Morpholino-based correction of hypomorphic ZAP70 mutation in an adult with combined immunodeficiency
Authors: Christina Gavino, Marija Landekic, Jibin Zeng, Ning Wu, Sungmi Jung, Ming-Chao Zhong, Alexis Cohen-Blanchet, Mélanie Langelier, Odile Neyret, Duncan Lejtenyi, Claudia Rochefort, Judith Cotton-Montpetit, Christine McCusker, Bruce Mazer, André Veillette, Donald C. Vinh
Journal: Journal of Allergy and Clinical Immunology (2017)

The ζ-associated protein of 70 kDa (ZAP-70) is a cytoplasmic tyrosine kinase critical for intracellular signaling downstream of the T-cell receptor (TcR). Biallelic mutations in ZAP70 cause combined immunodeficiency (CID). Classically, ZAP-70 deficiency results from null alleles and presents with failure to thrive and severe/recurrent infections in infancy (ie, severe CID) that is marked by absent circulating CD8+ T cells, quantitatively normal yet dysfunctional CD4+ T cells accounting for hypo-/dysgammaglobulinemia, with quantitatively intact B cells and natural killer cells and requires hematopoietic stem cell transplant for survival beyond early childhood1Chan A.C. Kadlecek T.A. Elder M.E. Filipovich A.H. Kuo W.L. Iwashima M. et al.ZAP-70 deficiency in an autosomal recessive form of severe combined immunodeficiency.Science. 1994; 264: 1599-1601Crossref PubMed Google Scholar, 2Arpaia E. Shahar M. Dadi H. Cohen A. Roifman C.M. Defective T cell receptor signaling and CD8+ thymic selection in humans lacking zap-70 kinase.Cell. 1994; 76: 947-958Abstract Full Text PDF PubMed Scopus (439) Google Scholar, 3Gelfand E.W. Weinberg K. Mazer B.D. Kadlecek T.A. Weiss A. Absence of ZAP-70 prevents signaling through the antigen receptor on peripheral blood T cells but not on thymocytes.J Exp Med. 1995; 182: 1057-1065Crossref PubMed Scopus (84) Google Scholar, 4Elder M.E. Skoda-Smith S. Kadlecek T.A. Wang F. Wu J. Weiss A. Distinct T cell developmental consequences in humans and mice expressing identical mutations in the DLAARN motif of ZAP-70.J Immunol. 2001; 166: 656-661Crossref PubMed Google Scholar, 5Honig M. Schuetz C. Schwarz K. Rojewski M. Jacobsen E. Lahr G. et al.Immunological reconstitution in a patient with ZAP-70 deficiency following transfusion of blood lymphocytes from a previously transplanted sibling without conditioning.Bone Marrow Transplant. 2012; 47: 305-307Crossref PubMed Scopus (8) Google Scholar (see Table E1 in this article's Online Repository at www.jacionline.org). "Leaky" ZAP-70 deficiency has been reported only once: a 9-year-old boy with nonsevere skin and lung infections had a homozygous intronic mutation producing a novel splice acceptor site, resulting in predominant expression of a frame-shifted mutant protein but with residual levels of wild-type (WT) protein.6Picard C. Dogniaux S. Chemin K. Maciorowski Z. Lim A. Mazerolles F. et al.Hypomorphic mutation of ZAP70 in human results in a late onset immunodeficiency and no autoimmunity.Eur J Immunol. 2009; 39: 1966-1976Crossref PubMed Scopus (63) Google Scholar Recently, a sibling pair manifesting only early-onset autoimmune diseases, without opportunistic infections, was shown to be compound heterozygous for a hypoactive allele and a weakly hyperactive allele; the specific combination of these paired alleles was required for disease.7Chan A.Y. Punwani D. Kadlecek T.A. Cowan M.J. Olson J.L. Mathes E.F. et al.A novel human autoimmune syndrome caused by combined hypomorphic and activating mutations in ZAP-70.J Exp Med. 2016; 213: 155-165Crossref PubMed Scopus (20) Google Scholar Collectively, these cases show that residual levels of functional protein regulate the clinical phenotype of ZAP-70 deficiency. It further suggests that, in distinction to null alleles, different therapeutic approaches may be needed for "leaky" mutations that permit survival beyond the typical time frame and for whom hematopoietic stem cell transplant