title: Rapid and sensitive detection of calreticulin type 1 and 2 mutations by real-time quantitative PCR.
abstract: BACKGROUND
  The majority of patients with JAK2 V617F-negative essential thrombocythemia or primary myelofibrosis harbor mutations involving the calreticulin (CALR) gene. These mutations are located in CALR exon 9 and lead to a frameshift with subsequent alteration of the CALR protein C-terminus. They have emerged as valuable molecular markers for the diagnosis of clonal myeloproliferative diseases. Although a variety of CALR mutations have been described, two mutations, denoted type 1 and type 2, account for around 85 % of cases. The type 1 mutation encompasses a 52 bp deletion and the type 2 mutation a 5 bp TTGTC insertion.
  
  
  METHODS
  This work describes the development and testing of quantitative real-time PCRs (qPCRs) for detecting these two mutations.
  
  
  RESULTS
  The final type 1 CALR qPCR displayed a sensitivity of <0.1 % mutant alleles and the type 2 CALR qPCR had a sensitivity of <0.01 % mutant alleles. Additionally, two new CALR mutations are reported.
  
  
  CONCLUSIONS
  These sensitive and specific qPCRs should be helpful in establishing the diagnosis and in monitoring minimal residual disease in patients during or after therapy.
authors: Charité, Med. Klinik für Hämatologie, Onkologie, Tumorimmunologie, Hindenburgdamm 30, 12200, Berlin, Germany.
authors: Michael
authors: M
authors: Zinke
authors: Charité, Med. Klinik für Hämatologie, Onkologie, Tumorimmunologie, Hindenburgdamm 30, 12200, Berlin, Germany.
authors: Vanasa
authors: V
authors: Nageswaran
authors: Max-Planck-Genomzentrum, Cologne, Germany.
authors: Richard
authors: R
authors: Reinhardt
authors: Charité, Med. Klinik für Hämatologie, Onkologie, Tumorimmunologie, Hindenburgdamm 30, 12200, Berlin, Germany. thomas.burmeister@charite.de.|Labor Berlin, Hematology-Tumor Genetics, Berlin, Germany. thomas.burmeister@charite.de.
authors: Thomas
authors: http://orcid.org/0000-0003-4843-2876
authors: T
authors: Burmeister
mesh_terms: D037282:Calreticulin / Q000235:genetics*; D006801:Humans; D053614:Janus Kinase 2 / Q000235:genetics; D016254:Mutagenesis, Insertional; D009154:Mutation; D009196:Myeloproliferative Disorders / Q000175:diagnosis* / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000379:methods; D017384:Sequence Deletion
chemical_list: C576483:CALR protein, human; D037282:Calreticulin; C507924:JAK2 protein, human; D053614:Janus Kinase 2