Poster Exhibition

Human and Medical Genetics

Bioengineering, Biotechnology and Bioinformatics

Genetics of Natural Resources

Biomonitoring and Genetic Toxicology

Forensic Genetics

Human and Medical Genetics 

Marija Dusanovic Pjevic (University of Belgrade, Faculty of Medicine, Institute of Human Genetics, Belgrade, Serbia):

ANALYSIS OF THE ASSOCIATION BETWEEN TUMOR NECROSIS FACTOR-Α -308 G/A POLYMORPHISM AND THROMBOLYTIC THERAPY SIDE EFFECTS IN ACUTE ISCHEMIC STROKE PATIENTS

Abstract

Marija Dusanovic Pjevic 1 , Ljubica Vojvodic 2 , Milka Grk 1 , Tatjana Damnjanovic 1 , Milica Gulic 1 , Milica Pesic 1 , Nela Maksimovic 1 , Biljana Jekic 1
1 University of Belgrade, Faculty of Medicine, Institute of Human Genetics, Belgrade, Serbia
2 Euromedic Clinic, Belgrade, Serbia

Thrombolytic therapy with recombinant tissue plasminogen activator (rtPA) is the gold standard therapy for the treatment of acute ischemic stroke (AIS). However, due to the potential destructive effect of the rtPA on the extracellular matrix, it may lead to the blood-brain barrier (BBB) breakdown and hemorrhagic complications after this therapy. Tumor necrosis factor-α (TNF-α), as one of the inflammatory mediators, is associated with BBB
breakdown and increased risk of hemorrhagic complications after AIS treated with rtPA.
Polymorphism -308 G/A within the TNF-α gene affects TNF-α gene expression and consequently AIS patients’ recovery and the occurrence of rtPA-induce side effects, especially hemorrhagic. To explore the association between genotypes of the -308 G/A TNF-α gene polymorphism (rs1800629) and the occurrence of the rtPA therapy-induced side effects in the AIS patients. From 2016 to 2018, a total of 166 consecutive patients with AIS treated with rtPA at the St. Sava Hospital in Belgrade were enrolled in the study. Patients’ outcome was determined using the Modified Rankin Scale (mRS) 3 months after the AIS commencement. At hospital admission, all patients underwent neurological and laboratory assessments. The favorable outcome has been defined with scores 0-1 and unfavorable with scores 2-6. Additionally, rtPA-induced side effects were followed during the hospitalization.
Genotypisation was performed using the polymerase chain reaction in real time (Real-Time PCR) method. Statistical analysis was performed by SPSS software version 22.0 (SPSS Inc, Chicago, Illinois, USA). The GG genotype was the most frequent among our patients (76.5%), whereas only one (0.6%) patient had an AA genotype. We have observed no association between any genotypes with thrombolytic therapy outcome and rtPA-induced side effects.
However, after grouping genotypes by the recessive model (GG VS. GA+AA), the GG genotype showed borderline evidence of association with occurrence of the hemorrhagic transformation (p=0.054). Polymorphism -308 G/A within TNF-α gene might have an influence on hemorrhagic complications after rtPA therapy.
Keywords: acute ischemic stroke, thrombolytic therapy, rtPA, hemorrhagic transformation, TNF-α -308 G/A polymorphism
Correspondence: marija_dusanovic@hotmail.com

Milka Grk (University of Belgrade, Faculty of Medicine, Institute of Human Genetics, Belgrade, Serbia):

ANALYSIS OF IL10RB GENE HAPLOTYPES IN PATIENTS WITH SYSTEMIC LUPUS ERYTHEMATOSUS AND RHEUMATOID ARTHRITIS

Abstract

Milka Grk 1 , Rada Miskovic 2 , Ivica Jeremic 3 , Milica Basaric 3 , Marija Dusanovic Pjevic 1 , Milica Gulic 1 , Biljana Jekic 1 , Nela Maksimovic 1 , Tatjana Damnjanovic 1 , Danijela Miljanovic 4 , Ivana Lazarevic 4 , Andja Cirkovic 5 , Ana Banko 4
1 University of Belgrade, Faculty of Medicine, Institute of Human Genetics, Belgrade, Serbia
2 Clinical Center of Serbia, Clinic of Allergology and Immunology, Belgrade, Serbia
3 University of Belgrade, Faculty of Medicine, Institute of Rheumatology, Belgrade, Serbia
4 University of Belgrade, Faculty of Medicine, Institute of Microbiology and Immunology, Virology Laboratory, Belgrade, Serbia
5 University of Belgrade, Faculty of Medicine, Institute for Medical Statistics and Informatics, Belgrade, Serbia

Interleukin 10 receptor (IL10R) is a tetrameric receptor composed of two IL10RA subunits and two IL10RB subunits. This receptor is part of the Interleukin 10 (IL10) pathway and might be important for maintaining immune homeostasis. As IL10 exerts its action via IL10 receptors the IL10RB gene haplotypes could potentially affect this interaction. We will access the frequency of IL10RB haplotypes in patients with Systemic Lupus Erythematosus
(SLE) and with Rheumatoid Arthritis (RA). This is the beginning of a larger study in which we want to analyse associations between selected polymorphisms/haplotypes and susceptibility to SLE and RA. Genotipisation for 63 patients with SLE and 48 patients with RA was performed using TaqMan assays, and amplification reaction was performed in ABI7500 RealTime PCR machine (Applied Biosystems, Foster, CA) according to manufacturer’s
instructions. For this study, we selected three polymorphisms within the IL10RB gene (rs999788, rs2834167, and rs1058867). Haplotype analysis was performed using Haploview software. Haplotype block was defined between rs999788 and rs2834167 IL10RB gene polymorphisms (r2=0.65, D’=0.96) after use of the Confidence intervals LD method. Patients with Systemic Lupus Erythematosus were harbouring IL10RB gene CAA, CAG, TGA, CGA and
TGG haplotypes (41.9%; 27.8%; 12.9%; 7.9%; 7.7%, respectively), while patients with Rheumatoid Arthritis had CAA, CAG, TGG, TGA and CGG haplotypes (40.6%; 32.3%; 12.5%; 9.4%; 5.2%, respectively). There was no statistically significant difference between frequencies of IL10RB haplotypes in SLE and RA patients. According to our study, IL10RB gene haplotype frequencies do not differ between SLE and RA patients. It is necessary to
compare these two groups of patients with the healthy control group.
Keywords: IL10RB, systemic lupus erythematosus, rheumatoid arthritis
Correspondence: milkagrk@gmail.com

