Ann Lab Med.  2016 Jul;36(4):291-299. 10.3343/alm.2016.36.4.291.

Screening PCR Versus Sanger Sequencing: Detection of CALR Mutations in Patients With Thrombocytosis

Affiliations
  • 1Department of Laboratory Medicine, Gachon University Gil Medical Center, Incheon, Korea. jyahn66@naver.com, pwpark@gilhospital.com
  • 2Department of Internal Medicine, Gachon University Gil Medical Center, Incheon, Korea.

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.

Keyword

CALR; Screening PCR; Sanger sequencing; Fragment analysis
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