A novel zeta-associated protein 70 homozygous mutation causing combined immunodeficiency presenting as neonatal autoimmune hemolytic anemia.

Biallelic mutations in the zeta-associated protein 70 (ZAP70) gene cause combined immunodeficiency (CID). Neonatal screening for severe CID in Israel is implemented since 2015. We report on clinical, flow cytometry, and genetic data of an unusual ZAP70 deficiency patient. A 10-week-old Bedouin female presented with severe autoimmune hemolytic anemia. Cytomegalovirus (CMV) negative packed cell therapy was given without improvement; indexes of hemolysis worsened. At this time, thrombocytopenia was noted. The patient was treated with single dose of 1 g/kg intravenous immunoglobulin with rapid resolution of hemolysis. Serum immunoglobulin concentrations were normal; flow cytometry revealed severe CD8 lymphocytopenia. Lymphocyte proliferation test demonstrated reduced response to concanavalin A and phytohemagglutinin. Gated T cells were negative for intracellular ZAP70. A genetic analysis revealed a missense homozygous c.1388C > T (p.A463V) mutation, confirming the diagnosis of ZAP70 deficiency. She later on developed urinary tract infection due to ESBL producing E. coli treated with amikacin and severe CMV infection that partially responded to ganciclovir therapy and at 7 months of age, she successfully underwent allogeneic hematopoietic stem cell transplantation. Neonatal screening by T cell receptor excision circles (TRECs) for SCID was normal, yet very low TRECs were recorded at the time of CID diagnosis. Normal neonatal screening for SCID does not rule out the diagnosis of CID due to ZAP70 deficiency. This type of CID can present with autoimmunity as the sole initial manifestation of the disease.

Clinical, Immunological, and Genetic Features in 49 Patients With ZAP-70 Deficiency: A Systematic Review.

Background: Zeta-Chain Associated Protein Kinase 70 kDa (ZAP-70) deficiency is a rare combined immunodeficiency (CID) caused by recessive homozygous/compound heterozygous loss-of-function mutations in the ZAP70 gene. Patients with ZAP-70 deficiency present with a variety of clinical manifestations, particularly recurrent respiratory infections and cutaneous involvements. Therefore, a systematic review of ZAP-70 deficiency is helpful to achieve a comprehensive view of this disease. Methods: We searched PubMed, Web of Science, and Scopus databases for all reported ZAP-70 deficient patients and screened against the described eligibility criteria. A total of 49 ZAP-70 deficient patients were identified from 33 articles. For all patients, demographic, clinical, immunologic, and molecular data were collected. Results: ZAP-70 deficient patients have been reported in the literature with a broad spectrum of clinical manifestations including recurrent respiratory infections (81.8%), cutaneous involvement (57.9%), lymphoproliferation (32.4%), autoimmunity (19.4%), enteropathy (18.4%), and increased risk of malignancies (8.1%). The predominant immunologic phenotype was low CD8+ T cell counts (97.9%). Immunologic profiling showed defective antibody production (57%) and decreased lymphocyte responses to mitogenic stimuli such as phytohemagglutinin (PHA) (95%). Mutations of the ZAP70 gene were located throughout the gene, and there was no mutational hotspot. However, most of the mutations were located in the kinase domain. Hematopoietic stem cell transplantation (HSCT) was applied as the major curative treatment in 25 (51%) of the patients, 18 patients survived transplantation, while two patients died and three required a second transplant in order to achieve full remission. Conclusion: Newborns with consanguineous parents, positive family history of CID, and low CD8+ T cell counts should be considered for ZAP-70 deficiency screening, since early diagnosis and treatment with HSCT can lead to a more favorable outcome. Based on the current evidence, there is no genotype-phenotype correlation in ZAP-70 deficient patients.

ZAP70 deficiency promotes reverse cholesterol transport through MAPK/ERK pathway in Jurkat cell.

