Marija Stankovic

A novel role for cardiac ankyrin repeat protein Ankrd1/CARP as a co-activator of the p53 tumor suppressor protein

The muscle ankyrin repeat protein (MARP) family member Ankrd1/CARP is a part of the titin-mechanosensory signaling complex in the sarcomere and in response to stretch it translocates to the nucleus where it participates in the regulation of cardiac genes as a transcriptional co-repressor. Several studies have focused on its structural role in muscle, but its regulatory role is still poorly understood. To gain more insight into the regulatory function of Ankrd1/CARP we searched for transcription factors that could interact and modulate its activity. Using protein array methodology we identified the tumor suppressor protein p53 as an Ankrd1/CARP interacting partner and confirmed their interaction both in vivo and in vitro. We demonstrate a novel role for Ankrd1/CARP as a transcriptional co-activator, moderately up regulating p53 activity. Furthermore, we show that p53 operates as an upstream effector of Ankrd1/CARP, by up regulating the proximal ANKRD1 promoter. Our findings suggest that, besides acting as a transcriptional co-repressor, Ankrd1/CARP could have a stimulatory effect on gene expression in cultured skeletal muscle cells. It is probable that Ankrd1/CARP has a role in the propagation of signals initiated by myogenic regulatory factors (MRFs) during myogenesis.

Genetic Risk Factors for Arterial Ischemic Stroke in Children: A Possible MTHFR and eNOS Gene-Gene Interplay?

In order to investigate the influence of genetic factors in childhood stroke, we compared the distributions of mutations/ polymorphisms affecting hemostasis and/or endothelial function (factor V [FV] Leiden, factor II [FII] G20210A, methylenetetrahydrofolate reductase [MTHFR] C677T, angiotensin-converting enzyme [ACE] insertion/deletion [ID], and endothelial nitric oxide synthase [eNOS] G894T) among children with stroke and controls. A total number of 26 children with arterial ischemic stroke and a control group of 50 healthy children were included in the study. No statistically significant differences in allelic and genotypic distribution were detected in comparisons between groups. However, when combined genotypes were analyzed, statistical significance was observed for the association of MTHFR CT and eNOS TT gene variants. The results of our study suggest that this genotype combination represents a risk factor of 7.2 (P = .017) for arterial ischemic stroke in children.

The CFTR M470V Gene Variant as a Potential Modifier of COPD Severity: Study of Serbian Population

Chronic obstructive pulmonary disease (COPD) is a complex disease influenced by genetic and environmental factors. Cystic fibrosis transmembrane conductance regulator (CFTR) protein is an important component of the lung tissue homeostasis, involved in the regulation of the rate of mucociliary clearance. As it is known that certain CFTR variants have consequences on the function of CFTR protein, the aim of this study was to examine the possible role of F508del, M470V, Tn locus, and R75Q variants in COPD development and modulation. Total number of 86 COPD patients and 102 control subjects were included in the study. Possible association between COPD susceptibility, severity, and onset of the disease and allele or genotype of four analyzed CFTR variants was examined. No associations were detected between COPD development, onset of the disease and tested CFTR alleles and genotypes. However, VV470 genotype was associated with mild/moderate COPD stages in comparison to severe/very severe ones (OR = 0.29, 95%CI = 0.11-0.80, p = 0.016). Our study showed that patients with VV470 genotype had a 3.4-fold decreased risk for the appearance of severe/very severe COPD symptoms, and the obtained results indicate that this genotype may have a protective role. These results also suggest the importance of studying CFTR gene as a modifier of this disease.

Matrix metalloproteinases gene variants in idiopathic disseminated bronchiectasis.

Background The excess of matrix metalloproteinases (MMPs) might be associated with the airways destruction or dilatation in bronchiectasis. The functional promoter polymorphisms of MMP1 and MMP9 genes, involved in the extracellular matrix remodeling, might increase the expression of MMPs leading to the development of bronchiectasis. Methods Detection of MMP1 G-1607GG and MMP9 C-1562T gene variants was performed on 37 patients with idiopathic disseminated bronchiectasis and 102 control subjects. We also described a novel method for simple and rapid detection of MMP1 G-1607GG polymorphism. Results The frequency of -1607GG allele was significantly higher in the group of patients than in control subjects (P = 0.014). The heterozygote genotype showed association with bronchiectasis (odds ratio, 5.3; 95% confidence intervals, 1.4-20.0). The association was even stronger in homozygotes for -1607GG allele (odds ration, 8.7; 95% confidence intervals, 1.9-41.0). The allelic and genotype frequencies of MMP9 C-1562T variant did not show significant differences between the groups. Conclusions This is the first report concerning a role of MMP1 G-1607GG and MMP9 C-1562T variants in pathogenesis of idiopathic disseminated bronchiectasis. The results of our study revealed the association of -1607GG allele and the lack of association of MMP9 C-1562T variant with the disease.

Alpha-1-antitrypsin deficiency in Serbian adults with lung diseases.

