Branko Tomic

Novel ORC4L Gene Mutation in B-Cell Lymphoproliferative Disorders

B-cell lymphoproliferative disorders are characterized by marked genetic, morphological, and clinical heterogeneity. The identification of prognostic markers could help to develop risk-adapted treatment strategies. Because proliferation of cells is essential for tumor growth, analysis of the cell cycle might give additional information on tumor progression and clinical behavior. Because initiation of DNA replication represents a significant step in cell division, it is worthwhile to focus the attention to the origin recognition complex (ORC), protein complex essential for initiation of DNA replication. Studies have already shown that ORC-associated factors give a more accurate assessment of cell proliferation than previous markers for many types of malignancies, but so far there have been no studies of eventual role of ORC4L in B-cell lymphoproliferative disorders. Here, we describe 3 patients with B-cell lymphoproliferative disorders (2 with non-Hodgkin lymphoma and 1 with nonsecretory multiple myeloma) carrying a novel A286V mutation within ORC4L gene. All 3 patients were in the advanced stage of disease, but their response to the chemotherapy treatment was good and they achieved complete clinical remission in a relatively short period. Although the functional relevance of this mutation has not yet been elucidated, our observation raises a possibility that A286V mutation, which is constitutively present in these patients, might represent a favorable prognostic marker in B-cell lymphoproliferative disorders.

Human initiation protein Orc4 prefers triple stranded DNA

In higher eukaryotes mechanism of DNA replication origin recognition and binding by origin recognition complex (ORC) is still unknown. Origin transfer studies have shown that origin sites are genetically determined, containing functionally interchangeable modules. One of such modules from the human lamin B2 origin of replication has the ability to adopt unorthodox structure partly composed of intramolecular triplex. Sequences involved in triplex formation coincide with ORC binding sites both in vitro and in vivo. To explore potential significance of unorthodox DNA structures in origin recognition by ORC, we tested DNA binding properties of human ORC subunit 4 (HsOrc4) which has independent DNA binding activity in vitro and similar binding characteristics as ORC holocomplex. Our results demonstrated that DNA binding activity of HsOrc4 depends on length and structure of DNA with triplex being the protein’s preferred binding target. Such feature could play part in origin selection through directing ORC to DNA sequence prone to adopt unorthodox structure.

Formation of noncanonical DNA structures mediated by human ORC4, a protein component of the origin recognition complex.

Many genomic sequences, DNA replication origins included, contain specific structural motifs prone to alternative base pairing. Structural rearrangements of DNA require specific environmental conditions and could be favored by chemical agents or proteins. To improve our understanding of alternative conformations of origins and the manner in which they form, we have investigated the effect of DNA-binding, AAA+ protein human ORC4 on single-stranded origin DNA or various oligonucleotides. Here we demonstrate that human ORC4 stimulated formation of inter- and intramolecular T.A.T triplexes and created novel structures, such as homoadenine duplexes. Adenine-based structures were held together by Hoogsteen hydrogen bonds, as demonstrated on 7-deaza-dAMP- or dAMP-containing substrates, and characterized by increased thermal stability. Adenine pairing occurred only in the presence of human ORC4, in a neutral buffer supplemented with ATP and Mg (2+) ions. The protein mutant that could not bind ATP was inactive in this reaction. Since the action of human ORC4 could be biologically important, its potential impact on DNA replication is discussed.

Clinical and biochemical characterization of the prothrombin Belgrade mutation in a large Serbian pedigree: new insights into the antithrombin resistance mechanism

Essentials Prothrombin Belgrade mutation leads to antithrombin resistance. Clinical and biochemical phenotypes in a large family with this mutation were investigated. In carriers, we detected decreased factor II activity and increased endogenous thrombin potential. Prothrombin Belgrade mutation represents a strong prothrombotic risk factor.

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 19911GG 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.

Determination of transgene copy number in stably transfected mammalian cells by PCR-capillary electrophoresis assay.

The quantitative determination of transgene copy number in stably transfected mammalian cells has been traditionally estimated by Southern blot analysis. Recently, other methods have become available for appraisal of gene copy number, such as real-time PCR. Herein we describe a new method based on a fluorescently labeled PCR, followed by capillary electrophoresis. We amplified our target gene (prothrombin) and the internal control originating from genomic DNA (18S rRNA) in the same PCR tube and calculated the mean peak height ratio of the target:control gene for every cell clone sample. With this approach we identified stably transfected cell clones bearing the same transgene copy number. The results of our assay were confirmed by real-time PCR. Our method proves to be fast, low-cost, and reproducible compared with traditionally used methods. This assay can be used as a rapid screening tool for the determination of gene copy number in gene expression experiments.

THE PREVALENCE OF PAI-1 4G/5G POLYMORPHISM IN WOMEN WITH FETAL LOSS – FIRST DATA FOR A SERBIAN POPULATION / UČESTALOST POLIMORFIZMA PAI-1 4G/5G KOD ŽENA SA SPONTANIM POBAČAJEM - PRVI PODACI ZA SRPSKU POPULACIJU

