Nekodirajuće RNK kao perspektiva u dijagnostici i lečenju kardiovaskularnih bolesti

Kardiovaskularne bolesti (KVB) su klasa oboljenja koja obuhvataju srce i/ili krvne sudove i nastaju složenim sadejstvom genetičkih i stečenih faktora. Razvoj molekularne biologije omogućio je nove uvide u fundamentalne mehanizme koji dovode do KVB, kao i napredak u dijagnostici, prognostici i lečenju. Novi pristupi u dijagnostici i lečenju KVB podstaknuti su istraživanjima koja se odnose na nekodirajuće RNK - duge nekodirajuće RNK i kratke nekodirajuće RNK. Ova klasa molekula, ne samo da se povezuje sa različitim mehanizmima koji dovode do razvoja KVB, nego se prepoznaje njihov potencijal kao biomarkera, farmakogenetičkih faktora, novih meta lekova i novih alatki u lečenju bolesti. Upotreba nekih od njih u lečenju ljudi je i odobrena od strane relevantnih agencija. Posebnu pažnju privlače studije koje se odnose na nekodirajuće RNK poreklom iz ekstracelularnih vezikula, dodatno potvrđujući potencijal nekodirajuće RNK kao leka budućnosti, ne samo u tretmanu KVB.Cardiovascular diseases (CVDs) are group of diseases which encompass heart and/or blood vessels and they originate from complex coaction of genetic and acquired factors. Development of molecular biology has enabled new insights into fundamental mechanisms which lead to CVDs, as well as progress in diagnostics, prognosis and treatment. New approaches to diagnostics and treatment of CVDs have been encouraged by researches which are related to non-coding RNA, long non-coding and short non-coding RNA. Not only is this group of molecules being associated with different mechanisms which lead to CVDs, but their potential to be biomarkers, pharmacogenetic factors, new drug targets and new treatment tools is being recognised. Relevant agencies have approved some of them to be used for human treatments. Studies related to non-coding RNAs deriving from extracellular vesicles are getting special attention, additionally confirming non-coding RNAs potential as drug of the future, not limited just to CVDs

An Overview of Genetic Risk Factors in Thrombophilia

Thrombophilia is a multifactorial disorder, involving both genetic and acquired risk factors that affect the balance between procoagulant and anticoagulant factors and lead to increased tendency to thrombosis. The concept that thrombophilia could be associated with genetic defects was first proposed in 1965 after the discovery of familiar antihrombin III deficiency. Further family studies showed that deficiency of protein C or protein S also increased thrombotic risk. In the coming years the advent in DNA technology, especially the invention of PCR reaction, played an important role in the identification of the exact nature of these deficiencies and opened new possibilities in the genetic research of thrombophilia. The breakthrough came with the discovery of activated protein C resistance and Factor V Leiden mutation. Shortly afterwards a mutation in the 3' untranslated region of Factor II gene (FII G20210A) associated with increased concentration of factor II in plasma, was described. Large epidemiologic studies have conformed that these two common mutations represent significant risk factors for thrombophilia. In the last decade several prothrombotic genetic risk factors have been described, including genes variants associated with increased levels of coagulation factors, defects of natural coagulation inhibitors, defects of the fibrinolytic system and hyperhomocysteinemia. These genetic defects or their combination have been extensively studied in an attempt to elucidate the possible association with increased thrombotic tendency. The large-scale DNA analysis systems are now becoming available, opening a new era in the genetic studies of thrombophilia. New technology will enable many genes to be studied in a single patient bringing us closer to the "personalized" medicine

The c.-1639g>A polymorphism of the VKORC1 gene and his influence on the therapeutic response during oral anticoagulants use

