Metilacijski in transkriptomski biooznačevalci pri Huntingtonovi bolezni

Zadel, Maja (author), Peterlin, Borut (mentor)

Huntingtonova bolezen (HB) je avtosomna dominantna nevrodegenerativna bolezen, ki jo povzroča mutacija gena za protein huntingtin htt in nastopi v starosti od 30 do 45 let. Klinično se kaže kot upadanje kognitivnih in motoričnih funkcij, ki po od 15 do 20 letih vodijo v smrt bolnika. Raziskave kažejo, da je ekspresija genov spremenjena v različnih tkivih bolnika s HB ter da lahko spremembe v transkriptomu striatuma bolnikov s HB povežemo s spremembami v krvi. Naša hipoteza je bila, da bomo s transkriptomsko analizo in analizo globalne metilacije DNA v krvi nosilcev mutacije lahko pokazali na specifične biooznačevlace za HB, ki bi se uporabili kot surogatni biooznačevalci dogajanja v možganih. Transkriptomska analiza vzorcev polne krvi nosilcev mutacije za HB in zdravih preiskovancev je med 740 statistično značilno spremenjenimi transkripti pokazala na 3 nove gene (SPG7, C21orf2, PCSK7), ki smo jih povezali s patogenetskimi mehanizmi, značilnimi za nevrodegenerativne bolezni. Sistematična analiza naših rezultatov in še 4 transkriptomskih raziskav je pokazala na ujemanje v 15 diferenčno izraženih genih, od katerih jih je 6 povezanih z razvojnimi nevrološkimi motnjami. Poleg analize transkriptoma smo analizirali tudi globalno metilacijo DNA pri nosilcih mutacije za HB in zdravih preiskovancev. Tako smo pri skupini predsimptomatskih in simptomatskih bolnikov pokazali na 3 signifikantne gene (CLDN16, NCT2, DDC), kjer je bil gen DDC že povezan z nevropsihiatričnimi motnjami, a ne s HB. Kljub navedenemu se omenjeni 3 geni niso izkazali za dovolj specifične in zanesljive biooznačevalce za HB. Z integrativno analizo smo preučili povezavo med spremembami v metilaciji DNA in spremembami v transkriptomu. Analiza je pokazala na 12 genov, a nobeden od njih ni dosegel signifikantnosti. Skupen je bil gen FBXL5, ki je vključen v ubikvitinacijo proteinov in aktivacijo imunskega sistema in bi ga tako lahko povezali s sistemskim odzivom na bolezen. Nadalje so bili v skupini tudi 4 geni (POP5, GRAP, UPS5, SEC24C), ki obratno korelirajo med metilacijo in ekspresijo RNA. Analiza globalne metilacije skupaj s transkriptomsko analizo ni pokazala na povezano delovanje transkriptoma in globalne metilacije DNA in je tako premalo, da bi jih lahko postavili kot specifične in zanesljive biooznačevalce HB.

• Language: Slovenian
• Keywords: Huntingtonova bolezen, transkriptomska analiza, analiza metilacije, polna kri, biooznačevalci, patogenetski mehanizmi
• Work type: Doctoral dissertation
• Typology: 2.08 - Doctoral Dissertation
• Organization: BF - Biotechnical Faculty
• Year: 2019
• PID: 20.500.12556/RUL-107359
• COBISS.SI-ID: 931447
• Publication date in RUL: 03.04.2019

Secondary language

• Language: English
• Title: Methylation and transcriptomic biomarkers for huntington disease

Abstract

Huntington’s disease (HD) is an autosomal dominant neurodegenerative disorder caused by a single gene mutation in protein-coding gene huntingtin htt. Clinical charascteristics of the disease include cognitive decline and progressive motor impairment. The disease manifests at the age of 30 to 45 years and is fatal after 15–20 years of progressive neurodegeneration. Gene expression is altered in a variety of tissues in HD. Recent studies have shown that changes in the transcriptome of the striatum in HD patients could be linked to transcriptomic changes in the blood. Thus, we hypothesized that analysis of transcriptomics and global DNA methylation in the blood of HD mutation carriers may reveal specific signatures that could serve as biomarkers of HD pathogenesis and could be used as surrogate biomarkers. Our transcriptomic analysis included HD mutation carriers and healthy controls with 740 significantly changed transcripts. There were 3 genes (SPG7, C21orf2, PCSK7) to be linked to pathogenetic mechanisms in neurodegenerative diseases. Systemic analysis of our results and 4 other transcriptomic studies showed 15 commons differentially expressed genes where 6 genes were linked to neurological disturbances. Analysis of global DNA methylation was performed separately due to clinical status. When comparing a presymptomatic and symptomatic group of patients 3 new genes (CLDN16, NCT2, DDC) were significantly differentially methylated and were not linked to HD yet. Groups of symptomatic or presymptomatic patients compared to healthy controls and group of HD mutation carriers also compared to healthy controls gave no significant genes in this respect. An integrative analysis was performed to see connections between changes in the methylation status of DNA and transcriptomic changes. There were 12 common genes not significantly changed. Even though, there was one common gene found (FBXL5) to be involved in ubiquitination of proteins and immune response, which could be linked to the systemic response of the disease. Using integrative analysis, we studied how the methylation of DNA and transcriptomics are linked. Analysis revealed 12 non-significant genes, where we found gene FBXL5 to be in common and is involved in ubiquitination of proteins and immune system activation. In this respect could be linked to systemic response to the disease. Further, we found an additional 4 non-significant genes (POP5, GRAP, UPS5, SEC24C), which have a reverse correlation between RNA expression and methylation. Integrative analysis finally did not reveal specific and stable biomarkers for predicting HD.

• Keywords: Huntington’s disease, transcriptomic analysis, methylation analysis, whole blood, biomarkers, pathogenetic mechanisms