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Lymphocyte Disturbance in The [Asp521Asn] ZAP70Mutation and An Overview of All Phenotype/GenotypeWen-I Lee  ( wen2707@gmail.com )

Chang Gung University and Children's Hospital https://orcid.org/0000-0003-2683-3169Yung-Feng Lin 

National Health Research InstitutesChing-Huang Ho 

National Health Research InstitutesShih-Hsiang Chen 

Primary Immunode�ciency Care and Research (PICAR) InstituteMei-Hsin Hsu 

Chang Gung University College of MedicineRen-Chin Wu 

Chang Gung Memorial Hospital and Chang Gung UniversityWan-Fang Lee 

Chang Gung Memorial HospitalTang-Her Jaing 

Chang Gung Memorial HospitalHuang Jing-Long 

Chang Gung Memorial HospitalShih-Feng Tsai 

National Health Research Institutes

Research Article

Keywords: immune dysregulaiton, autoimmune, ZAP70, Omenn syndrome, hematopoietic stem celltransplantation, Combined T and B immunode�ciency

Posted Date: May 20th, 2021

DOI: https://doi.org/10.21203/rs.3.rs-494192/v1

License: This work is licensed under a Creative Commons Attribution 4.0 International License.   Read FullLicense

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AbstractBackground. Activated Zeta (ζ)-chain-associated protein kinase 70 (ZAP70) phosphorylates the TCRαβ:CD3:ζ-complex to diversify the initial TCR signal. Patients with the ZAP70 mutations could present with the phenotype ofimmune dysregulation.

Methods. We identi�ed the �rst Taiwanese boy with the ZAP70 mutation, assessed downstream signaling,investigated lymphocyte disturbance and analyzed all available phenotype/genotype for optimal treatment.

Results. With the [Asp521Asn]ZAP70 mutation, the infant who had hypogammaglobulinemia, eosinophilia,isolated CD8 lymphopenia, and impaired lymphocyte proliferation experienced recurrent pneumonia, refractorydiarrhea, transient hematuria and autoimmune hepatitis. Decreased CD3/CD28 downstream phosphorylation ofAKT, ZAP70 and Ca2+ in�ux related to the Th2-skewing T follicular helper cells, expanded transitional B, increasedCD21-low B cells and lower Treg cells. To speculate clinical course for effective treatment, we overviewed 45available published patients. Except for two asymptomatic post-transplant infants, common pathogens were oralcandida (n=9), PJP (n=7), CMV (n=5), varicella (n=4), parain�uenza (n=3) and disseminated BCG (n=3). Theirphenotypes of immune dysregulation mainly included IBD-like diarrhea (n=12), dermatitis (n=7),hepatosplenomegaly (n=3) and nephritic syndrome (n=3). Founder-effect or hot-spot mutations (32/90 alleles)involving the kinase domain clustered on intron 12 [1624-11G>A] (in 24) and exon 12[1520C>T] (in 8). Eleven diedof sepsis (n=3), CMV pneumonitis (n=2), viral encephalitis, disseminated measles-vaccine infection, ARDS, HLH-like, EBV-lymphoproliferative disorder, and lymphoma each. Whatever developing opportunistic infections(p=0.2240), immune dysregulation (p=0.5268), or failure to thrive (p=0.5215), those who receiving HSCT hadsigni�cantly better prognosis (p=0.0003).  

Conclusions. The Asp521Asn-ZAP70 hinders TCR-CD3 downstream phosphorylation and disturbs lymphocytesubgroup “pro�les” leading to autoimmune/autoinlfmmation. The lymphocyte disturbance should be validated inlarge-scale studies. Receiving HSCT is a signi�cantly better survival factor, rather than mutation types,opportunistic infections, or immune dysregulation. 

IntroductionZeta (ζ)-chain-associated protein kinase 70 (ZAP70) belonging to a member of the spleen tyrosine kinase (SYK)family is a non-receptor protein tyrosine kinase mainly expressed in intracellular T cells.1 Upon antigenrecognition, T cell antigen receptors (TCR) activate lymphocyte-speci�c protein tyrosine kinases (LCK) that in turnphosphorylate ZAP70 and immunoreceptor tyrosine-based activation motifs (ITAM) of the TCRαβ:CD3:ζ-complex.2 The activated ZAP70 is recruited into phosphorylated ITAM of the TCRαβ:CD3:ζ-complex to modulatecore adaptor proteins and act as a T-cell master kinase by amplifying and diversifying the initial TCR signal.3

Different from the Zap70−/− murine model which has been shown to block both emigrant CD4 and CD8 T cellsfrom the thymus,4 ZAP70-de�cient human CD4 T cells can maintain su�cient quantitative emigration viacompensation with a higher SYK expression through downstream residual TCR signaling, although this is notseen with CD8 cells.5 Such selective CD8 lymphopenia was �rst reported by Roifman et al in 1989,6 and theharmful genetic defect of Zap70 mutations was identi�ed in 1994.7 With CD8 lymphopenia, a poor response tomitogens and increased susceptibility to opportunistic infections, such patients are diagnosed with combinedimmunode�ciency disease (CID) and can also exhibit immune dysregulation mimicking Omenn syndrome.8,9

