Vpliv urejenosti sladkorne bolezni tipa 1 na metilacijo DNA

Čugalj Kern, Barbara (avtor), Bratina, Nataša (mentor), Kovač, Jernej (komentor)

Ozadje: Dolgotrajna hiperglikemija povzroča mikro- in makrovaskularne zaplete, ki so glavni vzrok obolevnosti in smrtnosti, povezane s sladkorno boleznijo. Vedno več je dokazov, ki kažejo, da dolgotrajna hiperglikemija vpliva na razvoj vaskularnih zapletov preko epigenetskih mehanizmov, kot so metilacija DNA, modifikacije histonov in mikroRNA. Metilacija DNA je že bila povezana z zapleti, kot so diabetična retinopatija, diabetična ledvična bolezen in diabetično stopalo. Metilacija DNA pa še ni bila raziskana pri osebah s sladkorno boleznijo tipa 1 (T1D) brez klinične manifestacije zapletov in z različnimi urejenostjo sladkorne bolezni. Metode: V študijo smo vključili 146 preiskovancev s SB1, ki so imeli bolezen vsaj 5 let, bili stari med 10 in 21 let ter niso imeli znakov zapletov. Njihovo urejenost sladkorne bolezni smo ocenili na podlagi povprečnih vrednosti glikiranega hemoglobina (HbA1c) in variabilnosti ravni glukoze (GV), ki je bila določena kot koeficient variacije. Na podlagi teh vrednosti smo preiskovance razdelili v skupine, ki smo jih nato primerjali med seboj (HbA1c<7% proti HbA1c>8% in GV<40% proti GV>50%). DNA smo izolirali iz celotne krvi in združili glede na povprečne vrednosti HbA1c in GV. Različno metilirane regije smo določili z metodo MEDIP na združenih vzorcih. Za potrditev identificiranih regij smo na združenih vzorcih udeležencev z najbolj skrajnimi vrednostmi HbA1c in GV izvedli obogatitev s Cas9 in nato obogatene vzorce sekvencirali s tehnologijo tretje generacije. Različno metilirane pozicije in regije na celotnem genomu smo določili s sekvenciranje tretje generacije na združenem vzorcu preiskovancev z najbolj skrajnimi vrednostmi HbA1c. Rezultati: Z metodo MEDIP smo med skupinama HbA1c<7% in HbA1c>8% določili 14 različno metiliranih regij, od katerih je bilo 11 hipometiliranih in 3 hipermetiliranih v skupini HbA1c>8%. Med skupinama GV<40% in GV>50% smo določili 4 različno metilirane regije, dve hipometilirani in dve hipermetilirani v skupini GV>50%. Te regije nismo potrdili na združenih vzorcih preiskovancev z najbolj skrajnimi vrednostmi HbA1c in GV. S sekvenciranjem celotnega genoma tretje generacije smo med skupinama z najbolj skrajnimi vrednostmi HbA1c določili 8385 različno metiliranih CpG pozicij. Ta mesta so bila anotirana 1802 genom. Več teh genov je bilo že prej povezanih z zapleti. Geni, anotirani hipometiliranim pozicijam, so bili obogateni v 48 signalnih poteh. Sosednje CpG pozicije smo združili in dobili 80 različno metiliranih regij. Zaključki: Dolgotrajna hiperglikemija pri posameznikih je povezana z nastankom različno metiliranih CpG pozicij in regij, ki se nahajajo v ključnih genih in signalnih poteh, ki so že bili raziskani v povezani z zapleti sladkorne bolezni. Nadaljnje raziskave bi lahko razkrile CpG pozicije, ki bi jih lahko uporabili kot zanesljive označevalce za odkrivanje posameznikov z večjim tveganjem za razvoj zapletov, povezanih s sladkorno boleznijo, še preden se pojavijo klinični simptomi, tudi pri posameznikih s strogo uravnavano sladkorno boleznijo.

• Jezik: Slovenski jezik
• Ključne besede: Sladkorna bolezen tipa 1, DNA metilacija, uravnavanje sladkorne bolezni, zapleti sladkorne bolezni
• Vrsta gradiva: Doktorsko delo/naloga
• Organizacija: MF - Medicinska fakulteta
• Leto izida: 2024
• PID: 20.500.12556/RUL-165266
• Datum objave v RUL: 29.11.2024

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Impact of different levels of glycemic control in type 1 diabetes on DNA methylation

Izvleček

Background: Prolonged hyperglycemia causes micro- and macrovascular complications, which are the main cause of diabetes-related morbidity and mortality. Growing evidence suggests that prolonged hyperglycemia influences the development of vascular complications through epigenetic mechanisms, such as DNA methylation, histone modifications, and microRNA. DNA methylation was already associated with diabetes-related complications of type 1 diabetes (T1D), such as diabetic retinopathy, diabetic nephropathy, and diabetic foot. DNA methylation was not yet studied in individuals with T1D without clinical manifestation of diabetes-related complications with different levels of glycemic control. Methods: 146 participants with type T1D for at least 5 years, aged between 10 and 21, and without clinical manifestation of diabetes-related complications were included. Their glycemic control was assessed with the mean values of glycated hemoglobin (HbA1c) and glycemic variability (GV), which was determined as coefficient of variation. Based on their values, the participants were divided into groups that were then compared one to another (HbA1c < 7% vs. HbA1c > 8% and GV < 40% vs. GV > 50%). The participants DNA was isolated from the whole blood and pooled together according to their mean values of HbA1c and GV. Differentially methylated regions were determined with MEDIP on pooled samples. For the conformation of identified regions, Cas9 enrichment and third-generation of sequencing was performed on pooled samples of participants with the most extreme values of HbA1c and GV. Finally, third-generation of sequencing was used to determine differentially methylated sites and regions on whole genome on a pooled sample of participants with most extreme values of HbA1c. Results: With MEDIP, 14 differentially methylated regions were detected between groups HbA1c<7% and HbA1c>8%, including 11 hypomethylated and 3 hypermethylated according to group HbA1c>8%. Between groups GV<40% and GV>50%, 4 differentially methylated regions were identified, two hypomethylated and two hypermethylated according to CV>50%. These regions were not conformed on pooled samples of participants with the most extreme values of HbA1c and GV. With whole genome third-generation sequencing, 8385 differentially methylated CpG sites were identified between groups with the most extreme values of HbA1c. These sites were annotated to 1802 genes. Several of these genes have previously already been associated with diabetes-related complications. Genes annotated to hypomethylated sites were enriched in 48 signaling pathways. Merging neighboring CpG sites resulted in 80 differently methylated regions. Conclusions: Prolonged hyperglycemia in individuals is associated with differentially methylated sites and regions located in critical genes and pathways with a known role in diabetes-related complications. Further research could reveal the exact CpG sites that could be used as reliable biomarkers for detecting individuals with higher risk for development of diabetes-related complications before the manifestation of clinical symptoms, even in individuals with T1D and strictly managed glycemic control.

• Ključne besede: Type 1 diabetes, DNA methylation, glycemic control, diabetes-related complications