Balkan Journal of Medical Genetics

TRANSLOCATION t(3;12)(q26;q21) IN JAK2V617F POINT MUTATION NEGATIVE CHRONIC IDIOPATHIC MYELOFIBROSIS: A CASE REPORT
Mešanović S. Šahović H. Perić M.
*Corresponding Author: Semir Mešanović, Ph.D., University Clinical Center Tuzla, Polyclinic for laboratory diagnostic, Department of Pathology, Trnovac bb, 75000 Tuzla, Bosnia and Hezegovina. Tel.: +387-35-303-509. E-mail: semir.mesanovic@ukctuzla.ba
page: 63

CASE PRESENTATION

with leucopenia, thrombocytosis and anemia was hospitalized at the Department of Hematology, Oncology and Radiotherapy, University Clinical Center Tuzla, Tuzla, Bosnia and Herzegovina. A complete blood cell count revealed: white blood cells (WBC) 2.0 × 109/L, hemoglobin (Hb) 8.8 g/dL, erythrocytes 3.2 × 1012/L and platelets 880.0 × 109/L. The clinical symptom of the patient was diagnosed as suspected chronic MPN. Physical examination was abnormal with a very increased spleen size. A bone marrow biopsy showed myelofibrosis with reticulin grade 3 and a marked increase of small to mediumsized dysplastic CD61 (+) megakaryocytes. The cytogenetic analysis and JAK2V617F mutation testing were undertaken at the Polyclinic for Laboratory Diagnostic, University Clinical Center Tuzla, Tuzla, Bosnia and Herzegovina. Cytogenetic Karyotype Analysis. A bone marrow aspirate was introduced into complete RPMI 1640 medium (Euroclone, Milano, Italy) with 10.0% fetal bovine serum (FBS) (Euroclone) and two different culture methods were used: one culture was harvested on the same day, while the second culture was harvested 24 hours after the first one. After harvesting, chromosomes were prepared and GTG-banded for karyotyping according to standard laboratory protocol. These banded preparations were analyzed on an Olympus BX 51 microscope with digital camera Olympus DP12 (Olympus Corporation, Tokyo, Japan). The description of karyotype followed the recommendations of the International System for Human Cytogenetics Nomenclature, ISCN 2005 [10]. Balanced translocation between 3q and 12q was detected in 18 metaphases, while a normal female karyotype was found in two metaphases. Her karyotype was 46,XX, t(3;12)(q26;q21)[18]/46,XX[2] (Figure 1). Allele - Specific Oligonucleotide - Polymerase Chain Reaction. Genomic DNA was extracted from the whole peripheral blood of the patient using the QIAmp DNA Mini Kit (Qiagen, Hilden, Germany) according to manufacturer’s instructions. Screening for the JAK2V617F point mutation was performed by allele-specific olignonucleotide-polymerase chain reaction (ASO-PCR) on 80 ng DNA prepared from the peripheral blood specimen according to the method of Baxter et al. [5]. The method uses one 1 μM common reverse primer and two 0-5 μM forward primers (Life Technologies, Grand Island, NY, USA) (Table 1). The first forward primer was specific for the mutant allele sequence (203 bp), while the second amplified a 364 bp product that served as an internal PCR control (Figure 2). The patient’s DNA was amplified in a 36-cycle PCR reaction as follows: an initial denaturation step at 94 °C for 11 min., followed by 36 cycles of 30 seconds at 94 °C, 30 seconds at 56 °C, 30 seconds at 72 °C, and a final extension step at 72 °C for 7 min. The PCR products were electrophoresed on 2.0% agarose gel, and the fragments were visualized by ethidium bromide under UV transilluminator. The result of the PCR experiment was the absence of the mutant allele sequence (Figure 2, lane 4). Based on all performed clinical and laboratory tests, especially the bone marrow biopsy, a diagnosis of CIMF was made at that time.

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