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Notably, some patients only present with isolated autoimmune disorders without infection and are �nally found tohave hypomorphic ZAP70 mutations by whole exome sequencing (WES).10,76 Herein, we present a 2-year-old boywith recurrent wheezing, chronic diarrhea, gross hematuria and elevated liver enzymes who was referred to ourPICAR (Primary Immunode�ciency Care And Research) Institute where we identi�ed a homozygous ZAP70mutation for the �rst time of Taiwan aboriginal ethnicity. To clarify what phenotype and adequate interventionbene�cial to survival prognosis, we investigated the function of this ZAP70 substitute and survival analysis of allavailable patients from a PubMed search 7,9,10, 21, 26-52,76

Materials And MethodsEthics Statement

All human samples were obtained under protocols approved by the Institutional Review Board at Chang GungMemorial Hospital and met the Institutional Review Board standards (IRB-201901385A3 and IRB-202001665A3)for ethical conduct of research with human subjects in accordance with the Declaration of Helsinki. Allexperimental protocols in this study, and the patient’s parents or guardians provided informed consent. Allmethods were performed in accordance with the relevant guidelines and standard regulations.

Basic immunologic function assessments

To induce lymphocyte proliferation, peripheral blood mononuclear cells (PBMCs; 105/well) were incubated withdifferent concentrations of phytohemagglutinin (PHA), pokeweed (PWM), ConA, and CD3/CD28 for 3 days, or theCandida antigen and Bacillus Calmette-Guérin vaccine for 7 days, followed by incubation with [3H]-thymidineovernight.15,16 Carboxy�uo rescein diacetate succinimidyl ester (CFSE) was added to stimulate PHA, and theproliferation index of mitotic cells was analyzed using FlowJo software 7.2.5 (TreeStar®). The lymphocytesubsets of T follicular helper cells, Th17 cells and Treg cells, transitional B cells and CD21-low B cells wereassessed to correlate with his autoimmune phenotype.

Candidate gene approach

CD8 lymphopenia and impaired lymphocyte proliferation can occur in patients with MHC class I de�ciency.17

Because of a normal MHC class I expression (Supplemental Fig. 1), the candidate ZAP70 gene responsible for CIDor leaky SCID phenotype was sequenced. Total RNA was extracted from the PBMCs using TRIzol (Life Tech.,Carlsbad, CA), followed by RT-PCR. The two pairs of primer sequences of the ZAP70 gene were based on humangenome sequences NM_001079 and designed to cover the whole coding region: ZAP70-102F: ATT CAG AAC CGGCTC TCC AT, ZAP70-1305R: CAC GTC GAT CTG CTT CTT GC; ZAP-70 1091F: AGC CAG CAC GCA TAA CGT CC,ZAP70-2176R: CAG CTG TGT GTG GAG ACA AC. At the same time, the genomic DNA in exon 12 (forward: TGAACA CAT GGT CAC CTG; backward: TGG TGT GTT GGA GAG CTG) was con�rmed based on NT_005403.18.

Flowcytometry for candidate protein expressions and intracellular Ca2+ �ux downstream CD3/CD28 signaling

For phospho-�ow, PBMCs were stimulated with anti-CD3 (10 μg/ml) and anti-CD28 (10 μg/mL) for an adequateperiod, then �xed (BD Biosciences®, Cyto�x), stained for surface antigens of CD4 or CD8, permeabilized (BDBiosciences®, Perm buffer III) and stained with anti-ZAP70 antibodies (eBiosciences), phospho-ZAP70 (BDBiosciences®, BD Phos�ow), and phospho-AKT (BD Biosciences®, BD Phos�ow).18

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l intracellular Ca2+ ; d by dTo study Ca2+ �ux, CD4 T cells were further enriched using a human CD4 T cellnegative selection kit (Biolegend, San Diego, CA). The cells were then washed twice with loading buffer HBSS with25 mM HEPES (ThermoFisher, Eugene, OR). Fluo-4-AM (ThermoFisher, Eugene, OR) was loaded at a �nalconcentration of 5 mM and the samples were incubated for 30 min in the dark at 37°C. After washing twice withloading buffer, the cells were either untreated or stimulated with anti-CD3 (Biolegend, San Diego, CA) plus anti-CD28 (Biolegend, San Diego, CA) in the presence of rabbit anti-mouse IgG (Jackson, West Grove, PA), each at 10mg/mL. The �uorescent Ca2+ signal was detected using �ow cytometry (BD, FACSCanto II). The �ow data werethen analyzed using FlowJo software version 7.6.1. l intracellular Ca2+ ; d by d18,19

Whole genome sequencing (WGS)

