In 20 families with 27 patients with AR-HIES, a total of 21 patients had biallelic deletions or intragenic small mutations involving DOCK8. Four subjects harbored large homozygous deletions, one had a large compound heterozygous deletion, seven had exonic deletions and nine suffered homozygous point mutations predicted to impair DOCK8 protein expression or function.
The fact that we were not able to demonstrate exonic mutations in the remaining six AR-HIES families may be due to difficulties in detecting slight changes, e.g. compound heterozygous mutations, in a 48 exon gene. Moreover, mutations may lie in intronic regions or in the regulatory regions of the gene. We will pursue this by in depth analysis of patients’ cDNA and protein expression.
By genomic sequence analysis, Griggs et al. had mapped DOCK8
to chromosome 9p2413
. They determined that DOCK8
contains 47 exons spanning 190 kb; more recently, however, the Ensembl database (www.ensembl.org
) provides evidence for ten different transcripts and 48 exons. Northern blot analysis detected DOCK8
expression in human placenta, lung, kidney, and pancreas and to a lesser degree in brain, heart, and skeletal muscle14
DOCK8 is one of 11 members of the dedicator of cytokinesis (DOCK) protein family15
. DOCK8 interacts with GTP- and GDP-bound forms of Ccd42 and Rac1, and Cdc42 family members TCL and TC10 in a yeast 2-hybrid assay14
. The protein contains DOCK Homology Region-1 (DHR-1, or CZH1) and DHR-2 (CZH2) domains, characteristic of DOCK180-related proteins. In some DOCK180-related family members, these domains bind phospholipid and carry out guanine nucleotide exchange factor (GEF) function, respectively15,16
. Although the GEF activity of DOCK8 remains to be determined, the high DHR-2 domain homology of the DOCK-C subfamily suggests that like orthologs DOCK6 and DOCK7, DOCK8 may be a GEF specific for Cdc42 and/or Rac117
Figure 7 Hypothetical function of DOCK8. DOCK8 is likely to function as a guanine-nucleotide exchange factor (GEF) for the Rho-GTPases Cdc42 and Rac1, turning them into the active, GTP-bound form upon receptor engagement (e.g. receptor tyrosine kinases, antigen (more ...)
DOCK family proteins play roles in regulation of cell migration, morphology, adhesion and growth. Immunofluorescence localized transiently transfected and endogenous DOCK8 to the cytoplasm at cell edges forming lamellipodia, which increased when cells were treated with PDGF or fetal calf serum. Transient transfection of a C-terminal fragment of DOCK8 containing the DHR-2 domain resulted in formation of vesicular structures, suggesting that DOCK8 may play a role in the organization of filamentous actin14
Patients with AR-HIES have atopic eczema, severe susceptibility to viral infections, and deficient TH17 cell function12
. Our present studies demonstrate that DOCK8 deficiency impairs CD4+
T cell proliferative responses, consistent with a critical role for this protein in T cell activation and effector functions. It can be speculated, based on the protein domain structure, that DOCK8 may fulfill an important cytoskeletal function relevant to T cell activation, such as in the formation of the immunological synapse. Other immune deficiencies, such as the Wiskott-Aldrich syndrome and severe combined immunodeficiency due to lack of Coronin-1A, also involve deficient function of important cytoskeleton-regulating proteins 18,19
The mechanisms by which abnormal T cell effector functions develop in DOCK8-deficient patients, including impaired TH17 differentiation, remains to be established. However, a role for DOCK8 in the maintenance of memory TH17 cells can be inferred, as it was that component of the TH17 response that appeared most affected in DOCK8-deficient subjects12
. It is conceivable that TH17 deficiency in AR-HIES maybe symptomatic of a more widespread derangement of TH cell differentiation, an eventuality that is currently under investigation. The presence of atopic dermatitis in AR-HIES suggests the action of immune dysregulatory mechanisms, although an additional contribution from a defective barrier function may also be involved. Overall, we propose that DOCK8 may play a critical role in cytoskeletal organization, and that its deficiency might result in impaired T cell activation and effector responses.
Two unrelated patients have been previously described with mental retardation and developmental disability associated with heterozygous
disruption of DOCK8
by deletion and by a translocation breakpoint, respectively. Immunologic abnormalities were not described in these patients. Mapping of the critical region shared by the two patients showed truncation of the longest isoform of DOCK8
. However, these patients had single copy deletions of DOCK8
along with disruptions of other genes13,20
, which may explain why their phenotype differed from those of our patients.
After the initial submission of our work, Zhang and colleagues published homozygous and compound heterozygous mutations in DOCK8
in a cohort of twelve patients from eight families11
. One of their families (#8 in Zhang et al.) is family ARH011 in our manuscript. They described the phenotype as a “combined T- B- NK-immunodeficiency” and presented evidence of decreased T, B and NK cell numbers. Our assessment of these published cases would suggest that they also suffer from an AR form of HIES, given that the major diagnostic criteria of elevated IgE, eosinophilia, recurrent pneumonia and skin eczema together with a susceptibility to viral infections were fulfilled in all but one patient. However, and in contrast to the findings by Zhang et al.11
, studies on the cohort presented herein demonstrate a selective decrease in CD4+
T cell numbers in many but not all patients. The CD8+
T cell population is less affected, while B and NK cells are usually within the normal range. Furthermore, whereas Zhang et al. found a selective defect in CD8+
T cell activation, our findings are consistent with a more comprehensive T cell activation defect involving both
T cell subsets. Such a global defect in T cell activation may explain the severe clinical phenotype of AR-HIES.
DOCK8 deficiency is characterized by recurrent infections of the upper and lower respiratory tract, susceptibility to severe, recurrent and mutilating viral infections (especially by Molluscum contagiosum and Herpes viruses), a severe dermatitis that resembles atopic dermatitis and may often be superinfected, elevated IgE levels and eosinophilia. Other clinical features such as asthma, allergies, CNS symptoms, or autoimmune phenomena are variably associated. Whether there is a genotype-phenotype correlation with regard to the size of the deletion or whether or not other deleted genes next to DOCK8 contribute to the phenotype needs to be determined in future studies. It is remarkable that the typical feature of cyst-forming pneumonia (pneumatoceles), as frequently seen in STAT3-mutated HIES, is not typical for DOCK8 deficiency and can be used as a discriminating feature.
Subtelomeric deletions often arise at sites of interspersed repetitive genomic sequences such as Alu, LINE, long terminal repeats, and simple tandem repeats21,22
. The region surrounding DOCK8
is particularly rich in these sequences, with 264 such sequences in the region from the p terminus to the end of DOCK8
(Alu database). In particular, there are Alu
-Jb sequences near the breakpoints of our patients as mapped by microarray analysis. Abnormal recombination or transposable element activity could contribute to the high frequency of deletions seen. Careful analysis of the breakpoints of these deletions will be helpful in delineating which functions of DOCK8 are affected by the deletions, potenitally shedding light on the role of DOCK8 in immune and integument function.
Homozygous mutations in DOCK8 were identified in most patients with autosomal recessive hyper-IgE syndrome. Hence patients with a phenotype of elevated IgE, eosinophilia, and recurrent skin boils, pneumonia, and viral infections (especially molluscum contagiosum and herpes) should be suspected of having mutations in DOCK8.
Twenty-one of 27 patients with the autosomal recessive hyper-IgE syndrome had homozygous deletions or point mutations in DOCK8. After STAT3, DOCK8 mutations represent the second most frequent cause of hyper-IgE syndromes.