Development and validation of CALR mutation testing for clinical diagnosis.
OBJECTIVE
To validate a diagnostic assay for detecting CALR mutations in the clinical setting.


METHODS
Traditional polymerase chain reaction (PCR) was performed on DNA previously extracted from 60 specimens (30 bone marrow aspirates [BMAs] and 30 peripheral blood [PB] samples) from 55 patients. Nearly all reported CALR mutations are insertions or deletions in exon 9. Therefore, we performed amplicon sizing by capillary electrophoresis and fragment length analysis (FLA) to determine mutation status. Mutations were confirmed by Sanger sequencing.


RESULTS
Fourteen samples from 10 patients with JAK2 and MPL wild-type myeloproliferative neoplasms were positive for CALR mutation. Detected mutations included a 52-base pair (bp) deletion (n = 6), a 5-bp insertion (n = 2), a 31-bp deletion (n = 1), and a 61-bp deletion (n = 1). Sanger sequencing of 15 samples showed 100% concordance. Matched patient PB and BMA samples (n = 5) harbored identical mutations, and samples run multiple times (n = 8) showed 100% reproducibility.


CONCLUSIONS
We conclude that CALR mutations may be quickly and accurately detected by FLA of PCR amplicons by capillary electrophoresis. These methods are routine procedures for most molecular laboratories and should allow for straightforward incorporation of the CALR assay into the clinical diagnostic testing menu.
From the Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA.
Cheryl L
CL
Maier
From the Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA.
Kevin E
KE
Fisher
From the Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA.
Heather H
HH
Jones
From the Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA.
Charles E
CE
Hill
From the Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA.
Karen P
KP
Mann
From the Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA. linsheng.zhang@emory.edu.
Linsheng
L
Zhang
D019046:Bone Marrow Neoplasms / Q000175:diagnosis* / Q000235:genetics; D037282:Calreticulin / Q000235:genetics*; D004252:DNA Mutational Analysis / Q000379:methods*; D005838:Genotype; D006801:Humans; D009196:Myeloproliferative Disorders / Q000175:diagnosis* / Q000235:genetics; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D037282:Calreticulin
Amplicon sizing; CALR; Clinical assay validation; Fragment length analysis