Bernard-Soulier syndrome caused by a dinucleotide deletion and reading frameshift in the region encoding the glycoprotein Ib alpha transmembrane domain.

We investigated the molecular genetic and biosynthetic basis of Bernard-Soulier syndrome in a severely affected white woman. Flow cytometric analysis showed a severe deficiency of glycoprotein (GP) Ib, GP IX, and GP V on the surface of her platelets. Similarly, GP Ib alpha was undetectable by immunoblot analysis of platelet lysates. Surprisingly, a large quantity of a 70-kD protein (which probably represents a GP Ib alpha degradation product) was found in the patient's plasma in much greater quantities than in the plasma of an unaffected individual. To analyze the molecular lesion responsible for the disorder, we amplified and sequenced gene segments corresponding to the entire coding regions of the GP Ib alpha, GP Ib beta, and GP IX genes. The patient was homozygous for a specific GP Ib alpha allele that contained two tandem VNTR repeats in the region encoding the macroglycopeptide (C variant) and three differences from the published GP Ib alpha gene sequence. Two mutations were unlikely to be involved in the disorder: the substitution of a single base (T --> C) in the second nucleotide of exon 2, which is in the 5' untranslated region of the GP Ib alpha transcript, and a silent mutation in the third base of the codon for Arg342 (A --> G) that does not change the amino acid sequence. The third mutation was a deletion of the last two bases of the codon for Tyr492 (TAT). This mutation causes a frameshift that alters the GP Ib alpha amino acid sequence, beginning within its transmembrane region. The mutant polypeptide contains 81 novel amino acids and is 38 amino acids shorter than its wild-type counterpart. The new sequence changes the hydrophobic nature of the transmembrane domain and greatly decreases the net positive charge of what had been the cytoplasmic domain. The deletion mutation was introduced into the GP Ib alpha cDNA, alone and in combination with the 5' mutation, and expressed in Chinese hamster ovary (CHO) cells. The deletion alone severely reduced GP Ib alpha expression on the cell surface. Expression was not decreased further by addition of the 5' mutation, confirming that the deletion was the cause of the Bernard-Soulier phenotype. Stable cell lines expressing the mutant polypeptide secreted large amounts of the polypeptide into the medium, suggesting that the mutant anchors poorly in the plasma membrane. Nevertheless, a fraction of the mutant was able to associate with GP Ib beta, as demonstrated by their coimmunoprecipitation with a GP Ib beta antibody.