Calreticulin Mutation Survey by High Resolution Melting Method Associated with Unique Presentations in Essential Thrombocythemic Patients
Yi-Chang Liu1,2, Ching-Ping Lee1, Tsung-Jang Yeh1, Yuh-Ching Gau1, Chieh-Yu Hsieh1, Ya-Lun Ke1, Jeng-Shiun Du1, Ming-Hui Lin1, Hui-Ching Wang1, Shih-Hao Tang1, Shih-Feng Cho1,2, Chi-En Hsiao1, Jui-Feng Hsu1, Samuel Yien Hsiao4, Chin-Mu Hsu1,3 and Hui-Hua Hsiao1,2,3.
of Hematology and Oncology, Department of Internal Medicine, Kaohsiung
Medical University Hospital, Kaohsiung, Taiwan.
2 Faculty of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan.
3 Center for Liquid Biopsy, Kaohsiung Medical University, Kaohsiung, Taiwan.
4 University of Rutgers-Camden, New Jersey, USA.
Received: December 27, 2020
Accepted: April 2, 2020
Mediterr J Hematol Infect Dis 2020, 12(1): e2020022 DOI 10.4084/MJHID.2020.022
| This is an Open Access article distributed
under the terms of the Creative Commons Attribution License
(https://creativecommons.org/licenses/by-nc/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
To the Editor,
CALR is a highly conserved endoplasmic reticulum calcium-binding chaperone that is related to calcium homeostasis, cell adhesion, and immune response. Until now, all CALR mutations have been found at exon 9, which encodes the C-domain region where there is a domain for Ca2+ to bind.[5-9,14,15] Of the more than 50 mutations found, all were exclusively on JAK2 V617F-negative patients. Most of the mutations correspond to a 52 kb deletion (del 1092-1143, type 1) or a 5bp insertion (1151 ins TTGTC, type 2), resulting in premature terminations from these frameshift mutations.[7,8] From previous studies, the CALR mutations had a unique clinical presentation compared with JAK2 V617F mutation; therefore, it is essential to survey these mutations in Philadelphia chromosome and/or BCR-ABL negative MPNs.[10-14]
High resolution melting (HRM) method using the saturating dsDNA binding dye for melting curve analysis is a rapid and labor-saving method for mutation screening. In this study, we used the HRM method to screen for these mutations in our ET patients by distinguishing the specific curve types. We also reviewed the clinical presentations of patients to find the relationship between mutations and clinical phenotypes.
Materials and Methods
JAK2 V617F mutation survey. The JAK2 V617F mutation was identified by amplification refractory mutation system (ARMS).
CALR mutation survey. For the HRM survey, the reaction was performed by the ABI ViiA7 machine. Briefly, 20 ng of DNA and 5 μM primers were amplified with the final volume of 20 μl with HRM reaction kit (MeltDoctor HRM Master Mix, Thermo fisher scientific, Waltham, MA, USA). The primer sets were designed (Primer Express® Software 3.0, Thermo fisher scientific, Waltham, MA, USA) with forward: 5’-GGCCTCTCTACAGCTCGTCCTT-3’ and reverse 5’-ACGTCCGTCGTCTCTTTGTT-3’. The amplification conditions are as follows: an initial denaturation step at 95 °C for 10 min, followed by 40 cycles of denaturation at 95 °C for 15 s and annealing/extension at 62 °C for 1 min, with the acquisition of fluorescent signals at the end of each extension step. The fluorescent signals for HRM analysis were detected at 0.2 °C intervals, with hold-time for 10 s, between 95-60 °C. The selected primer set amplified 213 bp amplicon, and results were analyzed as fluorescence versus temperature graphs by software with normalized, temperature-shifting melting cures display as a difference plot.
For sequencing analysis, the DNA samples were carried out polymerase chain reaction (PCR) used with primer sets of forward: 5’-ACAACTTCCTCATCACCAACG-3’ and reverse: 5’-GGCCTCAGTCCAGCCCTG-3’. The amplicons were performed with BigDye Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific, Waltham, MA, USA) by bidirectional sequencing and analyzed by ABI 3730 XL DNA sequencing analyzer (Applied Biosystems, CA, USA). The sequencing results were compared to reference sequences of the CALR gene available on GenBank (www.ncbi.nlm.nih.gov/genbank/) to verify the difference.
Statistical Analysis. Statistical analyses were performed by SPSS software ver.19 for Windows (SPSS Inc., Chicago, IL, USA). The chi-square test was used to test categorical variables, while the Student’s t-test compared continuous variables. A p-value of < 0.05 considered statistically significant.
|Table 1. CALR* mutation types and frequencies in 21 ET# patients.|
The clinical presentation revealed that there was no significant difference over gender between JAK2 and CALR mutations. However, patients with CALR mutations were younger and had a higher platelet count than patients with JAK2 mutation significantly (p<0.05) (Table 2). Patients with JAK2 mutations had significantly higher leukocytes and hemoglobin levels and more thrombotic events than patients with CALR mutations (p<0.05).
|Table 2. Characteristics of patients with different mutations.|
The incidence of CALR mutation in our patients was similar to previous reports. However, the frequency of type 1 mutation was lower, with only half of the mutated patients presenting either type 1 or 2 mutations. The distribution was different from other reports.[7,8,10,13,14,15] Recently, Keaney and Li et al., reported a lower incidence of type 1 mutation in myelofibrosis patients.[10,12] These data might suggest that there is a population difference in CALR mutations and highlight the need for further mutation survey outside type 1 and 2.
HRM system using the saturating dsDNA binding dye for mutation survey and genotyping without the need for costly labeled oligonucleotides is an important step forward for mutation survey. In previous studies with HRM methods, it could identify type 1 and 2 mutations effectively and rapidly[13,15] were also shown in our studies. Our results were able to detect 6 other mutation types concurrently and clearly by the HRM method relating to the CALR mutations even when the mutation is outside type 1 or 2.
Our previous reports showed typical presentations of JAK2 V617F mutation patients, characterized by frequent leukocytosis and thrombosis, here, the clinical presentations of the two groups with either JAK2 or CALR mutation were similar to previous reports.[5,11,14] Patients with JAK2 mutations had a higher WBC count, hemoglobin level, and thrombotic events, while patients with CALR mutations were younger and had a significantly higher platelet count. Some reports also demonstrated different clinical manifestations between type 1 and 2 CALR mutations;[10,14-15] however, due to the small number of studied cases, no differences were shown in this study.
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