BACKGROUND: Lots of studies have demonstrated that immune cells could regulate reverse cholesterol transport (RCT). However, neither T cell receptor (TCR) signalling nor Zeta-chain associated protein 70 (ZAP70) have been demonstrated to be associated with RCT. To investigate this association, we used a ZAP70-deficient Jurkat-derived mutant, P116 cell line, to detect the effect of ZAP70 on RCT and inflammatory response. ZAP70 deficiency improved cholesterol efflux capacity by 14%. Meanwhile, mRNA and proteins expression of RCT regulatory proteins such as ABCA1, ABCG1 and SR-BI were increased in P116 cells. ZAP70-deficiency had no influence on LXR-α and PPAR-γ. Regarding the inflammatory response, the mRNA expression and secretion of pro-atherosclerotic cytokines, TNF-α, IFN-γ, IL-2 and IL-6, were significantly decreased in the ZAP70-deficient cell line. Activation of MAP kinases cascades, as determined by of ERK, JNK and p38 MAPK phosphorylation, were found to be inhibited in the absence of ZAP70. Specific inhibition of ERK, JNK and p38 MAPK activity was also found to decreased TNF-α, IFN-γ, and IL-6 secretion. However, only the ERK inhibition was observed to reduce IL-2 secretion, improve cholesterol efflux capacity and increase expression of ABCA1, ABCG1 and SR-BI without increasing LXR-α and PPAR-γ. Using ChIP assay to detect the binding of LXR-α to LXRE, which promotes the expression of ABCG1, we found that inhibiting ERK improved binding without increasing LXR-α levels. Thus, we speculate that ZAP70-deficiency may improve RCT and decrease the inflammatory response of T cells. Furthermore, these effects are probably achieved via ERK signalling pathway.

Correction of T cell deficiency in ZAP-70 knock-out mice by simple intraperitoneal adoptive transfer of thymocytes.

The tyrosine kinase zeta chain-associated protein of 70 kDa (ZAP-70) plays a key role in T cell development and signalling. In the absence of ZAP-70, T cell development is arrested in the CD4+ CD8+ double-positive stage, thus ZAP-70 homozygous knockout (ZAP-70-/- ) mice have no mature T cells in their peripheral lymphoid organs and blood, causing severe immunodeficiency. We investigated the early kinetics and long-term effects of wild-type thymocyte transfer on T cell repopulation in ZAP-70-/- mice. We used a single intraperitoneal (i.p.) injection to deliver donor thymocytes to the recipients. Here, we show that after i.p. injection donor thymocytes leave the peritoneum through milky spots in the omentum and home to the thymus, where donor-originated CD4- CD8- double-negative thymocytes most probably restore T cell development and the disrupted thymic architecture. Subsequently, newly developed, donor-originated, single-positive αß T cells appear in peripheral lymphoid organs, where they form organized T cell zones. The established chimerism was found to be stable, as donor-originated cells were present in transferred ZAP-70-/- mice as late as 8 months after i.p. injection. We demonstrate that a simple i.p. injection of ZAP-70+/+ thymocytes is a feasible method for the long-term reconstitution of T cell development in ZAP-70-deficient mice.

Novel compound heterozygous mutations in ZAP70 in a Chinese patient with leaky severe combined immunodeficiency disorder.

In humans, the complete lack of tyrosine kinase ZAP70 function results in combined immunodeficiency (CID), with abnormal thymic development and defective T cell receptor (TCR) signaling of peripheral T cells, characterized by the selective absence of CD8+ T cells. So far, 15 unique ZAP70 mutations have been identified in approximately 20 patients with CID, with variable clinical presentations. Herein, we report the first case from China of novel compound heterozygous mutations in ZAP70 (c.598-599delCT, p.L200fsX28; c.847 C>T, R283H). The patient suffered from early-onset and recurrent infections, but showed normal growth and development without signs of failure to thrive, thus presenting as leaky SCID. The patient also had clinical manifestations of autoimmunity, such as eczematous skin lesion, inflammatory bowel disease (IBD), and intractable diarrhea, suggesting compromised T cell tolerogenic functions. Residual ZAP70 expression was identified. Immunological analysis revealed the selective absence of CD8+ T cells in the periphery and the presence of CD4+ T cells that failed to respond to phytohemagglutinin. Stimulation with lectin from pokeweed mitogen also failed to stimulate B cell proliferation in the patient. The frequency of Tfhs and Tregs in the patient was lower compared with the normal reference. Compared with the age-matched healthy control, the level of IL-17 was higher and the levels of IFN-γ, IL-4, and IL-21 were lower. Infants with selected CD8 deficiency and severe autoimmune disorders or exaggerated inflammation should be screened for ZAP70 deficiency.

