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|Coordinate||78,565,945 bp (GRCm38)|
|Base Change||T ⇒ G (forward strand)|
|Gene Name||RAS-related C3 botulinum substrate 2|
|Chromosomal Location||78,559,169-78,572,783 bp (-)|
|MGI Phenotype||Homozygotes for a targeted null mutation exhibit peripheral blood lymphocytosis, reductions in peritoneal B-1a lymphocytes, marginal zone lymphocytes, and IgM-secreting plasma cells, decreased levels of serum IgM and IgA, and abnormal T cell migration.|
|Amino Acid Change||Aspartic acid changed to Alanine|
|Institutional Source||Beutler Lab|
|Gene Model||predicted sequence gene model|
AA Change: D65A
|Predicted Effect||possibly damaging
PolyPhen 2 Score 0.955 (Sensitivity: 0.79; Specificity: 0.95)
|Phenotypic Category||decrease in B1 cells, decrease in B1a cells in B1 cells, increase in B1b cells|
|Alleles Listed at MGI|
|Mode of Inheritance||Autosomal Semidominant|
|Last Updated||12/08/2016 11:13 AM by Katherine Timer|
|Record Created||11/24/2015 3:29 PM|
The Big_bend phenotype was identified among N-ethyl-N-nitrosourea (ENU)-mutagenized G3 mice of the pedigree R0751, some of which showed a reduced frequency of B1 cells (Figure 1) and B1a cells in B1 cells (Figure 2) as well as an increased frequency of B1b cells (Figure 3), all in the peripheral blood.
|Nature of Mutation|
Whole exome HiSeq sequencing of the G1 grandsire identified 72 mutations. All of the above anomalies were linked by continuous variable mapping to mutations in Rac2, Eif3l, and Chadl on chromosome 15. The mutation in Rac2 was presumed to be causative because the Big_bend phenotypes mimic other known alleles of Rac2 (see MGI for a list of Rac2 alleles). The mutation in Rac2 is an A to C transition at base pair 78,565,945 (v38) on chromosome 15, or base pair 6,839 in the GenBank genomic region NC_000081 encoding the Rac2 gene. The strongest association was found with an additive model of linkage to the normalized frequency of B1 cells, wherein three variant homozygotes departed phenotypically from 20 homozygous reference mice and 14 heterozygous mice with a P value of 1.058 x 10-6 (Figure 4). A substantial semidominant effect was observed in the B1 and B1b assays, but a recessive effect was observed in the B1a cells in B1 cells assay.
The mutation corresponds to residue 332 in the mRNA sequence NM_009008 within exon 3 of 7 total exons.
The mutated nucleotide is indicated in red. The mutation results in an aspartic acid (D) to alanine (A) substitution at position 65 (D65A) in the Rac2 protein, and is strongly predicted by PolyPhen-2 to be damaging (score = 0.955) (1).
Rac2 is a member of the Rac subfamily of Rho guanosine triphosphatases (Rho GTPases). Rho GTPases have several conserved domains including five GTP binding and hydrolysis domains (G-boxes; G1-G5), two switch regions (switch I and II), a polybasic domain, and a prenylation site [Figure 5; (2)]. G-boxes function in GDP binding and exhibit GTPase activity (3). In Rac2, these regions correspond to amino acids 10-17 (G1), Thr35 (G2), 57-61 (G3), and 115-118 (G4), and 157-160 (G5). The Rac proteins each have two highly conserved switch regions, switch I (amino acids 27-40) and switch II (amino acids 56-71), situated on either side of the bound nucleotide. Both switch regions are sites of interactions between the Rac proteins and guanine nucleotide exchange factors (GEFs) and guanine nucleotide-dissociation inhibitors (GDIs) as well as with downstream protein targets (4). The polybasic region of Rac2 (RQQKRP; amino acids 183-188) is required for its function as a regulator of NAPDH oxidase.
The mutation in Big_bend results in an aspartic acid (D) to alanine (A) substitution at position 65. Amino acid 65 is within an undefined region between the G3 and G4 regions.
For more information about Rac2, please see the record for bingo.
