Patient 1, a six-month-old boy, was initially admitted to our hospital in April 2014 with thrombocytopenia, lung infections, bleeding, and ecchymosis. The complete blood count showed the following findings: hemoglobin (Hb), 110 g/L (120–140 g/L), white blood cell (WBC), 6.74 × 109/L (3.5–9.5 × 109/L), platelet (PLT) count, 14 × 109/L (125–350 × 109/L); and mean platelet volume (MPV), 6.9 fl (7.4–11.0 fl). Owing to fever, thrombocytopenia and upper respiratory tract infection, the patient was diagnosed as ITP. After anti-infective therapy with dexamethasone sodium phosphate and γ-globulin, his PLT count rose to 164 × 109/L and respiratory tract infection was under control. The patient was then discharged from the hospital. Two months later, his PLT count decreased to 42 × 109/L, and he experienced recurrent infections, intermittent bleeding, and frequent eczema again. Blood examination showed leukocytosis of 22.82 × 109/L with hematophagocytes in the bone marrow aspirate. The liver function test indicated the following findings: alanine transaminase (ALT), 113.5 U/L (9.0–50.0 U/L), aspartate aminotransferase (AST), 2 U/L (15.0–40.0 U/L), gamma glutamyl transferase (γ-GT), 137 U/L (10.0–60.0 U/L), triglycerides (TG), 2.67 mmol/L (0.56–1.70 mmol/L); cholesterol (CHO), 6.16 mmol/L (3.11–5.20 mmol/L). The results of other laboratory examinations were presented in Table 2.
Patient 2, a younger male cousin of patient 1, presented with the same clinical symptoms at 3 months of age. The laboratory test showed the following results: WBC count, 11.5 × 109/L, Hb, 103 g/L and PLT count, 6 × 109/L. He suffered from thrombocytopenia, intermittent bleeding, and umbilical hemorrhage and was also diagnosed with ITP. The PLT count reached 94 × 109/L after treatment with γ-globulin, dexamethasone sodium phosphate and platelet transfusions. One month later, he was admitted to the hospital because of cough, expiratory dyspnea, diarrhea, and pulmonary hemorrhage. His WBC count was 16.1 × 109/L and PLT count was 44 × 109/L. He experienced frequent infections and died due to a fungal infection at three years of age.
Figure 1 shows the pedigree of the patients’ families. Their family history revealed that their mothers’ brother had presented with thrombocytopenia and died of infections at 3 years of age and that their mothers’ uncle was thrombocytopenic before death. No abnormalities were observed in the other family members. Therefore, the proband and his younger cousin were suspected to have WAS. On the basis of the medical and family history, they were assigned scores of 4 and 5, respectively, using a previously described scoring system [10, 14].
Informed consent was obtained from the patients’ parents, legal guardians and family members before enrollment in the study. This study was approved by the Research Ethics Committee of the Second Hospital of Hebei Medical University. Ethylenediaminetetraacetic acid (EDTA)-anticoagulated venous blood samples (2 ml) were extracted from patients, their parents and carriers for mutation analysis. Heparinized venous blood samples (5 ml) were obtained from patients and carriers in their families for WASp detection.
Flow cytometry and western blot analysis
PBMCs were isolated from the blood samples. Intracellular staining with an anti-WASp monoclonal antibody (mAb) was performed as described previously . Cells were incubated with 0.25 mg/ml purified mouse anti-human WASp mAb (BD pharmingen, Franklin Lakes, NJ, USA) or 0.5 mg/ml isotype-matched control mouse IgG2a mAb (BioLegend, San Diego, CA, USA) and reacted with 1:100 diluted fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG2a (Affinity Biosciences, Jiangsu, China). The samples were analyzed on a BD FACSCanto II (Becton Dickinson, Franklin, Lakes, NJ, USA), using FlowJo V10 software (Becton Dickinson).
Western blotting of WASp and actin in PBMCs was performed using anti-WAS mAb (1:500; BD Pharmingen, Franklin Lakes, NJ, USA) and anti-β-actin control antibodies (1:3000; Servicebio, Wuhan, China), as described previously .
Total genomic DNA was extracted from PBMCs and amplified using PCR . The PCR products were sequenced by the next-generation sequencing to screen for inherited platelet disorders and immunodeficiency diseases . All PCR products were sequenced using NovaSeq 6000 Genetic Analyzer (Illumina, San Diego, CA, USA). Sanger sequencing was performed on the other family members for familial segregation . The forward primer (5′-CTGTCATGAGGCAGGAAGGAC-3′) and reverse primer (5′-CATCTGGATGAGTCTTTGGTTCTG-3′) were designed using Primer Premier 5. Similarity analysis was performed using the NCBI BLAST program (http://www.ncbi.nlm.nih.gov/BLAST/) to identify novel mutations. Sequences were aligned with WAS (NCBI reference sequence: NG_007877.1 and NM_000377.3) coding sequences.
Construction of mutant and wild-type plasmids and cell preparation
We constructed the expressing plasmid pET-01 containing the part-length gDNA of WAS, which included exons 1, 2, and 3 and introns 1 and 2 as the WT. The mutation in pET-01 was constructed by site-direct mutagenesis to generate an exon mutant group and the intron mutation in pET-01 was designed to generate an intron mutant group. Both mutations involved in pET-01 were constructed to generate a combined mutant group to verify the pathogenicity of compound mutations.
Recombinant mutant plasmid was constructed with the Transformer mutagenesis kit (Clonetech, Palo Alto, CA, USA). The sequence between the two unique restriction sites, XhoI and BamHI, of each construct was verified by sequencing. Empty Vector control and recombinant WAS containing the candidate mutation in the four groups were transiently expressed in COS-7 cells (SV40 transformed African green monkey kidney cells, ATCC CRL 1651). Transfection of eukaryotic COS-7 Cells with recombinant vector DNA was performed using the DEAE-dextran method .
cDNA synthesis was performed using the HiFiScript gDNA Removal cDNA Synthesis Kit (Cowin Biotech, Jiangsu, China). RT-qPCR was performed using CFX connect (Bio-Rad). PCR was performed with the following parameters: 1 cycle of 15 min at 95 °C; 40 cycles of 10 s at 95 °C, 30 s at 58 °C and 30 s at 72 °C. Ten mircoliters of PCR product were mixed with the sample buffer including gel dye markers and analyzed on an agarose gel.
Detection of the expression in mutant and wild-type recombinant plasmids in COS-7 cells
PCR products (20 μl of each PCR products including the amplified exons identified on the agarose gel) were digested with the restriction endonucleases XhoI and BamHI. Restricted DNA fragments were cloned into vectors. The inserted exons were amplified using the PCR primers 5′-CTCCAGGACCACGAGAACC-3′ and 5′-CCGTAAAGGCGGATGAAGTA-3′. The PCR products from the five groups were subjected to polyacrylamide gel electrophoresis (three replicates for each group). The cDNA products in the four groups were recovered and directly sequenced. The results were analyzed using Mygenostics software, and mutations were identified compared with the genomic gene of WAS (NG-007877.1) in GenBank's human genome database.