Long-Term Outcomes of Hematopoietic Stem Cell Transplantation for ZAP70 Deficiency.

ZAP70 deficiency is a rare T + B + NK+ combined immunodeficiency with limited outcome data to help guide decisions around hematopoietic stem cell transplant (HSCT). We sought to understand the long-term clinical and immunologic outcomes of both conditioned and unconditioned HSCT for ZAP70 deficiency following transplant from a variety of graft sources. We performed a retrospective, single center review of all cases of HSCT for genetically confirmed ZAP70 deficiency since 1992. At a median of 13.5-year post-HSCT, 8/8 (100 %) patients are alive. Three received unconditioned bone marrow transplants from human leukocyte antigen (HLA)-matched siblings and achieved stable mixed donor-recipient T cell chimerism but low B cell (4-9 %) and absent to near-absent myeloid donor engraftment. Despite this, all three have normal immunoglobulin levels, have developed specific protective antibody responses to post-HSCT vaccinations, and have discontinued immunoglobulin replacement. Five patients received myeloablative conditioning (three T cell-depleted haploidentical and two unrelated cord blood) and have full donor chimerism for T and B cells and myeloid lineages. One patient experienced primary graft failure after serotherapy only. CD8 T cell count is normal in 5/8, high in 1/8, and low in 2/8. Infectious complications in 5/5 and autoimmune thrombocytopenia in one patient resolved post-HSCT. Mitogen proliferation to phytohemagglutinin was normal after HSCT in 8/8 patients. In total, seven have discontinued immunoglobulin replacement. In conclusion, HSCT using a variety of graft sources and approaches, including unconditioned matched sibling donor transplant, is a life-saving therapy for ZAP70 deficiency, providing excellent long-term immune function and resolution of clinical problems.

A novel human autoimmune syndrome caused by combined hypomorphic and activating mutations in ZAP-70.

A brother and sister developed a previously undescribed constellation of autoimmune manifestations within their first year of life, with uncontrollable bullous pemphigoid, colitis, and proteinuria. The boy had hemophilia due to a factor VIII autoantibody and nephrotic syndrome. Both children required allogeneic hematopoietic cell transplantation (HCT), which resolved their autoimmunity. The early onset, severity, and distinctive findings suggested a single gene disorder underlying the phenotype. Whole-exome sequencing performed on five family members revealed the affected siblings to be compound heterozygous for two unique missense mutations in the 70-kD T cell receptor ζ-chain associated protein (ZAP-70). Healthy relatives were heterozygous mutation carriers. Although pre-HCT patient T cells were not available, mutation effects were determined using transfected cell lines and peripheral blood from carriers and controls. Mutation R192W in the C-SH2 domain exhibited reduced binding to phosphorylated ζ-chain, whereas mutation R360P in the N lobe of the catalytic domain disrupted an autoinhibitory mechanism, producing a weakly hyperactive ZAP-70 protein. Although human ZAP-70 deficiency can have dysregulated T cells, and autoreactive mouse thymocytes with weak Zap-70 signaling can escape tolerance, our patients' combination of hypomorphic and activating mutations suggested a new disease mechanism and produced previously undescribed human ZAP-70-associated autoimmune disease.

SYK expression endows human ZAP70-deficient CD8 T cells with residual TCR signaling.

