Clinical heterogeneity can hamper the diagnosis of patients with ZAP70 deficiency
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- Turul, T., Tezcan, I., Artac, H. et al. Eur J Pediatr (2009) 168: 87. doi:10.1007/s00431-008-0718-x
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One of the severe combined immunodeficiencies (SCIDs), which is caused by a genetic defect in the signal transduction pathways involved in T-cell activation, is the ZAP70 deficiency. Mutations in ZAP70 lead to both abnormal thymic development and defective T-cell receptor (TCR) signaling of peripheral T-cells. In contrast to the lymphopenia in most SCID patients, ZAP70-deficient patients have lymphocytosis, despite the selective absence of CD8+ T-cells. The clinical presentation is usually before 2 years of age with typical findings of SCID. Here, we present three new ZAP70-deficient patients who vary in their clinical presentation. One of the ZAP70-deficient patients presented as a classical SCID, the second patient presented as a healthy looking wheezy infant, whereas the third patient came to clinical attention for the eczematous skin lesions simulating atopic dermatitis with eosinophilia and elevated immunoglobulin E (IgE), similar to the Omenn syndrome. This study illustrates that awareness of the clinical heterogeneity of ZAP70 deficiency is of utmost importance for making a fast and accurate diagnosis, which will contribute to the improvement of the adequate treatment of this severe immunodeficiency.
KeywordsSevere combined immunodeficiencyZAP70T-cell receptor signalingT-cells
Severe combined immunodeficiency
ζ-chain-associated protein kinase of 70 kDa
Hematopoietic stem cell transplantation
Materials and methods
Flow cytometric immunophenotyping
The cell surface expression of CD3, CD4, and CD8 was assayed on whole-blood samples by flow cytometry using a FACSCalibur (BD Biosciences, San Jose, CA). Patient material was obtained according to the medical ethics guidelines of Hacettepe University Children’s Hospital, the Meram Medical Faculty of the Selçuk University, and Erasmus MC. Antibodies were obtained from BD Biosciences. ZAP70 surface expression was determined using the monoclonal antibody 1E7.2 (eBioscience, San Diego, CA).
Molecular analysis of the ZAP70 gene
Polymerase chain reaction (PCR) was performed to amplify the ZAP70 exons (MIM 176947; NCBI L05148); the primer sequences are available upon request. In each 25 μl of PCR reaction, 5 pmol of 5′ and 3′ oligonucleotides, 25 ng DNA, and 0.5 u AmpliTaqGold™ polymerase template and buffer PCR System (Applied Biosystems, Roche Molecular Systems Inc., Branchburg, NJ) were used. PCR products were used directly for sequencing with BigDye Terminator mix (Applied Biosystems) and 3.3 pmol 5′ or 3′ primers. All sequencing was performed on an ABI Prism 3100 fluorescent sequencer (Applied Biosystems).
Ca2+ flux assay
Thawed peripheral blood mononuclear cells (1 × 106) were incubated with 6 μg/ml of calcium-sensitive fluorescent dye Indo-1 (Molecular Probes) to evaluate the calcium fluxes after stimulation. The levels of free intracellular calcium were measured in CD4+ T-cells with a FACSVantage sorter (BD Biosciences) before and after stimulation with 12.5 μg/ml of functional-grade purified OKT3 CD3 antibody (eBioscience). Subsequently, 2 μg/ml of ionomycin (Molecular Probes) was added to control for the intracellular loading of Indo-1, as previously described .
Clinical characteristics and immunological parameters of the three patients with ZAP70 deficiency from unrelated families of Turkish origin
Age of onset (months)
Age of diagnosis (months)
A 3-month-old male sibling died of LRTI
A 2-month-old female cousin died of unknown reason
Recurrent LRTI (wheezing)
Tonsillary tissue (+)
Disseminated BCG infection
Perianal and perineal ulcers
Tonsillary tissue (+)
Tonsillary tissue (−)
Bilateral inguinal lymphadenopathy
Anti B 1/8 (+)
Anti A (−), Anti B (−)
Anti A 1/4
(−) (after 2nd dose)
Anti polio titers
In vitro lymphocyte proliferation test/ (SI)**
Anti CD3: 10%
Well with IVIG and prophylactic antibiotics, HSCT has not been performed yet
Died of multiorgan failure due to secondary hemophagocytic syndrome developed during follow-up
Still under antituberculous therapy with clinical improvement, waiting for HSCT
Patient 2 (ZAP70–2) had experienced recurrent gastroenteritis, lower respiratory tract infections, and oral moniliasis accompanied by failure to thrive, frequently requiring hospitalization since 3 months of age (Table 1).
On admission at the age of 13 months, both her weight and height were below the third percentile. She had visible tonsils and palpable lymph nodes. A scar tissue on the left deltoid formed following a BCG vaccination was visible. There were multiple ulcerative perianal and genital lesions. Her family tree is shown in Fig. 2.
Bone marrow aspiration performed during follow-up because of the development of hepatomegaly and bicytopenia showed hemophagocytosis. Viral serology, PCR studies, and bacterial cultures could not define an etiologic agent. The patient was considered to have secondary hemophagocytic syndrome and was put on immunosuppressive therapy. The CD8+ T-cell count was strongly reduced (Table 1). The patient’s clinical status deteriorated progressively and she died of multiorgan failure one month before HLA haploidentical transplantation could be done.