at older age may be associated with excess risk.8Pai S.Y. Logan B.R. Griffith L.M. Buckley R.H. Parrott R.E. Dvorak C.C. et al.Transplantation outcomes for severe combined immunodeficiency, 2000-2009.N Engl J Med. 2014; 371: 434-446Crossref PubMed Scopus (569) Google Scholar We expand the clinical phenotype of hypomorphic ZAP-70 deficiency and provide proof-of-concept that mutation-targeted therapy can improve immune function. We identified a 33-year-old male (P1) born from consanguineous Coptic parents (see Fig E1, A, in this article's Online Repository at www.jacionline.org). An older brother died at 16 months from suspected viral encephalitis; an older sister and parents were well. P1 had a history of failure to thrive, adrenal insufficiency, and inflammatory enterocolitis starting in infancy, recurrent respiratory tract infections starting at 9 months, and intermittent mucocutaneous candidiasis, recurrent herpes simplex virus stomatitis, and varicella-zoster virus infections 3 times. Investigations at the time revealed a visible thymus, normal immunoglobulin isotypes with poor response to vaccines, and persistent CD8+ lymphocytopenia (1% to 3% of total T cells age-adjusted reference range). Beginning at approximately 3 years of age, serial testing demonstrated increasing proportion of CD8+ T cells (8% to 10% of total lymphocytes). Further investigations were suggestive of ZAP-70 deficiency (Fig E1, B and C). However, a reference laboratory reported identifying no mutation in ZAP70. Thus, bone marrow transplantation was not pursued. IgA nephropathy at age 16 years eventually required renal transplant from his mother at age 30 years. The subsequent years were marked by EBV viremia/lymphoproliferative disorder (with recurrence of viremia ∼6 months after completing rituximab), cytomegalovirus viremia (with recurrence following cessation of (val)ganciclovir), polyomaviremia (BK and JC viruses), and epidermodysplasia verruciformis-like lesions (due to human papilloma virus-23) (see Fig E2 in this article's Online Repository at www.jacionline.org), which prompted reevaluation for an underlying immunodeficiency. He is currently aged 35 years, on immunoglobulin replacement for respiratory tract infection prophylaxis, and his viral infections are managed conservatively. Whole-exome sequencing revealed no known etiology for the immunodeficiency, other than a homozygous c.1272C>T variant in ZAP70 (NM_001079; hg19). The germline c.1272C>T variant, while synonymous (p.G355G), is predicted in silico (Human Splicing Finder) to create a novel donor splice site within exon 9 (DSSmut) that is stronger than the native junctional one (DSSwt) (Fig 1, A). This new splice site is predicted to create a 19-bp deleted product with a premature stop codon in exon 10. Sanger sequencing confirmed the homozygous c.1272C>T variant in P1; the unaffected family members were heterozygous (Fig 1, B). RT-PCR from P1's PBMCs confirmed the predicted splicing effect, demonstrating a predominant mutant isoform with trace levels of WT isoform (Fig 1, C and D). The heterozygous family members possessed both isoforms but with higher WT levels; unrelated healthy controls expressed only the WT isoform. Sequencing of gel-eluted bands confirmed the 19-bp deletion in the mutant isoform (data not shown). Thus, c.1272C>T variant creates a mutant mRNA isoform through aberrant splicing; in homozygous state, the mutant isoform predominates over WT in P1. To determine the impact of the predominant aberrant ZAP70 mRNA isoform on protein production, we performed immunoblot analysis on P1's PBMCs and confirmed reduced ZAP-70 levels, as was seen in the original childhood investigations (Fig E1, C). Plasmids encoding the open-reading frame of WT ZAP-70 (ZAP-70wt) or the 19-bp deleted variant (ZAP-70mut) were stably transfected into ZAP-70–deficient Jurkat P116 cells, and