Analiza mikro RNA, izoliranih iz urinskih zunajceličnih veziklov bolnikov s Fabryjevo boleznijo

Ivanuša, Nuša (author), Trebušak Podkrajšek, Katarina (mentor), Levstek, Tina (comentor)

Fabryjeva bolezen je redka genetska bolezen, za katero je značilna zmanjšana aktivnost encima α-galaktozidaze A, ki vodi v kopičenje substratov encima, kar se odraža v prizadetosti več organskih sistemov. Heterogena klinična slika otežuje hitro diagnostiko, ki pa je zaradi napredovanja bolezni ključnega pomena. Trenutni diagnostični algoritem temelji na merjenju aktivnosti α-galaktozidaze A in analizi gena GLA. Urinski zunajcelični vezikli izvirajo iz ledvic in vsebujejo tovor, med drugimi tudi mikroRNA (miRNA), ki odraža fiziološke in patofiziološke procese v ledvicah. MiRNA iz urinskih zunajceličnih veziklov zato predstavljajo obetaven označevalec razvoja in napredovanja nefropatije pri bolnikih s Fabryjevo boleznijo. Namen magistrske naloge je opredeliti izražanje šestih različnih miRNA, izoliranih iz urinskih zunajceličnih veziklov, in oceniti njihov pomen pri nastanku in napredovanju nefropatije pri bolnikih s Fabryjevo boleznijo. Za prepis RNA v komplementarno DNA bomo uporabili reverzno transkripcijo, sledila bo kvantitativna verižna reakcija s polimerazo za določitev izražanja miRNA z merjenjem pomnoženega produkta v realnem času. V raziskavo je bilo vključenih 33 bolnikov s Fabryjevo boleznijo, od tega devet moških in 24 žensk, in 33 po spolu in starosti ujemajočih se kontrolnih preiskovancev. Pri bolnikih s stabilno ledvično funkcijo, bolnikih z napredujočo nefropatijo in pri kontrolni skupini smo določali izražanje šestih izbranih miRNA (miR-15a-5p, miR-30c-5p, miR-30d-5p, miR-221-3p, miR-365a-3p, let-7f-5p), izoliranih iz urinskih zunajceličnih veziklov. Ugotovili smo statistično značilno razliko v izražanju miR-30d-5p in miR-30c-5p med bolniki z napredujočo nefropatijo in kontrolno skupino ter med bolniki z napredujočo nefropatijo in bolniki s stabilno ledvično funkcijo. Ker imata visoko diagnostično učinkovitost, bi miR-30d-5p in miR-30c-5p lahko bili uporabni za prepoznavanje bolnikov z večjo verjetnostjo za hitrejše napredovanje nefropatije. V prihodnosti bi bilo smiselno iskanje dodatnih označevalcev, katerih izražanje je spremenjeno že pred razvojem nefropatije. To bi omogočilo zgodnejše in s tem učinkovitejše zdravljenje.

• Language: Slovenian
• Keywords: Fabryjeva bolezen, nefropatija, miRNA, urinski zunajcelični vezikli, kvantitativna verižna reakcija s polimerazo z reverzno transkripcijo
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2024
• PID: 20.500.12556/RUL-158188
• Publication date in RUL: 29.05.2024

IVANUŠA, Nuša, 2024, Analiza mikro RNA, izoliranih iz urinskih zunajceličnih veziklov bolnikov s Fabryjevo boleznijo [online]. Master’s thesis. [Accessed 14 April 2025]. Retrieved from: https://repozitorij.uni-lj.si/IzpisGradiva.php?lang=eng&id=158188

Secondary language

• Language: English
• Title: Analysis of microRNAs extracted from urinary extracellular vesicles in patients with Fabry disease

Abstract

Fabry disease is a rare genetic disorder characterized by reduced activity of the enzyme α-galactosidase A, which leads to an accumulation of the enzyme's substrates, resulting in multiple organ systems being affected. Prompt diagnostics, which is crucial due to the progressive development of the disease, is made difficult by the heterogenous clinical picture. The current diagnostics algorithm is based on the measurement of α-galactosidase A activity and GLA gene analysis. Urinary extracellular vesicles originate from the kidneys and contain a cargo, including microRNAs (miRNAs), that reflects physiological and pathophysiological processes in the kidney. MiRNAs from urinary extracellular vesicles therefore present a promising marker for the development and progression of nephropathy in patients with Fabry disease. The aim of this master’s thesis was to characterize the expression of six miRNAs isolated from urinary extracellular vesicles and to assess their relevance in the development and progression of nephropathy in Fabry patients. Reverse transcription was used to transcribe RNA into complementary DNA, followed by quantitative polymerase chain reaction to determine the expression of miRNAs by real-time measurement of the amplified products. The study included 33 Fabry patients, nine males and 24 females, and 33 sex- and age-matched control subjects. The expression of six selected miRNAs (miR-15a-5p, miR-30c-5p, miR-30d-5p, miR-221-3p, miR-365a-3p, let-7f-5p) isolated from urinary extracellular vesicles was determined in Fabry patients and control subjects. We found a statistically significant difference in the expression of miR-30d-5p and miR-30c-5p between patients with progressive nephropathy and control subjects, and between patients with progressive nephropathy and patients with stable kidney functions. Due to a high diagnostic efficiency, miR-30d-5p and miR-30c-5p could be used to identify patients with a higher probability of faster progression of nephropathy. In the future, it would be worthwhile to search for additional markers whose expression is already altered before the development of nephropathy. This would allow treatment to be initiated quickly enough to increase the effectiveness of treatment.

• Keywords: Fabry disease, nephropathy, miRNA, urinary extracellular vesicles, quantitative polymerase chain reaction with reverse transcription