Autosomal recessive human ZAP70 deficiency is a rare cause of combined immunodeficiency (CID) characterized by defective CD4 T cells and profound CD8 T cell lymphopenia. Herein, we report two novel patients that extend the molecular genetics, the clinical and functional phenotypes associated with the ZAP70 deficiency. The patients presented as infant-onset CID with severe infections caused by varicella zoster virus and live vaccines. Retrospective TCR excision circle newborn screening was normal in both patients. One patient carried a novel non-sense mutation (p.A495fsX75); the other a previously described misense mutation (p.A507V). In contrast to CD4 T cells, the majority of the few CD8 T cells showed expression of the ZAP70-related tyrosine kinase SYK that correlated with residual TCR signaling including calcium flux and degranulation. Our findings highlight the differential requirements of ZAP70 and SYK during thymic development, peripheral homeostasis as well as effector functions of CD4 and CD8 T cells.

Silent brain infarcts in two patients with zeta chain-associated protein 70 kDa (ZAP70) deficiency.

Zeta-chain associated protein 70 kDa deficiency (ZAP70) is a form of severe combined immunodeficiency (SCID). It is caused by defects in the signaling pathways associated with T-lymphocyte activation. ZAP70 deficiency is characterized by a marked reduction in peripheral CD8+ T-cells. In this report, we described two patients with ZAP70 deficiency who presented with recurrent infections, lung tuberculosis (TBC), congenital nephrotic syndrome (CNS), and silent brain infarcts (SBIs) as a common feature. The first patient initially presented with recurrent infections and TBC as in a classic SCID patient. At the age of 4, he was interned with febrile seizure. Cranial magnetic resonance imaging (MRI) showed SBIs. The second patient, an 8-month-old boy, presented with congenital nephrotic syndrome caused by cytomegalovirus (CMV) and he had also SBIs.

Insight into the therapeutic aspects of 'Zeta-Chain Associated Protein Kinase 70 kDa' inhibitors: a review.

Zeta-Chain Associated Protein Kinase 70 kDa (ZAP-70), a member of Syk family (non-receptor protein tyrosine kinase family), has an imperative function in the immune cell signaling in T cells. Its role in T-cell development has been established by the severe combined immune deficiency syndrome in ZAP-70 deficient humans. Moreover, defects in T-cell activation and downstream signaling events were observed in T-cells that lack ZAP-70. Thus, the crucial role of ZAP-70 in the development and activation of T-cell and its predominant expression in T-cells make it a logical target for the treatment of pathological conditions related to abnormal T-cell responses. The present review article portrays the domain structure of ZAP-70 along with its implication in T-cell signaling. Additionally, varied ZAP-70 inhibitors published in different patents and papers have also been reviewed.

A novel Zap70 mutation with reduced protein stability demonstrates the rate-limiting threshold for Zap70 in T-cell receptor signalling.

Loss of ζ-associated protein 70 (Zap70) results in severe immunodeficiency in humans and mice because of the critical role of Zap70 in T-cell receptor (TCR) signalling. Here we describe a novel mouse strain generated by N-ethyl-N-nitrosourea mutagenesis, with the reduced protein stability (rps) mutation in Zap70. The A243V rps mutation resulted in decreased Zap70 protein and a reduced duration of TCR-induced calcium responses, equivalent to that induced by a 50% decrease in catalytically active Zap70. The reduction of signalling through Zap70 was insufficient to substantially perturb thymic differentiation of conventional CD4 and CD8 T cells, although Foxp3(+) regulatory T cells demonstrated altered thymic production and peripheral homeostasis. Despite the mild phenotype, the Zap70(A243V) variant lies just above the functional threshold for TCR signalling competence, as T cells relying on only a single copy of the Zap70(rps) allele for TCR signalling demonstrated no intracellular calcium response to TCR stimulation. This addition to the Zap70 allelic series indicates that a rate-limiting threshold for Zap70 protein levels exists at which signalling capacity switches from nearly intact to effectively null.

Intrathymic progenitor cell transplantation across histocompatibility barriers results in the persistence of early thymic progenitors and T-cell differentiation.