Because bi-genetic mutations have been reported in rare disorders, WGS was performed to explore other geneticdefects responsible for his autoimmune phenotype, especially those not yet reported for hepatitis. His DNA wasextracted from whole blood and WGS was performed on an Illumina NoveSeq 6000 system (Illumina Inc., SanDiego, CA) using a PCR-free protocol. The data were processed using CLC Genomic Workbench 11 software(Qiagen). Brie�y, paired-end reads were removed from low quality bases (Q < 30, Phred scale) and aligned to ahuman reference genome (GRCh37/hg19). The mapping parameters were set to default except for mappinglength which was set to 0.9 and mapping similarity which was also set to 0.9, and the mapped reference genomewas only read once. Variants were �ltered based on sequencing coverage (removed if the depth was < 10) and bycomparisons with a common variant database (dbSNP version 150).20-22

Clinical features and survival prognosis of all patients with ZAP70 mutations

To understand the whole disease course, we searched for all reported patients with ZAP70 mutations in a Medlinesearch. Survival curves were estimated using Kaplan-Meier analysis to compare their prognoses based onopportunistic infections (Oi), failure to thrive (Ft), immune dysregulation (Id) including autoimmune disorders,chronic or IBD-like diarrhea, skin lesions, lymphoproliferative disorders and lymphoma, and whether or not theyreceived HSCT. The �rst follow-up day was de�ned as the initial presentation date of the patients with ZAP70mutations. The last follow-up day and duration were de�ned according to those reported in the studies. Allanalyses were performed using GraphPad Prism software, and a p value of <0.05 was de�ned as beingstatistically signi�cant.

ResultsCase demonstration

This 1-year 4-month-old boy was born to aboriginal parents via cesarean section without any adverse events. Hewas hospitalized for pneumococcus pneumonia three times from the age of 6 months, and experienced frequentcoughing and wheezing thereafter. Respiratory syncytial virus, enterovirus and adenovirus infections causedrecurrent wheezing. Neither infective cultures nor reverse transcription polymerase chain reaction ampli�cation(RT-PCR) in bronchial lavage revealed any evidence of pneumocystis jirovecii pneumonia (PJP), aspergillosis,fungus and mycobacterium. Because of his failure to thrive since 2 months of age, he only received hepatitis B,diphtheria-pertussis-tetanus and Hib vaccines and avoided any live attenuated vaccines in spite of his normalTREC value (252 copies) on his neonatal Guthrie card. When he was 13 months old, Pseudomonas aeruginosacaused severe colitis, sepsis and hypovolemic shock that met the diagnosis criteria of Shanghai fever.11

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Ceftazidime, amikin, cipro�oxacin and hydration rescued his life-threatening infection. A hematologic evaluationshowed mild neutropenia (1034/mm3), normal lymphocyte count (3290/mm3), eosinophilia (12%), anemia (Hb7.6 g/dL) and thrombocytosis (platelets 760 k/UL). Hypogammaglobulinemia (IgG 74.8 mg/dL, IgM 24.6 mg/dL,but higher IgE 1430 IU/mL), a profound decrease in CD8 lymphocytes to 5% (CD4 46%, NK 16%, and CD19 24%)and obviously impaired lymphocyte proliferation (Supplemental Table 1) were compatible with combined B and Tcell immunode�ciency. Regular immunoglobulin infusions (0.8g/kg per month) and prophylactics forbronchiectasis (25 mg/kg/day Augmentin), PJP (5mg/kg/day Trimethoprim-Sulfamethoxazole) and fungalinfections (5 mg/kg/day �uconazole) were given until successful HSCT engraftment.

During the waiting period, his ALT and AST levels increased to over 3000, but levels of ALP, γ-GT, and bilirubinremained normal. Any kinds of antibiotics with the potential for hepatic toxicity were discontinued, and RT-PCR forhepatitis viral load excluded EBV, CMV, HSV, hepatitis A, B, C and E. A liver biopsy did not favor medication-relatedor viral hepatitis. Liver enzyme levels often returned to normal after monthly immunoglobulin infusion. Mildlyelevated anti-smooth muscle antibodies (1:40) supported autoimmune hepatitis, but negative for anti-nuclear(ANA) and anti-liver-kidney-microsomal (LKM) antibodies. Gross hematuria once occurred, but urine cultures andRT-PCR ampli�cation for adenovirus and BK virus were negative. One month later, he had chronic diarrhea andacute exacerbation leading to abdominal distension, ileus, and moderate ascites. Under the impression of sepsisfrom colonic pathogens (especially previous pseudomonas aeruginosa), hypovolemic shock and severehematochezia (Fig. 1A) with an Hb level down to 4 g/dL, aggressive antibiotic treatment was given over 2 weekswith a continuous blood transfusion for 7 days. Abdominal CT, angiography and Meckel’s scan did not reveal anysigni�cant �ndings for bleeders. Colonoscopy showed small white nodules of 5 x 5 mm in size protruding into theintestinal lumen (Fig. 1B). Mucosa nodular lymphocyte aggregation and submucosa oozing-like edema have beenreported to be a possible prodrome of in�ammatory bowel diseases (Fig. 1C and 1D) that resemble intestinallymphoproliferation in patients with activated phosphoinositide 3-kinase δ syndrome (APDS)12 and commonvariable immunode�ciency (CVID)13,14

With regular immunoglobulin infusion and adequate prophylactics,

n the near future, ing donor is ready to hepatitis, hematuria and chronic . kinds of antibiotics low-doseprednisolone (0.5mg/kg/day) was added for his phenotype of immune dysregulation including hepatitis,hematuria and chronic diarrhea. Fortunately, he received an HSCT from an HLA-matching sibling donor when hewas 1 year and 11 months of age. At present, he was 3 years 6 months and free of IVIG infusion and GvHDmedication.

