A novel mutation in the transmembrane region of glyco-protein IX associated with Bernard-Soulier syndrome.

We describe here a novel mutation in glycoprotein (GP) IX transmembrane region in a patient with Bernard-Soulier syndrome (BSS). Flow cytometric analysis of the patient's platelets showed that GP Iband GP IX were expressed at decreased levels. Sequence analysis of the gene coding for GP IX revealed a homozygous (G to A) transition at nucleotide 2113, resulting in a Ala 140 (GCC) to Thr (ACC) replacement in the mature peptide, whereas no defects were found in the coding region of the GP Iband GP Ibgene. Allele-specific restriction enzyme analysis using HPYCH4 III revealed that the patient was homozygous and her mother and brother were heterozygous for the defect, and excluded the possibility that the mutation was a polymorphism of GP IX. To clarify the effect of this mutation on the surface expression of the GP Ib/IX complex, we introduced this mutation into the cDNA of GP IX by site-directed mutagenesis and performed in vitro transfection studies with plasmids harboring GP Ib, GP Iband wild-type GP IX or mutant GP IX. Mutant GP IX decreased the surface expression of GP Iband GP IX, whereas both immunostaining and immunoblotting of the transfected Chinese hamster ovary (CHO) cells showed abundant GP Iband GP IX in the cytoplasm of the CHO cells transfected with plasmids harboring GP Ib, GP Iband wild-type GP IX or mutant GP IX These findings indicate that the Ala14-->Thr mutation in the transmembrane region of GP IX does not induce intracellular GP Ib/IX complex degradation, but prevents its insertion in the cytoplasmic membrane of platelets and CHO cells.