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

Prenatal diagnostics of cystic fibrosis by DNA analysis

Cistična fibroza je najčešće autozomno recesivno oboljenje, koje se u opulaciji belaca javlja saučestalošću od 1/2000 do 1/4000 novorođenčadi. Oboljevaju osobe kod kojih je mutirani CFTR gen prisutan u homozigotnom stanju. S obzirom na težinu ove bolesti. kao i visok rizik za rađanje obolelog deteta (1 :4) kod roditelja koji su heterozigotni nosioci mutiranog gena, prenatalna dijagnostika je od ključnog značaja. U okviru ovog istraživanja analizirano je 105 visokorizičnih porodica, sa ukupno 325 članova. Cilj analiza bio je utvrđivanje informativnosti ovih porodica za dijagnostiku cistične fibroze u narednoj trudnoći. Sprovođena je analiza na prisustvo mutacija u CFFR genu (ΔF508, G542X, G551D, i R553X) bilo direktno, bilo na osnovu polimorfizama dužina restrikcionih fragmenata (RFLP). Ovi podaci su poslužili i za određivanje učestalosti određenih mutacija u našoj populaciji, što je neophodna priprema za genetičko skrinovanje. Od analiziranih porodica 49,5% je bilo informativno, 32,4% poluinformativno, a 18,1% neinformativno za direktnu mutacionu analizu na prisustvo najčešće, ΔF508 mutacije. Od porodica neinformativnih za direktnu mutacionu analizu, 82.9% je bilo potpuno informativno za RFLP analizu. Od analiziranih porodica, 1/3 se javila na prenatalnu dijagnozu u narednoj trudnoći. U ispitivanoj populaciji porodica sa rizikom za rađanje deteta sa cističnom fibrozom, sa barem jednim rođenim obolelim detetom, utvrđeno je da je ΔF508 mutacija zastupljena sa frekvencijom od 67.2%, G542X sa 6.4%, G551D sa 0.6%, dok mutacija R553X nije detektovana. Zbirna učestalost ovih mutacija (73.4%) nije dovoljna za populaciono skrinovanje na mutacije u CFTR genu, u našoj populaciji. Na osnovu sprovedenih istraživanja predlaže se pristup u prenatalnoj dijagnozi i genetičkom savetovanju rizičnih porodica. koji uključuje skrining rizičnih porodica na prisustvo ΔF508 mutacije. Za informativne porodice, preporučuje se prenatalna dijagnoza direktnom detekcijom ove mutacije u narednoj trudnoći. U neinformativnim ili poluinformativnim porodicama sprovodi se RFLP analiza, i porodicama za koje se utvrdi da su informativne preporučuje se prenatalna dijagnoza RFLP analizom u narednoj trudnoći. dok se za neinformativne porodice preporučuje prenatalna dijagnostika ove bolesti određivanjem nivoa mikroviiarnih enzima u amnionskoj tečnosti.Cystic fibrosis is one of the most common autosomal recessive diseases (from 1 in 2000 to 1 in 4000 live births gives rise to an affected child). Affected persons are homozygous for the mutated CFTR gene. Regarding the severity of this disease, and a high risk (1/4) of couples heterozygous for the mutated gene to have an affected child, it is essential to perform prenatal diagnostics. In this study, 105 (325 members) high risk families were screened for the presence of mutated CFTR gene. The purpose of this study was to detect families informative for diagnostics of cystic fibrosis in further pregnancies. Both direct mutation analysis (for the presence of ΔF508, G542X, G551D and R553X) and indirect molecular genetic analysis for the presence of mutated CFTR gene (RFLPs) were performed. These data gave us too, the frequency of certain mutations in Yugoslav population which is necessary initial step for planning the population genetic screening program. Among analyzed families, 49.5% were fully informative, 32.4% partly informative, and 18.1% noninformative, for direct detection of the most common ΔF508 mutation The rest of the families were analyzed by RFLPs, and 82.9% were fully informative for RFLPs analysis in the next pregnancies. In studied families, at a known risk for cystic fibrosis, having at least one affected child, the frequency of ΔF508 was 67.2%, G542X 6.4%, G551D 0.6%, while R553X was not detected. Cumulative frequency of these mutations (73.4%) is not enough for establishing the population screening program for mutations within CFTR gene in Yugoslav population. As a final result of our study, we propose a proper approach in prenatal diagnosis and genetic counseling in cystic fibrosis in high risk families. The first step is the screening for the ΔF508. For informative families, prenatal diagnosis in the next pregnancy is recommended. In families partly informative or noninformative for the presence of the ΔF508, RFLPs analysis should be performed. In families, informative for RFLPs analysis, the prenatal diagnosis using this method is recommended in the next pregnancies. ln noninformative families, the microvilar enzyme testing inbthe amniotic fluid is the recommended method of prenatal diagnosis of cystic fibrosi

