YF Hui, SY Chan, YL Lau

Aims: Wiskott-Aldrich syndrome (WAS) is a rare primary immunodeficieny. The afflicted patients suffer from thrombocytopenia with small platelets, ezcema, defects of both T and B cell immunity, and increased risk of malignancies. Mutations in WASP gene at Xp 11.23 was found responsible for this genetic disorder. It was found that mutations predominate in the amino-terminal region of the gene. Single base substitutions are the commonest type of mutations, while small insertions! deletions are rare. Our project is aimed at characterizing mutations in 4 Chinese patients with WAS. All of them were treated by bone marrow transfusion (BMT) before this project commenced.

Methods: Genomic DNA of the patients both before and after BMT were analysed by single-stranded conformation polymorphism (SSCP) and PCR-sequencing.

Results: After screening each exon (exon/intron boundary sequences included) by SSCP analysis, exon 10 of Patient CK's pre-BMT DNA was the only DNA fragment shifted abnormally. It was found to be caused by a small deletion of 11 base-pair in the exon. Completely normal exon 10 was shown from his DNA post-BMT by PCR-sequencing. This suggested a complete replacement of his bone marrow with the normal one after transplant, or that the abnormal gene was at a level beyond detection by PCR. The deletion caused frameshift in translation and a premature stop codon at the carboxyl-terminal part of the protein. This kind of deletion was not reported in previous studies from other countries. PCR amplification of exon 3&4 of Patient MW was unsuccessful. It is possible that a small genomic deletion may be responsible for this. SSCP analysis of exons of Patient CS & CP failed to reveal any abnormally shifted exon fragments. Sequencing of their exon 3, which was reported to be the most common mutated exon and contained a mutational hotspot at codon 86, showed absence of mutations.

Conclusion: Our preliminary results suggest the idea of mutational hotspot at codon 86 may be invalid in Chinese WAS patients, and a more diverse mutational pattern is to be expected.