Lucija Zunic (Genom Ltd, Zagreb, Croatia) 

PANEL ANALYSIS OF EVIDENCE-BASED INFERTILITY GENES IN SERTOLI CELL-ONLY SYNDROME PATIENTS

Abstract

Lucija Žunić, Monika Logara Klarić 1,2 , Tihana Marić 3 , Lovro Trgovec-Greif 2 , Filip Rokić 2 , Ana Merkler 4 , Robert Belužić 2 , Oliver Vugrek 2 , Ana Katušić Bojanac 3 , Maja Barbalić 1,5
1 Genom Ltd, Zagreb, Croatia
2 Laboratory for Advanced Genomics, Division of Molecular Medicine, Rudjer Boskovic Institute, Zagreb, Croatia
3 Department of Medical Biology, University of Zagreb, School of Medicine, Zagreb, Croatia
4 Department of Laboratory Diagnostics, University Hospital Center Zagreb, Zagreb, Croatia
5 Department of Medical Biology, University of Split, School of Medicine, Split, Croatia

Sertoli cell-only syndrome (SCOS) is the most severe form of azoospermia that is characterized by a complete lack of spermatogenic cells in almost all seminiferous tubules. Apart from well-established genetic determinants that are responsible for infertility such as Klinefelter syndrome, CFTR variants and Y-chromosome microdeletions, it is hard to clearly define the causative role of several hundred candidate genes that were reported as a
possible cause of infertility. We selected 92 evidence-based genes associated with infertility and examined data derived from whole-exome sequencing in 6 SCOS individuals. Likely causative variants that passed our filtering criteria for functional impact were detected in 3 patients. Two patients had likely causative variant in the PKD1 gene that were only 37 bp apart. Apart from the PKD1 variants, causative variants were detected in CHD7 and SCYP3
genes. All those genes have an autosomal dominant effect on male infertility. However, only CHD7 and SCYP3 have likely causative role in isolated forms of male infertility. Our findings suggest that panel testing of infertile men could add to the diagnostic yield, however, the construction of a panel consisting of only real causative genes is crucial.
Keywords: male infertility, SCOS, genetics, NGS panel, gene disease association
Correspondence: E-mail: lucija@genom.hr

Azra Licina Sinanovic (Association for Nutrition and Dietetics “Food for Health”, Tuzla, Bosnia and Herzegovina):

GENETIC DIVERSITY OF THE MALE POPULATION OF THE  BIJELO POLJE MUNICIPALITY THROUGH THE PRISM OF THE Y CHROMOSOME

Abstract

Azra Licina Sinanovic 1 , Fehim Licina 2
1 Association for Nutrition and Dietetics "Food for Health", Tuzla, Bosnia and Herzegovina
2 Association of the family Licina, Podgorica, Montenegro

Genetic genealogy becomes more popular in recent years. Several types of commercial tests are available for testing certain genetic markers which could use in genealogy researches. For genealogists it is especially interesting Y chromosome test, which test the diversity of selected molecular genetic markers located on the Y chromosome, which is only exists in males. The aim of this paper is to collect, summarize and analyze data on the genetic
diversity of the male population of the four largest national confessions, classified in two groups based on religion, of Bijelo Polje municipality, Montenegro. The paper presents the results of data were obtained by testing 83 samples of DNA materials taken from the male inhabitants of Bijelo Polje (53 Bosniak and Muslim confessions – group 1 and 30 Montenegrin and Serbian confessions – group 2). The results of the research point out that
the two national confessions of Bijelo Polje municipality can be found within seven haplogroups: I2, I1, J2, E2, R1b, R1a and G2, while T1 haplogroup is found only in one sample of group 1. The most common haplogroup is I2 identified in 31.33% of samples. At the second place is I1 identified in 18.28%, while in the third place is haplogroup E2 identified in 13.25% of samples. J2 and R1b are in the four and fifth place, while the other
three haplogroups are present in a lower percent. According to the national confession, the most common haplogroup for group 1 is I2 identified in 35.85%, while I1, J2 and E2 identified per 13.21% in each. R1a is identified in 11.32% while the other three haplogroups are present in a lower percent. For the group 2, on the first place is I1 identified in 30%, while on the second place is I2 identified in 23.33%. R1b and E2 are in therd and fourt place
with 13.33% while other three haplogroups are present in a lower percent. T1 haplogroup is not identified in this population. These results will contribute to popularization of the genetic genealogy as a new tool in genealogical research.
Keywords: genetic genealogy, haplogroup, Bijelo Polje, Y chromosome
Correspondence: azra79vet@hotmail.com

Alma Mudrov (University of Tuzla, Faculty of Pharmacy, Tuzla, Bosnia and Herzegovina):

KAUFMAN OCULOCEREBROFACIAL SYNDROME: PHENOTYPIC LANDSCAPE AND LITERATURE REVIEW

Abstract

Alma Mudrov 1 , Belma Mutapcic 2 , Anesa Alic 3 , Amra Fejzic 1
1 University of Tuzla, Faculty of Pharmacy, Tuzla, Bosnia and Herzegovina
2 University of Tuzla, Faculty of Medicine, Department of Health Science, Tuzla, Bosnia and Herzegovina
3 University of Tuzla, Faculty of Science and Mathematics, Department of Biology, Tuzla, Bosnia and Herzegovina