Alpha-1-Antitrypsin in Pathogenesis of Hepatocellular Carcinoma

Context: Alpha-1-antitrypsin (A1AT) is the most abundant liver-derived, highly polymorphic, glycoprotein in plasma. Hereditary deficiency of alpha-1-antitrypsin in plasma (A1ATD) is a consequence of accumulation of polymers of A1AT mutants in endoplasmic reticulum of hepatocytes and other A1AT-producing cells. One of the clinical manifestations of A1ATD is liver disease in childhood and cirrhosis and/or hepatocellular carcinoma (HCC) in adulthood. Epidemiology and pathophysiology of liver failure in early childhood caused by A1ATD are well known, but the association with hepatocellular carcinoma is not clarified. The aim of this article is to review different aspects of association between A1AT variants and hepatocellular carcinoma, with emphasis on the epidemiology and molecular pathogenesis. The significance of A1AT as a biomarker in the diagnosis of HCC is also discussed. Evidence Acquisitions: Search for relevant articles were performed through Pub Med, HighWire, and Science Direct using the keywords "alpha-1-antitrypsin", "liver diseases", "hepatocellular carcinoma", "SERPINA1". Articles published until 2011 were reviewed. Results: Epidemiology studies revealed that severe A1ATD is a significant risk factor for cirrhosis and HCC unrelated to the presence of HBV or HCV infections. However, predisposition to HCC in moderate A1ATD is rare, and probably happens in combination with HBV and/or HCV infections or other unknown risk factors. It is assumed that accumulation of polymers of A1ATD variants in endoplasmic reticulum of hepatocytes leads to damage of hepatocytes by gain-of-function mechanism. Also, increased level of A1AT was recognized as diagnostic and prognostic marker of HCC. Conclusions: Clarification of a carcinogenic role for A1ATD and identification of pro-inflammatory or some still unknown factors that lead to increased susceptibility to HCC associated with A1ATD may contribute to a better understanding of hepatic carcinogenesis and to the development of new drugs. Published by Kowsar Corp, 2012. cc 3.0

Molecular basis of thrombophilia

Trombofilija nastaje kao rezultat kompleksne interakcije između negenetičkih i genetičkih faktora rizika koji hemostaznu ravnotežu pomeraju u smeru hiperkoagulacije i dovode do pojave tromboze. Veoma značajan faktor rizika za nastanak trombofilije je deficijencija inhibitora koagulacije: antitrombina, proteina C ili proteina S. Veliki korak u razumevanju genetičke osnove i molekularne dijagnostike trombofilije napravljen je otkrićem rezistencije na aktivirani protein C i faktor V Leiden mutacije. Ubrzo je otkrivena i varijanta u 3'-nekodirajucem regionu gena za faktor II (FII G20210A), za koju je pokazano da dovodi do povišene koncentracije protrombina u plazmi. Ove dve genske varijante su najučestaliji genetički faktori rizika za nastanak trombofilije. Nedavno je opisana nova mutacija u genu za protrombin (c.1787G gt T) za koju je pokazano da dovodi do rezistencije na antitrombin, odnosno do smanjene mogućnosti inaktivacije mutiranog trombina od strane antitrombina, sto predstavlja novi mehanizam za nastanak trombofilije. U toku poslednjih decenija, opisan je veliki broj genetičkih faktora rizika za nastanak trombofilije, uključuju}i one koji dovode do: nedostatka inhibitora koagulacije, povećanog nivoa ili smanjene inaktivacije koagulacionih faktora ili defekata sistema za fibrinolizu. Međutim, većina njih nije od dijagnostičke važnosti zbog njihovog malog ili još uvek nepoznatog uticaja na etiologiju trombofilije. Primena novih tehnologija koje omogućavaju analizu velikog broja gena kod jednog pacijenta otvoriće mogućnost individualnog utvrđivanja genetičkih faktora rizika, samim tim i adekvatan terapeutski pristup.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 thrombotic tendency. The severe forms of thrombophilia are caused by a deficiency of natural anticoagulants: antithrombin, protein C and protein S. The advances in DNA technology played an important role in the identification of the exact nature of these deficiencies and opened up new possibilities in genetic research and molecular diagnostics of thrombophilia. The major breakthrough came with the discovery of activated protein C resistance and the Factor V Leiden gene mutation. Shortly afterwards, a variant in the 3' untranslated region of the Factor II gene (FII G20210A) associated with an increased concentration of Factor II in plasma was described. These two gene variants represent the most common thrombophilic genetic risk factors. Recently, a novel prothrombin mutation (c.1787G gt T) was identified in a Japanese family with juvenile thrombosis. This mutation leads to impaired inhibition of mutant thrombin by antithrombin, proposing a new mechanism of thrombophilia named resistance to antithrombin. In the last decade, several prothrombotic genetic risk factors have been described, including gene variants associated with defects of natural coagulation inhibitors, increased levels of coagulation factors or their impaired inhibition and defects of the fibrinolytic system. However, most of them are not of diagnostic value, due to their minor or unknown impact on the thrombotic risk. Large-scale DNA analysis systems are now becoming available, opening a new era in the genetic studies of the molecular basis of thrombophilia

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 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

Ankrd1-mediated signaling is supported by its interaction with zonula occludens-1

The muscle ankyrin repeat protein Ankrd1 is localized in a mechanosensory complex of the sarcomeric I-band. It is involved in signaling pathways activated in response to mechanical stretch. It also acts as a transcriptional cofactor in the nucleus, playing an important role in cardiogenesis and skeletal muscle differentiation. To investigate its regulatory function in signaling we employed protein array methodology and identified 10 novel Ankrd1 binding partners among PDZ domain proteins known to act as platforms for multiprotein complex assembly. The zonula occludens protein-1 (ZO-1) was chosen for further analysis since its interaction with Ankrd2 had already been demonstrated. Both Ankrd2 and Ankrd1 have similar functions and localize in the same regions. We confirmed the interaction of Ankrd1 with ZO-1 protein and determined their subcellular distribution in HeLa cells, showing their colocalization in the cytoplasm. Our findings corroborate the role of Ankrd1 in intracellular signaling

Clinical presentation of mild cystic fibrosis in a Serbian patient homozygous for the CFTR mutation c.1393-1G gt A

We present a case of a 19-year old male with uncommon initial clinical cystic fibrosis (CF) presentation and a rare CFTR genotype, homozygote for c.1393-1G gt A mutation (legacy name 1525-1G gt A)