Homozygous mutants are defective in the migration of T and B lympohcytes in response to chemokines, and thus display immune defects such as lymphocytopenia, atrophy of lymphoid follicles and loss of marginal-zone B cells.
Figure 1.Capitol_reefmice exhibited increased IgE secretion. IgE 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.
Figure 2.Capitol_reefmice exhibited increased IgE secretion after immunization with ovalbumin adminstered with aluminum hydroxide. IgE 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 capitol_reef phenotype was identified among G3 mice of the pedigree R4742, some of which showed increased secretion of total IgE (Figure 1) and ovalbumin-specific IgE after immunization with ovalbumin administered with aluminum hydroxide (Figure 2).
Nature of Mutation
Figure 3.Linkage mapping of the increased OVA-specific IgE using a recessive model of inheritance. Manhattan plot shows -log10 P values (Y-axis) plotted against the chromosome positions of 49 mutations (X-axis) identified in the G1 male of pedigree R4742. 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 49 mutations. Both of the above phenotypes were linked by continuous variable mapping to a mutation in Dock2: a A to C transversion at base pair 34,294,170 (v38) on chromosome 11, or base pair 489,723 in the GenBank genomic region NC_000077 within the acceptor splice site of intron 31. The strongest association was found with a recessive model of linkage to the normalized amount of OVA-specific IgE, wherein two variant homozygotes departed phenotypically from 13 homozygous reference mice and 14 heterozygous mice with a P value of 3.568 x 10-11 (Figure 3).
The effect of the mutation at the cDNA and protein level have not examined, but the mutation is predicted to result in an in-frame skipping of the 59-nucleotide exon 32 (out of 52 total exons).
Genomic numbering corresponds to NC_000077. The acceptor splice site of intron 31, which is destroyed by the capitol_reef mutation, is indicated in blue lettering and the mutated nucleotide is indicated in red.
Figure 4. Domain structure of mouse DOCK2. DOCK2 contains an N-terminal SH3 domain. SH3-containing DOCK proteins have been shown to interact physically with the scaffolding proteins engulfment and cell motility protein 1 (ELMO1) and ELMO2, significantly promoting Rac activation. The DHR-1 domain shares weak homology to the C2 domain. The large DHR-2 domain interacts with the nucleotide-free form of Rac. The location of the capitol_reef mutation is indicated. Click on the image to view other mutations found in DOCK2. Click on each mututation for more specific information.
DOCK2 belongs to the DOCK180 superfamily of guanine nucleotide exchange factors (GEFs) that have been shown to activate members of the Rho family of small GTPases [Figure 4; (1-4)]. Members of the DOCK A and B groups contain an N-terminal SH3 domain. Two domains are shared amongst all DOCK proteins, the catalytic DHR-2 (DOCK homology region 2) or CZH-2 (CDM-zizimin homology 2) domain (amino acids 1114-1620 in DOCK2) and the DHR-1 or CZH-1 domain (amino acids 420-662 in DOCK2) (2;4). Several DOCK proteins, including DOCK2, appear to be localized to the plasma membrane via interaction of the DHR-1 domain with phosphatidylinositol (3,5)-biphosphate [PtdIns(3,5)P2] and phosphatidylinositol (3,4,5) P3 (PIP3) signaling lipids (5;6). The DHR-2 domains of several DOCK family members interact with the nucleotide-free form of Rac and/or Cdc42 (2;4). DOCK2 has an N-terminal SH3 domain (amino acids 8-69). The capitol_reef mutation is within the splice acceptor site of intron 31 and is predicted to result in deletion of exon 32. Exon 32 encodes amino acids 1058-1076, which is an undefined region between the DHR-1 and DHR-2 domains.
For more information on Dock2, see the record forfrazz.
Rho GTPases are known regulators of the actin cytoskeleton and affect multiple cellular activities including cell morphology, polarity, migration, proliferation and apoptosis, phagocytosis, cytokinesis, adhesion, vesicular transport, transcription and neurite extension and retraction. Regulation of Rho GTPase activity involves the GEFs that promote the exchange of GDP for GTP, the GTPase-activating proteins (GAPs) that enhance the GTPase activity of Rho proteins, and the Rho guanine nucleotide-dissociation inhibitors (RhoGDIs) that sequester Rho GTPases in a GDP-bound state. DOCK2 functions in the polarization and migration of immune cell subsets. DOCK2 functions downstream of chemokine receptors to regulate Rac activation and migration of B and T lymphocytes, neutrophils, plasmacytoid dendritic cells (pDCs), and hematopoietic stem cells, but not monocytes or other myeloid cell types (6-10). The immune cells affected by DOCK2 deficiency display aberrant homing, activation, adhesion, polarization and migration. DOCK2-deficient lymphocytes fail to respond normally to the chemokines CCL21, CCL19, CXCL13, and CXCL12, resulting in impaired homing to secondary lymphoid organs and aberrant morphology of these tissues (8). Dock2 -/ -mice display a number of immunological phenotypes including lack of marginal zone B cells, reduced numbers of mature CD4+ T cells and the major subset of natural killer T (NKT) cells expressing the semi-invariant Vα14 T cell receptor (TCR), and aberrant TCR signaling (8;11;12). Similar to Dock2-/ - mice, the Arches mice exhibit reduced frequencies of peripheral CD4+ T cells, indicating loss of function in DOCK2Arches.
capitol_reef(F):5'- TTTGGGTCATCACACAGGCAC -3'
capitol_reef(R):5'- TCAGACCCTCTCCATGACTG -3'
capitol_reef_seq(F):5'- CAGGCACAGACTGACATATGCTG -3'
capitol_reef_seq(R):5'- GCTGTGGAACAACTATTTTCATCTGG -3'