Kaufman oculocerebrofacial syndrome is a distinct condition characterized by intellectual disability, distinctive pattern of craniofacial features and eye abnormalities. It is inherited in an autosomal recessive manner and caused by biallelic mutations in the UBE3B gene. Kaufman oculocerebrofacial syndrome is a rare disorder, and the knowledge on its phenotypic and molecular characteristics is still expanding. We performed an extensive literature review in order to pinpoint all of the phenotype/genotype patterns, evaluate relevant functional studies and expand on the existing phenotype of this condition.
Keywords: Kaufman oculocerebrofacial syndrome, UBE3B
Correspondence: anesaalic92@gmail.com

Aldijana Avdic (University of Tuzla, Faculty of Natural Sciences and Mathematics, Department of Biology, Tuzla, Bosnia and Herzegovina):

MOLECULAR-GENETIC CHARACTERIZATION OF ApoE GENE POLYMORPHISM AND VNTR POLYMORPHISM OF eNOS GENE IN HUMAN POPULATION OF TUZLA CANTON

Abstract

Aldijana Avdic 1 , Naida Lojo-Kadric 2 , Jasmin Ramic 2 ,Lejla Pojskic 2 , Vesna Hadžiavdic 1
1 University of Tuzla, Faculty of Natural Sciences and Mathematics, Department of Biology, Tuzla, Bosnia and Hercegovina
2 University of Sarajevo, Institute for genetic engineering and biotechnology, Sarajevo, Bosnia and Herzegovina

Multifactorial diseases such as cardiovascular diseases, caused by genetic and external factors, represents a source of research at the molecular level. In multifactorial diseases, genetic polymorphisms represent genetic markers that can potentially have predictive significance for modulating the effect of genetic predisposition. The aim of the study was to determine the allelic and genotypic frequencies of ApoE and eNOS genes and to accurately assess the association of polymorphic alleles of ApoE and eNOS genes with cardiovascular diseases in the human population of Tuzla Canton. Based on the distribution analysis of ApoE gene genotypes in the group of subjects with cardiovascular diseases, the highest frequency of 32.0% was determined for the genotype E3/E4. In the control subjects group, the genotype E3/E3 had the highest frequency, which was determined in 34.5% of subjects.
By analyzing the distribution of eNOS gene genotypes in the group of subjects with cardiovascular diseases, the highest frequency was recorded for genotype bb and amounted to 30.5%, and for the same genotype in the control group, a frequency of 36.5% was determined. Based on the correlation between the observed polymorphisms of the ApoE gene and the eNOS gene and cardiovascular diseases, the association of the combined genotypes of the ApoE and eNOS genes with cardiovascular diseases has also been analyzed.
In the group of subjects with cardiovascular diseases, the highest frequency of 37.0% was recorded for the combined genotype E3/E4 of ApoE and genotype bb of eNOS gene. The research indicated the association of ApoE gene genotypes and cardiovascular diseases, as well as the association of combined ApoE and eNOS gene genotypes with cardiovascular diseases. eNOS gene polymorphisms can certainly be observed in correlation with other risk
factors for development of cardiovascular diseases.
Keywords: polymorphism, ApoE, eNOS, cardiovascular diseases, Tuzla Canton
Correspondence: aldijana.tursunovic@untz.ba

Maja Milosevic Markovic (University of Belgrade, School of Dental Medicine, Department of Human Genetics, Belgrade, Serbia):

THYMUS SERPYLLUM AND MENTHA PIPERITA ESSENTIAL OILS AFFECT SHH AND NOTCH SIGNALING PATHWAYS IN BASAL CELL CARCINOMA IN VITRO

Abstract

Maja Milosevic Markovic 1 , Milos Lazarevic 1 , Milica Jaksic Karisik 1 , Branko Dožić 2 , Branislav M Milovanovic 3 , Nada V Petrovic 4 , Slobodan S Petrovic 4 , Jelena Milasin 1
1 University of Belgrade, School of Dental Medicine, Department of Human Genetics, Belgrade, Serbia
2 University of Belgrade, School of Dental Medicine, Department of Pathology, Belgrade
3 University of Belgrade, University Hospital Medical Center Bezanijska Kosa, Medical Faculty, Department of Cardiology, Belgrade, Serbia
4 Development and Production Center BIOSS – PS and Others, Belgrade, Serbia

Basal cell carcinoma (BCC) is the most common skin cancer and the most frequently occurring form of all cancers. Although it rarely metastizes, BCC may be locally aggressive and recurrent. Among other treatment modalities, phytotherapy has also been considered in the management of BCC. Thymus serpyllum and Mentha piperita have shown significant antitumor effects on several different types of cancers. Hence, the aim of the present study
was to examine whether essential oils of these two plants affect Sonic Hedgehog and Notch signaling pathways, known to play key roles in BCC pathogenesis. Primary cultures were generated from five BCC tumor tissues and their distant resection margins (>5 mm) which served as controls. The cells were cultivated in humidified atmosphere under standard conditions until achieving 80% of confluence, when passaging was done. After reaching the
5 th passage tumor cells were treated with 262 μg/ml essential oil of Thymus serpyllum and 556 μg/ml essential oil of Mentha piperita. RNA isolation was performed from treated and un-treated tumor cells and from healthy control cells by standard procedure followed by cDNA synthesis with reverse transcriptase. The relative expression of Sonic Hedgehog signaling cascade (SHH, PTCH1, SMO and GLI1) and Notch signaling pathway (Notch 1 and
Jagged 1) molecules were determined by real-time PCR. After the treatment with Thymus serpyllum there was a significant decrease of SMO and GLI1 expression, and a slight decrease of SHH gene expression in treated compared to un-treated cells. After exposure to Mentha piperita essential oil there was a significant increase of PTCH1, Notch 1 and Jagged 1 expression. This study showed that Thymus serpyllum acts through downstream cascade
of Sonic Hedgehog signal pathway while Mentha piperita acts through upstream parts of Sonic Hedgehog pathway and Notch signaling which can be useful in the therapy of basal cell carcinoma.
Keywords: basal cell carcinoma, Sonic Hedgehog signaling pathway, Notch signaling pathway
Correspondence: maja.milosevic@stomf.bg.ac.rs

