Sholikah Tri Agusti, Hutajulu Susanna Hilda, Sulistyawati Dewi, Aning Sumartiningsih, Fatmawati Sri, Syifarahmah Anditta, Widayati Kartika, Kurnianda Johan, Paramita Dewi Kartikawati
Biomedical Science Study Program, Faculty of Medicine, Universitas Gadjah Mada, Yogyakarta, Indonesia.
Departement of Histology, Faculty of Medicine, Universitas Sebelas Maret, Surakarta, Indonesia. Email:
Asian Pac J Cancer Prev. 2017 May 1;18(5):1343-1348. doi: 10.22034/APJCP.2017.18.5.1343.
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.
在多项关于BCR-ABL融合基因的研究中，已在BCR基因的几个外显子中发现多态性碱基。大多数多态性对BCR-ABL蛋白的一级结构没有任何影响。核苷酸变化通常位于靠近融合区域的区域，因此可能影响引物退火。我们之前的工作未能使用多重PCR从200例BCR-ABL阳性慢性髓性白血病（CML）患者样本中扩增出15例，多重PCR是我们机构用于检测BCR-ABL转录本的标准方法。这种失败被认为是由于序列变异导致引物退火出现问题。迄今为止，印度尼西亚从未对CML患者的BCR-ABL融合基因断点类型进行过序列分析。因此，本研究的目的是对使用标准方法未显示扩增的几个样本进行序列分析。
使用15个样本通过两步PCR进行定性扩增，在第二次PCR中使用内部引物来确定BCR-ABL融合基因的断点类型。将第二次PCR产物用作模板进行序列分析，并将结果与基因库中的结果进行比较。
15个样本中分别有7个和5个被确认为主要的b3a2和主要的b2a2。1个样本具有b3a2和b2a2的组合，2个样本具有b3a2和b2a2的组合以及一个500bp处的额外片段。序列分析显示主要b3a2断点处有3个序列变异。其中一个先前已被报道（c.3296T>C），但其他两个（c.3245C>T和c. 3359T>C）是新发现的。500bp处的片段被确认为b3a2且与基因库中的b3a2序列相似。
本研究在BCR-ABL融合病例的BCR基因中发现了两个新的基因变异。
