Povezanost polimorfizmov v genih za glutation peroksidazo in glutation disulfid reduktazo s koncentracijo 8-hidroksi-deoksigvanozina v urinu

Vukoja, Jure (author), Marc, Janja (mentor), Prodan Žitnik, Irena (co-mentor)

Endogeni 8-hidroksi-deoksi gvanozin (8-OHdG) nastaja kot posledica reakcije hidroksilnega radikala z deoksigvanozinskimi ostanki, njegova prisotnost pa lahko povzroči transverzijske mutacije GC>TA, ki vodijo v polimorfizme ter hitrejše krajšanje telomer. Uporablja se tako kot diagnostični kot prognostični kazalnik stanj, ki so posledica oksidativnega stresa v telesu. Izloča se z urinom, kar omogoča neinvaziven odvzem in merjenje koncentracije pri posamezniku. Glutation-peroksidaze so družina encimov, ki organizem ščitijo pred oksidativnimi poškodbami preko redukcije vodikovega peroksida. Po drugi strani pa encimi glutation-reduktaze, čigar vloga je redukcija oksidirane oblike glutationa – GSSG nazaj v sulfhidrilno obliko – GSH, na ta način posredno delujejo kot obrambni element proti oksidativnemu stresu. Namen magistrskega dela je bil primerjati metodi RFLP in PCR v realnem času ter ugotoviti morebitno povezanost polimorfizmov rs1050450 G<A v genu za glutation-peroksidazo (GPx) in rs2978663 A<G v genu za glutation disulfid-reduktazo (GSR) s koncentracijo 8-hidroksi-deoksi gvanozina v urinu. V ta namen smo z metodo PCR v realnem času s specifičnimi TaqMan sondami določili genotip obeh polimorfizmov rs1050450 G<A in rs2978663 A<G pri 175 nosečnicah z območja Slovenije in Hrvaške, starih med 19 in 44 let, ki so sodelovale v projektu PHIME in za katere smo imeli podatke o urinski koncentraciji 8-hidroksi-deoksi gvanozina. S primerjavo metod RFLP in PCR v realnem času za polimorfizem rs2978663 A<G v genu za GSR smo ugotovili, da dajeta enake rezultate, vendar je izbira metode PCR v realnem času bolj smiselna, saj je hitrejša, cenovno ugodnejša, obenem pa z manj koraki dovoljuje manj možnosti za napake. Za polimorfizma rs1050450 G<A in rs2978663 A<G s pomočjo statistične analize nismo dokazali vpliva genotipa na koncentracijo 8-OHdG v urinu kot kazalcem oksidativnega stresa. Zaključimo lahko da predstavlja nivo 8-OHdG neodvisen kazalec oksidativnega stresa, ki ni povezan s prisotnostjo/odsotnostjo genetskih sprememb rs1050450 in rs2978663 v genih za glutation-peroskidazo in glutation-reduktazo.

• Language: Slovenian
• Keywords: 8-hidroksi-deoksi gvanozin, glutation, glutation-peroksidaza, glutation-reduktaza, oksidativni stres
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2021
• PID: 20.500.12556/RUL-124430
• Publication date in RUL: 21.01.2021

Secondary language

• Language: English
• Title: Correlation between glutathione peroxydase and glutathione-disulfide reductase gene polymorphisms and 8-hydroxydeoxyguanosine urine concentration

Abstract

Endogenous 8-hydroxydeoxyguanosine (8-OHdG) is a product of a reaction between a hydroxyl radical and deoxyguanosine groups and can cause transversion mutations GC>TA which lead to polymorphisms and accelerated shortening of telomeres. It is used both as a diagnostic and prognostic marker for oxidative stress-related illnesses. It is excreted from the body with urine, which allows for a non-invasive sampling and measuring of the concentration. Glutathione peroxidases are a family of enzymes which protect the organism from oxidative damage by reducing hydrogen peroxide to water. A decrease in glutathione peroxidase activity as a consequence of genetic mutations leads to increased oxidative stress in the body and the illnesses caused by it, including different cancers. Glutathione reductases reduce the oxidized form of glutathione – GSSG back into its reduced state – GSH, which works as an antioxidant in cells and therefore provides a defense against oxidative stress. The goal of this Master’s thesis was to compare the methods of RFLP and real-time PCR as well as to find a possible connection between the polymorphisms rs1050450 G<A of the glutathione-peroxidase gene (GPx) and rs2978663 A<G of the glutathione disulfide-reductase gene (GSR) and an increase of urinary 8-hydroxydeoxyguanosine. To achieve this, we used Real-time PCR with TaqMan probes to determine the genotypes of both polymorphisms in 175 samples collected from pregnant women aged 19 to 44 that live in Slovenia or Croatia and took part in the PHIME project, for which we also had urine concentrations of 8-OHdG. By comparing the RFLP and real-time PCR methods for rs2978663 A<G polymorphism, we determined that the results are identical while using the method of real-time PCR is more sensible as it is faster, cheaper and includes less steps, which allows for less possibilities of human error. Using statistical analysis, we were not able to confirm an influence of polymorphisms on urinary 8-OHdG concentration, which was used as a marker of oxidative stress. We concluded that urinary 8-OHdG is an independent marker of oxidative stress which is not related to the presence or absence of polymorphisms rs1050450 and rs2978663 in the genes for Glutathione-peroxidase and Glutathione disulfide-reductase.

• Keywords: 8-hydroxydeoxyguanosine, glutathione, glutathione-peroxidase, glutathione disulfide-reductase, oxidative stress