Immune functional assessment

Obvious decreases in mitogen and antigen proliferation were shown by [3H]-thymidine incorporation(Supplemental Table 1). Compared with the parallel control, PHA stimulation and lymphocyte proliferation of less10% of normal was de�ned as being “absent” function and was considered to be an indication for HSCT. Suchprofound impairment of lymphocyte proliferation was also demonstrated by a CFSE evaluation of theproliferation index (Fig. 2A).

Compared to the parallel control, the lymphocyte subsets of T follicular helper cells shifted to Th2 differentiation(Th2+Th17/ Th1 = 2.50 vs. 1.05 in the control) (Fig. 2B), with an increase in transitional B cells (CD19+IgM++IgD++

= 51.30% vs. 5.05%) (Fig. 2C), CD21-low B cells (CD19+CD21 low = 22.30% vs. 5.05%) (Fig 2D), and a higher

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Th17/Treg ratio (1.8/0.4 vs. 3.1/4.9 in Supplemental Fig. 2 predominantly ascribed to lower Treg cells) wereconsistent with those CVID, Wiskott-Aldrich syndrome, or APDS patients who often present with the phenotype ofimmune dysregulation. For memory cells, memory B cells (non-switched, switched and exhausted) and memory Tcells (effector) were obviously diminished (Fig. 2E and 2F) and likely contributed to the increased susceptibility toinfections.

Genetic analysis

Using a candidate gene approach, Sanger sequencing revealed that the 1561st nucleotide G was homozygouslyreplaced by A in exon 12 leading to a missense mutation of Asp 521 Asn in the ZAP70 gene (Fig. 3). We furtherassessed the effect of 521Asn-ZAP70 on CD3/CD28 downstream signaling, and also utilized WGS to investigatewhether a gene other than ZAP70, or even bi-genetic mutations23 were related to his complex phenotype including�uctuating higher levels of liver enzymes, gross hematuria and hematochezia. However, only the same mutationwas consistently identi�ed instead of revealing any other responsible novel gene (Supplemental Table 2). Hisparents and older sibling were all carriers.

ZAP70 expression and intracellular Ca2+ dynamic in�ux downstream the CD3CD28 signaling

In an in-depth study of the mutant 521Asn-ZAP70 function, we investigated the expressions of phosphorylatedtranslation factors of ZAP and AKT in downstream signaling. Compared to that in the control, the intracellularexpression of 521Asn-ZAP70 obviously decreased in CD4 cells and CD8 cells without/with CD3CD28 stimulationfor 10 min (Fig. 4). In parallel, the downstream signal expressions of phosphorylated-ZAP70 and -AKT were alsodecreased (Fig. 4). Thus, such alterations attenuated its binding capacity and signal pathway. Asp521-ZAP70 isan acidic residue which forms hydrogen bonds with H459 and R460 and stabilizes an activation loop, a crucialcomponent of kinase function.24,25 Changes in Asp521Asn can cause a conformational change in the activationloop and impact signaling cascades, compatible with the SIFT predicted scoring system (Score 0 meaningdamage, http://sift.jcvi.org/) and PolyPhen2 (Score 1 also meaning damage,http://genetics.bwh.harvard.edu/pph2/index.shtml).

We then conducted calcium in�ux assays to evaluate the downstream function of ZAP70 in T cell activation. Asshown in Figure 5A, calcium in�ux in the patient’s T cells was nearly undetectable when stimulated. Compared tothe patient (Fig. 5A), the results of calcium in�ux in the carrier was evident (Fig. 5B), even though the in�ux ofmaximum calcium concentration was not as high as in the normal control (Fig. 5C). To further compare thedifferences between these three subjects, kinetic plots of each calcium in�ux were superimposed to reveal thefunctional de�cit (Fig. 5D). After stimulation, the response time and ascending slope of the calcium in�ux in thecarrier were nearly the same as those in the control. However, the carrier did not reach the same level of calciumconcentration as the control, and the descending slope was also signi�cantly faster than that of the control.

Genotype, phenotype and survival analysis

A PubMed search for the key words “Zap70 mutation” and “immunode�ciency” excluding studies which did notidentify the gene revealed 45 patients from 35 families, including 33 patients associated with founder-effect orconsanguineous families and the parents of our patient who originated from the indigenous Payuan region(Supplemental Table 3). Their ethnicity included 13 Mennonite Canadians, 11 Turkish, 6 Caucasians, 4 Japanese,2 Cops, 2 Mexican, 2 Indians, 1 Iranian, 1 Chinese, 1 Taiwanese and 2 unknown. Among a total of 90 alleles, there

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were missense mutations in 47, splicing mutations in 31, deletions in 7, insertion in 2, nonsense in 1 allele andundetectable mRNA in one patient. The most common mutation was in intron 12 (23 alleles), exon 12 (23 alleles),exon 9 (10 alleles), exon 10 (8 alleles), exon 3 (6 alleles), exon 11 and exon 13 (4 alleles each), exon 5 (3 alleles),exon 7 (2 alleles), intron 13 (2 alleles) and exon 6, intron 5 and exon 2 (one each), but one without detectablemRNA. Two founder effect or hot-spot mutations were located at intron 12 [1624-11G>A] in 23 alleles and exon 12[1520C>T] in 8 alleles, all of which affected the kinase domain.

