Polycythemia rubra vera (PRV) is a myeloproliferative neoplasm characterized by uncontrolled proliferation of the erythroid lineage, most commonly resulting from mutations in the JAK2 gene. In the majority of cases, the JAK2 V617F mutation is present, whereas patients who are negative for this mutation frequently harbor mutations in exon 12 of the JAK2 gene. Although the proportion of patients with exon 12 mutations is relatively small, their identification is of great clinical importance, as it enables confirmation of the diagnosis of PRV and timely initiation of appropriate treatment. The aim of this master thesis was to determine the presence of exon 12 JAK2 mutations in patients with clinical suspicion of PRV who tested negative for the JAK2 V617F mutation, and to evaluate the usefulness of high-resolution melting (HRM) analysis for genotyping exon 12 of the JAK2 gene. A total of 184 samples from patients with suspected PRV who were negative for the JAK2 V617F mutation were included in the study. Following PCR amplification of the target region, HRM analysis was performed to identify samples with altered melting profiles. Samples showing deviation from the reference melting curve were subsequently analyzed by Sanger sequencing. HRM analysis identified seven samples (3.8%) with altered melting profiles among the 184 analyzed samples. Subsequent Sanger sequencing confirmed the presence of exon 12 JAK2 mutations in two samples, representing 28.6% of HRM-positive samples and 1.09% of all analyzed samples. The findings demonstrate that HRM analysis is an effective, rapid, and cost-efficient screening method for genotyping and identifying samples with potential mutations; however, it does not enable definitive mutation characterization on its own. Therefore, HRM results must be confirmed by Sanger sequencing, which allows precise identification of genetic alterations. The combination of both approaches represents a reliable and clinically useful model for the molecular diagnosis of PRV.
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