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

Data mining for long-non coding RNAs deregulated in colon cancer through analysis of Gene Expression Omnibus database

Colorectal cancer (CRC) is one of the most commonly diagnosed cancers worldwide. Lack of specific CRC symptoms is a challenge for clinicians, as the symptoms overlap with other non-cancerous diseases, leading to 20-25% of newly diagnosed CRC patients already having liver metastasis. Thus, discovering reliable early-disease biomarkers is of high importance. Non-coding RNAs (ncRNAs) have been demonstrated to be involved in CRC development and progression. Long non-coding RNAs (lncRNAs) can interact with RNA, DNA and proteins, forming complexes that are involved in regulation of gene expression via multiple mechanisms, affecting every stage of colon carcinogenesis and making them top candidates for novel biomarker discovery. The aim of our study was to conduct data mining of Gene Expression Omnibus (GEO) database by using “colon cancer” and “ncRNA” keywords, and identify differentially expressed lnRNAs present in different GEO datasets. GEO database which collects submitted high-throughput gene expression data was queried for all datasets that studied colon cancer and ncRNA. Over 60 datasets were manually inspected in order to identify those where analysis of colon and normal tissue originating from the same patient was done. Each dataset was analyzed by GEO2R software to discover differentially expressed lncRNAs. LncRNAs were considered significant if they appeared in more than one GEO dataset. Parts of lncRNAs sequences available in GEO2R analysis results were run through BLAST in order to identify full length lncRNAs. Five GEO datasets matched our criteria. We discovered 12 sequences that appeared in more than one dataset and we identified them through BLAST analysis. Six sequences originated from lncRNAs (RYR3 divergent transcript, long intergenic non-protein coding RNA 595, TOX divergent transcript, FLVCR2 antisense RNA 1, LHRI_LNC744.1 lncRNA gene, and ELFN1 antisense RNA 1), while six sequences represented partial sequences of various mRNAs. Four lncRNAs were down-regulated in colon cancer; one was upregulated, while one showed different expression patterns in different GEO datasets. In this study, we have identified six lncRNAs that have potential significance for colorectal cancer etiology and will be a subject of further in silico and in vitro study.Book of abstract: 4th Belgrade Bioinformatics Conference, June 19-23, 202

The frequency of allele CCR5Δ32 in a Serbian population

Uvod: Nosioci alela CCR5Δ32 su relativno rezistentni na infekciju HIV-om. Postoji nekoliko hipoteza o poreklu i održanju ovog alela u ljudskoj populaciji. Pretpostavlja se da je mutacija Δ32 nastala u populaciji severne ili istočne Evrope i da se potom proširila ka jugu. Iako je učestalost CCR5Δ32 određena u mnogim evropskim populacijama, dodatni podaci su neophodni za formiranje sveobuhvatne slike o distribuciji CCR5Δ32 u Evropi. Zbog toga smo u našoj studiji odredili učestalost CCR5Δ32 u srpskoj populaciji, za koju do ovog rada nisu postojali takvi podaci. Metode: DNK je izolovana iz periferne krvi 352 osobe. U reakciji lančanog umnožavanja korišćeni su prajmeri specifični za gen CCR5. Dobijen je proizvod od 263 bp na osnovu matrice 'wild type', sekvence CCR5 gena, a proizvod od 231 bp na osnovu okrnjene sekvence gena CCR5 (CCR5Δ32). Ukupna učestalost alela CCR5Δ32 u srpskoj populaciji iznosi 4,55%. Rezultati: Od ukupnog broja analiziranih osoba, identifikovano je 8,52% heterozigotnih i 0,57% homozigotnih nosilaca za ovaj alel. Zaključak: Utvrđena učestalost alela CCR5Δ32 u srpskoj populaciji je neočekivano niska, u poređenju sa učestalošću u ostalim slovenskim populacijama.Background: The mutant CCR5Δ32 allele confers resistance to HIV infection. Several hypotheses regarding its origin and persistence in the human population have been proposed. It is assumed that the Δ32 mutation was introduced in Northern or Eastern Europe and that it spread to the south. Although the frequency of CCR5Δ32 was determined in numerous European nations and regions, further data are needed to complete the puzzle of CCR5Δ32 distribution within the continent. Methods: To this end, CCR5Δ32 frequency was determined in a Serbian population (sample size 352). DNA was extracted from peripheral whole blood and polymerase chain reaction specific for CCR5 gene was performed. A reaction product of 263 bp was obtained from the wild­type CCR5 sequence and a product of 231 bp was obtained from the truncated CCR5Δ32 sequence. Results: Overall allele frequency of CCR5Δ32 is 4.55%; 0.57% of individuals in the examined population are homozygous and 8.52% are heterozygous for CCR5Δ32. Conclusions: The determined frequency of the CCR5Δ32 allele in a Serbian population is unexpectedly low, considering ethnically related populations

