Glanzmann’s Thrombasthenia Patients with No Mutations in Both the ITGA2B and ITGB3 Genes as Identified by Conformation Sensitive Gel Electrophoresis (CSGE)

Glanzmann’s Thrombasthenia (GT) is an autosomal recessive inherited platelet function disorder that is due to defect platelet aggregation in response to multiple physiologic agonists. The defect may be because of mutations in the genes encoding either ITGA2B or ITGB3 that result in qualitative or quantitative abnormalities of the platelet receptor aIIbb3. A total of 45 unrelated GT patients were analyzed for mutations in all the exons of ITGA2B and ITGB3 genes by a mutation screening technique, Conformation Sensitive Gel Electrophoresis (CSGE). Mutation was identified in 36 out of 45 patients; and in 9 patients no causative gene alterations were identified in both the genes. Only polymorphisms were identified in 5 patients; however, 4 patients did not show any sequence variation in both the genes. Though their mutation status was not identified, their hematological parameters, platelet aggregation, flow cytometry and western blot revealed that they were definite GT (Table 1). Of these 9 patients with no mutations, 5 patients had family history of bleeding; that included death episode, due to prolonged bleeding in all four families and bleeding complication in sibling in one family. The clinical manifestations included epistaxis, Gum bleeding and petechiae in 8 patients and Echymotic spots in 6 patients. The major bleeding complication of gastro- intestinal bleed and eye bleed was seen in 4 patients and 2 patients respectively; one patient had hematuria. Among the 9 patients with no mutation, a blood transfusion was required in 8 patients. Normal values of Prothrombin time, Activated partial thromboplastin time and platelet count in these patients excluded the possibility of other factor deficiencies and thrombocytopenia. Platelet aggregation was absent with all the four agonists in all these patients. Platelet aIIbb3 analysis by flow cytometry classified 5 patients as type I, one as type II and 3 patients as type III GT. Western blot analysis revealed complete absence of both aIIb and b3 in 6 patients. In remaining 3 patients, one had trace amount of aIIb and reduced amount of b3; another had mild amount of b3 with no trace of aIIb, the third patient had abnormal aIIb protein. GT in these 9 patients may be due to defect in a regulatory element affecting the transcription of these two genes or abnormalities in mechanisms that are responsible for post-translational modifications and trafficking of integrin subunits. Moreover, the strategy to detect mutations in these patients was based on CSGE, whose sensitivity is not 100%. Even though the CSGE technique was carefully set up and tested with known mutations, making it unlikely that nucleotide substitutions were missed in these patients, the literature reports that the sensitivity of the CSGE technique is approximately 80%. The above data shows that these patients need to be considered as definite GT and treated based on Clinical, hematological parameters and flow cytometry, even though they did not show mutations. Hematological evaluation protein analysis GT No Age/Sex CG FH Bld Tfn Bleeding Symptoms P/S PC BT (min) CR (%) Platelet aggregation FCM Western blot Polymorphisms ADP ADR AA Collag. Risto. aIIb/b3 aIIb b3 4 15/M N Y Y P, Ep, GB, HU Isolated plt N 8′ 20 Abs Abs Red Abs Red 20.4% AN N IIb c.2188-7C>G IIIa c.1126-30delT IIIa c.1545C>A IIbc.2188-7C>G 5 19/F Y Y Y P, Ep, GB, Mr Isolated plt N >15′ 5 Abs Abs Abs Abs N 0.13% Abs Red IIb c.2187+34_42 del 9bp 6 44/M N Y Y P, ES, Ep, GB, GI Isolated plt N >15′ 0 Abs Abs Abs Abs N 7.72% Red Red Nil 7 12/M N Y Y P, ES, Ep, GB Isolated plt N >15′ 12 Abs Abs Abs Abs N 1.02% Abs Red Nil 8 46/M N Y Y P, ES, Ep, GB, GI Isolated plt N >15′ 0 Abs Abs Abs Abs N 0.27% Abs Abs Nil 13 4/M Y N N P, ES, GB Isolated plt N >15′ 0 Abs Abs Abs Abs N 20% Abs Abs IIbc.2188-7C>G IIbc.2188-47C>T IIb c.1600+100delT IIIa c.2040T>C 21 14/M N N Y ES, Ep, GB, GI, EB Isolated plt N >15′ 0 Abs Abs Abs Abs N 0.2% Abs Abs Nil 22 11m/M Y N Y P, ES, Ep, GI, EB Isolated plt N 9′ 0 Abs Abs Abs Abs N 21.7% Abs Abs IIb c.2188-7C>G IIb c.3063C>T 33 1/F Y N Y P, Ep, GB Isolated N >15′ 0 Abs Abs Abs Abs N 3.5% Abs Abs IIb c.2188-7C>G Table 1: The clinical and hematological parameters along with polymorphisms identified in GT patients with no mutations Note: CG: Consanguinity, Bld Tfn: Blood transfusion, P/S: Peripheral smear, PC: Platelet count, BT: Bleeding time, CR: Clot retraction, FCM: Flow cytometry, ADP: Adenosine 5′-diphosphate, ADR: Adrenaline, AA: Arachidonic acid, Collag: Collagen, Risto: Ristocetin sulphate, P: Purpura, Ep: Epistaxis, GB: Gum bleed, HU: Hematuria, Mr: Menorrhagia, ES: Epistaxis, GI: Gastrointestinal bleed, EB: Eye bleed