Screening PCR Versus Sanger Sequencing: Detection of CALR Mutations in Patients With Thrombocytosis.
10.3343/alm.2016.36.4.291
- Author:
Ji Hun JEONG
1
;
Hwan Tae LEE
;
Ja Young SEO
;
Yiel Hea SEO
;
Kyung Hee KIM
;
Moon Jin KIM
;
Jae Hoon LEE
;
Jinny PARK
;
Jun Shik HONG
;
Pil Whan PARK
;
Jeong Yeal AHN
Author Information
1. Department of Laboratory Medicine, Gachon University Gil Medical Center, Incheon, Korea. jyahn66@naver.com, pwpark@gilhospital.com
- Publication Type:Original Article ; Comparative Study
- Keywords:
CALR;
Screening PCR;
Sanger sequencing;
Fragment analysis
- MeSH:
Adult;
Aged;
Base Sequence;
Bone Marrow/metabolism;
Calreticulin/chemistry/*genetics/metabolism;
DNA Mutational Analysis;
Female;
Follow-Up Studies;
Genotype;
Humans;
Janus Kinase 2/chemistry/genetics/metabolism;
Male;
Middle Aged;
Mutation;
Myeloproliferative Disorders/complications/*diagnosis/genetics;
Polymerase Chain Reaction;
Thrombocytosis/complications/*diagnosis
- From:Annals of Laboratory Medicine
2016;36(4):291-299
- CountryRepublic of Korea
- Language:English
-
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.