Infections were the most common presentations of combined T and B immunode�ciency. The identi�edpathogens over two eventful episodes included candida (9 episodes), PJP (n=7), CMV (n=5), varicella (n=4),parain�uenza (n=3) and BCG vaccine (n=3) that was re�ected by their CD8 lymphopenia as they were mainlyunable to resist viral pathogens rather than bacteria (Table 1). To compare survival based on their phenotypes, wede�ned the phenotype of immune dysregulation encompassing autoimmune disorders as the patients whodeveloped in�ammatory bowel disease (IBD)-like chronic diarrhea, eczematous dermatitis, hepatosplenomegaly,lymphoadenopathy, hemolytic anemia, nephritic syndrome or brain infarction and lymphoma (Table 2).

In complex-phenotype comparisons (in Table 3 and Supplemental Table 4), we classi�ed the major phenotypes ofthese patients as “opportunistic infection (Oi)”, “immune dysregulation encompassing autoimmune disorders (Id)”and “failure to thrive (Ft)”. The onset age and published age of the patients with the Oi-Id-Ft phenotype weresigni�cantly older than those with the Oi-Id phenotype (p=0.0399; p= 0.0143) because failure to thrive (Ft) oftenappeared after 3 months old, but not signi�cantly in each-other mutual comparisons (Table 3). The oldest patientwas 33 years old at time of writing and presented with the Oi-Id-Ft phenotype.46 In the strict sense, no relationshipbetween genotype and phenotype existed as the recent review.77 

For Kaplan-Meier survival analysis, those receiving HSCT showed an obviously and signi�cantly higher survival(p=0.0003) than without, but not in the other comparisons of opportunistic infections (p=0.2240), immunedysregulation (p=0.5268), and failure to thrive (p=0.5215). (Fig. 6). 

DiscussionPatients with ZAP70 mutations have hypogammaglobulinemia, CD8 lymphopenia and impaired lymphocyteproliferation, therefore suffered from recurrent infections. As well as approximate 60% (26/43) developed thephenotype of immune dysregulation, our patient with the [Asp521Asn] ZAP70 mutation experienced chronicdiarrhea, non-virus autoimmune hepatitis (with positive anti-smooth antibodies), hematuria and eosinophilia witha relatively high level of IgE, mimicking Omenn syndrome. Omenn syndrome is a T-B-NK+ SCID subgroup �rstidenti�ed by hypomorphic RAG mutations interrupting T and B cell receptor recombination and mainlyorchestrating oligoclonal T cells to in�ltrate multiple organs of intestine, liver and kidney.53 Based on Freiburg13

and EUROclass14 classi�cation, markedly increased proportion of CD21-low B cells (more than 10%), reducedswitched memory B cells (less than 2%), and expanded transitional B cells (IgM++CD38++ more than 9%) trend todevelop autoimmune cytopenia, lymphadenopathy, splenomegaly, and granulomatous disease.54 Furthermore, animbalance between Treg and Th17cells (the ratio of Th17/Treg) is a feature of autoimmunity in systemic lupuserythematosus,57 vasculitis (such as Kawasaki disease)58 and exacerbated asthma.59-61 Taken together,expanded transitional B cells, augmented CD21-low B cells, Tfh shift to Th2 deviation and higher Th17/Treg ratiodue to lower Treg cell contribute to immune dysregulation of autoimmunity/autoin�ammation as well as reducedswitched memory B cells and TEM cells 55,56 that also increase susceptibility to infections.

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Immune imbalance could affect peripheral and central tolerance, trigger autoimmune/autoin�ammation processand possibly trend to malignancy transformation.8,62 Thus, in a broad sense, primary immune “dysfunction ordysregulation” (PID) is a more appropriate term than “de�ciency” since that the four aspects of autoimmunity,autoin�ammation, malignancy and refractory allergy have diversi�ed the PID phenotype.63 Patients withmutations of the DCLRE1C, IL7RA, RMRP, ADA, LIG4, IL2RG, and AK2 genes and those responsible for DiGeorgesyndrome are observed to resemble Omenn syndrome and listed in the 2014 PID classi�cation.64,65 Such “Omenn”syndrome combining T cell de�ciency and immune dysregulation has been omitted in the 2017 classi�cationbecause advanced WGS/WES66 reveal novel mutations of the LRBA,67CTLA4,68,69, PPIK3CD70 and PIK3R171,72

genes in previously termed “Omenn” syndrome. Nevertheless, the ZAP70 gene could be still a potential candidategene for Omenn-like syndrome after validation by a large cohort study.

