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|Mutation Type||critical splice donor site (1 bp from exon)|
|Coordinate||36,608,577 bp (GRCm38)|
|Base Change||C ⇒ T (forward strand)|
|Gene Name||ATPase, class VI, type 11C|
|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.|
|Amino Acid Change|
|Institutional Source||Australian Phenomics Network|
Ensembl: ENSMUSP00000033480 (fasta)
|Gene Model||not available|
|Phenotypic Category||anemia, B cells - decreased, hematopoietic system, homeostasis/metabolism, immune system, liver/biliary system|
|Alleles Listed at MGI|
|Mode of Inheritance||X-linked Recessive|
|Last Updated||2016-05-13 3:09 PM by Stephen Lyon|
|Record Created||2011-02-07 12:25 PM by Nora G. Smart|
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).
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.
|Nature of Mutation|
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
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.
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.
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.
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.
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.|
1. Nelms, K. A., and Goodnow, C. C. (2001) Genome-Wide ENU Mutagenesis to Reveal Immune Regulators. Immunity. 15, 409-418.
2. Yabas, M., Teh, C. E., Frankenreiter, S., Lal, D., Roots, C. M., Whittle, B., Andrews, D. T., Zhang, Y., Teoh, N. C., Sprent, J., Tze, L. E., Kucharska, E. M., Kofler, J., Farell, G. C., Broer, S., Goodnow, C. C., and Enders, A. (2011) ATP11C is Critical for the Internalization of Phosphatidylserine and Differentiation of B Lymphocytes. Nat. Immunol.. 12, 441-449.
3. Siggs, O. M., Arnold, C. N., Huber, C., Pirie, E., Xia, Y., Lin, P., Nemazee, D., and Beutler, B. (2011) The P4-Type ATPase ATP11C is Essential for B Lymphopoiesis in Adult Bone Marrow. Nat. Immunol.. 12, 434-440.
4. Fleming, H. E., and Paige, C. J. (2001) Pre-B Cell Receptor Signaling Mediates Selective Response to IL-7 at the Pro-B to Pre-B Cell Transition Via an ERK/MAP Kinase-Dependent Pathway. Immunity. 15, 521-531.
5. Ogilvy, S., Metcalf, D., Print, C. G., Bath, M. L., Harris, A. W., and Adams, J. M. (1999) Constitutive Bcl-2 Expression Throughout the Hematopoietic Compartment Affects Multiple Lineages and Enhances Progenitor Cell Survival. Proc. Natl. Acad. Sci. U. S. A.. 96, 14943-14948.
6. Mertsching, E., Grawunder, U., Meyer, V., Rolink, T., and Ceredig, R. (1996) Phenotypic and Functional Analysis of B Lymphopoiesis in Interleukin-7-Transgenic Mice: Expansion of pro/pre-B Cell Number and Persistence of B Lymphocyte Development in Lymph Nodes and Spleen. Eur. J. Immunol.. 26, 28-33.
7. Goodnow, C. C., Crosbie, J., Adelstein, S., Lavoie, T. B., Smith-Gill, S. J., Brink, R. A., Pritchard-Briscoe, H., Wotherspoon, J. S., Loblay, R. H., and Raphael, K. (1988) Altered Immunoglobulin Expression and Functional Silencing of Self-Reactive B Lymphocytes in Transgenic Mice. Nature. 334, 676-682.
|Science Writers||Nora G. Smart|
|Illustrators||Diantha La Vine|
|Authors||Mehmet 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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