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

• Jeong JH
• Lee HT
• Seo JY
• Seo YH
• Kim KH
• Kim MJ
• Lee JH
• Park J
• Hong JS
• Park PW
• Ahn JY

• KMID: 2373547
• DOI: http://doi.org/10.3343/alm.2016.36.4.291

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

MeSH Terms

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
Calreticulin
Janus Kinase 2

Figure

• 

  Fig. 1 Screening PCR design and results. (A) Three primers were designed for the detection of wild-type CALR (product: 357 bp), CALR type 1 mutation (product: 302 bp), or CALR type 2 mutation (product: 272 bp) in one reaction. The different product sizes, depending on mutant types, are shown in (B). In the case of type 2 mutation, the zygosity cannot be classified by gel electrophoresis. (C) Wild-type CALR, type 1, type 2, and both type 1 and type 2 mutations were detected by the PCR. Lane 28; wild-type, homozygous, lane 48; type 1 mutation, heterozygous, lane 62; type 1 and type 2 mutations, heterozygous, lane 45; type 2 mutation, indeterminate.Abbreviations: (A) F1: forward primer 1 (5'-GCA GCA GAG AAA CAA ATG AAG G-3'), F2: forward primer 2 (5'-GCA GAG GAC AAT TGT CGG A-3'), R: reverse primer (5'-AGA GTG GAG GAG GGG AAC AA-3'), (B) Homo: homozygous; Hetero: heterozygous, and (C) M: 100-bp ladder; 28, 48, 62, and 45: sample numbers.

• 

  Fig. 2 Sensitivity of screening PCR in the detection of CALR mutations. (A) The minimal quantity of genomic DNA required for the reaction was approximately 10 ng for the type 1 mutation, 1 ng for the type 2 mutation, and <0.1 ng for both mutations. (B) Sensitivities in cases with different tumor burdens (50% mutant, 25% mutant, 12.5% mutant, 6.3% mutant, 3.2% mutant, 1.6% mutant, and 0.8% mutant) were determined by using reference sequences. Screening PCR can detect CALR type 1 and type 2 mutants with a maximal sensitivity of 3.2% and <0.8%, respectively, (C) compared with 6.3% or higher sensitivity by Sanger sequencing.

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