ABSTRACT
BACKGROUND: Approximately 15% to 24% of essential thrombocythemia (ET) and 25-35% of primary myelofibrosis cases carry a mutation in the calreticulin (CALR) gene. Sanger sequencing, qPCR, high resolution melt or targeted next generation sequencing usually used to detect these mutations are expensive and require costly equipment. Nevertheless, type 1 CALR mutations are detectable by using polymerase chain reaction (PCR) and agarose gel electrophoresis. AIM: To offer the use of the allele-specific reverse transcription (RT) PCR for rapid low-cost detection of the type 2 mutation in the CALR gene. MATERIALS AND METHODS: Allele-specific primers designed for detecting type 2 mutation (5-bp insertion; c.1154_1155 ins TTGTC) of the CALR gene were used for allele-specific RT-PCR analysis of cDNA of the patient with JAK2-, MPL-negative ET, whose mutation in CALR gene has been identified by Sanger sequencing. RT-PCR samples were analyzed by agarose gel electrophoresis. RESULTS: The type 2 mutation (K385fs*47 ins5) in CALR gene was detected by Sanger sequencing in JAK2- and MPL-negative ET patient. The cDNA obtained was then re-analyzed by using allele-specific RT-PCR with newly designed primers. Normal and type 2 mutation alleles of the CALR gene were detected by gel electrophoresis. The results of allele-specific RT-PCR were consistent with the data of Sanger sequencing. CONCLUSION: Allele-specific RT-PCR analysis may be used for the fast low-cost detection of the major type 2 mutation (ins 5) of the CALR gene in patients with MPNs.
Subject(s)
Myeloproliferative Disorders , Neoplasms , Thrombocythemia, Essential , Alleles , Calreticulin/genetics , DNA, Complementary , Humans , Janus Kinase 2/genetics , Mutation , Myeloproliferative Disorders/diagnosis , Myeloproliferative Disorders/genetics , Neoplasms/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Thrombocythemia, Essential/geneticsABSTRACT
AIM: To obtain polyclonal antibodies against recombinant proteins recognizing Bcr domain and fusion region of Bcr-Abl and analyze the patterns of intracellular distribution of Bcr and Bcr-Abl proteins in K562 cells of chronic myelogenous leukemia. METHODS: The coding sequences of DH and PH domains of Bcr-Abl were cloned, and the recombinant proteins were expressed in E. coli. The rabbit polyclonal antibodies were produced and used for immunocytochemical study of Bcr and Bcr-Abl localization in K562 cells. RESULTS: The gene constructs containing sequences coding for DH and PH domains of Bcr-Abl have been obtained. The antibodies with relative specificity to corresponding recombinant proteins differ by the patterns of their intracellular reactivity with Bcr- and Bcr-Abl related structures. While Bcr protein is located predominantly perinuclearly, antibody against hybrid Bcr-Abl protein is reacted with the structures in cell periphery, namely on cell membranes. CONCLUSION: Antibodies against DH and PH domains of Bcr-Abl react with proteins located differently in chronic myelogenous leukemia cells. The difference in intracellular localization of Bcr and Bcr-Abl may be attributable to the different domains interacting with different multiprotein complexes.
Subject(s)
Leukemia, Myelogenous, Chronic, BCR-ABL Positive/metabolism , Proto-Oncogene Proteins c-bcr/metabolism , Animals , Antibodies , Antibody Specificity , Blotting, Western , Cell Line , Cell Membrane/metabolism , Cytoplasm/metabolism , Fusion Proteins, bcr-abl/chemistry , Fusion Proteins, bcr-abl/immunology , Fusion Proteins, bcr-abl/metabolism , Humans , Immunohistochemistry , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics , Neoplasms , Protein Structure, Tertiary , Proto-Oncogene Proteins c-bcr/chemistry , Proto-Oncogene Proteins c-bcr/immunology , Rabbits , Recombinant Proteins/metabolismABSTRACT
Molecular-biological approaches was applied for detection of Philadelphia chromosome in patients with chronic myeloid and acute lymphoblastic leukemias. It includes bcr-genomic probes and polymerase chain reaction with specific bcr/abl primers. The latter is more preferable for clinic use. Different diagnostic methods are compared and leukemias etiology problems are discussed.
Subject(s)
Leukemia, Myelogenous, Chronic, BCR-ABL Positive/diagnosis , Precursor Cell Lymphoblastic Leukemia-Lymphoma/diagnosis , Adult , Base Sequence , Child , DNA/blood , DNA/genetics , DNA Primers , Fusion Proteins, bcr-abl/genetics , Genes, abl/genetics , Genetic Markers , Humans , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics , Molecular Sequence Data , Philadelphia Chromosome , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , RNA/blood , RNA/geneticsABSTRACT
BspRI and HinfI polymorphism of DNA in the inhabitants of Ukraine was analyzed with probe based on M13 phage. In the region 3 - 8 kb, the mean probabilities of coincidence of to marker bands comprised 0.22 for BspRI polymorphism and 0.34 for HinfI polymorphism. The indexes of similarity by Lee between the parents comprised 0.44 for BspRI polymorphism and 0.45 for HinfI polymorphism. The characteristics obtained may be used for the correct description of DNA fingerprints in the inhabitants of Ukraine.
