Use of CSGE, TspRI- RFLP and Western Blot in Carrier Detection in an Indian Family with Type I Glanzmann Thrombasthenia.

Glanzmann Thrombasthenia (GT) is an inherited, autosomal recessive, bleeding disorder which is characterized by absent/reduced platelet Glycoprotein IIb/IIIa. The sub classification of GT into Types I, II and III is based on the levels of GPIIb/IIIa by flow cytometry. Type I is the most severe form of GT and is found to be most common in north Indian population. Since not much study is available on carrier detection based on western blot analysis, it is suggested to confirm the defect in carriers by molecular diagnosis. Here we present a carrier status using TspRI in a family with Glanzmann Thrombasthenia patient. Glanzmann Thrombasthenia was diagnosed in a patient with bleeding manifestations accompanied by absent platelet aggregation, secondary to ADP, ADR, Arachidonic acid and collagen. The patient was sub typed as Type I based on flow cytometry analysis as he had absent GPIIb/IIIa. Patient’s DNA was analyzed for mutation in both the gpIIb and gpIIIa genes by CSGE, followed by sequencing. The patient was found to have mutation, CTG>CCG at exon 12 of GPIIb gene. The mutation caused amino acid change from Leu to Pro in the GPIIb protein. The same mutation was looked for in all the family members (Both parents and two siblings) using CSGE and by TspRI- RFLP analysis. Both the parents and siblings were heterozygous for this mutation, where as patient was homozygous (Fig 1). As this mutation is not present in the normal individuals and is not reported earlier, this considers being a novel mutation. Presence of abnormal protein in the family members was revealed by western blot analysis for GPIIb (Fig 2). The same mutation is being looked for in more number of patients with Glanzmann Thrombasthenia using TspRI- RFLP. So far, a total of two out of 25 GT patients found to carry this mutation. It is possible that abnormal GPIIb protein by western blot in family members may reflect their carrier status. It is also postulated that western blot and CSGE of GPIIb and IIIa in parents/siblings may detect carrier status in Glanzmann Thrombasthenia.

Fig 1:

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Carrier detection by restriction digestion using TspRI

Fig 1:

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Carrier detection by restriction digestion using TspRI

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Fig 2:

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Immunoblot followed by chemiluminescent detection shows absent/reduced protein in patient and abnormal band pattern in the family members

Fig 2:

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Immunoblot followed by chemiluminescent detection shows absent/reduced protein in patient and abnormal band pattern in the family members

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