Mutation Screening of GPIIb and GPIIIa Exons by Conformation Sensitive Gel Electrophoresis

• Published in: Blood 2007-11, Vol.110 (11), p.3218-3218
• Main Authors: Kannan, Meganathan, Ahmad, Firdos, Yadav, Birendra K., Kumar, Rajive, Saxena, Renu
• Format: Article
• Language: English
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood.V110.11.3218.3218

Glanzmann's Thrombasthenia (GT) is an inherited platelet function disorder characterized by a severe reduction in, or absence of, platelet aggregation in response to multiple physiologic agonists due to abnormalities of platelet glycoprotein GPIIb and / or GPIIIa. The clinical complications in GT include life long bleeding with easy bruising, epistaxis, menorrhagia and gastrointestinal bleeding. GT is caused by mutations in the genes encoding GPIIb or GPIIIa that result in qualitative or quantitative abnormalities of the platelet membrane proteins. For the detection of mutations affecting GPIIb and GPIIIa genes, screening method like single stranded conformation polymorphism (SSCP) is being used in most of the developing countries like India, but is a less sensitive technique. The current study was done to screen for mutations in GT patients using conformation sensitive gel electrophoresis (CSGE), an enhanced method of mutation screening. The work was carried out on 45 GT patients that included 25 males and 20 females with an age ranging from 5 months to 46 years. GT was diagnosed in these patients on the basis of clinical and hematological parameters. Platelet receptor GPIIb/IIIa protein was analyzed first by flow cytometry followed by western blot. Flow cytometry allowed the characterization of GT in to types I, II and III and western blot allowed measuring the presence of aIIb and b3 protein in the platelet lysates. Platelet GPIIb/IIIa analysis by flow cytometry revealed 62.2%, 15.5% and 22.2% of GT to have types I, II and III respectively. Western blot showed the absent or reduced amount of aIIb or b3 protein. Nucleotide changes in either of GPIIb or GPIIIa gene was screened first by CSGE followed by sequencing of defective exons. A total of 25 exons of GPIIb and 12 exons of GPIIIa showed band sift by CSGE. 5 out of 30 exons (Exons 2, 3, 7, 27 and 29) of aIIb gene and 3 out of 15 exons (Exons 4, 8 and 11) of GPIIIa gene showed no band shift by CSGE. Sequencing revealed mutations in 36 of 45 (80%) unrelated GT patients. Of these, 22 patients (48.8%) showed defect in aIIb gene and 14 patients (31.1%) showed defect in b3 gene. In 9 out of 45 (20%) GT patients, no gene alterations were identified and these patients though their mutation status were not identified, their hematological tests including platelet aggregation and flow cytometry revealed that they were definite GT. GPIIb gene was the most common defective gene identified in GT patients in India.