A novel human autoimmune syndrome caused by combined hypomorphic and activating mutations in ZAP-70 - PubMed (original) (raw)

Case Reports

. 2016 Feb 8;213(2):155-65.

doi: 10.1084/jem.20150888. Epub 2016 Jan 18.

Divya Punwani 1, Theresa A Kadlecek 2, Morton J Cowan 1, Jean L Olson 3, Erin F Mathes 4, Uma Sunderam 5, Shu Man Fu 6, Rajgopal Srinivasan 5, John Kuriyan 7, Steven E Brenner 8, Arthur Weiss 2, Jennifer M Puck 9

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Case Reports

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

Alice Y Chan et al. J Exp Med. 2016.

Abstract

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.

© 2016 Chan et al.

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Figures

Figure 1.

Figure 1.

Pedigree and clinical phenotype of the affected siblings. (A) Family pedigree showing ZAP-70 genotypes. (B–D) Renal biopsy images from patient II-2. (B) Hematoxylin and eosin stain showing slightly increased glomerular mesangial matrix without hypercellularity or segmental sclerosis; no tubular, interstitial, or vascular abnormalities were noted. Bar, 100 µm. (C) Immunofluorescence demonstrating 1–2+ IgG staining in mesangium and capillary loops. Bar, 50 µm (D) Electron microscopy showing widespread foot process effacement (red arrows), with inset showing lucencies within the glomerular basement membrane (white triangles). Bar, 2 µm. (E–H) Bullous pemphigoid from patient II-2 (E and F); and from patient II-3 before (G) and 5 wk after (H) hematopoietic cell transplantation.

Figure 2.

Figure 2.

ZAP-70 mutations. (A) Sanger sequence showing heterozygous mutations in II-2 (RefSeq accesssion nos. NM_001079.3 [ClinVar reference nos. SCV000258321 and SCV000258322] and NP_001070.2). (B) ZAP-70 protein diagram, showing N- and C-SH2 domains, interdomains I-A and I-B, and kinase domain. Mutation sites in the family are in red; previously reported mutations in combined immunodeficiency patients (all of which are loss-of-function) are in blue. (C) Three-dimensional structural representations of ZAP-70 (PyMOL software). Residues shown in red are R192 in the ITAM peptide phosphotyrosine binding site of the C-SH2 domain (magnified at left); and R360 in the kinase domain (blue), near amino acids Y315 and Y319 (yellow), known to participate in the autoinhibitory site (magnified at right).

Figure 3.

Figure 3.

Association with the TCR ζ-chain and kinase activity of the mutant ZAP-70 proteins. (A) Blot of whole-cell lysates (WCL) and anti-TCR ζ-chain immunoprecipitates from 293T cells transfected with Lck (∼56 kD), FLAG-ζ-chain (∼18 kD), and WT, R37K/R190K, R192W, or R360P ZAP-70 (70kD). (left two panels) Protein expression levels of ZAP-70, Lck, or FLAG-ζ-chain from WCLs and levels of these proteins associated with ζ-chain immunoprecipitates. (right two panels) Phosphotyrosine (pTyr) immunoblots of WCL and ζ-chain immunoprecipitates. Ig heavy chain (Ig HC) is notated in the ζ-chain IPs. (B) Protein structure of the autoinhibitory site in the ZAP-70 kinase domain, highlighting residues known (Y315 and Y319) or proposed (E324, D327, R360, and K362) to participate in autoinhibition. R360, mutated in our patient, lies in the autoinhibitory region. (C) Immunoblots on WCLs from 293T cells transfected with LAT and WT and mutant ZAP-70 (mutants include YYAA, R360P, E324A, D327A, K362A, and K362E). (top) Expression of ZAP-70 and LAT; (bottom) pTyr blotting to show phosphorylated LAT. (D) After 24 h of doxycycline treatment to induce WT or R360P ZAP-70 protein expression in ZAP-70 deficient P116 rtTA cell lines, cells were stimulated with anti-TCR antibody for 0, 2, 5, or 10 min. Immunoblots of WCLs are shown for ZAP-70, pY319, and pY493 ZAP-70, Slp-76, pY128 Slp-76, LAT, pY132, and pErk. Data are representative of four independent experiments.

Figure 4.

Figure 4.

Activation states of cells transfected with mutant ZAP-70. (A) Basal CD69 expression in P116 cells transfected with ZAP-70 constructs. Cell populations gated on ZAP-70 expressing cells (left) and their corresponding CD69 expression (right, blue, ZAP-70−; red, ZAP-70+). (B) CD69 expression in transfected cells with equal ZAP-70 expression, unstimulated (thin lines), or stimulated (bold lines) with anti-TCR antibody C305 or PMA plus ionomycin (shading). (C) Phospho-Erk expression in PBMCs from the father (open squares), healthy sister (open diamonds), and mother (open circles) of the affected patients and from four controls (Xs), stimulated with graded dilutions of anti-CD3 antibody for 15 min. Error bars, SEM. Data are representative of four independent experiments. Note that affected patients’ cells were not available due to their having received hematopoietic stem cell transplants before identification of their gene defect. (D) CD69 expression by mean fluorescence intensity (MFI) in P116 cells either untransfected (hatched bar) or transfected with indicated amounts of plasmid vectors containing no insert or ZAP-70 cDNA constructs, as shown. All transfections contained 20 µg of plasmid DNA. Black bars, WT with or without R360P; gray bars, R192W with or without R360P. White bar, R360P plus vector with no insert. *, P < 0.05, unpaired Student’s t test. Data are representative of three independent experiments.

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