IDENTIFICATION OF A HOMOZYGOUS SINGLE-BASE PAIR DELETION IN THE GENE CODING FOR THE HUMAN PLATELET GLYCOPROTEIN IB-ALPHA CAUSING BERNARD-SOULIER SYNDROME

Bernard-Soulier Syndrome (BSS) is a hereditary bleeding disorder which is caused by the absence or the dysfunction of the platelet glycoprot ein Ib/IX/V (GP Ib/IX/V) complex, the major receptor for von Willebran d factor (vWf). BSS is characterized by the presence of giant platelet s that show a reduced binding of vWf. Although BSS is a well-character ized disease, and many cases have been described in the literature, th e molecular genetic basis of this disorder has been studied in only a few patients We have studied the genetic basis of the defect in a BSS patient. Flow cytometric analysis of the platelet membrane glycoprotei ns revealed a significant decrease or absence of GP Ib alpha on the pl atelet surface, and low levels of GP V and GP M. In subsequent immunop recipitation experiments, we confirmed the presence of GP V (although in significantly decreased amounts) on the platelet surface. These res ults indicated a defect in the GP Ib alpha chain. Genomic DNA coding f or GP Ib alpha was amplified, using the polymerase chain reaction (PCR ). Subsequent direct sequence analysis demonstrated a homozygous delet ion of T-317 resulting in a frameshift deletion and predicting a subst itution of Arg for Leu(76). This deletion causes a shift in the readin g frame, predicting a premature stop codon after 19 altered amino-acid s, leading to a severily truncated molecule. The molecular genetic def ect found in this patient differed from the mutations observed in thre e other BSS patients described in the literature. This points to a mar ked hetereogeneity of this disease. The single basepair deletion creat ed a target site for the restriction enzyme HhaI. This allowed us to p erform PCR-ASRA (Allele-Specific Restriction enzyme Analysis) on all a vailable family members. Both parents and the daughter of the patient appeared to be heterozygous for the deletion, while the homozygosity o f the propositus for the mutant allele was confirmed.