Background/Aim. A single nucleotide polymorphism c.- 1639G>A in the promoter region of vitamin K-epoxide reductase (VKORC1) gene has been found to account for most of the variability in response to oral anticoagulants (OA). The aim of the study was to determine the incidence and the effect of c.-1639G>A polymorphism on the acenocoumarol dosage requirements in the group of patients under stable anticoagulation, and to estimate the variability in response to OA. Methods. Our study included 200 consecutive patients requiring low (n = 43), medium (n = 127) and high (n = 30) acenocoumarol dose. Results. Out of 43 low dose patients, 40 (93 %) carried the A allele. The A allele was less frequent in the group of 30 patients requiring high dose: among these patients 13 (43.3%) carried the A allele in the heterozygous form and none of them carried AA genotype. The patients with GG genotype required 2.6 times higher dose than the patients carriers of AA genotype (p < 0.0001). In 33 patients (16.5%) the overdose occurred during the initiation of anticoagulant therapy and in 11 patients (5.5%) it was associated with bleeding. Out of the group of 33 overdosed patients, 27 and 6 patients carried AA and GA genotype, respectively (p < 0.000001). Conclusion. VKORC1 significantly influenced OA dose and predicted individuals predisposed to unstable anticoagulation. The carriers of AA genotype required 2.6 time lower doses of OA than the carriares of GG genotype. Pharmacogenetic testing could predict a high risk of overdose among 28.5 % of our patients - carriers of AA genotype, before anticoagulation therapy initiation

The 'VKORC1' and 'CYP2C9' gene variants as pharmacogenetic factors in acenocoumarol therapy in Serbian patients: Consideration of hypersensitivity and resistance

Uvod/Cilj Terapija kumarinima predstavlja jedan od najboljih modela za primenu farmakogenetike. Doprinos faktora koji utiču na terapiju kumarinima može značajno da varira između etničkih grupa, što opravdava sprovođenje studija specifičnih za populaciju. Cilj ove studije je bio da se analizira uticaj najvažnijih genetičkih faktora (geni VKORC1 i CYP2C9) koji utiču na terapiju kumarinima kod bolesnika iz Srbije. Metode Sprovedena je retrospektivna studija koja je obuhvatila 207 bolesnika na terapiji acenokumarolom. Genetičke analize su vršene direktnim sekvenciranjem. Analiziran je uticaj na dozu acenokumarola varijanti (VKORC1*2, CYP2C9*2, CYP2C9*3) koje izazivaju preosetljivost i varijanti gena VKORC1 koje izazivaju rezistenciju na kumarine. Višestruka regresiona analiza je korišćena u cilju dizajniranja matematičkog modela za predviđanje individualne doze leka na osnovu kliničko-demografskih i genetičkih podataka. Rezultati Studija je potvrdila značajan uticaj analiziranih genetičkih faktora na održavanje doze acenokumarola. Dizajniran je matematički model za predviđanje individualne doze acenokumarola i njegov nekorigovani R2 je bio 61,8. Prilikom testiranja, naš model je dao R2 vrednost od 42,6 i pokazao bolje predviđanje u poređenju sa modelom koji su dali drugi autori. Kod analiziranih bolesnika pronađeno je devet različitih varijanti u kodirajućem regionu gena VKORC1. Među nosiocima ovih varijanti 78% je bilo potpuno rezistentno, te nije bilo moguće postići terapeutski efekat čak ni sa visokim dozama acenokumarola. Zaključci Populacioni model za predviđanje individualne doze acenokumarola može pokazati prednosti u odnosu na modele koji se koriste na generalizovan način. Takođe, VKORC1 varijante koje izazivaju rezistenciju na kumarin treba uzeti u obzir prilikom planiranja terapije.Introduction/Objective Coumarin therapy represents one of the best models for applying pharmacogenetics. The contribution of factors influencing coumarin therapy can vary significantly between ethnic groups, which justifies conducting population-specific studies. The aim of this study was to analyze the influence of the most important genetic factors (VKORC1 and CYP2C9 genes) that affect coumarin therapy in patients from Serbia. Methods A retrospective study involving 207 patients on acenocoumarol therapy was conducted. Genetic analyses were performed by direct sequencing. Influence on acenocoumarol dose of variants (VKORC1, CYP2C9*2, CYP2C9*3) causing hypersensitivity and VKORC1 variants causing resistance to acenocoumarol were analyzed. Multiple regression analysis was used to design a mathematical model for predicting individual drug dosage based on clinical-demographic and genetic data. Results The study confirmed significant influence of the analyzed genetic factors on acenocoumarol maintenance dose. We designed mathematical model for predicting individual acenocoumarol dose and its unadjusted R2 was 61.8. In the testing cohort, our model gave R2 value of 42.6 and showed better prediction in comparison with model given by other authors. In the analyzed patients, nine different variants in the VKORC1 coding region were found. Among carriers of these variants 78% were completely resistant, and it was not possible to achieve therapeutic effect even with high doses of acenocoumarol. Conclusions Population-specific model for prediction individual dose of acenocoumarol, may show advantages over protocols that are used in a generalized manner. Also, VKORC1 variants which cause coumarin resistance should be considered when planning therapy

