Bernard-Soulier Syndrome Caused by a Dinucleotide Deletion and Reading Frameshift in the Region Encoding the Glycoprotein Iba Transmembrane Domain

We investigated the molecular genetic and biosynthetic badeletion of the last two bases of the codon for Tyr492 (TAT). This mutation causes a frameshift that alters the GP Iba sis of Bernard-Soulier syndrome in a severely affected white woman. Flow cytometric analysis showed a severe defiamino acid sequence, beginning within its transmembrane region. The mutant polypeptide contains 81 novel amino ciency of glycoprotein (GP) Ib, GP IX, and GP V on the surface of her platelets. Similarly, GP Iba was undetectable by imacids and is 38 amino acids shorter than its wild-type counterpart. The new sequence changes the hydrophobic nature munoblot analysis of platelet lysates. Surprisingly, a large quantity of a 70-kD protein (which probably represents a GP of the transmembrane domain and greatly decreases the net positive charge of what had been the cytoplasmic domain. Iba degradation product) was found in the patient’s plasma in much greater quantities than in the plasma of an unafThe deletion mutation was introduced into the GP Iba cDNA, alone and in combination with the 5* mutation, and exfected individual. To analyze the molecular lesion responsible for the disorder, we amplified and sequenced gene segpressed in Chinese hamster ovary (CHO) cells. The deletion alone severely reduced GP Iba expression on the cell surface. ments corresponding to the entire coding regions of the GP Iba, GP Ibb, and GP IX genes. The patient was homozygous Expression was not decreased further by addition of the 5* mutation, confirming that the deletion was the cause of the for a specific GP Iba allele that contained two tandem VNTR repeats in the region encoding the macroglycopeptide (C Bernard-Soulier phenotype. Stable cell lines expressing the mutant polypeptide secreted large amounts of the polypepvariant) and three differences from the published GP Iba gene sequence. Two mutations were unlikely to be involved tide into the medium, suggesting that the mutant anchors poorly in the plasma membrane. Nevertheless, a fraction of in the disorder: the substitution of a single base (T r C) in the second nucleotide of exon 2, which is in the 5* untranslated the mutant was able to associate with GP Ibb, as demonstrated by their coimmunoprecipitation with a GP Ibb antiregion of the GP Iba transcript, and a silent mutation in the third base of the codon for Arg342 (A r G) that does not body. q 1997 by The American Society of Hematology. change the amino acid sequence. The third mutation was a