Analiza novih molekularnih poti in genov povezanih z eritrocitozo

Vičič, Ana (author), Debeljak, Nataša (mentor)

Eritrocitoza je bolezensko stanje, za katero je značilna kronično povišana količina rdečih krvnih celic (eritrocitov) in povišana raven hematokrita ter hemoglobina. Glede na to, kje je motnja, ločimo primarno in sekundarno eritrocitozo, glede na vzrok razvoja bolezni pa pridobljeno in prirojeno. Najpogosteje je eritrocitoza posledica ne-genetskih zunanjih dejavnikov ali drugih kroničnih bolezni, redkeje pa pridobljenih ali prirojenih različic v nukleotidnem zaporedju. Družinska eritrocitoza (ECYT) je redka prirojena hematološka bolezen. Diagnosticiramo jo z analizo nukleotidnega zaporedja devetih genov vključenih v uravnavanje eritropoeze (EPOR, EPO), homeostazo zaznavanja kisika (VHL, EGLN1, EPAS1) in afiniteto vezave kisika na hemoglobin (HBB, HBA1, HBA2 in BPGM). Več kot 60 % bolnikov s sumom na ECYT ostaja neopredeljenih (idiopatska eritrocitoza), saj genetskega ozadja ne moremo pojasniti s trenutno diagnostično metodo. Visok delež neopredeljenih bolnikov kaže, da obstajajo še ne poznani potencialni geni in molekularni mehanizmi, vključeni v razvoj eritrocitoze. V prvem delu naloge smo s pregledom podatkovnih zbirk STRING in Reactome iskali interakcijske partnerje že znanih genov, vpletenih v eritrocitozo. Za vsak na novo opredeljen interakcijski partner smo na podlagi števila interakcij z različnimi tarčnimi geni ocenili njegov potencialen prispevek za razvoj eritrocitoze. Največje število interakcij (4) so imeli GRB2, SOCS3 in JAK2, drugo največje število interakcij (3) pa so imeli EP300, HIF1A, HIF3A, PLCG1, CBL, KIT, STAT5A in PTPN6. V drugem delu smo optimizirali metodo PCR za pomnoževanje in sekvenčno analizo nukleotidnega zaporedja 12. eksona gena HIF1A. Slednjega smo izbrali na podlagi bioinformacijske analize, v kateri je bil HIF1A eden izmed genov z maksimalnim številom eksperimentalno potrjenih interakcij s tarčnimi geni. Pomembna je bila tudi analiza literature, ki je pokazala povezavo med specifičnimi genetskimi različicami v 12. eksonu HIF1A in simptomi eritrocitoze. Optimalna reakcija PCR z izbranim parom začetnih oligonukleotidov (HIF1A-int11F-1 in HIF1A-int12R-1) je bila brez dodatkov, s temperaturo prileganja 65 °C, časom podaljševanja 15 s in 30 cikli. Optimizirana metoda PCR bi lahko bila uporaben dodatek k rutinskemu diagnostičnemu postopku opredelitve ECYT v Sloveniji, ki bi nekoliko povečal sposobnost opredelitve vzroka bolezni in s tem izbor ustreznega zdravljenja.

• Language: Slovenian
• Keywords: Družinska eritrocitoza, idiopatska eritrocitoza, analiza genske interakcije, sekvenčna analiza, HIF1A.
• Work type: Bachelor thesis/paper
• Organization: FKKT - Faculty of Chemistry and Chemical Technology
• Year: 2022
• PID: 20.500.12556/RUL-140538
• COBISS.SI-ID: 130868995
• Publication date in RUL: 15.09.2022

Secondary language

• Language: English
• Title: Analysis of new molecular pathways and genes involved in erythrocytosis

Abstract

Erythrocytosis is a disease with a characteristically high red blood cell count (RBC count), high hematocrit and high levels of hemoglobin. We recognize primary and secondary erythrocytosis according to the location of the defect and acquired and congenital based on its origin. Most commonly, erythrocytosis is due to non-genetic external factors or other chronic illnesses. Rarely it originates form acquired of congenital gene variants. Familial erythrocytosis (ECYT) is a rare hereditary hematological disorder. It is diagnosed by screening nine genes involved in regulation of erythropoiesis (EPOR, EPO), homeostasis of oxygen detection (VHL, EGLN1, EPAS1) and affinity of hemoglobin for oxygen (HBB, HBA1, HBA2 in BPGM). More than 60 % of patients with suspected ECYT remain undiagnosed (idiopathic erythrocytosis), as the current diagnostic method fails to explain its genetic background. This indicates that there are other, not yet identified potential genes and molecular mechanisms with an important role in development of the disease. In the first part of this work, we analysed databases STRING and Reactome in search of interaction partners of already known genes, involved in erythrocytosis. For every newly identified interaction partner we evaluated its potential contribution to development of erythrocytosis based on the number different target genes it interacted with. Genes GRB2, SOCS3 and JAK2 had the maximum number of interactions (4). Genes EP300, HIF1A, HIF3A, PLCG1, CBL, KIT, STAT5A and PTPN6 had the second most interactions (3). In the second part we optimized the PCR method for amplification of the nucleotide sequence of exon 12 in HIF1A gene. We chose this gene based on the previous bioinformatic analysis, where HIF1A was one of the genes with the maximum number of experimentally determined interactions with target genes. In addition, analysis of literature showed that there was a link between specific gene variants of exon 12 HIF1A and symptoms of erythrocytosis. Optimal PCR reaction with a selected pair of oligonucleotide primers (HIF1A-int11F-1 in HIF1A-int12R-1) was without aditional reactants, with 65 °C annealing temperature, 15 s extention time and 30 cycles. The optimized PCR method could be a useful addition to the routine diagnostic procedure in Slovenia, that would enhance our ability to explain and treat this disease.

• Keywords: Familial erythrocytosis, idiopathic erythrocytosis, analysis of gene interactions, sequential analysis, HIF1A.