This 1-year 4-month-old boy was born to aboriginal parents via cesarean section without any adverse events. He was hospitalized for pneumococcus pneumonia three times from the age of 6 months, and experienced frequent coughing and wheezing thereafter. Respiratory syncytial virus, enterovirus and adenovirus infections caused recurrent wheezing. Neither infective cultures nor reverse transcription polymerase chain reaction amplification (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 normal TREC value (252 copies) on his neonatal Guthrie card. When he was 13 months old, Pseudomonas aeruginosa caused severe colitis, sepsis and hypovolemic shock that met the diagnosis criteria of Shanghai fever.11 Ceftazidime, amikin, ciprofloxacin and hydration rescued his life-threatening infection. A hematologic evaluation showed mild neutropenia (1034/mm3), normal lymphocyte count (3290/mm3), eosinophilia (12%), anemia (Hb 7.6 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 T cell immunodeficiency. Regular immunoglobulin infusions (0.8g/kg per month) and prophylactics for bronchiectasis (25 mg/kg/day Augmentin), PJP (5mg/kg/day Trimethoprim-Sulfamethoxazole) and fungal infections (5 mg/kg/day fluconazole) 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 bilirubin remained normal. Any kinds of antibiotics with the potential for hepatic toxicity were discontinued, and RT-PCR for hepatitis viral load excluded EBV, CMV, HSV, hepatitis A, B, C and E. A liver biopsy did not favor medication-related or viral hepatitis. Liver enzyme levels often returned to normal after monthly immunoglobulin infusion. Mildly elevated 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 and RT-PCR amplification for adenovirus and BK virus were negative. One month later, he had chronic diarrhea and acute exacerbation leading to abdominal distension, ileus, and moderate ascites. Under the impression of sepsis from colonic pathogens (especially previous pseudomonas aeruginosa), hypovolemic shock and severe hematochezia (Fig. 1A) with an Hb level down to 4 g/dL, aggressive antibiotic treatment was given over 2 weeks with a continuous blood transfusion for 7 days. Abdominal CT, angiography and Meckel’s scan did not reveal any significant findings for bleeders. Colonoscopy showed small white nodules of 5 x 5 mm in size protruding into the intestinal lumen (Fig. 1B). Mucosa nodular lymphocyte aggregation and submucosa oozing-like edema have been reported to be a possible prodrome of inflammatory bowel diseases (Fig. 1C and 1D) that resemble intestinal lymphoproliferation in patients with activated phosphoinositide 3-kinase δ syndrome (APDS)12 and common variable immunodeficiency (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-dose prednisolone (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 he was 1 year and 11 months of age. At present, he was 3 years 6 months and free of IVIG infusion and GvHD medication.
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 less 10% of normal was defined as being “absent” function and was considered to be an indication for HSCT. Such profound impairment of lymphocyte proliferation was also demonstrated by a CFSE evaluation of the proliferation 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 Th17/Treg ratio (1.8/0.4 vs. 3.1/4.9 in Supplemental Fig. 2 predominantly ascribed to lower Treg cells) were consistent with those CVID, Wiskott-Aldrich syndrome, or APDS patients who often present with the phenotype of immune dysregulation. For memory cells, memory B cells (non-switched, switched and exhausted) and memory T cells (effector) were obviously diminished (Fig. 2E and 2F) and likely contributed to the increased susceptibility to infections.
Using a candidate gene approach, Sanger sequencing revealed that the 1561st nucleotide G was homozygously replaced by A in exon 12 leading to a missense mutation of Asp 521 Asn in the ZAP70 gene (Fig. 3). We further assessed the effect of 521Asn-ZAP70 on CD3/CD28 downstream signaling, and also utilized WGS to investigate whether a gene other than ZAP70, or even bi-genetic mutations23 were related to his complex phenotype including fluctuating higher levels of liver enzymes, gross hematuria and hematochezia. However, only the same mutation was consistently identified instead of revealing any other responsible novel gene (Supplemental Table 2). His parents and older sibling were all carriers.
ZAP70 expression and intracellular Ca2+ dynamic influx downstream the CD3CD28 signaling
In an in-depth study of the mutant 521Asn-ZAP70 function, we investigated the expressions of phosphorylated translation factors of ZAP and AKT in downstream signaling. Compared to that in the control, the intracellular expression of 521Asn-ZAP70 obviously decreased in CD4 cells and CD8 cells without/with CD3CD28 stimulation for 10 min (Fig. 4). In parallel, the downstream signal expressions of phosphorylated-ZAP70 and -AKT were also decreased (Fig. 4). Thus, such alterations attenuated its binding capacity and signal pathway. Asp521-ZAP70 is an acidic residue which forms hydrogen bonds with H459 and R460 and stabilizes an activation loop, a crucial component of kinase function.24,25 Changes in Asp521Asn can cause a conformational change in the activation loop and impact signaling cascades, compatible with the SIFT predicted scoring system (Score 0 meaning damage, http://sift.jcvi.org/) and PolyPhen2 (Score 1 also meaning damage, http://genetics.bwh.harvard.edu/pph2/index.shtml).
We then conducted calcium influx assays to evaluate the downstream function of ZAP70 in T cell activation. As shown in Figure 5A, calcium influx in the patient’s T cells was nearly undetectable when stimulated. Compared to the patient (Fig. 5A), the results of calcium influx in the carrier was evident (Fig. 5B), even though the influx of maximum calcium concentration was not as high as in the normal control (Fig. 5C). To further compare the differences between these three subjects, kinetic plots of each calcium influx were superimposed to reveal the functional deficit (Fig. 5D). After stimulation, the response time and ascending slope of the calcium influx in the carrier were nearly the same as those in the control. However, the carrier did not reach the same level of calcium concentration as the control, and the descending slope was also significantly faster than that of the control.
Genotype, phenotype and survival analysis
A PubMed search for the key words “Zap70 mutation” and “immunodeficiency” excluding studies which did not identify the gene revealed 45 patients from 35 families, including 33 patients associated with founder-effect or consanguineous 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 were missense mutations in 47, splicing mutations in 31, deletions in 7, insertion in 2, nonsense in 1 allele and undetectable 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 detectable mRNA. 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 immunodeficiency. The identified pathogens over two eventful episodes included candida (9 episodes), PJP (n=7), CMV (n=5), varicella (n=4), parainfluenza (n=3) and BCG vaccine (n=3) that was reflected by their CD8 lymphopenia as they were mainly unable to resist viral pathogens rather than bacteria (Table 1). To compare survival based on their phenotypes, we defined the phenotype of immune dysregulation encompassing autoimmune disorders as the patients who developed inflammatory 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 classified the major phenotypes of these 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 were significantly older than those with the Oi-Id phenotype (p=0.0399; p= 0.0143) because failure to thrive (Ft) often appeared after 3 months old, but not significantly in each-other mutual comparisons (Table 3). The oldest patient was 33 years old at time of writing and presented with the Oi-Id-Ft phenotype.46 In the strict sense, no relationship between genotype and phenotype existed as the recent review.77
For Kaplan-Meier survival analysis, those receiving HSCT showed an obviously and significantly higher survival (p=0.0003) than without, but not in the other comparisons of opportunistic infections (p=0.2240), immune dysregulation (p=0.5268), and failure to thrive (p=0.5215). (Fig. 6).