AIM Alpha-1-antitrypsin (A1AT) is the main inhibitor of neutrophil elastase, and severe alpha-1-antitrypsin deficiency (A1ATD) is a genetic risk factor for early-onset emphysema. Despite the relatively high prevalence of A1ATD, this condition is frequently underdiagnosed. Our aim was to determine the distribution of the A1ATD phenotypes/alleles in patients with lung diseases as well as in the Serbian population. METHODS The study included the adults with chronic obstructive pulmonary disease (COPD) (n=348), asthma (n=71), and bronchiectasis (n=35); the control was 1435 healthy blood donors. The A1ATD variants were identified by isoelectric focusing or polymerase chain reaction-mediated site-directed mutagenesis. RESULTS PiMZ heterozygotes, PiZZ homozygotes, and Z allele carriers are associated with significantly higher risk of developing COPD than healthy individuals (odds ratios 3.43, 42.42, and 5.49 respectively). The calculated prevalence of PiZZ, PiMZ, and PiSZ was higher in patients with COPD (1:202, 1:8, and 1:1243) than in the Serbian population (1:5519, 1:38, and 1:5519). CONCLUSION The high prevalence of A1ATD phenotypes/allele in our population has confirmed the necessity of screening for A1ATD in patients with COPD. On the other hand, on the basis of the estimated number of those with A1ATD among the COPD patients, it is possible to assess the diagnostic efficiency of A1ATD in the Serbian population.

Prothrombotic genetic risk factors in stroke: a possible different role in pediatric and adult patients.

The role of thrombophilia in the pathogenesis of stroke is still controversial, especially in the pediatric stroke. In order to examine the role of common thrombophilic mutations in children and adults with stroke, a case–control study was carried out in a group of 80 children and 73 younger adult patients. The control groups encompassed 100 healthy children and 120 healthy blood donors. Our results showed no significant differences in the frequency of factor V (FV) Leiden, FII G20210A, and methylenetetrahydrofolate reductase (MTHFR) C677T variants between patient groups and corresponding controls. According to our results, carriers of 677CT genotype have 3.62 higher risks to develop stroke in children than in adults (P < .001). The obtained data indicate that heterozygosity for MTHFR C677T variant represents a possible important risk factor for pediatric stroke and suggest a different role of this gene variant in etiology of stroke in pediatric and adult patients.

Quantification of structural changes in acute inflammation by fractal dimension, angular second moment and correlation.

The aim of the study was to examine alteration and possible application of fractal dimension, angular second moment, and correlation for quantification of structural changes in acutely inflamed tissue.

Association of Functional Variants of Phase I and II Genes with Chronic Obstructive Pulmonary Disease in a Serbian Population / Udruženost Funkcionalnih Varijanti Gena Faze I I Ii Sa Hroničnom Opstruktivnom Bolešću Pluća U Srpskoj Populaciji

Summary Background: Chronic obstructive pulmonary disease (COPD) is a complex disorder characterized by increased oxidative stress. Functional genetic variants of phase I and II genes are implicated in oxidants-antioxidants imbalance and may be involved in COPD development. In this study, we aimed to investigate the role of cytochrome P450 (CYP), glutathione S-transferase (GST) and microsomal epoxide hydrolase (mEH) functional variants in the pathogenesis of COPD in a Serbian population. Methods: The genotypes of 122 COPD patients and 100 controls with normal lung function were determined for CYP1A1 *1A/*2A, CYP2E1 *1A/*5B, GSTM1 null, GSTT1 null GSTP1 Ile105Val, mEH Tyr113His and mEH His139Arg gene variants. Results: Results obtained showed that GSTM1 null variant was significantly more represented in COPD patients than in controls (61.5% vs. 47.0%; OR=1.80; p=0.042). Also, a significant difference was observed for combinations of GSTM1 null and GSTP1 105Val/(Val) (38.5% vs. 24.0%; OR=1.98; p=0.029), as well as for CYP1A1 *1A/*2A, GSTM1 null and mEH 113His/(His) genotypes (7.4% vs. 1.0%; OR=7.88; p=0.025). Conclusions: These are the first data concerning the analysis of the variants of phase I and II genes in the pathogenesis of COPD in a Serbian population. Results obtained in this study open up the possibility for thorough analyses of the role of genetic factors in COPD on larger cohorts. Also, they implicate the importance of previously described genetic associations with COPD in our population, as well as reveal a new one, not reported so far. Kratok sadržaj Uvod: Hronična opstruktivna bolest pluća (HOBP) jeste složeno oboljenje koje karakteriše povišen oksidativni stres. Funkcionalne varijante gena faze I i II ksenobiotičkog meta- bolizma mogu uticati na ravnotežu oksidanti-antioksidanti i mogu dovesti do razvoja HOBR Cilj ove studije je bio ispi- tivanje uloge funkcionalnih genskih varijanti u genima za citohrom P450 (CYP), glutation S-transferazu (GST) i mi- krozomalnu epoksidnu hidrolazu (mEH) u patogenezi HOBP u srpskoj populaciji. Metode: U ovoj studiji analizirane su genske varijante CYP1A1 *W*2A, CYP2E1 *W*5B, GSTM1 null, GSTT1 null, GSTP1 lle105Val, mEH Tyr113His i mEH His139Arg u grupi obolelih od HOBP koja je obuhvatala 122 ispita- nika i kontrolnoj grupi koja je obuhvatala 100 ispitanika sa normalnom funkcijom pluća. Rezultati: Dobijeni rezultati su pokazali da je GSTM1 null varijanta statistički značajno povišena u grupi obolelih od НОВР u poređenju sa kontrolnom grupom (61,5% i 47,0%; OR=1,80; p=0,042). Takođe, uočena je značajna razlika u zastupljenosti kombinacije genotipova GSTM1 null i GSTP1 105Val/(Val) (38,5% i 24,0%; OR=1,98; p=0,029), kao i kombinacije СУР1А1 *1A/*2A, GSTM1 null i mEH 113His/(His) (7,4% i 1,0%; OR=7,88; p=0,025). Zaključak: Ovo su prvi podaci o ulozi genskih varijanti gena faze I i II u patogenezi HOBP u srpskoj populaciji. Rezultati dobij|eni u ovoj studiji otvaraju mogućnostza detaljniju ana- lizu uloge genetičkih faktora u HOBP na većim grupama ispitanika. Pored toga, podaci dobijeni u našoj studiji po- tvrđuju važnost genetičkih determinanti povezanih sa HOBP u prethodnim studijama, ali takođe otkrivaju nove genetičke faktore, koji nisu objavljeni do sada.