Summary Background: Plasminogen activator inhibitor 1 (PAI-1) is an inhibitor of fibrinolysis. The PAI-1 4G/5G polymorphism is associated with elevated plasma levels of PAI-1. Over- expression of PAI-1 and impaired fibrinolysis in homozygous carriers of the 4G/4G PAI polymorphism may lead to abnor- mal placental formation and increased risk of fetal loss (FL). The aim of our study was to determine the frequency of this polymorphism in patients with FL in a Serbian population. Methods: The study was carried out in a group of 203 women (91 controls and 112 women with FL). The presence of PAI-1 4G/5G polymorphism was detected by PCR-RFLP analysis. Results: Slightly increased frequency of the PAI-1 4G/4G genotype was observed in the study group compared to the controls (32.1% vs. 30.8%). The frequency of PAI-1 was highest in women experiencing FL in the second trimester of pregnancy (50%), but this difference was not statistically sig- nificant. Conclusions: Our findings suggest that PAI-1 4G/4G might be a risk factor for FL occurring in the second trimester of pregnancy. Further studies are required in order to determine the role of PAI-1 4G/5G polymorphism in the etiology of FL. Kratak sadržaj Uvod: Inhibitor aktivatora plazminogena 1 (PAI-1) igra zna- čajnu ulogu u procesu inhibicije fibrinolize. Pokazano je da je PAI-1 4G/5G polimorfizam povezan sa povišenim nivoom PAI-1 proteina u plazmi. Povećana ekspresija PAI-1 i sma- njena fibrinoliza kod homozigotnih nosilaca PAI-1 4G/5G polimorfizma može dovesti do poremećaja tokom formiranja placente i povećanja rizika za spontane pobačaje (SP). U okviru ove studije analizirali smo učestalost PAI-1 4G/5G polimorfizma kod pacijentkinja sa spontanim pobačajima. Metode: Studija je obuhvatila grupu od 203 žene (91 u kon- trolnoj grupi i 112 žena sa SP). Prisustvo PAI-1 4G/5G poli- morfizama je detektovano PCR-RFLP analizom. Rezultati: Detektovana učestalost homozigotnih nosilaca PAI-1 4G/5G polimorfizma je bila nešto visa u grupi paci- jentkinja u odnosu na kontrolnu grupu (32,1% vs. 30,8%). Najviša učestalost je detektovana kod žena koje su imale SP u drugom trimestru trudnoće (50%), ali ova razlika nije bila statistički značajna. Zaključak: Rezultati naše studije ukazuju da PAI-1 4G/4G može biti faktor rizika za pojavu SP u drugom trimestru trud- noće. Potrebna su dalja ispitivanja u cilju određivanja uloge PAI-1 4G/4G polimorfizma u etiologiji spontanih pobačaja.

The effect of FII c.1787G>A (prothrombin Belgrade) mutation on prothrombin gene expression in vitro

The FII c.1787G>A (prothrombin Belgrade) is a novel prothrombotic mutation which leads to impaired inhibition of thrombin by antithrombin (antithrombin resistance). So far, the mechanism of this variant has not been fully elucidated. To investigate the effect of FII c.1787G>A mutation on the prothrombin gene expression, its functional analysis was performed in vitro. By Real-Time PCR, expression levels of FII gene variants were evaluated in Cos-7 cells transiently transfected with c.1787G (wild-type) and c.1787A prothrombin expression vectors, with no differences observed. The relative quantification of prothrombin protein amounts was accomplished by Western blot analysis, also with no differences observed. Therefore, the mechanism of FII c.1787G>A mutation does not alter prothrombin expression profile.

Are Prothrombotic Mutations a Time-to-Event Risk Factor?

Background Deep vein thrombosis (DVT) represents a common disorder involving genetic and acquired risk factors. It has been proposed that acquired risk factors are more important with aging than genetic factors, indicating different prevalence of prothrombotic mutations throughout the lifespan. Objective To determine the role of the most frequent prothrombotic genetic risk factors (Factor V [FV] Leiden and Factor II [FII] G20210A mutations) in first-time DVT etiology in patients of different ages. Method This retrospective study included 701 patients living in Serbia with diagnosed DVT as a first-time thrombotic event. Results Risk assessment for mutations as age-related markers showed no statistical difference (FV Leiden mutation-OR, 1.027; 95% confidence interval [CI], .87-1.22; P = .76 and FII G20210A mutation-OR, 0.940, 95% CI, .74-1.19; P = .61). Our results show similar mutation prevalence regardless of how old the patients were at the time of the first DVT occurrence. Conclusion Our results indicate that these 2 mutations cannot be used as prognostic marker for time-to-event first DVT in the Serbian population; however, further studies are required.

The 3’ End Prothrombin Gene Variants in Serbian Patients with Idiopathic Thrombophilia

Abstract Thrombophilia is a multifactorial disorder that arises from the interaction of acquired and genetic risk factors. Despite the significant efforts made to understand the etiology of this disease, there are still a certain number of patients suffering from idiopathic thrombophilia. The aim of this study was to screen the 3’ end of the prothrombin (FII) gene, which is susceptible to gain-of-function mutations due to its non canonical architecture, in patients with idiopathic thrombophilia and to determine its eventual role in the pathogenesis of thrombophilia. This study was carried out in 100 patients with idiopathic thrombophilia and 100 healthy controls. DNA variants in the 715 bp long region of the 3’ end of the prothrombin gene were identified by sequencing. In our study, we detected two variants: A19911G and C20068T. The frequency of the A19911G gene variant was slightly increased in the group of patients compared to controls, however with no statistically significant difference compared to controls [odds ratio (OR) = 1.06; 95% confidence interval (95% CI) 0.53-2.13]. Heterozygous carriers of the FII C20068T gene variant were four times more frequent in patients (4.0%) than in controls (1.0%), but this difference did not reach statistical significance (OR = 4.12; 95% CI 0.45-37.57). Our findings suggest that variant A19911G is not a significant risk factor, while C20068T may represent a potential risk factor for idiopathic thrombophilia. To confirm our results, further studies should be conducted in a larger cohort of patients.