Nora Puseljic (Institute of Emergency medicine OBŽ, University of Osijek, Faculty of Medicine, University Hospital Center, Department of pediatrics, Division of neurology, genetic, metabolic disease and endocrinology, Osijek, Croatia):

CLINICAL MANIFESTATIONS OF A PATIENT WITH MICRODUPLICATION 12q23 AND DELETION Xp22

Abstract

Nora Puseljic1, Nika Puseljic2, Visnja Tomac2,3, Ema Poznic4, Domagoj Majetic1, Silvija Puseljic2,3

 1 Institute of Emergency medicine OBŽ, Osijek, Croatia

2 University of Osijek, Faculty of Medicine, Osijek, Croatia

3 University Hospital Center, Department of pediatrics, Division of neurology, genetic, metabolic disease and endocrinology, Osijek, Croatia

4 Family medicine, DZO Osijek, Osijek

 

Microdeletion and microduplication syndromes are disorders caused by submicroscopic deletions or duplications of contiguous genes on particular parts of chromosomes. The aim was to present the clinical manifestations of microduplication 12q23 and deletion Xp22 present in one patient, which have not been described together so far. A 10-month-old girl, born from the first pregnancy, of healthy non-consanguine parents. There were no perinatal risk factors but several verified developmental defects were present – microcornea of ​​the left eye with bilateral corneal dystrophy and cataracts, cardiomyopathy, congenital deafness, and germinolysis of the brain. Elements of phenotypic dysmorphia: soft formation on the left parietal-occipital part, gothic palate, microretrognathia, cutaneous chin hemangioma, rectal diastase 1 cm, minor umbilical hernia, furrows four fingers of both palms. Neurological status: hypertonic, hyperreflexia, enlarged reflexogenic zone, feeding difficulties with poor weight progression. Ultrasound of the brain described asymmetry of the corpus callosum and possible dysgerminolysis. An ultrasound of the heart shows a thickened septum of the left ventricle, a trabeculated posterior wall with numerous recessions indicating a “spongy” myocardium. Magnetic resonance imaging of the brain shows hypoplasia of the corpus calosum, and a suspected extradural venous anomaly corresponding to the atretic parietal meningocele. Excluding TORCH infection and mitochondrial disorder, Chromosomal Microarray Analysis was performed and revealed microduplication 12q23 and deletion Xp22. A regular diet of B1 milk formula and antirachitic therapy is carried out. This presentation expanded the range of clinical manifestations and phenotypic features for microduplication 12q23 and deletion Xp22. In the literature, both mutations have been described separately whose clinical picture differs from the manifestations present when both mutations coexist in a single patient.

 

Keywords: microduplication 12q23, deletion Xp22, chromosomal microarray analysis

Correspondence: nora.puseljic@gmail.com

Maja Zekavica (University Medical Center “Zvezdara“, Department of Microbiology and Genetics, Belgrade, Serbia):

PRESENCE OF HLA-Cw*06 ALLELE IN PATIENTS WITH PSORIASIS VULGARIS – THE FIRST REPORT FROM OUR CENTER

Abstract

Maja Zekavica 1 , Tatjana Djordjevic Todorovic 2 , Natasa Stan 2 , Jelena Lalosevic 2 , Branka Nesic 1 , Ivana Grubisa 1
1 Univerity Medical Center “Zvezdara”, Department of Microbiology and Genetics, Belgrade, Serbia
2 University Medical Center "Zvezdara", Department of Dermatovenereology, Belgrade, Serbia

Psoriasis is a chronic, recurrent, inflammatory, systemic skin disease associated with a number of comorbidities. Psoriasis occurs worldwide with prevalence ranging between 0.09% and 11.4%. Estimated prevalence of about 2% of the Serbian population means that up to 140.000 individuals are affected. Both environmental and genetic factors contribute to its pathogenesis, its appearance and severity. Candidate genes have been identified in the
PSORS1 region of chromosome 6, most significantly the HLA-C gene in the region of the major histocompatibility complex. The HLA-C allele HLA-Cw*06 is strongly associated with the development of psoriasis in world’s population and with a more severe form presenting at a younger age. HLA-Cw*06 has also been shown to influence the response of psoriasis patients to biological treatments. To ascertain the association between the presence of HLA-Cw*06 allele in psoriasis vulgaris patients and the occurrence of the disease. Genomic DNA was isolated from peripheral blood cells of 100 participants (33 patients and 67 controls, age and gender matched) and genotyping for HLA-Cw*06 allele was performed using both PCR-SSP and PCR-RFLP methods: PCR-SSP was used to specifically match HLA-Cw*06, and PCR-RFLP was used to distinguish between homozygotes and heterozygotes. We found 9
heterozygotes and 3 homozygotes in cases (12 affected, or 36%) and 8 hetero- and 2 homozygotes in control group (10 affected or 14.92%). Albeit no significant difference between groups was found, association between allele and disease approached significance (P=0.06). Also, our analysis showed a nonsignificant 2.4-fold increase in risk for development of psoriasis in HLA-Cw*06 allele carriers (OR=2.4, 95%CI=0.95-6.22). Although the association between risk allele and disease is considered to be not quite statistically significant, our results showed that there is a risk for psoriasis development in HLA-Cw*06 carriers and that larger study group is needed to establish the true relationship between this allele and the disease.
Keywords: psoriasis vulgaris, HLA-Cw*06, genetic analysis, Serbian population
Correspondence: makse777@gmail.com