Rho GTPases integrate receptor-mediated signals through binding to effectors and regulators of the actin cytoskeleton and affect multiple cellular activities including cell morphology, polarity, migration, proliferation, apoptosis, phagocytosis, cytokinesis, adhesion, vesicular transport, and transcription. Rac2 functions in actin polymerization resulting in lamellopodial extension and membrane ruffling, directed migration, chemotaxis, and superoxide (O2−) production in phagocytic cells as well as cytoskeleton organization in red blood cells and osteoclasts (5-10). The Rac proteins regulate leukocyte migration by transducing signals from cell surface receptors (e.g., the Fcγ receptor, formylmethionyl-leucyl-phenylalanine (fMLP) receptor, and β2 integrins) to the actin and microtubule cytoskeletons through cytoplasmic effectors (e.g., tyrosine kinases, scaffolding/adapter proteins, nucleotide exchange proteins, and phosphatases) upon binding of GTP (11).
The Big_bend mice exhibited increased frequency of B1 cells and B1b cells as well as a reduced frequency of B1a cells in B1 cells in the peripheral blood. Rac2 is required for B cell development as well as for either B cell receptor (BCR) signal transduction and subsequent calcium mobilization or in determining the efficiency of BCR ligation (12;13). Rac2-deficient (Rac2-/-) mice exhibit a 30% reduction in B cell numbers due mainly be a reduced number of recirculating B lymphocytes in the bone marrow (12). Rac2-/- mice also display a lack of peritoneal B1 and marginal zone B cells (12). In the peripheral blood, Rac2-/- mice had an increase in total leukocyte number including both B and T cells (12). B cell numbers were reduced in the spleen due to a loss of mature and/or marginal zone B cells (12). In humans, mutations in RAC2 are linked to neutrophil (alternatively, phagocytic) immunodeficiency syndrome [NIS; OMIM: #608203; (14-16)] and decreased numbers of peripheral T and B cells. Patients with NIS have severe, recurrent infections, poor wound healing, and exhibit reduced neutrophil migration, azurophilic granule secretion, and superoxide production (14-16).
The alterations in the frequencies of B1, B1b, and B1a cells in B1 cells indicate a loss of Rac2Big_bend function; however, some Rac2 function may remain or Rac1 may be compensating for the loss of Rac2 function and/or expression as other Rac2-/--associated phenotypes were not observed in the Big_bend mice.
Big_bend(F):5'- TGCCTGGCACACACTGAGAAAG -3'
Big_bend(R):5'- GTTGTGGAAACAGCCAGTTGCAC -3'
Big_bend_seq(F):5'- CTCGGGGCATGGTGATACTC -3'
Big_bend_seq(R):5'- GGTCACAGTCCCTCTCTGAG -3'
1. Adzhubei, I. A., Schmidt, S., Peshkin, L., Ramensky, V. E., Gerasimova, A., Bork, P., Kondrashov, A. S., and Sunyaev, S. R. (2010) A Method and Server for Predicting Damaging Missense Mutations. Nat Methods. 7, 248-249.
2. Hirshberg, M., Stockley, R. W., Dodson, G., and Webb, M. R. (1997) The Crystal Structure of Human rac1, a Member of the Rho-Family Complexed with a GTP Analogue. Nat Struct Biol. 4, 147-152.
3. Bourne, H. R., Sanders, D. A., and McCormick, F. (1991) The GTPase Superfamily: Conserved Structure and Molecular Mechanism. Nature. 349, 117-127.
4. Yamauchi, A., Marchal, C. C., Molitoris, J., Pech, N., Knaus, U., Towe, J., Atkinson, S. J., and Dinauer, M. C. (2005) Rac GTPase Isoform-Specific Regulation of NADPH Oxidase and Chemotaxis in Murine Neutrophils in Vivo. Role of the C-Terminal Polybasic Domain. J Biol Chem. 280, 953-964.
5. Gu, Y., Filippi, M. D., Cancelas, J. A., Siefring, J. E., Williams, E. P., Jasti, A. C., Harris, C. E., Lee, A. W., Prabhakar, R., Atkinson, S. J., Kwiatkowski, D. J., and Williams, D. A. (2003) Hematopoietic Cell Regulation by Rac1 and Rac2 Guanosine Triphosphatases. Science. 302, 445-449.
