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Korean J Pediatr. 2004 Jul;47(7):726-734. Korean. Original Article.
Hwang DJ , Yang JW , Kim SY , Yi HK , Lee DY , Hwang PH .
Department of Pediatrics, College of Medical School, Chonbuk National University Jeonju, Korea.
Department of Biochemistry, College of Dental School, Chonbuk National University Jeonju, Korea.

OBJECTIVE: A probable diagnosis of Wiskott-Aldrich syndrome(WAS) should be considered in any boy presenting with unusual bleeding, congenital thrombocytopenia and small platelets. The definitive diagnosis of WAS is usually made by the detection of WASP gene mutation or a decrease or absence of the WAS protein(WASP) in blood cells using molecular genetic analysis. However, these methods are too time-consuming and difficult. In this study, therefore, we tried to compare various diagnostic methods and establish the molecular screening steps for the definitive diagnosis of WAS. METHODS: Peripheral blood was drawn from WAS patient with clinical characteristic symptoms, and analyzed with automated complete blood cell count analysis, immunological analysis, and molecular analysis. The morphologic change of WAS patient's blood cell membrane was examined by scanning electron microscopy(SEM). Protein analysis for the expression of WASP protein in PBMC cells was evaluated by FACS and Western immunoblot. Genetic analysis for the detection of a mutation of the WASP gene was performed by polymerase chain reaction-single strand conformational polymorphism analysis(PCR-SSCP) and direct sequencing of PCR products. RESULTS: In addition to microthrombocytopenia, our investigation revealed morphologic defects in WAS lymphocytes by SEM and abnormal mobility shifting in WAS patients by PCR-SSCP. Sequencing the WASP gene detected a specific single base mutation in exon 2, resulting in missense substitution of adenine for guanine 208(G208A, Gly70Arg). FACS and Western immunoblot demonstrated absent expressions of WASP in WAS patients and reduced expression of WASP in carrier when compared with normal controls. CONCLUSION: From these results, we suggested the following diagnostic approaches in patients suspected of having WAS. Protein-based analysis such as FACS and Western immunoblot should be employed as the first line of investigation. The second line genetic analyses should employ second- step approaches with localization of mutation by screening exons, typically by PCR-SSCP, followed by direct sequencing.

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