Distinct from the recent review summarized by Shari�nedjad et al,77 our study emphasized more on optimaltreatment. In practice, all of the patients with ZAP70 mutations received regular immunoglobulin infusion andprophylactics for opportunistic infections.73 Immunosuppressants such as prednisolone and thalidomide werenot uncommon for patients with IBD-like chronic diarrhea and recurrent intestinal stenosis.48 In an extreme case,nephrectomy was performed in one Turkish male patient who suffered from congenital nephritic syndrome,persistent hypertension and silent brain infarction.47 Receiving mini- or myeloablative conditioning for partial-matched donors or non-conditioning for sibling-matched donors, 19 patients were still alive despite of receiving 26HSCTs due to graft failure in some. The donors included 15 from bone marrow, 8 from peripheral stem cells, and 3from cord blood. Like our toddler boy, three receiving sibling-matched donors without conditioning are still well 20years post-transplant.28 Unexpectedly, one Turkish male infant developed cholangitic hepatitis and portal �brosis,possibly ascribed to persistent hepatitis and/or an adverse event of myeloablative conditioning (melphalan 140mg/m2 and �udarabine 160 mg/m2), which was resolved by a liver transplantation 2 years post-transplant.43

Notably, one male 33-year-old patient with a homozygous c1272 C>T mutation had a minor product of the wildform with a silent mutation p.G355G, but more truncation products from creating a donor splice (within exon 10)p.G355G fs10X losing the kinase domain. An antisense morpholino oligonucleotide (AMO) was designed torecognize this segment (20 nucleotides) and to prevent the aberrant splicing.46 In vitro, AMO treatment restoredCD3/CD28 signaling by predominantly deviating to wild-type transcription. AMO driving to intact transcription hasbeen utilized to treat inborn errors of muscle and metabolism.74

Newborn TREC screening using the Gathrie card may prompt diagnosis of patients with ZAP70 mutations. ACanadian Mennonite patient with the splicing mutation IVS 12 (-11) G>A had an obviously decreased TREC valuewhen he clinically deteriorated at around 8 months of age.51 Because the TREC value correlates to the number ofnaïve CD4CD45RA T cells, 75 the normal number of CD3+ and CD4+CD45RA+ lymphocytes could cover the CD8+lymphopenia and showed a normal TREC value in the newborn Guthrie card as our patient.

In contrast to the Iranian boy with the same missense mutation who suffered form autoimmune hemolyticanemia and immune thrombocytopenia anemia (in supplemental Table 3),52 our patient distinctly developedautoimmune hepatitis and transient gross hematuria (reversible gromerulonephritis) as well ashypogammaglobulinemia. These two boys also experienced IBD-like diarrhea. All of such immune dysregulationand in�ammation had good response to methylprednisolone and regular immunoglobulin infusion.

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This study should be interpreted in light of its limitations. First, patients with the same homozygous mutationscould have diverse phenotypes. In addition to our and Iranian boys with the same [Asp521Asn]ZAP70 mutation,the other mutation of 1624-11G>A [IVS 12 (-11) G>A] could cause three diverse features: opportunistic infection(Oi), combined immune dysregulation and failure to thrive (Id-Ft), and the complex of Oi-Id-Ft phenotypes. Furtherstudies are worth of investigating whether compensation mechanisms exist to rescue such defective ZAP70signaling to open an alternative therapeutic avenue. Second, the patients receiving HSCT had signi�cantly bettersurvivor despite the �rst graft failure and unrecognized in newborn TREC screening. Multidisciplinary care teamwith a certain level of successful HSCT should save all patients if suitable donors available. Third, bi-geneticdefects have been identi�ed in a blended syndrome with ZAP70 and RNF168 mutations.23 The genetic candidateone-by-one approach for complex phenotypes may not reveal the second genetic defect. More advancedsequencing techniques and analysis software may explore signi�cant genetic variance that is not recognized atpresent.

In conclusion, the [Asp521Asn] ZAP70 missense mutation decreased downstream TCR-CD3 phosphorylation ofAkt, ZAP70 and Ca2+ in�ux. The correspondent cellular phenotype of immune imbalance included Th2-skewing Tfollicular helper cells, lower Trg cell (higher Th17/Treg ratio), increased transitional B and CD21-low B cells. Thosewho presented with CD8-lymphopenia related CID phenotypes caused by the ZAP70 mutations and receivedsuitable HSCT had signi�cantly higher survival even though they suffered from graft failure and severe infection.

