MUTATIONS IN THE ALPHA(IIB) AND BETA(3) GENES THAT CAUSE GLANZMANN THROMBASTHENIA CAN BE DISTINGUISHED BY A SIMPLE PROCEDURE USING TRANSFORMED B-LYMPHOCYTES

Glanzmann thrombasthenia (GT) is caused by a defect in either glycopro tein (GP) IIb (alpha(IIB)) or GPIIIa (beta(3)) genes and therefore scr eening of both genes is required for mutation identification. The beta subunit of the GPIIb/IIIa complex (beta(3)) forms a complex with anot her alpha subunit (alpha(v)) yielding the alpha(v) beta(3) vitronectin receptor (VnR), GT patients with mutations in the GPIIIa gene that ca use diminished synthesis of GPIIIa are deficient in both GPIIb/IIIa an d VnR, whereas patients with mutations in the GPIIb gene are deficient in GPIIb/IIIa, yet express normal or increased VnR in their platelets . The presence or absence of VnR in platelet membranes of GT patients has therefore been used for distinguishing between mutations in the GP IIb, gene and mutations in the GPIIIa gene. However, the method of ass essing VnR in platelets is cumbersome and use of fresh platelets is in dispensible. In the present work we devised a procedure for detection of the VnR in B-lymphocytes transformed by Epstein-Bar virus (EBV). Th e transformed lymphocytes transcribed GPIIIa mRNA but not GPIIb mRNA a nd expressed VnR on their surface. Using flow cytometry analysis or im muno-precipitation and western blotting VnR was found in B-lymphocytes of GT patients bearing a well characterized mutation in the GPIIb gen e. In contrast, in B-lymphocytes of GT patients bearing 2 different mu tations in the GPIIIa gene no VnR was detectable. Thus, for determinin g which gene is mutated in a GT patient, EBV-transformed B-lymphocytes are useful and can as well be used for analyses of GPIIIa mRNA and ge nomic DNA. Ten ml of blood are sufficient for the procedure.