The expression of Muscle ankyrin repeat proteins in brown adipose tissue

MARP family members CARP, Ankrd2 and DARP are expressed in the striated muscle, while DARP protein is also detected in brown adipose tissue (BAT). Taking into account recent findings concerning the common origin of muscle and brown fat, expression of CARP and Ankrd2 in mouse BAT was investigated. We demonstrated Ankrd2 expression in both inactive and thermogenically active BAT, while CARP expression was not detected. Our findings suggest that the expression of Ankrd2 in BAT could be a part of the 'myogenic transcriptional signature', further supporting the evidence that muscle and brown adipose cells arise from the same myoblastic precursor

Genetic prediction of hemophilia a by int18/BclI RFLP analysis

Hemofilija A je X vezana bolest, koja se javlja sa učestalošću od 1 do 2 na 10000 muškaraca. Ona predstavlja najčešći koagulacioni poremećaj i izazvana je mutacijama u genu koji kodira osmi faktor koagulacije (FVIII). Nakon kloniranja gena za FVIII okarakterisan je veliki broj mutacija koje dovode do hemofilije A, što otežava direktnu DNK dijagnostiku ove bolesti. Indirektna DNK dijagnostika primenom polimorfizama u dužini restrikcionih fragmenata DNK (RFLPs) predstavlja alternativni pristup. Cilj ovog rada je bio da se utvrdi informativnost porodica u kojima se javlja hemofilija A za prenatalnu dijagnostiku u narednim trudnoćama. Korišćenjem reakcije lančanog umnožavanja DNK (PCR), analizirano je 67 uzoraka DNK članova, 24 porodice, u kojima se javlja hemofilija A. Umnožavan je polimorfan region u intronu 18, gena za VIII faktor koagulacije i obrađivan restrikcionim enzimom BclI. Utvrđeno je da je 12 porodica (50%) informativno za 18/BclI RFLP. Urađena je i jedna prenatalna dijagnoza, kod žene u prvom trimestru trudnoće, i pri tom je kod fetusa muškoga pola detektovan isti obrazac restrikcionih fragmenata kao kod obolelog od hemofilije. S obzirom da je indirektna DNK dijagnostika brz i ekonomski isplativ metod, ona predstavlja dijagnostičku strategiju izbora u većini porodica u kojima je indikovana genetička dijagnostika hemofilije A.Hemophilia A, an X linked genetic disease, is the most common coagulation disorder with an incidence of about 1-2 in 10 000 males. It is caused by mutations in the factor VIII coagulation gene. After cloning of the factor VIII gene, almost all types of mutations causing hemophilia A were characterized. The wide spectrum of different mutations in the factor VIII gene made direct DNA diagnosis of the disease not the method of choice. Indirect DNA diagnosis, using restriction fragment length polymorphisms (RFLPs) offers an alternative. The aim of our study was to provide carrier and prenatal diagnostics for affected families using the indirect approach. The genomic DNA of 67 members of 24 hemophilia A families were analyzed by polymerase chain reaction (PCR) amplification of the BclI polymorphic region at intron 18 of the factor VIII gene. Twelve families were informative for int18/BclI RFLP (50%). One prenatal diagnosis of hemophilia A was performed in the first trimester of gravidity, and the fetus was found to be a male affected by hemophilia A. Indirect DNA testing is straightforward, rapid and inexpensive to perform. Thus, in many families requiring genetic diagnosis of hemophilia A, as well as genetic counseling, the use of intragenic polymorphism analysis represents the diagnostic strategy of choice