AbbreviationsAPDS; activated phosphoinositide 3-kinase δ syndrome

CID, combined T and B cell immunode�ciency

CVID, common variable immunode�ciency

HSCT, hematopoietic stem cell transplantation

IBD, in�ammatory bowel disease

LPD, lymphoproliferative disorders

PBMCs, peripheral blood mononuclear cells

PJP, pneumocystis jirovecii pneumonia

PICAR, Primary Immunode�ciency Care And Research

PIDs, primary immunode�ciency diseases

RAPID, Resource of Asian Primary Immunode�ciency Diseases

RT-PCR, reverse transcription polymerase chain reaction ampli�cation

SNP, single nucleotide polymorphism

Tfh, T follicular helper cells

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WGS, whole gene sequencing

XIAP, X-linked inhibitor of apoptosis

ZAP70, ZETA-associated protein, 70-KD

DeclarationsACKNOWLEGEMENTS

The authors thank all of the patients and their families for their kind cooperation and their physicians for thereferrals. This study received grants from Chang-Gung Medical Research Progress (grants CMRPG3K0261 andCMRPG3K2231), National Science Council (grants MOST 104-2663-B-182A-005 and NMRPG3C6071; MOST 109-2314-B-182A-109 and NMRPG3K0361). Ministry of Health and Welfare (PMRPG3H0051) and the TaiwanFoundation for Rare Disorders (TFRD).

CONFLICT OF INTEREST

The authors declared that they have no con�ict of interest.

AUTHOR CONTRIBUTATIONS

LWI designed the study and organized the team; LWF, LYF, TSF and LWI performed the immunologic assessmentsand genetic analysis; LWF, LWI, CSH, HMH, JTH and HJL took care of the patients; WRC studied the pathology.

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2020;11:831.

TablesTable 1. Identi�ed pathogens in 43^ symptomatic patients with ZAP70 mutations 

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Pathogens and symptoms Patient number

Respiratory  

Recurrent sinopulmonary infections (non-mentioned pathogens) 18

Pneumocystis jirovecii pneumonia (PJP) 7

Cytomegalovirus (CMV) 5

Parain�uenza 3

Klebsiella pneumonia 2

Hemophilia in�uenza 1

Staphylococcus aureus 1

Mycobacterial tuberculosis (TB) pneumonia 1

Respiratory syncytial virus (RSV) 3

Adenovirus 2

In�uenza 1

Rhinovirus 1

Bronchiectasis 2

Mucocutaneous  

Oral candidiasis 9

Varicella 4

Papilloma (warts) 2

HSV stomatitis 1

Molluscum contagiosum 1

Epidermodysplasia verruciform-like lesions (HHV-23) 1

Gastrointestinal  

Recurrent infectious gastroenteritis (non-mentioned pathogens) 2

Norovirus 3

Rotavirus 2

Adenovirus 1

Coronavirus 1

Salmonella 1

Vaccine-related  

Disseminated BCG 3

Disseminated measles vaccine 1

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Neurotic  

Cryptococcal meningitis 1

Cerebellitis 1

Viral encephalitis 1

Polyomavirus John-Cunningham (JC) 1

Kidney  

BK virus 1

^Two patients diagnosed prenatally were asymptomatic and then received stem cell transplantation.

Table 2. The phenotype of immune dysregulation in 43^ symptomatic patients with ZAP70 mutations 

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Symptoms Patient number

Gastrointestinal 18      

In�ammatory bowel disease (IBD)-like chronic diarrhea   12    

Bloody diarrhea (hematochezia)   2    

Oral or perianal ulcer   2    

Hepatitis (cholangitis and anti-Sm 1:40 each)   2    

Cutaneous 15      

Eczema   7    

Follicular lesion   2    

Erythroderma   2    

Bullous pemphigoid   2    

Exfoliative dermatitis   1    

Ichthyosiform   1    

Lymphocytic 9      

Hepatosplenomegaly   3    

Lymphadenopathy   2    

EBV associated diffuse large B-cell lymphoma (DLBCL)   1    

EBV lymphoproliferative disorder   3    

Hematologic 6      

Hemolytic anemia   2    

Idiopathic thrombotic purpura (ITP)   2    

Hemophagocytic lymphohistiocytosis (HLH)   1    

Acquired hemophilia VIII autoantibodies   1    

Renal 4      

Nephrotic syndrome   3    

IgA nephropathy   1    

Cardiovascular 3      

Silent brain infarction   2    

Persistent hypertension   1    

^Two patients with early diagnosis after-birth were asymptomatic and then received stem cell transplantation.

 

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Table 3. The onset age, published age, phenotype, genotype and mortality in 43^ symptomatic patients withZAP70 mutations 

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Phenotype   Opportunistic Infection (Oi), Immune dysregulation (Id), and Failure to thrive(Ft)

Sub-phenotype Total Oi Oi-Id Oi-Id-Ft Oi-Ft Id Id-Ft

  43^ 15 12 6 2 7 1

Genotype   Splicing(15)Missense(11)

Deletion(4)

Missense(21)

Insertion(2)Splicing(1)

Splicing (8)Missense (2)Undetectable(2)

Missense(4)

Missense(9)Deletion(3)

Splicing(1)

Nonsense(1)

Splicing(2)

Onset mean age(months, range)

  5.400 ±0.7491

(1M-12M)

3.836 ±0.7949

(1D-5M)

8.333 ±2.418 (1M-6M)

5.500 ±2.500(3M, 8M)

7.571 ±2.671 (1-21M)

27M

Published age(months, range)

  66.07 ±26.13

(2M-31Y)