Physical examination on admission at 11 months of age showed that her weight, height, and head circumference were below the third percentile. She had widespread xerosis and ichthyosis, together with subcutaneous nodules. No tonsillary tissue was visible. There were bilateral fine rales on auscultation of the lungs and she had hepatomegaly and generalized lymphadenopathies of 1–2 cm in size. Computed tomography of the thorax revealed the absence of thymus, pneumonic consolidation of the right upper lobe, and a lymphadenopathy located in the azygoesophageal recess. In Fig. 2, her family tree is shown. The laboratory findings are shown in Table 1. Additionally, chimerism analysis ruled out the presence of maternal cells.
With the diagnosis of SCID, the patient was put on intravenous immunoglobulin (IVIG), antibacterial, antifungal, and antiprotozoal prophylaxis. During follow-up, splenomegaly and several new subcutaneous nodules developed located on the tibia, gluteal region, anterior chest wall, and the arm, including the left deltoid region where the BCG vaccine scar was located. Two separate biopsy specimens obtained from two of the nodules revealed granulomatous inflammation without caseification. No acid-resistant basil (ARB) was visible and the cultures were negative for Mycobacterium species. Since both the nodules and splenomegaly regressed by a multidrug regimen (isoniazide+rifampisin+pyrazinamide+streptomycin), she was considered to have mycobacteria (probably BCG) infection. She developed generalized erythrodermia with associative eosinophilia and an elevation in IgE levels presenting as Omenn phenotype. The patient is currently being prepared for an HLA identical hematopoietic stem cell transplantation (HSCT) from one of the siblings.
Flow cytometric analysis of the peripheral blood
Molecular analysis of the ZAP70 gene
Calcium flux study
To assess directly the signaling competence of CD4+ cells, we measured the mobilization of intracellular Ca2+ in the thawed PBMC of patients 1 and 3. OKT3 antibody was used to stimulate the classical TCR signaling pathway, leading to the intracellular mobilization of Ca2+. This antibody induces TCR cross-linking, but failed to mobilize significant amounts of intracellular Ca2+ from any of the patient’s T-cells, whereas biochemical stimulation with ionomycin could elaborate Ca2+ flux in all of the patient cells (data not shown).
Here, we present three patients from unrelated families with severe reduction in CD8+ T-cells and the presence of normal counts of nonfunctional CD4+ T-cells. These cells are defective in TCR activation, as demonstrated both by the absence of proliferative response to mitogens and impaired Ca2+ flux to CD3 (OKT3) stimulation. All three patients had missense mutations in the ZAP70 gene, leading to the absence of protein expression.
Although all three mutations lead to the total absence of ZAP70 protein expression, the clinical pictures of the patients showed a striking heterogeneity, discarding a straightforward genotype–phenotype relationship. The age of onset of the disease of the three patients was before six months of age, which is quite typical for SCID. In the literature, the age of onset is variable, however, being always before 12 months of age. Patients 2 and 3 had a quite severe phenotypes not compatible with life and typical for classical SCID and necessitated HSCT for survival. In contrast, patient 1 had a very mild clinical phenotype with recurrent attacks of self-limiting lower respiratory tract infections without failure to thrive. In the literature, only one patient has been reported as looking healthy with eczematous skin lesions and without any other associating symptoms .
Patient 3 had generalized progressive erythematous skin findings associated with eosinophilia and the elevation of IgE. The patient developed the clinical picture of Omenn syndrome. The patient’s T-cells were autologous, so not of maternal origin. Katamura et al. reported two similar cases who had CD4+ T-cells infiltrating the skin, which were activated and had a memory phenotype . These cells proliferated in vitro to a higher concentration of PHA and seemed to have elevated Syk expression levels. They suggested that Syk can compensate for ZAP70 during early T-cell development from double-positive (CD4+CD8+) thymocytes to single-positive (CD4+ or CD8+) thymocytes. This was apparently sufficient for reaching the threshold that leads to the CD4 but not to the CD8 lineage selection, but was insufficient for eliminating self-reactive T-cells, which later infiltrated the skin. A second explanation for skin changes in ZAP70-deficient patients came from Toyabe et al., who found that T-cells in these patients had the capacity to induce antigen-specific IgE production from B cells in response to TCR stimulation .
The humoral immunity is also variably affected among the ZAP70 patients, some having severe hypogammaglobulinemia requiring IVIG, some having normal levels of immunoglobulins, as in patient 2, or even hypergammaglobulinemia [1, 6, 8, 17]. In sporadic cases, specific antibody production can be preserved [17, 19]. Only in patient 1 was the protein antigen response selectively preserved for hepatitis B, whereas this was not the case in the two other patients.
In conclusion, this study illustrates the clinical heterogeneity of patients with a ZAP70 deficiency. These patients can present as classical SCID, but, sometimes, they present as healthy looking wheezy infants or can come to clinical attention for the eczematous skin lesions simulating atopic dermatitis with eosinophilia and elevated IgE similar to the Omenn syndrome. Awareness of the clinical heterogeneity of ZAP70 deficiency is of utmost importance for making a fast and accurate diagnosis, which will contribute to the improvement of the adequate treatment of this severe immunodeficiency.
The authors thank Mrs. Marieke Comans-Bitter for her preparation of the figures. This study has been supported by an ESID Scholarship of the European Society for Immunodeficiencies (TT) and by a Health Care Innovation grant (Zorgvernieuwingsproject) (JJMvD, MvdB).
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