immunoblot of cell lysates confirmed the absence of ZAP-70 protein with the mutant isoform (Fig 1, E). TcR signaling downstream of ZAP-70, assessed by phosphorylation of linker for activation of T cells, was absent following OKT3 stimulation of ZAP-70mut–transfected Jurkat P116, but intact with ZAP-70wt (see Fig E3, B, in this article's Online Repository at www.jacionline.org). These findings confirm that the mutant ZAP70 mRNA resulting from the c.1272C>T variant is null. Although the mutant donor splice site (DSSmut) is stronger than the native WT site (DSSwt), the latter is still intact and functionally able to generate WT ZAP70 mRNA and protein. In P1's homozygous mutant state, this is clearly inadequate for T-cell homeostasis. However, in heterozygous family members, ZAP-70 WT protein is sufficiently produced to maintain an otherwise well state. We hypothesized that blocking the stronger DSSmut in P1 would favor usage of DSSwt to increase WT ZAP-70 levels and consequently, T-cell function. To prove this, we designed an antisense morpholino oligonucleotide (AMO) to target the c.1272C>T variant while sparing the native exon-intron junction so as to block the mutant splice effect during processing of ZAP70 pre-mRNA (Fig 1, F). To confirm that the AMO specifically inhibited expression of the mutant ZAP70 isoform (ie, blocked its transition from pre-mRNA to mRNA), we treated P1's PBMCs and demonstrated correction of splicing (Fig 1, G). Optimization studies demonstrated maximal corrective effect starting at 48 hours using 5 μM AMO, producing approximately 50% WT mRNA (confirmed by sequencing; data not shown). A similar AMO-induced effect on mutant mRNA was seen on the sister's cells (data not shown); there were insufficient number of cells from P1's parents for testing. To verify the specificity of this effect, we treated cells from healthy controls and observed no effect (data not shown). Immunoblot analysis of P1's AMO-treated PBMCs demonstrated increased ZAP-70 expression, confirming that the correction of aberrant splicing results in augmented production of ZAP-70 protein (Fig 2, A and B). AMO-treated PBMCs demonstrated increased linker for activation of T cells activation following OKT3 stimulation compared with untreated cells (Fig 2, A and B), indicating that this restored ZAP-70 retains functionally intact TcR signaling. To determine whether the AMO could improve cellular function, we evaluated the expression of prototypical T-cell cytokine genes, IL2, IL4, IL10, and IFNG (IFN-γ) (Fig 2, C). AMO treatment alone increased IL2, IL4, and IL10 expression; IL2 and IL4 expression was further enhanced by CD3/CD28 stimulation (Fig 2, C). Treatment with AMO had no effect on IFN-γ expression (data not shown), likely due to the reestablished TH2-driven responses in the PBMCs polarizing away from TH1 responses. Last, AMO treatment reestablished the proliferative response to CD3/CD28 costimulation in P1's primary T cells, an effect seen in both CD4+ and CD8+ T cells (Fig 2, D and E; see Fig E4 in this article's Online Repository at www.jacionline.org). Collectively, these findings demonstrate that AMO treatment increases the expression of WT ZAP-70 protein, which is molecularly intact and capable of restoring key T-cell functional responses. Analysis of this hypomorphic ZAP-70–deficient patient uniquely illustrates several key points: (1) Clinically, the morbid natural history suggests a role for ZAP-70 in the functional control of double-stranded DNA viruses beyond the generation of CD8+ T cells, which requires further elucidation. Interestingly, the autoimmune diseases here (colitis; nephropathy) were also reported by Chan et al,1Chan A.C. Kadlecek T.A. Elder M.E. Filipovich A.H. Kuo W.L. Iwashima M. et al.ZAP-70 deficiency in an autosomal recessive form of severe combined immunodeficiency.Science. 