Objective: To identify a mutation G2113-->A in the glycoprotein (GP)IX gene associated with Bernard-Soulier syndrome (BSS) and to investigate BSS pathogenesis.
Methods: Allele-specific restriction enzyme was used to analyze the samples of patient, her mother, her brother and 40 healthy volunteers. Site-directed mutagenesis was performed to construct a expression vector PD-IXG2113A harboring the mutation G2113-->A. Chinese hamster ovary (CHO) cells were transiently cotransfected with plasmids harboring the entire coding region of GPIbalpha, GPIbeta and GPIX or mutant GPIX, respectively. Expression of GPIbalpha and GPIX in transfected CHO cells were analysed with flow cytometer. GPIbalpha and GPIX in the cytoplasma of transfected CHO cells were analysed by immunostaining and Western blotting.
Results: The patient was found to be homozygosity of the substitution, her mother and her brother be heterozygous. Expressions of GPIbalpha and GPIX in mutant CHO cells were remarkably reduced, but abundant in the cytoplasma.
Conclusion: The mutation of Ala139(GCC)-->Thr(ACC) in the GPIX did not affect synthesis and assembly of GPIb/IX complex but influence its anchoring and expression on the cell surface, which was responsible for BSS.