New Genetic Variation in BCR gene of Major B3a2 Breakpoint BCR-ABL Fusion Gene in Patients with Chronic Myelogenous Leukemia in Yogyakarta, Indonesia

Document Type : Research Articles

Authors

1 Biomedical Science Study Program, Faculty of Medicine, Universitas Gadjah Mada, Yogyakarta, Indonesia.

2 Departement of Histology, Faculty of Medicine, Universitas Sebelas Maret, Surakarta, Indonesia.

3 Departement of Internal Medicine, Faculty of Medicine, Universitas Gadjah Mada, Yogyakarta, Indonesia.

4 Division of Hematology and Medical Oncology, Department of Internal Medicine, Dr Sardjito Hospital, Yogyakarta, Indonesia.

5 Molecular Biology Laboratory, Faculty of Medicine, Universitas Gadjah Mada, Yogyakarta, Indonesia.

6 Medical Study Program, Faculty of Medicine, Universitas Gadjah Mada, Yogyakarta, Indonesia.

7 Departement of Histology and Cell Biology, Faculty of Medicine, Universitas Gadjah Mada, Yogyakarta, Indonesia.

Abstract

 
Background: Polymorphic bases in several exons of the BCR gene have been found in several studies of the BCR-ABL fusion gene . Most of the polymorphisms do not have any implications for the primary structure of the BCR-ABL protein. Nucleotide changes are often located in the area close to the fusion region, and therefore may influence primer annealing. Our previous work failed to amplify 15 of 200 samples from BCR-ABL positive chronic myelogenous leukemia (CML) patients using multiplex PCR, the standard method to detect BCR-ABL transcripts used in our institution. The failure was considered due to problems in primer annealing caused by sequence variations. Sequence analysis of BCR-ABL fusion gene breakpoint types in CML patients has never been hitherto performed in Indonesia. Therefore, the aim of this study was to perform sequence analysis of several samples that did not show amplification using the standard method. Methods: Fifteen samples were qualitatively amplified by two-step PCR using inner primers in the 2nd PCR to determine the breakpoint type of the BCR-ABL fusion gene. The 2nd PCR products were used as templates to perform sequence analysis, and the results were compared to those in genbank. Result: Seven and 5 of 15 samples were confirmed as major b3a2 and major b2a2, respectively. One sample featured a combination of b3a2 and b2a2, and 2 samples a combination of b3a2 and b2a2 with an additional fragment at 500bp. Sequence analysis showed 3 sequence variations in the major b3a2 breakpoint. One had been reported earlier (c.3296T>C) but the others (c.3245C>T and c.3359T>C) were novel. Fragments at 500bp were confirmed as b3a2 and similar sequence b3a2 in genbank. Conclusion: This study found two new genetic variations in the BCR gene in BCR-ABL fusion cases.

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