Comparative genome-wide DNA methylation studies of healthy human tissues and non-small cell lung cancer tissue
Date
2016-12-16
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Abstract
Metüleeritud tsütosiinil on suur roll imetajate organismi arengus. CpG dinukleotiidis asuvad tsütosiinid võivad olla kas metüleeritud või metüleerimata olekus. Kuna DNA metülatsioon on eluliselt vajalik organismi funktsioneerimiseks, tekitavad muutused epigenoomis erinevaid haiguslikke seisundeid, näiteks vähki. Ka põhjustab erinev metüleerimise tase inimestevahelist varieeruvust ravimite metaboliseerimises, mis võib kaasa tuua ebasoovitavaid ravimite kõrvaltoimeid.
Töös kirjeldati ülegenoomseid DNA metülatsioonimustreid ning nende mõju geeniekspressioonile erinevates inimkudedes, nii tervetes kui ka tuumoris. Inimese tervete kudede uuringute põhjal järeldasime, et promootorpiirkonna metülatsiooni ning geeniekspressiooni vahel on negatiivne seos ja DNA metülatsiooni mustrid peegeldasid kudede funktsioone. Koe-spetsiifiliste erinevalt metüleeritud regioonide (tDMR) analüüs leidis suurel hulgal piirkondi, mis paiknesid geenisiseselt, kuid ei suudetud leida seoseid, kuidas need tDMR-d põhjustavad koe-spetsiifilist transkriptsiooni.
Geenide, mis on seotud absorbtsiooni, jaotuse, metabolismi ning väljutusega (ADME) ekspressioonis on leitud suur indiviidide vaheline varieeruvus, mis omakorda mõjutab ravimvastust ja toksilisust. Uurides spetsiifiliselt maksakude, püüdsime määrata, kui suurel hulgal SNP-d ning DNA metülatsioon on võimelised üheskoos kirjeldama ADME geenide ekspressiooni varieeruvust maksas. Ootuspäraselt leidsime, et SNP genotüübi ning CpG metülatsioonitaseme kombineerimine võimaldab kirjeldada ekspressiooni variatsiooni paremini, kui kasutades ainult SNP-d või CpG metülatsiooni taset eraldiseisvalt.
Töö viimane osa keskendus DNA metülatsiooni kirjeldamisele varajase staadiumi mitteväikerakulise kopsuvähi (NSCLC) patsientidel. Leidsime suurel hulgal erinevalt metüleeritud CpG saite. Mitmed neist on tuntud vähiseoselised biomarkerid, kuid enamik kirjeldatud geenidest on uued kopsuvähi markerid. Leitud geenide ontoloogiaanalüüs näitas, et nende geenide funktsioonid olid seotud vähi progresseerumisega. Elulemusanalüüsi tulemusel leidsime CpG saite, mille metülatsiooni tasemed erinesid erinevates elulemusgruppides. Mõned neist geenidest võivad olla head prognostiliste markerite kandidaadid.
Methylated cytosines have a crucial role in mammalian development, and the proportion of methylated CpG sites can vary greatly over a genome. As DNA methylation is vital for the normal functioning of organism, changes in the epigenome can account for individual differences in drug responses or the incidence of severe diseases, especially cancer. The aim of the experimental work presented in this thesis was to describe methylation patterns and their effect on gene expression in different tissue types, both healthy and cancerous. Studies of healthy human tissues confirmed a clear correlation between DNA methylation in promoter regions and gene expression. The DNA methylation patterns also clearly reflected tissue-specific functions as demonstrated with hierarchical clustering and GO analyses of hypomethylated tissue-specific differentially methylated regions (tDMRs). The tDMR analysis revealed that a large number of methylated regions were within gene body regions, yet we were not able to show how these tDMRs mechanistically contribute to tissue-specific functions. Based on the observation that inter-individual variability in ADME gene expression affects drug efficacy, toxicity, and susceptibility to environmental toxins, we determined to what extent SNPs and DNA methylation can jointly explain variations in ADME gene expression in liver. As expected, the combination of SNP genotype and CpG site methylation levels data explained more of the observed expression variations than the use of SNP or methylation levels alone. The third part of the thesis concentrated on describing DNA methylation patterns in early-stage non-small cell lung cancer (NSCLC) patients. A number of differentially methylated CpG sites were found, most of the identified genes represent novel markers for NSCLC. GO analysis revealed that differentially methylated genes were closely related to cancer progression. Furthermore, a survival analysis identified a number of CpG sites whose methylation levels differed according to patient survival. Accordingly, the latter sites represent CpGs that could potentially serve as prognostic markers.
Methylated cytosines have a crucial role in mammalian development, and the proportion of methylated CpG sites can vary greatly over a genome. As DNA methylation is vital for the normal functioning of organism, changes in the epigenome can account for individual differences in drug responses or the incidence of severe diseases, especially cancer. The aim of the experimental work presented in this thesis was to describe methylation patterns and their effect on gene expression in different tissue types, both healthy and cancerous. Studies of healthy human tissues confirmed a clear correlation between DNA methylation in promoter regions and gene expression. The DNA methylation patterns also clearly reflected tissue-specific functions as demonstrated with hierarchical clustering and GO analyses of hypomethylated tissue-specific differentially methylated regions (tDMRs). The tDMR analysis revealed that a large number of methylated regions were within gene body regions, yet we were not able to show how these tDMRs mechanistically contribute to tissue-specific functions. Based on the observation that inter-individual variability in ADME gene expression affects drug efficacy, toxicity, and susceptibility to environmental toxins, we determined to what extent SNPs and DNA methylation can jointly explain variations in ADME gene expression in liver. As expected, the combination of SNP genotype and CpG site methylation levels data explained more of the observed expression variations than the use of SNP or methylation levels alone. The third part of the thesis concentrated on describing DNA methylation patterns in early-stage non-small cell lung cancer (NSCLC) patients. A number of differentially methylated CpG sites were found, most of the identified genes represent novel markers for NSCLC. GO analysis revealed that differentially methylated genes were closely related to cancer progression. Furthermore, a survival analysis identified a number of CpG sites whose methylation levels differed according to patient survival. Accordingly, the latter sites represent CpGs that could potentially serve as prognostic markers.
Description
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Keywords
DNA metüülimine, geeniekspressioon, koed, vähk (med.), kopsuvähk, biomarkerid, kantserogenees, kasvaja markerid, võrdlevuuringud, geneetilised assotsiatsiooniuuringud, DNA methylation, gene expression, tissues, cancer (medicine), lung cancer, biochemical markers, cancerogenesis, tumor markers, comparative research, candidate gene association study