Andreas Giannopoulos1, Niki Rougkala1,2, Theodoros Loupis1, Marina Mantzourani2, Nora-Athina Viniou2, Eleni Variami2, Theodoros P. Vassilakopoulos3, George Dryllis1,3, Ioannis Kotsianidis4, Theodora Gougopoulou5, Marianna Politou6, Kostas Konstantopoulos3 and George Vassilopoulos1,7.
1 Haematology Research Laboratory, Biomedical Research Foundation, Academy of Athens, Athens, Greece.
2 1st Department of Internal Medicine, Laikon Hospital, School of Medicine, University of Athens, Athens, Greece.
3 Department of Haematology and Bone Marrow Transplantation, Laikon Hospital, School of Medicine, University of Athens, Athens, Greece.
4 Department of Haematology, Democritus University of Thrace, Medical School, Alexandroupolis, Greece.
5 Haematology Clinic, Department of Internal Medicine, Faculty of Medicine, School of Health Sciences, University of Ioannina, Ioannina, Greece.
6 Laboratory of Haematology and Blood Transfusion Unit, Aretaieion Hospital, School of Medicine, University of Athens, Athens, Greece.
7 Department of Haematology, University of Thessaly Medical School, Larissa, Greece.
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Background and Objectives: Somatic mutations in the calreticulin gene (CALR)
are detected in approximately 70% of patients with essential
thrombocythemia (ET) and primary or secondary myelofibrosis (MF),
lacking the JAK2 and MPL mutations. To determine the prevalence of CALR
frameshift mutations in a population of MPN patients of Greek origin,
we developed a rapid low-budget PCR-based assay and screened samples
from 5 tertiary Haematology units. This is a first of its kind report
of the Greek patient population that also disclosed novel CALR mutants.
Material and Methods
|Table 1. Distribution of CALR exon 9 mutations detected in the cohorts of Greek ET and MF patients.|
|Table 2. Common and rare CALR exon 9 mutations detected in the cohorts of Greek ET and MF patients. Registration order according to increasing start site of mutation within the coding sequence. Mutations # 3, 6, 7, 9, 10 and 11 are novel and (as of May 8, 2017) are not included in the COSMIC v85 database.|
|Figure 1. A. HRM-A curves after normalization of temperature and fluorescence data, using the LightCycler 480 SW 1.5.1 software (ROCHE Diagnostics, Indianapolis, IN, US). The two most common CALR exon 9 mutations detected, are Type-1 deletion (L367fs*46) and Type-2 insertion (K385fs*47). These are grouped separately, due to variations in the pattern of their melting curves. Both melting curves differ significantly compared to the normal genotype sample. Each Type-1-like, Type-2-like and complex mutation identified provided a unique melting curve pattern, that diverges from a normal control. B. Agarose gel 3% dense electrophoresis of PCR products. Left to right: line 1, MW marker; line 2, a 34 bp deletion (E364fs*55); line 3, a Type-1 deletion of 52 bp (L367fs*46); line 4, a 37 bp deletion (L367fs*50); line 5, a 34 bp deletion (L367fs*52); line 6, a 31 bp deletion (D373fs*47); line 7, a complex mutation (D373fs*56); line 8, a complex mutation (D373fs*51); line 9, a 19 bp deletion (K375fs*49); line 10, the MW marker; line 11, a complex mutation (K377fs*55); line 12, a 19 bp deletion (K377fs*47); line 13, a complex mutation (E379fs*47); line 14, a 5 bp insertion (E386fs*46); line 15, a Type-2 insertion of 5 bp (K385fs*47); line 16, a normal CALR genotype sample; line 17, a MARIMO cell line DNA sample carrying a 61 bp deletion (L367fs*43) which was initially used as a study reference; line 18, a Non Template Control sample (NTC); line 19 the MW marker. A 100 bp molecular-weight size marker (MW) was used as a DNA ladder (Thermo-Fischer Scientific). C. Sanger sequencing chromatograms for sequence identification of the HRM-A previously detected CALR mutations. i. Top to bottom: a CALR normal genotype sample and a common Type-1 deletion (L367fs*46). ii. Top to bottom: a CALR normal genotype sample and a Type-2 insertion (K385fs*47). D. LoD analysis of the designed HRM-A protocol. A sample harboring a Type-1 deletion mutation (L367fs*46) was used as a reference. Starting at 50% of mutation allele burden, as determined after Sanger sequencing analysis, we ended up detecting as low as 2% of mutant alleles, through serial dilutions.|