1994; 264: 1599-1601Crossref PubMed Google Scholar who elegantly demonstrated that a fine molecular balance in ZAP-70 effect on T-cell signaling can be associated with autoimmunity only. (2) Genetically, it shows that synonymous genetic variants may be deleterious by modifying splicing, with hypomorphism resulting from a skewed net balance of amorphic versus residual WT protein. (3) Immunotherapeutically, it demonstrates that the accurate identification of causal genetic lesions permits a mutation-silencing approach that leads to molecular correction and recuperation of fundamental T- cell cytokine responses, at least in vitro. Although the breadth of T-cell functions could not be fully investigated pragmatically, to our knowledge this represents the first example in which AMO therapy successfully restored immune function in primary cells from a human with a genetically defined immunodeficiency. This approach could also extend to therapeutically modulate functional single-nucleotide polymorphisms in immune genes. Clearly, additional parameters will need to be addressed before this approach can be used in clinical practice (eg, pharmacokinetics and safety), although this strategy is in use for inborn errors of muscle or metabolism.9Winkler J. Oligonucleotide conjugates for therapeutic applications.Ther Deliv. 2013; 4: 791-809Crossref PubMed Scopus (119) Google Scholar Overall, the insight gained from this work opens new avenues for individualized, mutation-targeting therapy for CID. Subjects and the patient's family members provided informed consent on McGill University Health Centre institutional review board–approved research protocol (GEN10-256). Comprehensive medical histories, including review of all available outside records and serial clinical evaluations, and clinical immunologic laboratory testing were performed at the McGill University Health Centre. TH17 enumeration from whole blood was performed as previously described.E1Renner E.D. Rylaarsdam S. Anover-Sombke S. Rack A.L. 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Fast and accurate short read alignment with Burrows-Wheeler transform.Bioinformatics. 2009; 25: 1754-1760Crossref PubMed Scopus (31723) Google Scholar to the GRCh37 reference genome. Duplicate reads were marked with Picard tools.E7A set of command line tools (in Java) for manipulating high-throughput sequencing (HTS) data and formats such as SAM/BAM/CRAM and VCF. n.d. Available at: http://broadinstitute.github.io/picard/. Accessed May 16, 2016.Google Scholar Reads were realigned around indels with GATK IndelRealigner. Base quality scores were recalibrated with GATK BaseRecalibrator. Variant calling was performed with GATK HaplotypeCaller. Variant annotation was performed with SnpSiftE6Cingolani P. Patel V.M. Coon M. Nguyen T. Land S.J. Ruden D.M. et al.Using Drosophila melanogaster as a model for genotoxic chemical mutational studies with a new program, SnpSift.Front Genet. 2012; 3: 35Crossref PubMed Scopus (627) Google Scholar and SnpEff.E9Cingolani P. Platts A. Wang le L. Coon M. Nguyen T. Wang L. et al.A program for annotating and predicting the effects of single nucleotide polymorphisms, SnpEff: SNPs in the genome of Drosophila melanogaster strain w1118; iso-2; iso-3.Fly (Austin). 2012; 6: 80-92Crossref PubMed Scopus (6989) Google Scholar Only variants with coverage of at least 10 reads were kept for downstream analysis. To identify rare variants, the frequency of the variants in the Exome Aggregation ConsortiumE10Lek M. Karczewski K. Minikel E. Samocha K. Banks E. Fennell T. et al.Exome Aggregation ConsortiumAnalysis of protein-coding genetic variation in 60,706 humans.Nature. 