title: Screening PCR Versus Sanger Sequencing: Detection of CALR Mutations in Patients With Thrombocytosis.
abstract: BACKGROUND
  Mutations in calreticulin (CALR) have been reported to be key markers in the molecular diagnosis of myeloid proliferative neoplasms. In most previous reports, CALR mutations were analyzed by using Sanger sequencing. Here, we report a new, rapid, and convenient system for screening CALR mutations without sequencing.
  
  
  METHODS
  Eighty-three bone marrow samples were obtained from 81 patients with thrombocytosis. PCR primers were designed to detect wild-type CALR (product: 357 bp) and CALR with type 1 (product: 302 bp) and type 2 mutations (product: 272 bp) in one reaction. The results were confirmed by Sanger sequencing and compared with results from fragment analysis.
  
  
  RESULTS
  The minimum detection limit of the screening PCR was 10 ng for type 1, 1 ng for type 2, and 0.1 ng for cases with both mutations. CALR type 1 and type 2 mutants were detected with screening PCR with a maximal analytical sensitivity of 3.2% and <0.8%, respectively. The screening PCR detected 94.1% (16/17) of mutation cases and showed concordant results with sequencing in the cases of type 1 and type 2 mutations. Sanger sequencing identified one novel mutation (c.1123_1132delinsTGC). Compared with sequencing, the screening PCR showed 94.1% sensitivity, 100.0% specificity, 100.0% positive predictive value, and 98.5% negative predictive value. Compared with fragment analysis, the screening PCR presented 88.9% sensitivity and 100.0% specificity.
  
  
  CONCLUSIONS
  This screening PCR is a rapid, sensitive, and cost-effective method for the detection of major CALR mutations.
authors.affiliation: Department of Laboratory Medicine, Gachon University Gil Medical Center, Incheon, Korea.
authors.forename: Ji Hun
authors.initials: JH
authors.lastname: Jeong
authors.affiliation: Department of Laboratory Medicine, Gachon University Gil Medical Center, Incheon, Korea.
authors.forename: Hwan Tae
authors.initials: HT
authors.lastname: Lee
authors.affiliation: Department of Laboratory Medicine, Gachon University Gil Medical Center, Incheon, Korea.
authors.forename: Ja Young
authors.initials: JY
authors.lastname: Seo
authors.affiliation: Department of Laboratory Medicine, Gachon University Gil Medical Center, Incheon, Korea.
authors.forename: Yiel Hea
authors.initials: YH
authors.lastname: Seo
authors.affiliation: Department of Laboratory Medicine, Gachon University Gil Medical Center, Incheon, Korea.
authors.forename: Kyung Hee
authors.initials: KH
authors.lastname: Kim
authors.affiliation: Department of Laboratory Medicine, Gachon University Gil Medical Center, Incheon, Korea.
authors.forename: Moon Jin
authors.initials: MJ
authors.lastname: Kim
authors.affiliation: Department of Internal Medicine, Gachon University Gil Medical Center, Incheon, Korea.
authors.forename: Jae Hoon
authors.initials: JH
authors.lastname: Lee
authors.affiliation: Department of Internal Medicine, Gachon University Gil Medical Center, Incheon, Korea.
authors.forename: Jinny
authors.initials: J
authors.lastname: Park
authors.affiliation: Department of Internal Medicine, Gachon University Gil Medical Center, Incheon, Korea.
authors.forename: Jun Shik
authors.initials: JS
authors.lastname: Hong
authors.affiliation: Department of Laboratory Medicine, Gachon University Gil Medical Center, Incheon, Korea. pwpark@gilhospital.com.
authors.forename: Pil Whan
authors.initials: PW
authors.lastname: Park
authors.affiliation: Department of Laboratory Medicine, Gachon University Gil Medical Center, Incheon, Korea. jyahn66@naver.com.
authors.forename: Jeong Yeal
authors.initials: JY
authors.lastname: Ahn
mesh_terms: D000328:Adult; D000368:Aged; D001483:Base Sequence; D001853:Bone Marrow / Q000378:metabolism; D037282:Calreticulin / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004252:DNA Mutational Analysis; D005260:Female; D005500:Follow-Up Studies; D005838:Genotype; D006801:Humans; D053614:Janus Kinase 2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008297:Male; D008875:Middle Aged; D009154:Mutation; D009196:Myeloproliferative Disorders / Q000150:complications / Q000175:diagnosis* / Q000235:genetics; D016133:Polymerase Chain Reaction; D013922:Thrombocytosis / Q000150:complications / Q000175:diagnosis*
chemical_list: D037282:Calreticulin; D053614:Janus Kinase 2
keywords: CALR; Fragment analysis; Sanger sequencing; Screening PCR