Figure 1. Warts_and_all mice exhibit increased B to T cell ratios. Flow cytometric analysis of peripheral blood was utilized to determine B and T cell frequencies. 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. Warts_and_all mice exhibit decreased frequencies of peripheral T cells. Flow cytometric analysis of peripheral blood was utilized to determine 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 4. Warts_and_all mice exhibit decreased frequencies of peripheral CD4+ T cells. Flow cytometric analysis of peripheral blood was utilized to determine 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 5. Warts_and_all mice exhibit decreased frequencies of peripheral CD8+ T cells. Flow cytometric analysis of peripheral blood was utilized to determine 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. Warts_and_all mice exhibit decreased frequencies of peripheral naïve CD4 T cells in CD4 T cells . Flow cytometric analysis of peripheral blood was utilized to determine 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 7. Warts_and_all mice exhibit decreased frequencies of peripheral naïve CD8 T cells in CD8 T cells. Flow cytometric analysis of peripheral blood was utilized to determine 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 8. Warts_and_all mice exhibit increased frequencies of peripheral CD44+ CD4 T cells. Flow cytometric analysis of peripheral blood was utilized to determine 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 9. Warts_and_all mice exhibit increased frequencies of peripheral central memory CD4 T cells in CD4 T cells. Flow cytometric analysis of peripheral blood was utilized to determine 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 10. Warts_and_all mice exhibit increased frequencies of peripheral central memory CD8 T cells in CD8 T cells. Flow cytometric analysis of peripheral blood was utilized to determine 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 11. Warts_and_all mice exhibit increased frequencies of peripheral effector memory CD4 T cells in CD4 T cells. Flow cytometric analysis of peripheral blood was utilized to determine 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 12. Warts_and_all mice exhibit increased frequencies of peripheral effector memory CD8 T cells in CD8 T cells. Flow cytometric analysis of peripheral blood was utilized to determine 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 13. Warts_and_all mice exhibit increased CD44 expression on peripheral T cells. Flow cytometric analysis of peripheral blood was utilized to determine CD44 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.

Figure 14. Warts_and_all mice exhibit increased CD44 expression on peripheral CD4+ T cells. Flow cytometric analysis of peripheral blood was utilized to determine CD44 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.

Figure 15. Warts_and_all mice exhibit increased CD44 expression on peripheral CD8+ T cells. Flow cytometric analysis of peripheral blood was utilized to determine CD44 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.

Figure 16. Warts_and_all mice exhibit reduced B220 expression 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 warts_and_all phenotype was identified among G3 mice of the pedigree R6818, some of which showed increased B to T cell ratios (Figure 1) as well as reduced frequencies of B1 cells (Figure 2), T cells (Figure 3), CD4+ T cells (Figure 4), CD8+ T cells (Figure 5), naïve CD4 T cells in CD4 T cells (Figure 6), and naïve CD8 T cells in CD8 T cells (Figure 7) with concomitant increased frequencies of CD44+ CD4 T cells (Figure 8), central memory CD4 T cells in CD4 T cells (Figure 9), central memory CD8 T cells in CD8 T cells (Figure 10), effector memory CD4 T cells in CD4 T cells (Figure 11), and effector memory CD8 T cells in CD8 T cells (Figure 12), all in the peripheral blood. The expression of CD44 on peripheral blood T cells (Figure 13), CD4+ T cells (Figure 14), and CD8+ T cells (Figure 15) was increased. The expression of B220 on peripheral blood B cells was reduced (Figure 16).

Whole exome HiSeq sequencing of the G1 grandsire identified 61 mutations. All of the above anomalies were linked by continuous variable mapping to a mutation in Dock8: a T to C transition at base pair 25,169,501 (v38) on chromosome 19, or base pair 16,9991 in the GenBank genomic region NC_000085 in the splice donor site of intron 34, two-base pairs from exon 34. The strongest association was found with a recessive model of inheritance to the reduced T cell frequency phenotype, wherein six variant homozygotes departed phenotypically from 12 homozygous reference mice and eight heterozygous mice with a P value of 1.241 x 10^-9 (Figure 17).

The effect of the mutation at the cDNA and protein levels have not examined, but the mutation is predicted to result in skipping of the 97-base pair exon 34 (out of 48 total exons). The mutation would cause a frame-shifted protein product beginning after amino acid 1,415 of the protein, which is normally 2,100 amino acids in length, and termination after the inclusion of 19 aberrant amino acids.

