Hypomorphic function and somatic reversion of DOCK8 cause combined immunodeficiency without hyper-IgE

Loss-of-function mutations in DOCK8 are linked to hyper-IgE syndrome. Patients typically present with recurrent sinopulmonary infections, severe cutaneous viral infections, food allergies and elevated serum IgE. Although patients may present with a spectrum of disease-related symptoms, molecular mechanisms explaining phenotypic variability in patients are poorly defined. Here we characterized a novel compound heterozygous mutation in DOCK8 in a patient diagnosed with primary combined immunodeficiency which was not typical of classical DOCK8 deficiency. In contrast to previously identified mutations in DOCK8 which result in complete loss of function, the newly identified single nucleotide insertion results in expression of a truncated DOCK8 protein. Functional evaluation of the truncated DOCK8 protein revealed its hypomorphic function. In addition we found somatic reversion of DOCK8 predominantly in T cells. The combination of somatic reversion and hypomorphic DOCK8 function explains the milder and atypical phenotype of the patient and further broadens the spectrum of DOCK8-associated disease.

disease-related symptoms, molecular mechanisms explaining phenotypic variability in patients are poorly defined. Here we characterized a novel compound heterozygous mutation in DOCK8 in a patient diagnosed with primary combined immunodeficiency which was not typical of classical DOCK8 deficiency. In contrast to previously identified mutations in DOCK8 which result in complete loss of function, the newly identified single nucleotide insertion results in expression of a truncated DOCK8 protein. Functional evaluation of the truncated DOCK8 protein revealed its hypomorphic function. In addition we found somatic reversion of DOCK8 predominantly in T cells. The combination of somatic reversion and hypomorphic DOCK8 function explains the milder and atypical phenotype of the patient and further broadens the spectrum of DOCK8-associated disease.

Introduction
Bi-allelic loss-of-function mutations in the guanine-nucleotide exchange factor dedicator of cytokinesis 8 (DOCK8) cause autosomal recessive hyper-IgE syndrome. The vast majority of DOCK8-deficient patients present with combined immunodeficiency characterized by recurrent sino-pulmonary and/or gastrointestinal infections, severe cutaneous viral infections, severe atopy, eosinophilia and massively elevated serum IgE levels. Patients also have a predisposition to cancer. (1,2) Recent studies have highlighted the phenotypic variability of patients suffering from DOCK8-deficiency. (3,4) Patients with susceptibility to infection but less severe allergic disease were identified to carry a functional wild-type DOCK8 allele in lymphocyte subpopulations due to somatic reversion of the mutated DOCK8 alleles. (3) Here we report for the first time a patient with a hypomorphic mutation in DOCK8 presenting with recurrent bacterial infections, low serum IgM and IgG, CD4 lymphopenia and severely impaired vaccination responses, but without severe viral infections and severe atopy.

Methods
Detailed information can be found in the Supplementary Data.

Case presentation
The female patient is the only child of non-consanguineous, healthy parents. She presented aged eight with a two-year history of recurrent bacterial chest infections and radiological signs of early bronchiectasis. The patient also had a long-standing history of mild eczema and asthma requiring treatment with inhaled corticosteroids and betaagonists. All routine childhood immunizations were received uneventfully. Immunological evaluation (Table I)  Lymphocyte subset analysis demonstrated low CD4 + T cell numbers and low frequencies of CD27 + effector B cells (Table I). Following the failure of antibiotic prophylaxis alone to reduce the infection burden, immunoglobulin replacement therapy was commenced with a good clinical response. Sequence analysis of recombination-activating gene (RAG) 1, RAG2, and DNA cross-link repair 1C (DCLRE1C encoding Artemis) did not reveal any mutations. Therefore the patient was given a diagnosis of undefined primary combined immunodeficiency.