Early-onset ischaemic stroke in a patient with the novel F2 c.1824C>T gene variant and PAI-1 4G/4G, MTHFR 677TT genotype

Introduction. Ischemic stroke (IS) is a heterogeneous dis-order caused by several genetic and environmental risk factors. It was suggested that coagulation disorders cause 1-4% of cases with IS, especially in patients with early onset of IS. Case report. We describe a case of a young adult male who developed an unprovoked IS. Biochemical, immunological, and thrombophilia screening, as well as DNA sequencing, were performed in order to reveal molecular pathology underlying the stroke of the patient. Thrombophilia testing showed that patient was a homozygous carrier for PAI-1 4G/5G and MTHFR C677T mutations. Additional genetic analysis revealed the presence of the recently reported F2 c.1824C>T gene variant, located in the last exon of the pro-thrombin gene and has previously been shown to cause hy-perprothrombinemia, hypofibrinolysis, and altered fibrin clot phenotype. Conclusion. Our results suggest that the newly reported F2 c.1824C>T gene variant might have a synergistic effect with PAI 4G/4G and MTHFR 677TT genotype in the formation of altered fibrin clot phenotype characterized by thin, densely packed fibrin fibers, which makes clot less susceptible to fibrinolysis and greatly in-creases the risk for early ischemic stroke onset.Ishemijski moždani udar (IMU) je heterogeni poremećaj koji može biti uzrokovan genetskim faktorima rizika i faktorima sredine. Poremećaji koagulacije mogu biti uzročnici u 1-4% slučajeva IMU, naročito kod bolesnika kod kojih se IMU dogodi u mlađem životnom dobu. Prikaz bolesnika. Prikazan je slučaj bolesnika koji je u mlađem životnom dobu razvio IMU nepoznatog uzroka. Urađeni su biohemijski, imunološki i testovi za trombofiliju kao i sekvenciranje DNK sa ciljem da se utvrdi molekularna patologija koja je mogla biti u osnovi moždanog udara kod tog bolesnika. Testovima za trombofiliju utvrđeno je da je bolesnik homozigotni nosilac mutacija PAI-1 4G/5G i MTHFR C677T. Dodatnom genetičkom analizom otkriveno je prisustvo nedavno opisane F2 c.1824C>T genske varijante, koja se nalazi u poslednjem egzonu gena za protrombin i za koju je prethodno pokazano da izaziva hiperprotrombinemiju, hipofibrinolizu i izmenjeni fenotip fibrinskog ugruška. Zaključak. Naši rezultati ukazuju na to da bi nova F2 c.1824C>T genska varijanta mogla imati sinergistički efekat sa PAI 4G/4G i MTHFR 677TT genotipom u nastanku fibrinskog ugruška sa izmenjenim fenotipom, koji se odlikuje tankim, gusto upakovanim fibrinskim vlaknima, što čini ugrušak manje podložnim fibrinolizi i povećava rizik od nastanka IMU u ranijem životnom dobu

Impaired Fibrinolysis Is Linked With Digital Vasculopathy and Onset of New Digital Ulcers in Systemic Sclerosis (vol 49, pg 598, 2022)

J Rheumatol 2022; doi: 10.3899/jrheum.210931 In the Methods section, under the subheading “Follow-up and study outcome,” the last sentence should be as follows: “All new DUs were recorded by contacting all 39 patients once every 1–3 months during follow-up.” The error does not affect the results or conclusions of the study. This correction only applies to the April 1 First Release. The correct text appears in the print and online issues

Increased Expression of Extracellular Vesicles Is Associated With the Procoagulant State in Patients With Established Rheumatoid Arthritis

