Association between COMT, TNF-alpha, TNFR1, IL-1alpha and IL-10 genetic polymorphisms with a risk of early preeclampsia and itʼs complication

Preeklampsija (PE) se smatra jednim od vodećih uzročnika mortaliteta i morbiditeta majke, kao i perinatalnog mortaliteta i morbiditeta. Posebnu pažnju potrebno je posvetiti ranoj PE i teškom obliku PE, zbog potvrđene korelacije sa jako visokim stepenom morbiditeta i pojavom teških zdravstvenih komplikacija. Mehanizam nastanka PE je još uvek nepoznat. U poslednjih nekoliko godina se kao etiološka hipoteza izdvaja hipoteza kombinovanog prekomernog inflamatornog odgovora i disbalansa angiogenih faktora. Analizom epidemioloških podataka, utvrđeno je da genetski faktori predstavljaju jedan od ključnih faktora rizika za razvoj PE. Katehol-Ometil transferaza (COMT; EC 2.1.1.6) gen i geni inflamatornih citokina predstavljaju gene kandidate za PE sa izuzetnim potencijalom. Cilj istraživanja bio je da se ispita potencijalna povezanost između Val158Met COMT, TNF-α -857 C>T, TNFR1 36 A>G, IL-1α 4845 G>T i IL-10 -1082 A>G polimorfizama i rizika razvoja rane preeklampsije (PE) i njenih komplikacija. Ispitivana populacija je obuhvatila dve grupe. Prvu grupu je činilo 47 pacijentkinja sa ranom PE, dok je drugu grupu činilo 47 zdravih ispitanica (kontrolna grupa). Grupa pacijentkinja sa PE je podeljena u dve podgrupe prema dva različita kriterijuma, težini PE i težini novorođenčeta. Antenatalna nega je izvršena prema protokolu i vodičima dobre kliničke prakse. Deo analizirane EDTA pune krvi zamrznut je na temperaturu -70 °C za DNK ekstrakciju i genotipizaciju. Izolacija DNK urađena je komercijalnim setom za izolaciju DNK (Roche Diagnostics). Detekcija prisustva mutacije gena inflamatornih citokina i COMT enzima je urađena pomoću amplifikacije lančane reakcije DNK, PCR metodom pomoću GeneAmp PCR System 9700 (Applied Biosystems). Proizvod PCR reakcije za polimorfizam inflamatornih citokina je digestovan EXOsap IT enzimom, N1aIII enzimom (Hin1II Thermo SCIENTIFIC) za COMT polimorfizam. Za analizu je korišćen Sanger sekvencioni metod (inflamatorni citokini) odnosno elektroforeza i vizuelizacija Wilber Lourmat metodom (COMT). Poređenje opštih kliničkih karakteristika je vršeno t-testom ili Wilcox rank sum testom...Preeclampsia ( PE) is considered as a one of the leading causes of maternal and perinatal mortality and morbidity. Special attention should be paide on early and severe form of PE, due to confirmed correlation with a very high morbidity rate and the emergence of serious health complications. Mechanism of PE is still unknown . In the last few years hypothesis of combined excessive inflammatory response and imbalance of angiogenic factors was proposed as the etiological hypothesis. By epidemiological data analysis, it was shown that genetic factors are one of the key risk factors for the development of PE . Catechol -O - methyl transferase ( COMT ; EC 2.1.1.6 ) gene and genes of inflammatory cytokine are considered as PE candidates genes with exceptional potential. The goal of this study was to assess the potential association between Val158Met COMT, TNF-α -857 C>T, TNFR1 36 A>G, IL-1α 4845 G>T and IL-10 - 1082 A>G polymorphisms and risk of early-onset preeclampsia (PE) and its complications. The study included two groups. First group consisted of 47 early-onset PE patients and second group consisted of 47 healthy subjects (control group). PE group was divided into two subgroups according to two different criteria, PE severity and newborn weight. Antenatal care was carried out according to the protocol and good clinical practice guidelines. EDTA whole blood was frozen on -70 °C for DNA extraction and genotyping. DNA isolation was done with a DNA isolation kit (Roche Diagnostics). The detection of the gen mutation presence for inflammatory cytokines and COMT enzyme was performed with PCR method using GeneAmp PCR System 9700 (Applied Biosystems). The product of the PCR reaction forinflammatory cytokines polymorphism was digested by the EXOSAP IT enzyme, while product of PCR reaction for CMOT polymorphism was digested by N1aIII enzyme (Hin1II Thermo Scientific). Sanger sequential methods (inflammatory cytokines) or electrophoresis and visualization Wilber Lourmat method (COMT) were used for further analysis..