Donor hematopoietic stem cells (HSCs) can correct T-cell deficiencies in patients with severe combined immunodeficiency by replacing resident thymus cells. However, as those progenitors that naturally migrate to the thymus are not capable of supporting long-term thymopoiesis, a successful transplant is thought to require the ongoing migration of donor progenitors. We previously showed that the forced intrathymic administration of histocompatible HSCs can sustain long-term thymopoiesis in ZAP-70-immunodeficient mice. However, it is not known whether T-cell reconstitution across histocompatibility barriers is modulated by intrathymic vs intravenous administration of HSCs. In the absence of conditioning, long-term thymopoiesis by semiallogeneic progenitors was detected in mice transplanted via the intrathymic, but not the intravenous, route. In intrathymic-transplanted mice, ongoing thymopoiesis was associated with a 10-fold higher level of early thymic progenitors (ETPs). The enhanced reconstitution capacity of these intrathymic-derived ETPs was corroborated by their significantly augmented myeloid lineage potential compared with endogenous ETPs. Notably, though, myeloablative conditioning resulted in a reduced expansion of intrathymic-administered donor ETPs. Thus, in the absence of conditioning, the forced thymic entry of HSCs results in a sustained T-cell development across histocompatibility barriers, highlighting the capacity of the thymus to support cells with long-term renewal potential.

Identification of a novel mutation in ZAP70 and prenatal diagnosis in a Turkish family with severe combined immunodeficiency disorder.

Protein tyrosine kinases (PTKs) play an important role in T cell development and activation. In vitro and in vivo defects, resulting in variable deficiencies in thymic development and in T cell antigen receptor (TCR) signal transduction, in PTKs have been shown. ZAP70, one of those PTKs, is a 70-kDa tyrosine phosphoprotein and associates with the ζ chain and undergoes tyrosine phosphorylation following TCR stimulation. It is expressed in T and natural killer (NK) cells. Several mutations were shown to lead to an autosomal recessive form of severe combined immunodeficiency disease (SCID). Here, we present a family with a novel mutation in ZAP70. The proband, the second child of the first cousin parents of Turkish origin, was diagnosed with SCID having R514C mutation on homozygous state. She had decreased CD8(+) T and natural killer cells, normal CD4(+) T cells, high serum Ig E level, perivascular dermatitis and ichthyosis. This article presents clinical features of a novel mutation on ZAP70 and the first prenatal molecular diagnosis of ZAP70 deficiency. Different mutations in ZAP70 and related phenotypes reported in the literature are also discussed.

Characterization of ζ-associated protein, 70 kd (ZAP70)-deficient human lymphocytes.

BACKGROUND: ζ-associated protein, 70 kd (ZAP70), deficiency in human subjects results in a combined immunodeficiency characterized by normal numbers of circulating CD4 T cells and CD8 lymphocytopenia. Patients who live beyond infancy can also experience autoimmune manifestations. OBJECTIVES: We sought to further characterize the nature of the T-cell populations found in ZAP70-deficient patients and explored the mechanisms that might predispose them to autoimmunity. METHODS: T-cell development was assessed by examining T-cell receptor (TCR) gene rearrangements and thymopoiesis by measuring TCR exclusion circle levels. TCR repertoire on CD4 and CD8 T-cell populations was assessed by means of flow cytometry. T-cell gene expression patterns were examined by means of exonic microarray analysis and apoptotic responses by means of Annexin V binding and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling. RESULTS: Cells displaying recombination events from all stages of TCR gene rearrangement were present in the peripheral blood of ZAP70-deficient patients; however, the late TCRD-deleting rearrangement was significantly reduced. TCR exclusion circle levels were also found to be low. Surprisingly, all Vß families were detected in both CD4(+) and CD8(+) circulating T cells. Several Vß families were significantly overrepresented, which is reminiscent of autoimmune disorders. Levels of mRNA for cytotoxic T lymphocyte-associated antigen 4, TGF-ß, and IL-10 were found to be low, a signature of autoimmunity. Finally, Fas-mediated CD4 T-cell apoptosis was found to be reduced in vitro, and staining of thymus biopsy specimens revealed reduced thymocyte apoptosis. CONCLUSION: We show that in the absence of ZAP70, thymopoiesis is altered and differentiation to double-positive cells is hampered. Circulating T cells appear poorly regulated, do not differentiate into T(H)2 T cells, lack a number of inhibitory growth controls, and display reduced apoptosis, all predisposing patients to exaggerated inflammation and autoimmunity.