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BRAF V600 mutations in Langerhans cell histiocytosis with a simple and unique assay.

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  • معلومة اضافية
    • المصدر:
      Publisher: BioMed Central Country of Publication: England NLM ID: 101251558 Publication Model: Electronic Cited Medium: Internet ISSN: 1746-1596 (Electronic) Linking ISSN: 17461596 NLM ISO Abbreviation: Diagn Pathol Subsets: MEDLINE
    • بيانات النشر:
      Original Publication: [London] : BioMed Central, 2006-
    • الموضوع:
      Mutation* ; Polymerase Chain Reaction*; DNA Mutational Analysis/*methods ; Histiocytosis, Langerhans-Cell/*genetics ; Proto-Oncogene Proteins B-raf/*genetics; Adolescent ; Antigens, CD1/analysis ; Base Sequence ; Case-Control Studies ; Cell Line ; Child ; Child, Preschool ; Female ; Fixatives ; Formaldehyde ; Genetic Markers ; Genetic Predisposition to Disease ; Histiocytosis, Langerhans-Cell/diagnosis ; Histiocytosis, Langerhans-Cell/enzymology ; Humans ; Immunohistochemistry ; Infant ; Infant, Newborn ; Male ; Molecular Sequence Data ; Paraffin Embedding ; Phenotype ; Predictive Value of Tests ; Prognosis ; Reproducibility of Results ; Tissue Fixation
    • نبذة مختصرة :
      Background: BRAF (V-raf murine sarcoma viral oncogene homolog B1) is a serine-threonine protein kinase involved in cell survival, proliferation, and differentiation. The most common missense mutation of BRAF (mainly V600E) contributes to the incidence of various cancers, including Langerhans cell histiocytosis (LCH). BRAF inhibitors molecularly targeting the V600E mutation have been developed to counteract the effect of the mutation. To ensure the administration of effective pharmacotherapy, it is therefore imperative to develop an effective assay to screen LCH patients for the V600E mutation. However, tumor tissues of LCH typically contain many inflammatory cells which make a correct judgement of the mutation status difficult in the DNA sequence analysis.
      Results: In this study, we present a new, highly sensitive analyzing method combining PCR, restriction enzyme digestion, and a sequencing assay using DNA extracted from formalin-fixed paraffin-embedded (FFPE) tissue specimens. TspRI is a restriction enzyme that cleaves the sequence encompassing the wild-type BRAF codon 600 into two fragments, which cannot be used as a template for subsequent BRAF PCR amplification. We therefore evaluated the sensitivity of BRAF V600 mutation detection by amplifying the primary PCR product digested with TspRI and sequencing the secondary PCR products. The V600E mutation was detected in FFPE tissue samples from 32 LCH patients; our assay was able to identify mutations in four samples that gave inconclusive results, and ten that were negative, according to standard PCR and sequencing.
      Conclusions: We presented a new and highly sensitive method to detect BRAF V600 mutations. This screening method is expected to play an important role to select the most effective therapies.
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    • Contributed Indexing:
      Keywords: Formalin-fixed paraffin-embedded (FFPE); Langerhans cell histiiocytosis (LCH); TspRI; V-raf murine sarcoma viral oncogene homolog B1 (BRAF)
    • الرقم المعرف:
      0 (Antigens, CD1)
      0 (CD1a antigen)
      0 (Fixatives)
      0 (Genetic Markers)
      1HG84L3525 (Formaldehyde)
      EC 2.7.11.1 (BRAF protein, human)
      EC 2.7.11.1 (Proto-Oncogene Proteins B-raf)
    • الموضوع:
      Date Created: 20160421 Date Completed: 20161012 Latest Revision: 20220331
    • الموضوع:
      20240628
    • الرقم المعرف:
      PMC4837618
    • الرقم المعرف:
      10.1186/s13000-016-0489-z
    • الرقم المعرف:
      27094161