        <--exon 33     <--exon 34 intron 34-->         exon 35-->

4356 ……AAGCTGGACAA ……AACATCATCCAG gtgaggatgactcactcc…… GCAAGCTCCG……GGGTCCTGGTGA

1412 ……-K--L--D--K ……-N--I--I--Q-                      --Q--A--P-……-G--S--W--*-

         correct       deleted                                 aberrant

The donor splice site of intron 34, which is destroyed by the warts_and_all mutation, is indicated in blue lettering and the mutated nucleotide is indicated in red.

DOCK8 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 (1-4). The DOCK C subfamily (which includes DOCK8 and DOCK7; see the record for moonlight) has dual specificity for Rac and Cdc42 (1;3;5;6).  Two domains are shared amongst all DOCK proteins, the catalytic DHR-2 (DOCK homology region 2) or CZH-2 (CDM-zizimin homology 2) domain and the DHR-1 or CZH-1 domain (Figure 18).  The DHR-1 domain is located N-terminal to the DHR-2 domain (2;4).

The warts_and_all mutation results in abnormal splicing of Dock8 causing deletion of exon 34, which encodes amino acids 1,416 to 1,447 within the DHR-2 domain.

Please see the record for captain morgan for more information about Dock8.

The 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. Like DOCK2, DOCK8 is likely to regulate the activity of GTPases and thus be involved in cytoskeletal changes associated with various cellular processes. DOCK8 is proposed to serve as an effector downstream of CD19 and PI3K to promote G protein signaling events critical for integrin polarization at the synapse and for the survival of marginal zone B cells and germinal center (GC) B cells. 

During a T cell-dependent humoral immune response, CD4⁺ T helper cell subsets including T_FH, Th1 and Th2 cells migrate to the T cell/B cell borders in SLO, and interact with cognate antigen-specific B cells through the pairing of T cell and B cell surface ligands and receptors such as CD40 with its ligand (see the record for walla).  This interaction results in the secretion by T helper cells of certain cytokines known to promote B cell survival, proliferation, and antibody production (7;8).

In humans, DOCK8 deficiency results in an autosomal recessive form of hyper-IgE recurrent infection syndrome (HIES; OMIM #243700) (9;10).  Autosomal dominant HIES is characterized by recurrent Staphylococcus aureus skin abscesses, increased serum IgE, and abnormalities of the connective tissue, skeleton, and dentition (11).  The autosomal recessive form shares hyper-IgE, eosinophilia, and recurrent Staphylococcal infections, but is distinguished from autosomal dominant HIES by the lack of connective tissue and skeletal involvement (12). Patients with DOCK8 deficiency are unusually susceptible to viral infections and virally-caused cancers.  Reduced numbers of T, B, and NK cells have been reported along with a selective defect in CD8⁺ T cell activation (9).  However, another study suggests most patients have normal numbers of B and NK cells, a greater decrease in the CD4⁺ T cell population than the CD8⁺ T cell population, and a more comprehensive T cell activation defect involving both T cell subsets (10).  Both autosomal dominant and autosomal recessive HIES are linked to a lack of Th17 cell function including the failure to produce the interleukin 17 (IL-17) cytokine (12).  Th17 are a subset of CD4⁺ T helper cells that play an important role in the development of autoimmune diseases like rheumatoid arthritis, as well as being critical in the clearance of fungal and extracellular bacterial infections (13).    

The relatively normal initiation of antibody production by mice with Dock8 mutations suggests that the extrafollicular B cell clonal expansion, plasma cell formation and immunoglobulin class switching, which depends on interactions with T helper cells, is intact.  However, subsequent antibody responses and affinity maturation occurring in the GCs is significantly impaired. The humoral deficits are due to a defect in GC B cell survival and selection during the affinity maturation phase of GC responses (14).

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 409 nucleotides is amplified (chromosome 19, + strand):

1   gggttgtcat ctctggtacc tggccgagcc acacaatgct ttaatgggtg atgtgctttt
61  tgtttcttag aacaaaggca gagttagatc aagaagcctt gatcagtggc aacctggcta
121 cagaagctaa tttgatcatc ctggatatgc aggagaacat catccaggtg aggatgactc
181 actccccatc gggcccgctc gggggtgcca ttcttggaaa tagaaagggg gttcgtcatc
241 caaaattgaa aaagacacag gcgtgattct agaatgtata aaactaaagt gggaacgttt
301 ggaatttgca gcagttgcct cccttgcaga ttccagaggt gccaggcatc ctctggcttt
361 tctgactgct actcccttgg aaagagttag atgtttgagg agctgaccc

Primer binding sites are underlined and the sequencing primers are highlighted; the mutated nucleotide is shown in red.