Lejla Caluk Klacar (University of Sarajevo, Institute for Genetic Engineering and Biotechnology, Sarajevo, Bosnia and Herzegovina):

ASSOCIATION OF SELECTED THROMBOPHILIA GENETIC MARKERS WITH RECURRENT PREGNANCY LOSS IN BOSNIA AND HERZEGOVINA

Abstract

Lejla Caluk Klacar, Naida Lojo-Kadric, Jasmin Ramic, Nikolina Tomic, Naris Pojskic, Lejla Pojskic
University of Sarajevo, Institute for genetic engineering and biotechnology, Sarajevo, Bosnia and Herzegovina

This study aimed to analyze the proportion of selected gene polymorphisms in the risk assessment for thrombophilia in women with RPL compared to the control group of women with other complications in pregnancy, since hereditary thrombophilia is a risk factor for reproductive disorders, including infertility, recurrent pregnancy loss, and labor complications. The study included 97 women tested in the Laboratory of Human Genetics of the Institute for Genetic Engineering and Biotechnology, University of Sarajevo, in the period from 2012 to 2020. Materials used were 3 ml peripheral blood collected with EDTA coated tubes. All subjects were classified into two groups: women with RPL (N=45), and a group of women with other clinically significant complications in pregnancy (N=52). This study was approved by the Ethics Committee of the Institute for Genetic Engineering and Biotechnology, number 579/20. Genotyping of prothrombin (FII) G20210A, factor V Leiden (FVL, FV), MTHFR (C677T), PAI-1 (4G/5G), and factor XIII (FXIII) Val34Leu polymorphisms was performed by ASA-PCR reaction. ACE I/D polymorphism was genotyped using a single end- point PCR reaction. The results of the Fisher test and the Chi-square test (for allele and genotype associataions) were obtained using software MedCalc Statistical Software, and
statistical significances were set at a p-value of p<0.05. A statistically significant association (allele and genotype) between two examined groups was found at two gene loci, loci FII and FXIII. The p-value of the allele association for FII G20210A polymorphism is 0.019, while the p-value of the genotype association is 0.009. Notably, the p-value of allele association for FXIII Val34Leu polymorphism is 0.025, and genotype is 0.015. We did not find an association between FVL, MTHFR C677T, PAI-1 4G/5G, and ACE I/D polymorphisms with RPLs in the Bosnian population. The presence of thrombophilia polymorphisms may predispose women to recurrent pregnancy loss. This finding is significant but should be validated in larger dataset before translation into medical practice.
Keywords: thrombophilia, gene polymorphism, miscarriage, complications in pregnancy, risk factors
Correspondence: lejla.pojskic@ingeb.unsa.ba

Milica Gulic (University of Belgrade, Faculty of Medicine, Institute of Human Genetics, Belgrade, Serbia):

ANALYSIS OF THE ASSOCIATION BETWEEN I/D POLYMORPHISM WITHIN ACE GENE AND COVID-19 OUTCOME IN SERBIAN POPULATION

Abstract

Milica Gulic 1 , B Ljujic 2 , Nela Maksimovic 1 , Marija Dusanovic Pjevic 1 , Milka Grk 1 , I Rakovic 3 , B Popovska Jovicic 3 , V Volarevic 2,4 , Biljana Jekic 1
1 University of Belgrade, Faculty of Medicine, Institute of Human Genetics, Belgrade, Serbia
2 University of Kragujevac, Faculty of Medical Sciences, Department of Genetics, Kragujevac, Serbia
3 University of Kragujevac, Faculty of Medical Sciences, Department of Infectious diseases, Serbia
4 University of Kragujevac, Faculty of Medical Sciences, Department of Microbiology and Immunology, Kragujevac, Serbia

Coronavirus disease (COVID-19) is an infectious disease caused by highly infectious severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The clinical picture of the COVID-19 disease varies from asymptomatic to severe one. It was noticed that the patients with a severe form of the COVID-19 are more likely to have a history of hypertension, diabetes, and/or cardiovascular disease and receive Renin-Angiotensin-System (RAS) inhibitor
treatment. Hence, it is possible that RAS and Angiotensin-converting enzyme (ACE) has an important role in the pathogenesis of COVID-19. Insertion/deletion (I/D) polymorphism (rs4646994) within the ACE gene is one of the candidates with the potential to affect infection symptoms and mortality. To analyze whether genotypes of the I/D ACE gene polymorphism may influence outcome after COVID-19. The study included 129 patients with
COVID-19 disease treated from May to October 2020 at the Department of Infectious Diseases, Faculty of Medical Sciences, University of Kragujevac, Serbia. Each patient had confirmed PCR SARS-CoV-2 findings. At hospital admission, patients&#39; demographical and clinical data were obtained. To all patients’ chest X-rays, and, if required thoracic computed tomography (CT), as well as all significant laboratory analysis, were performed. Molecular-
genetic analyses were performed at the Institute of Human Genetics, Faculty of Medicine, University of Belgrade. Genotypisation of the ACE I/D polymorphism was performed by polymerase chain reaction (PCR). Statistical analysis was performed by SPSS software 22.0 (SPSS Inc, Chicago, Illinois, USA). Frequencies of DD, ID, II genotypes were 35.7%, 45.0%, and 19.4%, respectively. We have observed no association between analyzed ACE ID genotypes and the outcome after COVID-19 infection (p=0.662). However, after grouping genotypes (DD VS. II+ID), the patients with at least one I allele had statistically lower thrombocyte levels in serum than patients who were homozygotes for D allele (p=0.006). Additionally, patients with same genotypes (ID+II) had lower leucocyte levels compared to patients with DD genotype, but without reaching significance (p=0.069). I/D 67 polymorphism within ACE gene might have an influence on outcome laboratory parameters after COVID-19 infection.
Keywords: SARS-CoV-2, angiotensin-converting enzyme (ACE), Insertion/deletion (I/D) polymorphism
Correspondence: milicagulic05@gmail.com