2016; 536: 285-291Crossref PubMed Scopus (7176) Google Scholar database was verified. For Sanger sequencing, the ZAP70 gene (NG_007727.1) was PCR amplified from genomic DNA using primers designed to flank the respective regions (primers and sequencing conditions available on request). Sequencing was performed at the McGill University and Génome Québec Innovation Centre. Sequencing analyses were performed on Sequencher sequence analysis software (Gene Codes Corporation, Ann Arbor, Mich). Potential splice sites were predicted using the online tool Human Splicing Finder (www.umd.be/HSF/). The Myc-Flag–tagged ZAP-70 plasmid was from OriGene (Rockville, Md). Site-directed mutagenesis was used to create a 19-bp deletion at the end of exon 9 (New England Biolabs, Whitby, Ontario, Canada). Jurkat ZAP-70–deficient (p116) Jurkat cells were stably transfected with either WT or mutant ZAP-70 plasmid by electroporation followed by G418 selection (1 mg/mL). A 20-mer AMO was designed to target the de novo mutant donor splice site generated by the c.1272C>T mutation in the pre-mRNA of ZAP70, while sparing the native donor splice site. The AMO sequence was as follows: 5′-GCATGCGGTACACACCCTGG-3′. Vivo-morpholinos were used at a concentration of 5 μM. AMOs were designed and synthesized by Gene-Tools, LLC (Philomath, Ore). PBMCs were isolated as previously described.E11Gavino C. Cotter A. Lichtenstein D. Lejtenyi D. Fortin C. Legault C. et al.CARD9 deficiency and spontaneous central nervous system candidiasis: complete clinical remission with GM-CSF therapy.Clin Infect Dis. 2014; 59: 81-84Crossref PubMed Scopus (139) Google Scholar, E12Vinh D.C. Schwartz B. Hsu A.P. Miranda D.J. Valdez P.A. Fink D. et al.Interleukin-12 receptor beta1 deficiency predisposing to disseminated Coccidioidomycosis.Clin Infect Dis. 2011; 52: e99-e102Crossref PubMed Scopus (78) Google Scholar PBMCs were cultured in Opti-MEM Reduced Serum Media (Thermo Scientific, Waltham, Mass) and stimulated with 1 μg/mL OKT3 followed by 3 μg/mL secondary antibody or added to 96-well round-bottom plates coated with 3 μg/mL anti-CD28 and 1 μg/mL OKT3. There were insufficient number of cells from the sister and parents for functional testing. Samples were acquired on a BD FACSCANTO II flow cytometer. Single-color compensation samples were prepared using AbC Total Antibody Compensation Bead Kit (Thermo Scientific). Cells were labeled with CellTrace CFSE Cell Proliferation Kit (Thermo Scientific) and LIVE/DEAD Fixable Cell Stain Kit (Thermo Scientific) as per manufacturers' instructions. Compensation and analysis were performed on FlowJo version V10.2 Samples were separated on Bis-Tris Polyacyrlamide Gels (Thermo Scientific), transferred onto polyvinylidene difluoride membranes, and immunoblotted. Band densities were measured with ImageJ software (http://imagej.nih.gov/ij/, 1997-2014). Antibodies against phosphorylated linker for activation of T cells (Tyr171), and β-actin were from Cell Signaling Technology (Danvers, Mass). Total linker for activation of T cells antibody was from Santa Cruz Biotechnology (Dallas, Texas). Anti-Flag was from OriGene. Antibodies anti-CD3 (OKT3) and anti-CD28 were from Biolegend (San Diego, Calif). Antibodies against ZAP-70, Lck, Fyn, and Csk were as described previously.E13Latour S. Chow L.M. Veillette A. Differential intrinsic enzymatic activity of Syk and Zap-70 protein-tyrosine kinases.J Biol Chem. 1996; 271: 22782-22790Crossref PubMed Scopus (137) Google Scholar, E14Davidson D. Chow L.M. Fournel M. Veillette A. Differential regulation of T cell antigen responsiveness by isoforms of the src-related tyrosine protein kinase p59fyn.J Exp Med. 1992; 175: 1483-1492Crossref PubMed Scopus (133) Google Scholar Allophycocyanin-conjugated anti-human CD3 was from Thermo Scientific. Phycoerythrin-conjugated anti-human CD4 and APC-Cy7–conjugated CD8 were from BD Biosciences (Mississauga, Ontario, Canada). Total RNA was isolated using the RNeasy kit (Qiagen, Mississauga, Ontario, Canada) and reverse transcribed with the Maxima cDNA synthesis kit for RT-quantitative PCR (Thermo