The c.-1639g gt A polymorphism of the VKORC1 gene and his influence on the therapeutic response during oral anticoagulants use

Uvod/Cilj. Pojedinačni nukleotidni polimorfizam c.- 1639G gt A u promotorskom regionu gena za vitamin K epoksid- reduktazu (VKORC1), odgovoran je za varijabilnost odgovora u toku primene oralnih antikoagulanasa (OA). Cilj našeg istraživanja bio je da utvrdimo učestalost polimorfizma c.- 1639G gt A i njegov uticaj na dozu antikoagulansa acenokumarola, te da procenimo povezanost varijabilnosti u odgovoru na terapiju sa prisutnim polimorfizmom. Metode. U ispitivanje je bilo uključeno 200 bolesnika koji su primali OA (43 malu dozu, 127 srednju i 30 veliku dozu). Rezultati. Kod 40 (93%) bolesnika lečenih malom dozom OA, dokazano je prisustvo A-alela. U grupi koja je primala veliku dozu OA, 13 (43,3%) bolesnika bili su nosioci A-alela, heterozigoti (GA genotip), i nijedan od njih nije bio nosilac homozigotne varijante AA genotipa. Posmatrano u celini, u grupi sa AA genotipom doza održavanja OA bila je 10 mg nedeljno, sa GA 19 mg i sa GG genotipom 26 mg. Kod nosilaca GG genotipa bile su potrebne 2,6 puta veće doze antikoagulansa za postizanje terapijskog raspona INR u odnosu na nosioce AA genotipa (p lt 0,0001). Retrospektivnom analizom utvrđeno je da je 33 (16,5%) bolesnika u toku uvođenja terapije bilo predozirano, a kod 11 (5,5%) bolesnika predoziranost je bila udružena sa pojavom krvarenja. Od 33 predozirana bolesnika, 27 su bili nosioci AA genotipa, a šest nosioci GA genotipa (p lt 0,000001). Zaključak. Na individualnu osetljivost na antikoagulanse VKORC1 ima značajan uticaj. Nosiocima AA genotipa potrebne su 2,6 puta manje doze antikoagulansa za održavanje terapijskog raspona INR u odnosu na nenosioce. Farmakogenetski testovi mogli su da ukažu na visok rizik od predoziranja kod 28,5% naših bolesnika, nosioca AA genotipa, pre uvođenja terapije OA.Background/Aim. A single nucleotide polymorphism c.- 1639G gt A in the promoter region of vitamin K-epoxide reductase (VKORC1) gene has been found to account for most of the variability in response to oral anticoagulants (OA). The aim of the study was to determine the incidence and the effect of c.-1639G gt A polymorphism on the acenocoumarol dosage requirements in the group of patients under stable anticoagulation, and to estimate the variability in response to OA. Methods. Our study included 200 consecutive patients requiring low (n = 43), medium (n = 127) and high (n = 30) acenocoumarol dose. Results. Out of 43 low dose patients, 40 (93 %) carried the A allele. The A allele was less frequent in the group of 30 patients requiring high dose: among these patients 13 (43.3%) carried the A allele in the heterozygous form and none of them carried AA genotype. The patients with GG genotype required 2.6 times higher dose than the patients carriers of AA genotype (p lt 0.0001). In 33 patients (16.5%) the overdose occurred during the initiation of anticoagulant therapy and in 11 patients (5.5%) it was associated with bleeding. Out of the group of 33 overdosed patients, 27 and 6 patients carried AA and GA genotype, respectively (p lt 0.000001). Conclusion. VKORC1 significantly influenced OA dose and predicted individuals predisposed to unstable anticoagulation. The carriers of AA genotype required 2.6 time lower doses of OA than the carriares of GG genotype. Pharmacogenetic testing could predict a high risk of overdose among 28.5 % of our patients - carriers of AA genotype, before anticoagulation therapy initiation