Ana Djuranovic (University of Belgrade, Faculty of Medicine, Institute of Human Genetics, Belgrade, Serbia):

STUDY OF IL1B AND iNOS GENES POLYMORPHISMS AS A RISK FACTOR FOR THE DEVELOPMENT OF CEREBRAL PALSY

Abstract

Ana Djuranovic 1 , N Cerovac 2 , D Perovic 1 , Nela Maksimovic 1 , Tatjana Damnjanovic 1
1 University of Belgrade, Faculty of Medicine, Institute of Human Genetics, Belgrade, Serbia
2 Clinic of Neurology and Psychiatry for Children and Youth, Belgrade, Serbia

The most common cause of motor disabilities in childhood is cerebral palsy (CP). CP is a group of permanent neurological disorders that affect movement and muscle tone or posture. Hypoxic-ischemic Encephalopathy (HIE) is the most important cause of CP. The immune system and inflammation mediators are activated due to perinatal brain damage. Interleukin 1 beta (IL1B) and iNOS are involved in immune reponse and might be involved in
neurodegeneration. Polymorphisms in these genes may affect its expression and cause further damage. Aim of this study was to examine possible association between IL1B and iNOS polymorphisms and CP onset in children with perinatal asphyxia. Study included 152 patients aged 1 to 16 years with anamnesis of perinatal asphyxia. Detailed neurological evaluation and neuroimaging (ultrasound, computed tomography, magnetic resonance
imaging) were performed for all subjects. IL1B and iNOS polymorphisms were genotyped using rs2297518 and rs16944 TaqMan assays. There was no statistical significant difference in genotype distribution of IL1B polymorphism between patients who developed CP and patients who did not develop CP. Frequencies of iNOS polymorphisms in patients who developed CP were: 64.9% GG, 24.3% AG and 10.8% AA, while in the group of those who did
not develop CP were 59.1% GG, 39.4% AG, and 1.5% AA and there is statisticly significant difference between those groups (p=0.025). AA genotype was significantly more frequent in patients with CP (p=0.036, OR=0.1269, 95%CI 0.0154-1.0437). iNOS gene polymorphism could be a risk factor for CP in patients with perinatal asphyxia.
Keywords: cerebral palsy, IL1b gene, iNOS gene, perinatal asphyxia, SNP polymorphisms
Correspondence: djuranovic.ana@gmail.com

Dijana Majstorovic (University of Rijeka, School of Medicine, Department of Medical Biology and Genetics, Rijeka, Croatia):

FUNCTIONAL DNMT3B PROMOTER POLYMORPHISM (rs1569686) AND RISK FOR CONGENITAL HEART DEFECTS IN DOWN SYNDROME

Abstract

Dijana Majstorovic 2 , Anita Barisic 1 , Iva Bilic–Cace 3 , Mauro Stifanic 4 , Jadranka Vranekovic 1
1 University of Rijeka, School of Medicine, Department of Medical Biology and Genetics, Rijeka, Croatia
2 University Juraj Dobrila University of Pula, School of Medicine, Pula, Croatia
3 University Hospital Centre Rijeka, Department of Pediatrics, Rijeka, Croatia
4 University Juraj Dobrila University of Pula, Faculty of Natural Science, Pula, Croatia

The DNMT3B gene codes for DNA methyltransferase 3b (DNMT3b), a protein required for genome-wide de novo methylation during the early stage of embryonic development. Evidence suggested that impaired DNA methylation could be a risk factor for congenital heart defects (CHD). Down syndrome (DS) is one of the most common chromosomal abnormalities associated with congenital heart defects (CHD), with approximately 40 – 60% of cases showing cardiac defects. The most common CHD phenotypes in DS are congenital malformations of cardiac septa, such as atrioventricular septal defects (AVSDs), ventricular septal defects (VSDs), atrial septal defects (ASDs), and tetralogy of Fallot (TOF). The purpose of this study was to analyze the frequency of the phenotype of CHD as well as allele and genotypes association of functional DNMT3B promoter polymorphism −579 G &gt; T (rs1569686) on the development of CHD in DS. The case-control study included 102 CHD+ Down syndrome cases and 88 non-syndromic CHD cases. The median of the ages for CHD+DS cases was 2 years [0 – 27] and for the control group was 7 years [0 – 32].
Demographic data and phenotype of CHD were collected from medical records of participants after parents and guardians gave their written consent. Genomic DNA was isolated from different types of tissue using a commercial kit and quantified by spectrophotometry. Genotype analysis was performed by PCR-RFLP method. Statistically higher frequencies of AVSD and VSD were detected in a group of CHD+DS than controls (P=0.0044; P=0.0225). Study results showed a statistically lower frequency of the DNMT3B rs 1569686 TT genotype (OR = 0.40; 95% CI: 0.18 – 0.86; P=0.020) in CHD+DS cases compared with controls. AVSD and VSD were the most common CHD phenotypes in DS, which were consistent with the literature. Lower frequencies of DNMT3B (rs1569686) TT genotype in-group CHD+DS suggested that those genotype could decreased risk of CHD in DS individuals. Further ongoing studies will better explain those results.
Keywords: Down syndrome, Dnmt3b, congenital heart defects
Correspondence: dmajstorov@unipu.hr

Daniel Sosiński (Daniel’s Green Technology, Szczecin, Poland):