Scientific). Quantitative real-time PCR was performed using the Taqman quantitative PCR Gene Expression assay system with probes directed against IL2 (assay ID Hs00174114_m1), IL4 (assay ID Hs00174122_m1), IL10 (assay ID Hs00961622_m1), UBASH3A (assay ID Hs00955170_m1), and CD28 (assay ID Hs01007422_m1) (Thermo Scientific) on Applied Biosystem 7500 real-time PCR system. The mRNA input was normalized to the expression of the T-cell–specific housekeeping gene UBASH3A to account for any variability in T-lymphocyte composition. One experiment representative of 3 independent experiments performed is shown. Graphs and statistical analyses were generated with GraphPad Prism Version 6.00. Statistical significance was calculated according to an unpaired 2-tailed ratio student t test, with P < .05 considered significant.Fig E2A, Well-circumscribed pink macules on the trunk torso. B, Hematoxylin and eosin stain demonstrating enlarged superficial keratinocytes with distinctive blue-gray cytoplasm in association with prominent keratohyaline granules, consistent with HPV infection. C, Immunohistochemical pan-HPV stain.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E3A, A schematic representation of the ZAP-70 protein, with the asterisk denoting the location of the c.1272C>T mutation. B, Jurkat P116 cells were stimulated with OKT3 antibody and cell lysates were analyzed by immunoblot using antibodies specific for LAT and phospho-LAT. LAT, Linker for activation of T cells.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E4Gating strategy for assessment of T-cell proliferation of CFSE-labeled, CD3/CD28-stimulated cells. APC, Allophycocyanin; CFSE, carboxyfluorescein succinimidyl ester; FITC, fluorescein isothiocyanate; PE, phycoerythrin.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table E1Reports of ZAP-70 deficiencyCaseReferenceMutationEffect on proteinAge of onset; sexInfectionsAutoimmune/dysregulated inflammationImmunologic phenotypeOutcome1E15Monafo W.J. Polmar S.H. Neudorf S. Mather A. Filipovich A.H. A hereditary immunodeficiency characterized by CD8+ T lymphocyte deficiency and impaired lymphocyte activation.Clin Exp Immunol. 1992; 90: 390-393Crossref PubMed Scopus (30) Google Scholar, E16Chan A.C. Kadlecek T.A. Elder M.E. Filipovich A.H. Kuo W.L. Iwashima M. et al.ZAP-70 deficiency in an autosomal recessive form of severe combined immunodeficiency.Science. 1994; 264: 1599-1601Crossref PubMed Scopus (455) Google Scholar, E17Arpaia E. Shahar M. Dadi H. Cohen A. Roifman C.M. Defective T cell receptor signaling and CD8+ thymic selection in humans lacking zap-70 kinase.Cell. 1994; 76: 947-958Abstract Full Text PDF PubMed Scopus (507) Google ScholarCompound heterozygous<1 y-old M; MennoniteUpper respiratory tract Infections; Pneumonia; Oral ulcerations; PCP*Diarrhea, weight lossTotal CD3: normal.CD4+ T: normal.CD8+ T: absent.B: elevated.Immunoglobulin: IgG (↑); IgA (↑); IgM (N); IgE (NR)HLA-matched unrelated BMT at 20 mo with successful engraftment but died at 22 mo (unclear etiology)c.1763C>Ap.S518R.No protein detected in patients' cells.c.1624-11G>A [NM_001079.3]; G to A transition in intron 12 [g.98354447G>A; assembly GRCh37]Creation of stronger splicing acceptor site leading to addition of 3 amino acids (LEQ) in the catalytic domain. No protein detected in patients' cells2E15Monafo W.J. Polmar S.H. Neudorf S. Mather A. Filipovich A.H. A hereditary immunodeficiency characterized by CD8+ T lymphocyte deficiency and impaired lymphocyte activation.Clin Exp Immunol. 1992; 90: 390-393Crossref PubMed Scopus (30) Google Scholar, E16Chan A.C. Kadlecek T.A. Elder M.E. Filipovich A.H. Kuo W.L. Iwashima M. et al.ZAP-70 deficiency in an autosomal recessive form of severe combined immunodeficiency.Science. 