title: Role of RFLP using TspRI for carrier detection in Glanzmann's thrombasthenia: a report on two families.
abstract: Glanzmann's thrombasthenia (GT) was diagnosed in two patients who presented with bleeding manifestations accompanied by absent platelet aggregation, secondary to adenosine-5'-diphosphate, adrenaline, arachidonic acid and collagen. Flow cytometry analysis for GPIIb/IIIa expression was done using CD61 and CD41 markers in these patients and their family members including siblings. The patients were sub typed as Type I as he had absent glycoproteins (GP) IIb/IIIa. Family studies by flow cytometry showed reduced GPII/IIIa expression in both the parents and one sibling. However, western blot showed abnormal GPIIb protein in all the family members including siblings. It is possible that abnormal GPIIb protein by western blot in family members may reflect their carrier status. Patients' DNA was analyzed for mutation in both the GPIIb and GPIIIa genes by conformational sensitive gel electrophoresis (CSGE), followed by sequencing. CSGE showed defect in exon 12 and the promoter region of GPIIb. By sequence, it was confirmed that both the mutations were homozygous one was c.1028T>C and the other one was M33320.1:g.951G>A. For one of these mutations, restriction fragment length polymorphism (RFLP) was developed to look for the same mutation in all the family members. RFLP was developed using a restriction enzyme (TspRI) against the patient's mutation, c.1028T>C in exon 12 of GPIIb gene. RFLP followed by gel electrophoresis revealed that the mutation was heterozygous in all the family members. The findings by RFLP were double confirmed by direct DNA sequencing of the exon 12 in all the family members. Thus, TspRI marker may be used as a RFLP marker to predict the carriers in GT families, if the patients' mutation status is identified.
authors.affiliation: Department of Hematology, All India Institute of Medical Sciences, New Delhi, India.
authors.forename: M
authors.initials: M
authors.lastname: Kannan
authors.forename: B K
authors.initials: BK
authors.lastname: Yadav
authors.forename: F
authors.initials: F
authors.lastname: Ahmad
authors.forename: R
authors.initials: R
authors.lastname: Saxena
mesh_terms: D004262:DNA Restriction Enzymes; D006580:Genetic Carrier Screening / Q000379:methods*; D006801:Humans; D039421:Integrin alpha2 / Q000235:genetics*; D039661:Integrin beta3 / Q000235:genetics*; D008297:Male; D012150:Polymorphism, Restriction Fragment Length; D013915:Thrombasthenia / Q000175:diagnosis* / Q000235:genetics
chemical_list: C056564:ITGA2B protein, human; C061248:ITGB3 protein, human; D039421:Integrin alpha2; D039661:Integrin beta3; D004262:DNA Restriction Enzymes