| ObjectiveTo explore the clinical, immunological features and molecular analysis of 10 Chinese patients with Wiskott-Aldrich syndrome (WAS).Methods1. Ten patients met the clinical diagnosis criteria of WAS at Children′Hospital,Chongqing Medical Univrsity during 2007 to 2009 were enrolled. Clinical manifestations and scoring of the phenotype were reviewed and analyzed.2. Analysis of the peripheral blood, immunoglobulin test, lymphatic cellular sub-group, lymphocyte transformation test, nitro blue tetrazolium test (NBT), bone marrow examination were performed.3. Ten patients and their relatives′fresh whole blood were collected.4. The patients′peripheral blood mononuclear cells (PBMCs) were isolated by the Ficoll-Hypaque density gradient centrifugation and subjected to scanning electron microscope (SEM) test in Chongqing Medical Univrsity SEM core facility. 5. The patients and parents′PBMCs were isolated. Flow cytometry (FCM) was used to analyze the Wiskott-Aldrich syndrome protein (WASP) expression on PBMCs of ten patients.6. Total RNA extracted from fresh whole blood was used for WASP cDNA sythesis and amplification by reverse transcription polymerase chain reaction (RT-PCR) . Genomic DNA extracted from whole blood were used for the detection of WASP gene.7. WASP cDNA and genomic DNA were amplified with primer pairs designed to span each exon and exon/intron junction. PCR products were purified by TIANGEN DNA gel extraction kit and directly sequenced in forward and reversely. The sequences were subsequently compared with reported WASP sequences in the GeneBank. If a specific mutation was identified in WASP cDNA, the genomic DNA was amplified for WASP gene to confirm.Results1. Most patients had hematorrhea and petechiae as an initial symptom in early lifetime. All 10 patients had the presentation of persistent thrombocytopenia and small platelets, eczema, recurrent infections, presented as classic WAS phenotype. Three patients scored 5 had autoimmune disorders, including autoimmune hemolysis (AIHA) in two and retinoblastoma (RB) in one patient. Three patients were scored 4 due to severe eczema and infections, four were scored 3. 2. The clinical diagnosis was made earlier in patients with family history (9 months) than those without family history (12.9 months).3. Most patients showed elevated IgA (8/9) ,IgE (8/9) and IgG (7/9). Six patients showed decreased CD4+T lymphocyte (6/9) and other patients had normal lymphatic cellular sub-group. The function of T lymphocyte proliferation decreased in one patient(1/3), two patients (2/5) had a decrease of NBT.4. The bone marrow examinations of six patients varied.5. Five WAS patients had typical SEM anomalies on lymphocytes.6. Deficient WASP expressions were distinctly demonstrated in ten patients with WAS.7. Nine of the 10 patients had 7 different mutations including 4 nonsense mutations (155 C>T, R 41 X in 2 cases; 665 C>T,R 211 X in 2 cases), 2 missense mutations (*168 C>A, T 45 K;1487G>A,D485N/5′splice site), a deletion mutation (*747-748 del T, I238 Fs X260), a splice anomaly (Ivs9 +2 T>C) and a insertion mutation (*253 Ins A, C73X). Eight mutations located in coding region, one in noncoding region. Three novel mutations were identified marked with (*), four of seven mutations located in EVH1, two in BR, one located in VCA and GBD, respectively. Eight of nine mothers were analyzed and all of them were carriers.8. As well as prophylactic antibiotics and intravenous immunoglobulin infusion(IVIG), three patients received hematopoietic stem cell transplantation (HSCT) and one died from cytomegalovirus (CMV) pneumonitis.ConclusionsMale patients presenting with hematorrhea, early-onset thrombocytopenia associated with small platelets should be suspected WAS. Flow cytometric analysis of the WASP expression in PBMCs and further WASP gene sequence analysis are the key to prompt confirmed diagnosis and finding of carriers. HSCT is currently the mainstay of treatment for WAS, especially those with higher severity score. |
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