27.58 ±8.723

(6M-9Y)

150.7 ±62.84

(7M-33Y)

14.00 ±1.00

(7M,15M)

25.21 ±10.29

(8M-7Y)

3Y

Mortality 11 3 4 0 2 2 0

Causes (age; M:months; Y: years)

             

Sepsis-like 3 1 (2M) 2(8M;12M)

       

CMV pneumonitis 2   2 (6M;1Y)

       

Viral encephalitis 1 1 (16M)          

Disseminatedmeasles vaccineinfection

1 1 (8M)          

Acute RespiratoryDistress Syndrome

1       1 (15M)    

Hemophagocytic-Lymphohistiocytosis-like

1       1 (13M)    

EBV-associatedlymphoproliferativedisorder

1         1 (27M)  

lymphoma 1         1 (13M)  

^Two patients with early diagnosis after-birth were asymptomatic and then received stem cell transplantation.Thick meant a signi�cant comparison each other.

Page 21/26

Figures

Figure 1

(A) Abdominal CT showing gaseous dilation of bowel loops with �uid retention compatible with enterocolitis. (B)Multiple hyperemia and edematous mucosa with whitish lymphoid-like nodules. (C) Biopsies showed distortion ofcrypt architecture (crypt branching and irregular size and shape), which is a sign of chronicity. (D) A higher powerview of (C); the lamina propria showed edema and an increase in eosinophils with few lymphocytes and plasmacells.

Page 22/26

Figure 2

(A) Representative demonstration of PHA-stimulated lymphocyte proliferation in the patient showed an almostabsent and “standstill” pattern. (B) T follicular help cells (Tfh) were gated as CD4+CXCR5+ lymphocytes. Althoughin similar percentages (9.45% vs. 10.8%), the ratio of polarized Th2 (CXCR3-CCR6- left lower quadrant) plus Th17(CXCR3-CCR6+ right lower quadrant) / Th1 (CXCR3+CCR6- left upper quadrant) in the patient was higher than inthe control (47.9+18.8/26.6 =2.50 vs. 23.2+23.1/40.2=1.05), with the trend to develop autoimmune disorders. (Cand D) At CD19 gating, the patient had more transitional B cells (CD38++IgM++; 51.3% vs. 5.05%) and CD21-lowcells (22.3% vs. 7.14%) than the control. (C and D) For memory cells, the patient had hardly any kind of memory Bcells that contain switched memory (SM: IgD-CD27+ left upper quadrant; 0.12% vs. 20.8%), non-switched memory

Page 23/26

(NSM: IgD+CD27+ right upper quadrant; 1.15% vs. 24.7%) and exhausted memory (ExM: IgD-CD27- left lowerquadrant; 0.175% vs. 7.6%) (E) and decreased T effector memory cells (EM: CD45RO+CCR7- right lower quadrant;23.2% vs. 42.0%) (F). CM means T central memory cells and EMRA means terminally differentiated effectormemory cells re-expressing CD45RA, which is a marker usually found on naive T cells.

Figure 3

In exon 12 of the ZAP70 gene, the 1561th nucleotide G was replaced by A, causing amino acid Asp 521 to besubstituted by Asn. His parents and sibling were all carriers.

Page 24/26

Figure 4

Under TCR activation stimulated by CD3CD28 for 10, 30 and 60 mins, the most obvious augmentation ofdownstream phosphorylation was at 10 mins. Therefore, we decided to detect phosphorylated AKT and ZAP70 at10 mins. Compared to that in the healthy control who had augmentation after CD3CD28 stimulation, the patientwas observed to have obviously decreased ZAP70 expression, downstream phosphorylation of ZAP70 and AKT(all <3%) in CD4 and CD8 gating. Black lines meant ZAP70, phospho-ZAP70 and phosphor-AKT antibodies byFlowJo histogram. Gray-ground area meant respective isotypes. Duplicated experiments were done beforesteroids intervention.

Page 25/26

Figure 5

Flow cytometric analysis of kinetic changes in [Ca2+] after stimulation with CD3/CD28/RaM IgG. CD4+ T cellsfrom the patient (A), carrier (B) and control (C) were either untreated (left panel) or stimulated (right panel) withCD3/CD28/RaM IgG. The �uorescence data of the �uo-4-Ca2+ complex were recorded at the indicated time. (D)Kinetic changes in [Ca2+] in the patient, carrier and control were superimposed. Ca2+ in�ux kinetics calculatedusing FlowJo software based on median �uorescence data and smoothing.

Page 26/26

Figure 6

We de�ned the �rst follow-up day as the initial presentation date of the patients with ZAP70 mutations, and thelast follow-up day and duration according to those reported in the studies. Except two patients diagnosedprenatally were asymptomatic and then received stem cell transplantation, forty-three symptomatic patients withZAP70 mutations reached signi�cant differences by Kaplan-Meier survival analysis whether or not they receivedHSCT (transplantation; p=0.0003), immune dysregulation (immune dysregu. p=0.5268), opportunistic infections(p=0.2440), or failure to thrive (p=0.5212).

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