Identifikacija in funkcijska karakterizacija patoloških genetskih različic za monogenske bolezni človeka z analizo RNA-prepisov

Jus, Matevž (author), Hodžić, Alenka (mentor), Maver, Aleš (comentor)

Tehnologije sekvenciranja nove generacije omogočajo analizo večjega dela človeškega genoma, vključno z nekodirajočimi področji in predstavljajo pomemben napredek v diagnostiki monogenskih bolezni. Izziv predstavlja karakterizacija najdenih različic kot patoloških ali nepatoloških, opredelitev njihovega učinka ter mehanizma patogenosti. V raziskavi smo analizirali vzorce dveh skupin preiskovancev z nediagnosticiranimi redkimi boleznimi, tiste z ugotovljeno kandidatno različico z nejasnim kliničnim pomenom ter tiste brez ugotovljene različice. Z uporabo metod analize RNA-prepisov smo določili in razjasnili pojav, mehanizem in učinek najdenih patoloških različic, pri preiskovancih brez ugotovljenih različic pa smo v večini identificirali verjetno vzročne različice. Ugotovljenim različicam smo po smernicah American College of Medical Genetics in Association for Clinical Genomic Science opredelili patogenost in določili klinični pomen opredelitve. V vseh primerih je preiskava izboljšala klasifikacijo patogenosti in vzročnosti. V primerih patogenih in verjetno patogenih različic so te nastale kot posledica okvarjenega procesa spajanja eksonov. V štirih primerih smo zaznali pojav preskoka eksonov, v treh primerih smo zaznali premik bralnega okvirja, v po enem primeru pa smo zaznali pojav zadržanja introna ter pojav nastanka novih mest izrezovanj. Z opravljeno analizo RNA-prepisov smo izboljšali funkcijsko karakterizacijo, izboljšali njihovo klasifikacijo v smislu povezave z monogenskimi boleznimi ter razjasnili mehanizme njihovega nastanka. Z analizo izbranih genov smo izboljšali identifikacijo patoloških genetskih različic v nekodirajočih področjih pri bolnikih z nepojasnjenimi monogenskimi boleznimi.

• Language: Slovenian
• Keywords: genetika, monogenske bolezni, patološke različice, RNA-prepisi, cDNA, PCR, sekvenciranje nove generacije, funkcijska karakterizacija
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: BF - Biotechnical Faculty
• Place of publishing: Ljubljana
• Publisher: [M. Jus]
• Year: 2022
• PID: 20.500.12556/RUL-139734
• UDC: 575
• COBISS.SI-ID: 120874243
• Publication date in RUL: 07.09.2022

JUS, Matevž, 2022, Identifikacija in funkcijska karakterizacija patoloških genetskih različic za monogenske bolezni človeka z analizo RNA-prepisov [online]. Master’s thesis. Ljubljana : M. Jus. [Accessed 11 April 2025]. Retrieved from: https://repozitorij.uni-lj.si/IzpisGradiva.php?lang=eng&id=139734

Secondary language

• Language: English
• Title: Identification and functional characterisation of pathogenic genetic variants causing human monogenic conditions with interrogation of RNA splicing alterations using next-generation sequencing approaches

Abstract

Next-generation sequencing represents an important step in diagnostics of monogenic conditions, as it allows the analysis of the majority of the human genome, including non-coding regions. However, characterization of the pathogenic effect of variants identified by these approaches and their functional assessment remain a challenge. The aim of this research is analysis of cases with undiagnosed genetic conditions that harbor genetic variants of uncertain clinical significance, and cases in which conventional genetic approaches identified no causal variants. Using next-generation sequencing based RNA analysis, we explained the pathogenic and functional effects and mechanisms in cases with known suspect variants. Additionally, we were able to identify disease-causing variants in samples with unknown variants. Based on the functional data, we reassessed the pathogenicity of variants according to American College of Medical Genetics and Association for Clinical Genomic Science standards and we found that the clinical classification of pathogenicity has improved in every case. In four cases we confirmed exon skipping, in three cases a frameshift mutation, and one case of each intron retention and novel splice site introduction. In conclusion, using the RNA transcript analysis we improved the functional characterization and classification of pathogenicity as well as identified the mechanism of their development. We demonstrate that next-generation sequencing-based RNA analysis can improve the assessment of the pathogenic effect of variants in coding and non-coding regions of the genome.

• Keywords: genetics, monogenic conditions, pathologic variants, RNA-transcripts, cDNA, PCR, next-generation sequencing, functional characterization