Analysis of the CYP2C19 genotype associated with bleeding in Serbian STEMI patients who have undergone primary PCI and treatment with clopidogrel (vol 74, pg 443, 2018)

The correct Author names are shown in this paper

A successful outcome of pregnancy in a patient with congenital antithrombin deficiency

Uvod. Prisustvo urođene trombofilije predstavlja dodatni faktor rizika od nastanka venskog tromboembolizma u trudnoći, ali i komplikacija same trudnoće kao što su ponavljani gubitak trudnoće, abrupcija placente, intrauterino zaostajanje u rastu i razvoju, te rana preeklampsija. Trudnice sa trombofilijom, a posebno one sa nedostatkom antitrombina, u riziku su od nastanka obe vrste komplikacija u trudnoći. Prikaz bolesnice. U radu je prikazana trudnica sa urođenim nedostatkom antitrombina u toku njene prve trudnoće, čija majka je četiri puta imala duboke venske tromboze, tokom trudnoće i puerperijuma. Uz redovno laboratorijsko praćenje hemostaznih parametara i redovne akušerske kontrole sa praćenjem važnog parametra placentne vaskularizacije, čitava trudnoća protekla je bez komplikacija. Profilaktička terapija niskomolekularnim heparinom uvedena je od 20. nedelje trudnoće, a supstituciona terapija primenom koncentrata antitrombina, neposredno pre porođaja. Porođaj je protekao bez komplikacija i u 37. nedelji rođena je zdrava muška beba telesne težine 3,6 kg i dužine 52 cm, sa Abgar skorom 9/10. Zaključak. Pravovremeno postavljena dijagnoza trombofilije, redovne akušerske kontrole i praćenje hemostaznih parametara u toku trudnoće, kao i primena adekvatne profilaktičke i supstitucione terapije, doprinele su prevenciji mogućih maternalnih ili komplikacija same trudnoće kod bolesnice sa urođenim nedostatkom antitrombina.Background. Presence of inherited thrombophilia is an additional risk factor for maternal thromboembolism and certain adverse pregnancy outcomes, including recurrent fetal loss, placental abruption, intrauterine growth restriction and earlyonset severe preeclampsia. Pregnant women with thrombophilia, especially those with antithrombin (AT) deficiency, are at high risk of both kinds of complications. Case report. We presented a pregnant women with congenital antithrombin deficiency in the first pregnancy, whose mother had had four times pregnancy-related deep vein thrombosis, and antithrombin deficiency. With the regular laboratory monitoring of hemostatic parameters and gynaecology surveillance including the follow-up of placental vascular flow, the whole pregnancy proceeded without complications. The prophylactic therapy with low molecular weight heparin was introduced from the 20th week of gestation and one dose of substitution therapy with antithrombin concentrate was administrated before delivery. Pregnancy and labour were terminated without complications at the 37th week of gestation, resulting in the delivery of a healthy male newborn of 3.6 kg body weight, 52 cm long, and with the Apgar scores of 9/10. Conclusion. A timely made diagnosis of thrombophilia, accompanied with regular obstetrics check-ups and follow-ups of hemostatic parameters during pregnancy, as well as the use of adequate prophylactic and substitution therapy, are the successful tools for the prevention of possible maternal complications and pregnancy itself in our patient with congenital AT deficiency