20.500.12556/RUL-187169 Analiza vloge gena PIEZO1 pri razvoju eritrocitoz Analysis of the role of the PIEZO1 gene in the development of erythrocytosis Eritrocitoza predstavlja patološko stanje, ki ga opredeljuje dolgotrajno povišana masa eritrocitov v krvnem obtoku, kar se odraža v zvišanih vrednostih hemoglobina in hematokrita. Po klasični delitvi jo glede na mesto nastanka ločimo na primarno ali sekundarno, glede na etiologijo pa na pridobljeno oziroma prirojeno. Čeprav so v ozadju večine primerov zunanji dejavniki ali spremljajoče kronične bolezni, je lahko stanje posledica tudi genetskih sprememb. Prirojene oblike, znane kot družinske eritrocitoze (ECYT), v klinični praksi diagnosticiramo z genskim testiranjem devetih genov, vključenih v regulacijo zaznavanja kisika (VHL, EGLN1, EPAS1), signalizacijo zorenja hematopoetskih celic (EPOR in EPO) oziroma prenos kisika (HBB, HBA1, HBA2, BPGM). Vzrok bolezni pri več kot 70 % bolnikov ostaja nepojasnjen, kar nakazuje na obstoj še nepoznanih genov in molekularnih mehanizmov. Med novejšimi kandidatnimi geni ima pomembno vlogo gen PIEZO1, ki kodira mehansko občutljiv ionski kanal, odgovoren za mehanotransdukcijo ter vzdrževanje hidracije, volumna in prožnosti eritrocitov. Začetni del naloge je vključeval pregled različic v literaturi ter bioinformacijsko analizo njihove lege in vpliva na funkcijo proteina. Na podlagi pridobljenih podatkov in predhodnih rezultatov sekvenciranja naslednje generacije bolnikov z idiopatsko eritrocitozo smo izbrali tri tarčne regije gena PIEZO1, ki vključujejo ekson 18, ekson 37 ter eksone 42–44, v namen potrditve prisotnosti različic PIEZO1 pri treh bolnikih. Optimizirali smo pogoje pomnoževanja odsekov z verižno reakcijo s polimerazo. Uspešnost in specifičnost pomnoževanja smo preverili z agarozno gelsko elektroforezo, očiščene produkte pa analizirali s Sangerjevim sekvenciranjem. Pri vseh treh bolnikih smo s Sangerjevim sekvenciranjem potrdili štiri različice, predhodno identificirane z metodo sekvenciranja naslednje generacije: NM_001142864.4 (PIEZO1):c.2344G>A (p.Gly782Ser) in c.2423G>A (p.Arg808Gln) v eksonu 18, c.5195C>T (p.Thr1732Met) v eksonu 37 ter c.6380C>T (p.Thr2127Met) v eksonu 44. Poleg predhodno identificiranih različic smo pri enem bolniku odkrili tudi novo sinonimno različico c.6390G>A (p.Thr2130=) v eksonu 44, ki je bila opredeljena kot benigna. Potrjene različice se nahajajo v strukturno in funkcionalno pomembnih območjih proteina PIEZO1, vključno s transmembranskima enotama in območjem sidra, ki sodelujejo pri mehanski aktivaciji, stabilizaciji in prenosu mehanskih sil. Erythrocytosis is a pathological condition characterized by a persistent increase in red blood cell mass in the circulation, reflected by elevated hemoglobin and hematocrit levels. According to the classical classification, erythrocytosis is divided into primary and secondary forms based on the site of origin, and into acquired and congenital forms according to its etiology. Although most cases are associated with external factors or underlying chronic diseases, the condition may also result from genetic alterations. Congenital forms, known as familial erythrocytosis (ECYT), are diagnosed in clinical practice through genetic testing of nine genes involved in the regulation of oxygen sensing (VHL, EGLN1, EPAS1), hematopoietic cell maturation signaling (EPOR and EPO), or oxygen transport (HBB, HBA1, HBA2, BPGM). The cause of the disease remains unexplained in more than 70% of patients, suggesting the existence of additional, as yet unidentified genes and molecular mechanisms. Among the more recently proposed candidate genes, PIEZO1 has attracted considerable attention. It encodes a mechanosensitive ion channel involved in mechanotransduction and in maintaining red blood cell hydration, volume, and deformability. The initial part of the thesis included a review of variants in the literature and a bioinformatic analysis of their location and impact on protein function. Based on the obtained data and previous next-generation sequencing results from three patients with idiopathic erythrocytosis, three target regions of the PIEZO1 gene were selected, encompassing exon 18, exon 37, and exons 42–44. The conditions for their amplification by polymerase chain reaction were optimized. The efficiency and specificity of amplification were assessed by agarose gel electrophoresis, and the purified products were subsequently analyzed by Sanger sequencing. In all three patients, Sanger sequencing confirmed four variants previously identified by next-generation sequencing: NM_001142864.4 (PIEZO1):c.2344G>A (p.Gly782Ser) and c.2423G>A (p.Arg808Gln) in exon 18, c.5195C>T (p.Thr1732Met) in exon 37, and c.6380C>T (p.Thr2127Met) in exon 44. In addition to the previously identified variants, a novel synonymous variant c.6390G>A (p.Thr2130=) was identified in exon 44 in one patient and was classified as benign. The confirmed variants are located within structurally and functionally important regions of the PIEZO1 protein, including transmembrane units and the anchor region, which are involved in mechanical activation, stabilization, and the transmission of mechanical forces. gen PIEZO1 idiopatska eritrocitoza ionski kanal PIEZO1 gene idiopathic erythrocytosis ion channel true false false Slovenski jezik Angleški jezik Diplomsko delo/naloga 2026-09-09 09:50:01 2026-09-09 09:50:04 2026-10-23 12:08:51 0000-00-00 00:00:00 2026 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 25875 302252291 Zerovnik_Klara_-_Analiza_vloge_gena_PIEZO1_pri_razvoju_eritrocitoz.pdf Zerovnik_Klara_-_Analiza_vloge_gena_PIEZO1_pri_razvoju_eritrocitoz.pdf 1 942A6B059BF522126740B1F5DEA1ECB3 eb63f06e15e07449b632743ea308b162ef842dadc0ac116d6afdb7c56c93c46a 0de224bc-ac23-11f1-8bc5-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=246629 Fakulteta za kemijo in kemijsko tehnologijo 0 0 0