profiles after 1 or 12 min incubation in pro-B cells from Atp11c mutant (amb) or wild type (wt) mice lacking or carrying the Vav-Bcl2 transgene (black lines), compared to CD45.1- marked wt pro-B cells (shaded gray). (b) NDP-PS geometric mean fluorescence intensity in pro-B cells of the indicated genotypes at different incubcation times. Figure reproduced from reference (2).

Like other members of the P4-ATPase family, ATP11C may be involved in phospholipid transport and maintaining membrane asymmetry.  Bone marrow from Atp11camb/Y CD45.2 and Atp11c+/Y CD45.1 control animals was mixed and incubated for various amounts of time with the fluorescent phosphatidylserine (PS) analogue, NBD-PS. Any dye remaining in the exoplasmic leaflet was then extracted with lipid-free albumin and washed away. The mutant and control pro-B cell subsets were distinguished by antibody staining, and analysed on a flow cytometer. NBD-PS fluorescence in mutant and wild-type pro-B cells increased rapidly and approached saturation by 12 minutes at 37?C, but was homogeneously less in mutant pro-B cells analysed at 1 or 3 minutes (Figure 6a,b). This suggests that ATP11c is a functional PS flippase in developing B cells.

Primers Primers cannot be located by automatic search.
References
Science Writers Nora G. Smart
Illustrators Diantha La Vine
AuthorsMehmet Yabas, Charis E. Teh, Sandra Frankenreiter, Dennis Lal, Carla M. Roots, Belinda Whittle, Daniel T. Andrews, Yafei Zhang, Narci C. Teoh, Jonathan Sprent, Lina E. Tze, Edyta M. Kucharska, Jennifer Kofler, Geoffrey C. Farell, Stefan Broer, Christopher C. Goodnow, Anselm Enders
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