Results and Discussion
To identify the underlying disease cause, we undertook whole exome sequencing (WES) on the patient and both parents. A novel heterozygous frameshift variant was detected in DOCK8 in the patient and her mother. Sanger sequencing confirmed a singlenucleotide duplication [c.6019dupT (p.Tyr2007Leufs*12)] within the conserved DOCK homology region 2 (DHR2) domain of DOCK8, leading to a frameshift and premature stopcodon ( Figure 1, A and C, and Supplementary Table 1]. As autosomal recessive mutations in DOCK8 cause combined immunodeficiency, we screened for further variants in DOCK8. The deletion in DOCK8 is predicted to result in absence of any protein expression since the deletion includes the start codon. The frameshift mutation is predicted to result in the production of a truncated protein lacking 81 amino acids (~11kDa). Indeed, patient EBV cells expressed low amounts of a truncated DOCK8 protein, but not the full-length protein ( Figure 1D). We hypothesized that this truncated DOCK8 protein has hypomorphic function accounting for the milder clinical presentation of our patient.
Previous studies of DOCK8-deficient patients report impaired T cell proliferation. (1,2) At the age of ten years, both CD4 + and CD8 + patient T cells did not proliferate in response to mitogen (PHA) stimulation (Figure 2A), consistent with an inability of the truncated DOCK8 protein to transmit proliferative signals. Interestingly, when studying T cell proliferation at the time of WES (four years later), proliferation of both CD4 + and CD8 + patient T cells was present, although reduced compared to a healthy control ( Figure 2B). We however there is no data available on the development of malignancies post HSCT. In our patient, expression of truncated, partially functional DOCK8 in combination with somatic reversion in T cells is sufficient to maintain antiviral immunity, as shown by the absence of severe viral infections. Therefore, the risk versus benefit of HSCT is unclear in patients with less severe disease and demands careful consideration.

Conclusion
Here we reported a patient with atypical DOCK8 deficiency characterized by a much milder phenotype of the immunodeficiency compared to classical DOCK8 deficiency which further broadens the spectrum of DOCK8 associated diseases. As suggested by normal patient EBV B cell migration and initially absent T cell proliferation in addition to somatic reversion of DOCK8 predominantly in T cells, this relatively mild phenotype is the result of hypomorphic DOCK8 function and somatic reversion. [b] Serum IgE was measured after identification of the DOCK8 mutation on serum samples frozen before start of immunoglobulin replacement therapy.

Study approval
Anticoagulated peripheral blood samples were obtained from the patient, her parents and healthy individuals after the provision of informed, written consent.
The patient and parents gave written consent (12/SC/0044) and the studies were performed according to the Declaration of Helsinki.
The parent-child trio were consented for exome sequencing; however, their consent was restricted to genes related to their condition and not for whole exome sequence (WES) analysis. Consequently, the patients have not consented for all data generated by WES or array CGH to be publically available. Therefore, we are not able to provide WES data beyond the immune-related targeted analysis.

DNA isolation
DNA extraction was performed using the FlexiGene DNA kit (Qiagen) according to the manufacturer's instructions.

Flow cytometry
Immunophenotypes were determined using whole blood and standard protocols with

Peak height quantification in sequencing traces
To determine the percentage of wild-type and mutated DOCK8 transcripts in respective cDNA samples, Sanger sequencing traces were subjected to quantitative analysis using the ab1 Peak Reporter tool (http://apps.lifetechnologies.com/ab1peakreporter). Ab1 sequencing files were converted into numerical peak height data of base traces by applying the 'ratio of maximal signals in a 7-scan window' calculation provided by the software. wild-type or non-mutated bases are called at a rate of about 5% which gives a total sequencing base call background of 10%.

Pyrosequencing
The c.6019dupT variant was detected in the separated cell population by pyrosequencing.
PCR templates for pyrosequencing were amplified from cDNA (10 ng) using the biotinylated forward primer 5' TCCTGCTGATCCAAAACT 3' and reverse primer 5' GCCATTTTCCTTCTTACC 3'. Following PCR amplification, the biotinylated PCR products were placed in 96-well plates and bound to streptavidin-coated sepharose beads (GE Healthcare, Piscataway, NJ, USA).
The PCR products were denatured, and the non-biotinylated fragments were washed from the beads using the Pyromark Q96 Vacuum Workstation (Qiagen). The beads were then resuspended in annealing buffer (40µL) containing 0.4 pmol of the sequencing primer (5' TCAACTTGTTGTGATGTCG 3'). Pyrosequencing was performed using the Pyro Gold Q96 reagents (Qiagen), using dispensations based on the target sequence with the Pyromark Q96 system. The PCR templates and pyrosequencing reactions were performed in triplicate.
Raw data files were imported into excel for ratio calculations. "T" nucleotide phosphorescence signal ratios were calculated by dividing the phosphorescence signal of

Statistics
Analyses were performed with PRISM software (GraphPad Software, Inc.). Statistical hypotheses were tested using the unpaired 2-tailed t test. Differences were considered significant if the P value was less than 0.05.