title: Validation of a molecular diagnostic assay for CALR exon 9 indels in myeloproliferative neoplasms: identification of coexisting JAK2 and CALR mutations and a novel 9 bp deletion in CALR.
abstract: BACKGROUND
  The 2008 WHO criteria for the diagnosis and classification of myeloproliferative neoplasms (MPN) rely in part upon the assessment of mutations in JAK2 and MPL genes. Recently, mutations in calreticulin (CALR) have been identified in MPN lacking JAK2 and MPL mutations. We have validated a sensitive fragment analysis assay to detect CALR mutations.
  
  
  METHODS
  Genomic DNA from peripheral blood, bone marrow, and FFPE bone marrow clot preparations from 52 MPN specimens with known JAK2 and MPL mutation status and 29 non-MPN specimens was analyzed. CALR mutation testing was performed by fragment length analysis, and the results were confirmed by sequencing. Accuracy, precision, sensitivity, specificity, and robustness of the assay were determined.
  
  
  RESULTS
  Forty specimens (32 JAK2+, 2 JAK2-/MPL+, and 6 JAK2-/MPL-) were negative for CALR mutations. Twelve specimens had CALR mutations including 52 bp deletion (5), 5 bp insertion (6), and a novel 9 bp deletion (1). This 9 bp inframe deletion occurring at an allelic burden of 50% would delete three amino acids. One specimen with a 52 bp deletion also had JAK2 V617F mutation. All 29 non-MPN specimens were negative for CALR mutations. The assay accurately identified the mutation status of all 52 MPN specimens and had a coefficient of variation <3% for the fragment size and mutant peaks with a sensitivity of 5% for indels.
  
  
  CONCLUSIONS
  Fragment analysis is an accurate and sensitive method for the detection of CALR indels. The novel 9 bp deletion is likely a germline variant. Consequence of coexisting JAK2 V617F and CALR mutations requires careful interpretation.
authors.affiliation: Robert J. Tomsich Pathology and Laboratory Medicine Institute, Cleveland Clinic, Cleveland, OH, 44195, USA.
authors.forename: G
authors.initials: G
authors.lastname: Murugesan
authors.affiliation: Robert J. Tomsich Pathology and Laboratory Medicine Institute, Cleveland Clinic, Cleveland, OH, 44195, USA.
authors.forename: J
authors.initials: J
authors.lastname: Guenther-Johnson
authors.affiliation: Robert J. Tomsich Pathology and Laboratory Medicine Institute, Cleveland Clinic, Cleveland, OH, 44195, USA.
authors.forename: F
authors.initials: F
authors.lastname: Mularo
authors.affiliation: Robert J. Tomsich Pathology and Laboratory Medicine Institute, Cleveland Clinic, Cleveland, OH, 44195, USA.
authors.forename: J R
authors.initials: JR
authors.lastname: Cook
authors.affiliation: Robert J. Tomsich Pathology and Laboratory Medicine Institute, Cleveland Clinic, Cleveland, OH, 44195, USA.
authors.forename: T M
authors.initials: TM
authors.lastname: Daly
mesh_terms: D019943:Amino Acid Substitution; D001483:Base Sequence; D037282:Calreticulin / Q000235:genetics*; D005091:Exons; D005260:Female; D019337:Hematologic Neoplasms / Q000235:genetics*; D006801:Humans; D053614:Janus Kinase 2 / Q000235:genetics*; D008297:Male; D020125:Mutation, Missense; D009196:Myeloproliferative Disorders / Q000235:genetics*; D017384:Sequence Deletion
chemical_list: C576483:CALR protein, human; D037282:Calreticulin; C507924:JAK2 protein, human; D053614:Janus Kinase 2
keywords: Calreticulin; JAK2; coexisting; diagnos tic; myeloproliferative neoplasms; validation