[Allele-specific polymerase chain reaction and electrophoretic detection in the detection algorithm clinically significant somatic mutations in the gene of calreticulin (calr).]

Item request has been placed! ×
Item request cannot be made. ×
loading   Processing Request
Read More Add to Saved list
  • Additional Information
    • Source:
      Publisher: Meditsina Country of Publication: Russia (Federation) NLM ID: 9432021 Publication Model: Print Cited Medium: Print ISSN: 0869-2084 (Print) Linking ISSN: 08692084 NLM ISO Abbreviation: Klin Lab Diagn Subsets: MEDLINE
    • Publication Information:
      Original Publication: Moskva : "Meditsina", 1992-
    • Subject Terms:
      Electrophoresis* ; Polymerase Chain Reaction*; Calreticulin/*genetics ; Myeloproliferative Disorders/*diagnosis; Algorithms ; Alleles ; DNA Mutational Analysis ; Humans ; Mutation ; Myeloproliferative Disorders/genetics
    • Abstract:
      The detection of somatic mutations in the 9 exon of the calreticulin gene (CALR) is regulated by the clinical recommendations as a diagnostic criterion for chronic Ph-negative myeloproliferative neoplasms (MPN). Some methods of nucleic acids testing are used to identify CALR gene mutations with different requirements for special skills of personnel and expensive equipment. The purpose of this work is to compare the results of the detection of CALR gene mutations in venous blood samples by allele-specific RT-PCR with subsequent electrophoresis, fragment analysis and Sanger- or pyro- sequencing. We used 1284 blood samples of patients with suspected MPN and 20 blood donor samples. Mutations in the CALR gene of the I and II type were identified using PCR-RT with the original primers and TaqMan probes. Also, all samples were tested for mutations in the CALR gene by electrophoretic detection of PCR results in an agarose gel. The use of allele-specific RT-PCR followed by electrophoretic detection made it possible to determine clinically significant mutations in the CALR gene in 81 venous blood samples of JAK2- and MPL-negative patients, including 42 cases of type I mutation, 33 cases of type II mutation and 8 rare CALR mutations. Mutations in the 9 exon of the CALR gene were not detected in any of the 20 blood donor samples or in 121 blood samples of patients with polycythemia vera. In randomly selected 20 negative samples, CALR gene mutations were also not detected using Sanger sequencing. All positive samples were confirmed by fragment analysis, as well as with Sanger- sequencing and pyro- sequencing. The described combined approach to detect mutations of the CALR gene in peripheral blood samples can be used in clinical diagnostic laboratories that have a standard set of equipment for electrophoresis of nucleic acids and a PCR-RT. We also propose a confirmatory test based on the pyrosequencing of DNA using the system of genetic analysis "PyroMark Q24".
      Competing Interests: The authors declare no conflict of interest.
    • Contributed Indexing:
      Keywords: allele-specific RT-PCR; chronic myeloproliferative neoplasms; somatic mutations CALR
    • Accession Number:
      0 (CALR protein, human)
      0 (Calreticulin)
    • Publication Date:
      Date Created: 20190209 Date Completed: 20190722 Latest Revision: 20210503
    • Publication Date:
      20221213
    • Accession Number:
      10.18821/0869-2084-2018-63-8-588-592
    • Accession Number:
      30735328