CO-INHERITANCE OF GENETIC VARIANTS LINKED TO THE RISK OF PREGNANCY LOSS IN BOSNIAN WOMEN- OWN STUDIES REVIEW

Abstract

Daniel Sosiński 1 , Emir Mahmutbegovic 2 , Amina Valjevac 3 , Grażyna Adler 4
1 Daniel’s Green Technology, Szczecin, Poland
2 Institution of Health Protection of Women and Motherhood Canton Sarajevo, Sarajevo, Bosnia and Herzegovina
3 University of Sarajevo, Faculty of Medicine, Department of Human Physiology, Sarajevo, Bosnia and Herzegovina
4 Pomeranian Medical University, Department of Studies in Antropogenetics and Biogerontology, Szczecin, Poland

One of the issues in perinatal medicine is pregnancy loss, which occurs in up to 25% of women with clinically diagnosed pregnancy. The aetiology of pregnancy loss is an extremely complex interplay of: uterine, autoimmune, endocrine, metabolic, abnormal karyotype, antiphospholipid syndrome, thrombophilia, genetic and idiopathic factors. Genetic risk factors in etiopathogenesis of pregnancy loss still remain unclear, and about half of
pregnancies loss remains unknown. However, current knowledge indicates that a co-inheritance of risk factors, including multiple thrombophilic variants linked to secondary hypercoagulable states, have an association with adverse pregnancy outcome. For this purpose we determined, whether the co-inheritance of selected SNPs is linked to a higher risk of pregnancy loss. Each time, the same study group consisted of 154 women with pregnancy loss, mean age 33 (±5.4) y.o. and 154 mothers mean age 31.4 (±6.7) y.o. with at least one live-born child, as a control group were investigated. We reviewed SNPs: rs6025 FV, rs429358 and rs7412 ApoE, rs1799752 ACE, rs1799889 PAI-1, rs1799963 PT, rs1801133 MTHFR, rs9468 and rs1800547 INV 17q21.31, rs731236 and rs1544410 VDR, and rs10421768 HAMP, linked to thrombophilia and the risk of pregnancy loss, published in the years 2015-2019. All statistical analysis for this study were performed using the R CRAN computer software version 3.6.2 (R Core Team (2019). Both in group with and without pregnancy loss, co-inheritance for heterozygotes FV and homozygotes of other investigated genes was from 2 to 4 polymorphic alleles, average 2.5. The most common prevalence of co-inheritance was 2, 3 and 4 alleles. In women with pregnancy loss, there were: 27, 13 and 5, and without pregnancy loss: 34, 11 and 6, respectively. In women without co-inheritance of variants predisposing to thrombophilia (n = 107), the mean number of miscarriages was lower (1.3) compare to women with co-inheritance of these variants (n = 47), (1.6). There was no statistically significant difference between co-inheritance of investigated variants predisposing to thrombophilia and miscarriage occurrence in Bosnian women, (p ˃ 0.05).
Keywords: thrombophilia, variants of genes, co-inheritance, pregnancy loss
Correspondence: gra2@op.pl

Dzenana Klepo (International Burch University, Department of Genetics and Bioengineering, Sarajevo, Bosnia and Herzegovina):

GENETIC BACKGROUND OF FEMALE INFERTILITY

Abstract

Dzenana Klepo
International Burch University, Department of Genetics and Bioengineering, Sarajevo, Bosnia and Herzegovina

Infertility is a huge problem of modern society. According to numerous studies, today 10% of couples in the world population are infertile, and the next 10% have a problem called reduced fertility (subfertility). These changes in fertility of human population are evidently increasing. Recently data showes that infertility affects up to 15% of reproductive-aged couples worldwide. From the aspect of genetics, we can talk about genetic and non-genetic causes of infertility. However, today a large number of studies showes that the occurrence of infertility is very often result of a combination of these two factors, ie. that environmental factors often play the role of &quot;triggers&quot; of certain genetically determined processes, which actually result in development of infertility. When it comes to genetic causes of infertility, we distinguish infertility caused by chromosomal and/or genes abnormalities regarding hereditary material. Even women who do not have defects in chromosomal set, produce more than 20% of chromosomal abnormal eggs, and this percentage increases with aging. Chromosomal disorders related to infertility include
abnormalities in number of chromosome and changes in the their normal structure. This article describes the association of chromosomes changes with the manifestation of phenotypic infertility in following cases of sex chromosomes disorders: Turner syndrome, Mixed gonadal dysgenesis, Swyer syndrome, Y-cell line mosaic form, 46,XX testicular disorder of sex development, Triple X syndrome or  trisomy X or 47,XXX and autosomal
disorders like Robertsonian and reciprocal translocations, chromosome inversions and deletions. Also, article describes the occurrence of infertility in connection with disorders at the gene level, as is the case of: Fragile X chromosome-FRAXA syndrome, Kallman syndrome, Androgen insensitivity syndrome, gene disorders for β-subunit LH and FSH, gene disorders for LH and FSH receptors and XX female gonadal dysgenesis (XX-GD).
Keywords: female infertility, genetic causes, genetic abnormalities, human infertility
Correspondence: dz.h.klepo@gmail.com

Aleksandra Markovic (University of Kragujevac, Faculty of Science, Department of Biology and Ecology, Kragujevac, Serbia):

MICRONUCLEI FREQUENCY IN PATIENTS WITH ENDOMETRIAL CANCER IN RELATION TO STAGE OF DISEASE, AGE AND SMOKING HABITS

Abstract

Aleksandra Markovic 1 , Darko Grujicic 1 , Olivera Milosevic-Djordjevic 1,2
1 University of Kragujevac, Faculty of Science, Department of Biology and Ecology, Kragujevac, Serbia
2 University of Kragujevac, Faculty of Medical Sciences, Department of Genetics, Kragujevac, Serbia