The effects of caloric restriction against ethanol-induced oxidative and nitrosative cardiotoxicity and plasma lipids in rats

Caloric restriction (CR) prevents or delays a wide range of aging-related diseases possibly through alleviation of oxidative stress. The aim of our study was to examine the effect of CR on oxidative and nitrosative cardiac damage in rats, induced by acute ethanol intoxication. Male Wistar rats were divided into following groups: control; calorie-restricted groups with intake of 60–70% (CR60–70) and 40–50% of daily energy needs (CR40–50); ethanol-treated group (E); calorie-restricted, ethanol-treated groups (CR60–70 + E, CR40–50 + E). Ethanol was administered in five doses of 2 g/kg every 12 h, while the duration of CR was five weeks before ethanol treatment. Malondialdehyde level was significantly lower in CR60–70 + E and significantly higher in CR40–50 + E vs. control. Nitrite and nitrate level was significantly higher in CR40–50 + E compared to control group. Activity of total superoxide dismutase (SOD) and its isoenzyme, copper/zinc-SOD (Cu/ZnSOD), was significantly higher in CR60–70 + E and lower in CR40–50 + E vs. control. Activity of manganese-SOD (MnSOD), that is also SOD isoenzyme, was significantly lower in  CR40–50 + E compared to control group. Plasma content of sulfhydryl (SH) groups was significantly higher in CR60–70 group vs. control. Plasma concentration of total cholesterol, triacylglycerol, low-density lipoproteins and high-density lipoproteins was significantly lower in CR60–70 group compared to control values. Food restriction to 60–70% of daily energy needs has a protective effect on acute ethanol-induced oxidative and nitrosative cardiac damage, at least partly due to alleviation of ethanol-induced decrease in SOD activity, while restriction to 40–50% of energy needs aggravates lipid peroxidation and nitrosative stress.

Gene-environment interaction between the MMP9 C-1562T promoter variant and cigarette smoke in the pathogenesis of chronic obstructive pulmonary disease.

The aetiology of chronic obstructive pulmonary disease (COPD) is complex. While cigarette smoking is a well‐established cause of COPD, a myriad of assessed genetic factors has given conflicting data. Since gene‐environment interactions are thought to be implicated in aetiopathogenesis of COPD, we aimed to examine the matrix metalloproteinase (MMP) 9 C–1562T (rs3918242) functional variant and cigarette smoke in the pathogenesis of this disease. The distribution of the MMP9 C–1562T variant was analyzed in COPD patients and controls with normal pulmonary function from Serbia. Interaction between the C–1562T genetic variant and cigarette smoking was assessed using a case‐control model. The response of the C–1562T promoter variant to cigarette smoke condensate (CSC) exposure was examined using a dual luciferase reporter assay. The frequency of T allele carriers was higher in the COPD group than in smoker controls (38.4% vs. 20%; OR = 2.7, P = 0.027). Interaction between the T allele and cigarette smoking was identified in COPD occurrence (OR = 4.38, P = 0.005) and severity (P = 0.001). A functional analysis of the C–1562T variant demonstrated a dose‐dependent and allele‐specific response (P < 0.01) to CSC. Significantly higher MMP9 promoter activity following CSC exposure was found for the promoter harboring the T allele compared to the promoter harboring the C allele (P < 0.05). Our study is the first to reveal an interaction between the MMP9–1562T allele and cigarette smoke in COPD, emphasising gene‐environment interactions as a possible cause of lung damage in the pathogenesis of COPD. Environ. Mol. Mutagen. 57:447–454, 2016.