6. Kalfa, T. A., Pushkaran, S., Mohandas, N., Hartwig, J. H., Fowler, V. M., Johnson, J. F., Joiner, C. H., Williams, D. A., and Zheng, Y. (2006) Rac GTPases Regulate the Morphology and Deformability of the Erythrocyte Cytoskeleton. Blood. 108, 3637-3645.
7. Itokowa, T., Zhu, M. L., Troiano, N., Bian, J., Kawano, T., and Insogna, K. (2011) Osteoclasts Lacking Rac2 have Defective Chemotaxis and Resorptive Activity. Calcif Tissue Int. 88, 75-86.
8. Roberts, A. W., Kim, C., Zhen, L., Lowe, J. B., Kapur, R., Petryniak, B., Spaetti, A., Pollock, J. D., Borneo, J. B., Bradford, G. B., Atkinson, S. J., Dinauer, M. C., and Williams, D. A. (1999) Deficiency of the Hematopoietic Cell-Specific Rho Family GTPase Rac2 is Characterized by Abnormalities in Neutrophil Function and Host Defense. Immunity. 10, 183-196.
9. Yang, F. C., Atkinson, S. J., Gu, Y., Borneo, J. B., Roberts, A. W., Zheng, Y., Pennington, J., and Williams, D. A. (2001) Rac and Cdc42 GTPases Control Hematopoietic Stem Cell Shape, Adhesion, Migration, and Mobilization. Proc Natl Acad Sci U S A. 98, 5614-5618.
10. Yang, F. C., Kapur, R., King, A. J., Tao, W., Kim, C., Borneo, J., Breese, R., Marshall, M., Dinauer, M. C., and Williams, D. A. (2000) Rac2 Stimulates Akt Activation Affecting BAD/Bcl-XL Expression while Mediating Survival and Actin Function in Primary Mast Cells. Immunity. 12, 557-568.
11. Wheeler, A. P., Wells, C. M., Smith, S. D., Vega, F. M., Henderson, R. B., Tybulewicz, V. L., and Ridley, A. J. (2006) Rac1 and Rac2 Regulate Macrophage Morphology but are Not Essential for Migration. J Cell Sci. 119, 2749-2757.
12. Croker, B. A., Tarlinton, D. M., Cluse, L. A., Tuxen, A. J., Light, A., Yang, F. C., Williams, D. A., and Roberts, A. W. (2002) The Rac2 Guanosine Triphosphatase Regulates B Lymphocyte Antigen Receptor Responses and Chemotaxis and is Required for Establishment of B-1a and Marginal Zone B Lymphocytes. J Immunol. 168, 3376-3386.
13. Walmsley, M. J., Ooi, S. K., Reynolds, L. F., Smith, S. H., Ruf, S., Mathiot, A., Vanes, L., Williams, D. A., Cancro, M. P., and Tybulewicz, V. L. (2003) Critical Roles for Rac1 and Rac2 GTPases in B Cell Development and Signaling. Science. 302, 459-462.
14. Williams, D. A., Tao, W., Yang, F., Kim, C., Gu, Y., Mansfield, P., Levine, J. E., Petryniak, B., Derrow, C. W., Harris, C., Jia, B., Zheng, Y., Ambruso, D. R., Lowe, J. B., Atkinson, S. J., Dinauer, M. C., and Boxer, L. (2000) Dominant Negative Mutation of the Hematopoietic-Specific Rho GTPase, Rac2, is Associated with a Human Phagocyte Immunodeficiency. Blood. 96, 1646-1654.
15. Ambruso, D. R., Knall, C., Abell, A. N., Panepinto, J., Kurkchubasche, A., Thurman, G., Gonzalez-Aller, C., Hiester, A., deBoer, M., Harbeck, R. J., Oyer, R., Johnson, G. L., and Roos, D. (2000) Human Neutrophil Immunodeficiency Syndrome is Associated with an Inhibitory Rac2 Mutation. Proc Natl Acad Sci U S A. 97, 4654-4659.
|Science Writers||Anne Murray|
|Authors||Ming Zeng, Xue Zhong, and Bruce Beutler|
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