This study sought to identify different subpopulations of extracellular vesicles (EVs) in plasma from female patients with established rheumatoid arthritis (RA) in relation to the activation of coagulation and fibrin formation in these patients. Forty women were included in the study, 20 patients and 20 age-matched healthy controls. The mean disease duration in patients was 13.0 (5.0-25.0) years, with medium to high disease activity despite ongoing treatment with low-dose prednisolone and methotrexate. There were no differences between the investigated groups regarding the presence of traditional cardiovascular risk factors. The concentration of phosphatidylserine-positive (PS+) EVs; platelet (CD42a(+)), leucocyte (CD45(+)), monocyte (CD14(+)), and endothelial (CD144(+))-derived EVs; and EVs-expressing tissue factor (CD142(+)), P-selectin (CD62P(+)), and E-selectin (CD62E(+)) were determined by flow cytometry analysis. Overall hemostasis potential (OHP) was assessed to follow the hemostatic disturbances, including the parameters for overall coagulation potential (OCP) and overall fibrinolytic potential (OFP). Fibrin clot turbidity was measured together with clot lysis time, and scanning electron microscopy was performed. Increased concentrations of PS+, CD42a(+), CD142(+), CD45(+), CD14(+), and CD62P(+) EVs were found in plasma from patients with RA compared to healthy controls, and the concentrations of PS+, CD42a(+), CD14(+), and CD62P(+) EVs were positively correlated with the inflammatory parameters in RA patients. Positive correlations were also found between the levels of PS+ and CD42a(+) EVs and OCP as well as between the levels of PS+, CD42a(+), and CD62P(+)EVs and OHP. The levels of PS+, CD42a(+), CD14(+), CD62P(+), and CD62E(+) EVs were negatively correlated with OFP. Elevated levels of circulating EVs of different cell origins were found in patients with established RA, in relation to the inflammatory burden and coagulation activation in the disease

The frequency of allele CCR5Δ32 in a Serbian population

Background: The mutant CCR5Δ32 allele confers resistance to HIV infection. Several hypotheses regarding its origin and persistence in the human population have been proposed. It is assumed that the Δ32 mutation was introduced in Northern or Eastern Europe and that it spread to the south. Although the frequency of CCR5Δ32 was determined in numerous European nations and regions, further data are needed to complete the puzzle of CCR5Δ32 distribution within the continent. Methods: To this end, CCR5Δ32 frequency was determined in a Serbian population (sample size 352). DNA was extracted from peripheral whole blood and polymerase chain reaction specific for CCR5 gene was performed. A reaction product of 263 bp was obtained from the wild­type CCR5 sequence and a product of 231 bp was obtained from the truncated CCR5Δ32 sequence. Results: Overall allele frequency of CCR5Δ32 is 4.55%; 0.57% of individuals in the examined population are homozygous and 8.52% are heterozygous for CCR5Δ32. Conclusions: The determined frequency of the CCR5Δ32 allele in a Serbian population is unexpectedly low, considering ethnically related populations.Uvod: Nosioci alela CCR5Δ32 su relativno rezistentni na infekciju HIV-om. Postoji nekoliko hipoteza o poreklu i održanju ovog alela u ljudskoj populaciji. Pretpostavlja se da je mutacija Δ32 nastala u populaciji severne ili istočne Evrope i da se potom proširila ka jugu. Iako je učestalost CCR5Δ32 određena u mnogim evropskim populacijama, dodatni podaci su neophodni za formiranje sveobuhvatne slike o distribuciji CCR5Δ32 u Evropi. Zbog toga smo u našoj studiji odredili učestalost CCR5Δ32 u srpskoj populaciji, za koju do ovog rada nisu postojali takvi podaci. Metode: DNK je izolovana iz periferne krvi 352 osobe. U reakciji lančanog umnožavanja korišćeni su prajmeri specifični za gen CCR5. Dobijen je proizvod od 263 bp na osnovu matrice 'wild type', sekvence CCR5 gena, a proizvod od 231 bp na osnovu okrnjene sekvence gena CCR5 (CCR5Δ32). Ukupna učestalost alela CCR5Δ32 u srpskoj populaciji iznosi 4,55%. Rezultati: Od ukupnog broja analiziranih osoba, identifikovano je 8,52% heterozigotnih i 0,57% homozigotnih nosilaca za ovaj alel. Zaključak: Utvrđena učestalost alela CCR5Δ32 u srpskoj populaciji je neočekivano niska, u poređenju sa učestalošću u ostalim slovenskim populacijama.Projekat ministarstva br. 173005, br. 173008, br. 173013, br. 173035 i br. III4700

