20.500.12556/RUL-182636 Analiza mutacij v eksonu 12 gena JAK2 pri bolnikih s sumom na policitemijo rubra vera (PRV) magistrsko delo Analysis of mutations in exon 12 of the JAK2 gene in patients with suspected polycythemia rubra vera (PRV) M. Sc. thesis Policitemija rubra vera (PRV) je mieloproliferativna neoplazma, za katero je značilna nenadzorovana proliferacija eritroidne linije, najpogosteje kot posledica mutacij v genu JAK2. V večini primerov je prisotna mutacija JAK2 V617F, medtem ko so pri bolnikih brez te mutacije pogosto prisotne mutacije v eksonu 12 gena JAK2. Čeprav je delež bolnikov z mutacijami v eksonu 12 relativno majhen, je njihova identifikacija izjemno pomembna, saj omogoča potrditev diagnoze PRV in pravočasno uvedbo ustreznega zdravljenja. Namen magistrskega dela je bil ugotoviti prisotnost mutacij v eksonu 12 gena JAK2 pri bolnikih s kliničnim sumom na PRV, negativnih na mutacijo JAK2 V617F, ter ovrednotiti uporabnost analize talilne krivulje visoke ločljivosti (HRM) za genotipizacijo eksona 12 gena JAK2. V raziskavo smo vključili 184 vzorcev bolnikov s kliničnim sumom na PRV, ki so bili negativni na mutacijo JAK2 V617F. Po pomnoževanju s PCR tarčne regije smo izvedli analizo HRM, na podlagi katere smo identificirali vzorce s spremenjenim talilnim profilom. Vzorce z odstopajočim talilnim profilom smo nato dodatno analizirali s sekvenciranjem po Sangerju. Rezultati so pokazali, da je analiza HRM izmed 184 analiziranih vzorcev identificirala sedem vzorcev (3,8 %) z odstopajočim talilnim profilom. Nadaljnja analiza teh vzorcev s sekvenciranjem po Sangerju je potrdila prisotnost mutacij v eksonu 12 gena JAK2 pri dveh vzorcih, kar predstavlja 28,6 % vseh s HRM izločenih vzorcev in 1,09 % vseh analiziranih vzorcev. Ugotovitve potrjujejo, da HRM predstavlja učinkovito, hitro in stroškovno ugodno presejalno metodo za genotipizacijo in prepoznavanje vzorcev s potencialnimi mutacijami, vendar sama po sebi ne omogoča dokončne identifikacije njihove vrste. Zato moramo rezultate analize HRM potrditi s sekvenciranjem po Sangerju, ki omogoča natančno opredelitev mutacij. Kombinacija obeh pristopov tako predstavlja zanesljiv in klinično uporaben model molekularne diagnostike PRV. 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. policitemija rubra vera ekson 12 gena JAK2 analiza HRM sekvenciranje po Sangerju polycythemia rubra vera JAK2 exon 12 HRM analysis Sanger sequencing true false false M. Bogdan Slovenski jezik Angleški jezik Magistrsko delo/naloga 2026-05-20 07:15:11 2026-05-20 07:15:40 2026-05-21 04:57:08 0000-00-00 00:00:00 2026 0 Ljubljana 2026 Ljubljana 1 spletni vir (1 datoteka PDF (IX, [42] str.)) 0000-00-00 Zaloznikova Objavljeno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 616.155.191:575.224(043.2) 279773 278717955 57442.pdf 57442.pdf 1 49C7E90EEE66FB48818359FBBF6AF0CA 2fc44bd0c70f69612ba81285ebd419ca651c6f1df65f1076916ebc04445efa88 a0ebe2ce-540a-11f1-b0ab-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=234080 Biotehniška fakulteta 0 0 0