1994; 264: 1599-1601Crossref PubMed Scopus (455) Google Scholar, E17Arpaia E. Shahar M. Dadi H. Cohen A. Roifman C.M. Defective T cell receptor signaling and CD8+ thymic selection in humans lacking zap-70 kinase.Cell. 1994; 76: 947-958Abstract Full Text PDF PubMed Scopus (507) Google ScholarNeonate M (brother of above case); identified by immunophenotypic screen; MennoniteNone (kept in protective isolation).(Had CMV in urine and rotavirus in stool, but was asymptomatic)NRTotal CD3: decreased.CD4+ T: normal.CD8+ T: absent.B: elevated.Immunoglobulin: NRHLA-matched unrelated BMT at 4 mo; alive at age 2 y3E15Monafo W.J. Polmar S.H. Neudorf S. Mather A. Filipovich A.H. A hereditary immunodeficiency characterized by CD8+ T lymphocyte deficiency and impaired lymphocyte activation.Clin Exp Immunol. 1992; 90: 390-393Crossref PubMed Scopus (30) Google Scholar, E16Chan A.C. Kadlecek T.A. Elder M.E. Filipovich A.H. Kuo W.L. Iwashima M. et al.ZAP-70 deficiency in an autosomal recessive form of severe combined immunodeficiency.Science. 1994; 264: 1599-1601Crossref PubMed Scopus (455) Google Scholar, E17Arpaia E. Shahar M. Dadi H. Cohen A. Roifman C.M. Defective T cell receptor signaling and CD8+ thymic selection in humans lacking zap-70 kinase.Cell. 1994; 76: 947-958Abstract Full Text PDF PubMed Scopus (507) Google Scholar6 mo F; Mennonite; unrelated to above casesOtitis media with perforation; PCPNRTotal CD3: normal.CD4+ T: elevated.CD8+ T: absent.B: normal.Immunoglobulin: IgG (↓); IgA (↑); IgM (N); IgE (NR)HLA-matched related BMT at 11 mo; alive at 4 y4E15Monafo W.J. Polmar S.H. Neudorf S. Mather A. Filipovich A.H. A hereditary immunodeficiency characterized by CD8+ T lymphocyte deficiency and impaired lymphocyte activation.Clin Exp Immunol. 1992; 90: 390-393Crossref PubMed Scopus (30) Google Scholar, E16Chan A.C. Kadlecek T.A. Elder M.E. Filipovich A.H. Kuo W.L. Iwashima M. et al.ZAP-70 deficiency in an autosomal recessive form of severe combined immunodeficiency.Science. 1994; 264: 1599-1601Crossref PubMed Scopus (455) Google Scholar, E17Arpaia E. Shahar M. Dadi H. Cohen A. Roifman C.M. Defective T cell receptor signaling and CD8+ thymic selection in humans lacking zap-70 kinase.Cell. 1994; 76: 947-958Abstract Full Text PDF PubMed Scopus (507) Google Scholar, E18Roifman C.M. Hummel D. Martinez-Valdez H. Thorner P. Doherty P.J. Pan S. et al.Depletion of CD8+ cells in human thymic medulla results in selective immune deficiency.J Exp Med. 1989; 170: 2177-2182Crossref PubMed Scopus (65) Google Scholar5 mo F (sister of case 3); Mennonite; identified by immunophenotypic screenOral ulcerations with eczematous rash; PCP; chronic diarrhea with reovirus; oral thrushNRTotal CD3: normal.CD4+ T: elevated.CD8+ T: absent.B: normal.Immunoglobulin: IgG (N); IgA (N); IgM (N); IgE (NR)Partial HLA-matched unrelated BMT at age 24 mo; alive at 3 y5E19Elder M.E. Lin D. Clever J. Chan A.C. Hope T.J. Weiss A. et al.Human severe combined immunodeficiency due to a defect in ZAP-70, a T cell tyrosine kinase.Science. 1994; 264: 1596-1599Crossref PubMed Scopus (455) Google ScholarHomozygous: 1719_1931del13 with predicted frameshiftNo protein detected in patient's cellsNRNRNRCD4+ T: normal.CD8+ T: absent.B: normal.Immunoglobulin: NRNR6E20Gelfand E.W. Weinberg K. Mazer B.D. Kadlecek T.A. Weiss A. Absence of ZAP-70 prevents signaling through the antigen receptor on peripheral blood T cells but not on thymocytes.J Exp Med. 1995; 182: 1057-1065Crossref PubMed Scopus (93) Google Scholar, E21Mazer B. Harbeck R.J. Franklin R. Schwinzer R. Kubo R. Hayward A. et al.Phenotypic features of selective T cell deficiency characterized by absence of CD8+ T lymphocytes and undetectable mRNA for ZAP-70 kinase.Clin Immunol Immunopathol. 1997; 84: 129-138Crossref PubMed Scopus (13) Google ScholarMutation not defined but no mRNA detectedNo protein4.5 mo M; not MennoniteMultiple respiratory infections; laryngomalacia; enteritis due to coronavirus; pneumonitis due to human parainfluenza virus 3Diarrhea, fail