The 3 ' End Prothrombin Gene Variants in Patients With Different Thrombotic Events

Background: Prothrombin (FII) A19911G and C20221T gene variants are associated with increased prothrombin levels and potentially represent thrombotic risk factors. Objective: To determine the frequency of A19911G and C20221T FII gene variants in patients with thrombotic disorders and in women who have experienced pregnancy loss (PL). Methods: We determined the frequency of these variants in 133 patients with deep venous thrombosis (DVT), 80 patients with isolated pulmonary embolism (PE), 101 patients with idiopathic PL, and 180 control individuals. Results: The FII A19911G variant was more prevalent in patients with DVT and with PL compared with controls; however, these differences were not statistically significant. The 19911GG genotype was associated with increased risk of PE (odds ratio, 1.91; 95% confidence interval, 1.04-3.51). We did not detect carriers of the FII C20221T gene variant in this study. Conclusions: This is the first study, to our knowledge, that demonstrates the FII 1991199 genotype may represent a risk factor for isolated PE. Also, our results show that the FII C20221T is a rare variant in this population and therefore, routine thrombophilia screening should not include screening for this genotype

The prevalence of PAI-1 4G/5G gene variant in Serbian population

Uvod: Inhibitor aktivatora plazminogena 1 (PAI-1) ima značajnu ulogu u procesu inhibicije fibrinolize i normalne hemostaze. Prisustvo PAI-1 4G/4G genotipa uzrokuje povećanje ekspresije PAI-1. Povišen nivo PAI-1 u krvi povezan je sa brojnim bolestima kao što su tromboza, moždani udar, infarkt miokarda, spontani pobačaji, preeklampsija, insulinska rezistencija, dijabetes tipa 2, rak dojke i astma. U okviru ove studije određivana je učestalost PAI-1 4G/5G genske varijante kod zdravih ispitanika u srpskoj populaciji. Metode: Studija je obuhvatala grupu od 210 zdravih ispitanika (105 žena i 105 muškaraca). Prisustvo PAI-1 4G/5G genske varijante detektovano je PCR-RFLP metodom. Rezultati: Učestalost PAI-1 4G/4G genotipa iznosila je 34,76% i bila je povećana u odnosu na PAI-1 5G/5G genotip (19,05%), dok je najzastupljeniji genotip bio PAI-1 4G/5G (46,19%). Učestalost 4G alela bila je viša (0,58) u odnosu na 5G alel (0,42). Zaključci: Učestalost PAI-1 4G/5G genske varijante u srpskoj populaciji slična je sa okolnim populacijama. Rezultati ove studije su značajni, jer predstavljaju prve podatke za srpsku populaciju što će omogućiti dalja istraživanja o ulozi PAI-1 4G/5G genske varijante u patogenezi brojnih bolesti.Introduction: Plasminogen activator inhibitor 1 (PAI-1) has a major role in inhibition of firinolysis and normal haemostasis. The presence of the PAI-1 4G/4G genotype leads to increased expression of PAI-1. High blood level of PAI-1 is associated with many diseases such as thrombosis, cerebral insult, myocardial infarction, pregnancy loss, preeclampsia, insulin resistance, type 2 diabetes, breast cancer and asthma. In this study, the prevalence of PAI-1 4G/5G gene variant was determined in healthy subjects from Serbian population. Methods: The study was carried out in a group of 210 healthy subjects (105 women and 105 men). The presence of PAI-1 4G/5G gene variant was detected by PCR-RFLP analysis. Results: The prevalence of PAI-1 4G/4G genotype was 34.76% and it was increased compared to PAI-1 5G/5G genotype (19.05%). The most frequent was PAI-1 4G/5G genotype (46.19%). Allelic frequency for 4G allele was higher (0.58) compared to 5G allele (0.42). Conclusions: The prevalence of PAI-1 4G/5G gene variant in Serbian population is similar to the neighboring populations. Results of this study represent the first data for Serbian population. This study could be useful for further research where the role of PAI-1 4G/5G gene variant will be assessed in the pathogenesis of many diseases