Endometrial cancer is the most frequent malignant tumor, which is in the fourth place in terms of frequency, after breast cancer, colon and lung cancer. So, the aim of this study was to evaluate the chromosomal damage in peripheral blood lymphocytes of patients with newly diagnosed endometrial cancer with respect to stage of disease, age and smoking habits. The analyzed sample included 31 persons, 16 patients with endometrial cancer (8
with G1 and 8 with G2 degree of cancer) age from 52 to 79 (65.94±6.97) years and 15 healthy women age from 44 to 69 (53.40±7.15) years. To estimate the frequency of chromosomal damage, the cytokinesis-block micronucleus (CBMN) assay was used. The mean MN frequency was significantly higher in patients with endometrial cancer compared to healthy controls (19.38±2.87/1000 BN cells vs. 9.00±1.69/1000 BN cells, p&lt;0.001). There
was no significant difference in mean MN frequencies between patients with G1 and G2 degrees (19.13±2.30/1000 BN cells vs. 19.63±2.92/1000 BN cells, p&gt;0.05). The analysis of MN distribution per 1000 BN/cells person showed that the cells with 1MN (1.15%) were mostly presented in both patients and controls, while cells with 2MN were less common (0.11%). Cells with 3MN (0.02%) were seen only in a sample of patients. Taking into account
the factors that can affect the frequency of MN, using multifactorial linear regression analysis we found that health status (diagnosis) as well as degree of cancer significantly affected the frequency of MN (p&lt;0.001),while age, NDI values and cigarette smoking did not affect MN (p&gt;0.05). Analysis of nuclear division index (NDI) showed that patients had not significantly lower values compared to healthy controls (1.52±0.17 vs. 1.56±015; p&gt;0.05).
There was also no significant difference in NDI values between patients with different cancer degrees (1.49±0.16 for G1; 1.56±019 for G2; p&gt;0.05). Multifactorial linear regression analysis showed that health status, degree of cancer, MNi, age and cigarette smoking did not affect NDI values. Based on the results we can conclude that the patients with endometrial cancer had an increased frequency of MN frequency in human lymphocytes
which is in correlation with the stage of the disease. Keywords: endometrial cancer, peripheral blood lymphocytes, micronuclei, chromosomal damage.
Correspondence: aleksandra.markovic@pmf.kg.ac.rs

Jovana Tubic Vukajlovic (University of Kragujevac, Faculty of Science, Department of Biology and Ecology, Kragujevac, Serbia):

OXIDATIVE DNA DAMAGE IN PERIPHERAL BLOOD LYMPHOCYTES OF PATIENTS WITH CARDIOVASCULAR DISEASES

Abstract

Jovana Tubic Vukajlovic 1 , Ivan Simic 2,3 , Zorica Smiljanic 3 , Darko Grujicic 1 , Olivera Milosevic-Djordjevic 1,4
1 University of Kragujevac, Faculty of Science, Department of Biology and Ecology, Kragujevac, Serbia
2 University of Kragujevac, Faculty of Medical Sciences, Department of Internal Medicine, Kragujevac, Serbia
3 Clinical Center Kragujevac, Department of Cardiology, Kragujevac, Serbia
4 University of Kragujevac, Faculty of Medical Sciences, Department of Genetics, Kragujevac, Serbia

Cardiovascular diseases are multifactor progressive pathologies that seriously harm human health. Many risk factors for developing these diseases are related to lifestyle as well as to genetic factors. Thus, this study aimed to evaluate the association between potential risk factors for cardiovascular diseases and levels of oxidative DNA damage in peripheral blood lymphocytes of cardiovascular patients, including patients with acute coronary syndrome
and heart failure with reduced ejection fraction. The study included 30 persons, 20 cardiovascular patients (57.55 ± 4.65 years) and 10 healthy controls (55.40 ± 5.56 years). The frequency of DNA damage of individual cells expressed as genetic damage index (GDI) was analyzed using the alkaline comet assay. Cells were classified into five classes (0-4) depending on the degree of DNA damage. Increased levels of oxidative DNA damage were
observed in cardiovascular patients comparing to the healthy controls (1.36 ± 0.15 vs. 0.37 ± 0.05, p &lt; 0.001). However, when we analyzed only cardiovascular patients, we also noticed a difference in GDI values. Patients with health failure with reduced ejection fraction had significantly higher mean GDI value than patients with acute coronary syndrome (1.44 ± 0.16 vs. 1.28 ± 0.10, p &lt; 0.05). The number of undamaged cells decreased in cardiovascular patients comparing to healthy persons for about 1.5 times, while number of cells with tail (comet classes from 1 to 4) in cardiovascular patients was increased almost four times comparing to controls. Multiple linear regression analysis showed that health condition, drug therapy, family history of cardiovascular disease and triglyceride were predictors of DNA damage in patients with cardiovascular diseases (p &lt; 0.05), while age, gender, blood
pressure, cholesterol and cigarette smoking were not significant. We can conclude that increased level of genome instability was observed in cardiovascular patients, among which patients with health failure with reduced ejection fraction have higher level of DNA damage in peripheral blood lymphocytes than patients with acute coronary syndrome. This conclusion is correlated with disease severity and prognosis of treatment.
Keywords: acute coronary syndrome, DNA damage, peripheral blood lymphocytes, comet assay
Correspondence: jovana.tubic@pmf.kg.ac.rs

Jelena Vukajlovic (Mother and Child Health Care Institute of Serbia “Dr Vukan Cupic”, Laboratory of Medical Genetics, Belgrade, Serbia):

DETECTION OF P2RY8/CRLF2 REARRANGEMENT IN PEDIATRIC B-ALL PATIENTS FROM SERBIA

Abstract

Jelena Vukajlovic 1 , B Crnojevic 1 , N Ilic 1 , D Radivojevic 1 , M Kuzmanovic 2,3 , M Djurisic 1
1 Mother and Child Health Care Institute of Serbia “Dr Vukan Cupic”, Laboratory of Medical Genetics, Belgrade, Serbia
2 Mother and Child Health Care Institute of Serbia “Dr Vukan Cupic”, Department of Hematology and Oncology, Belgrade, Serbia
3 University of Belgrade, Faculty of Medicine, Belgrade, Serbia

B-cell acute lymphoblastic leukemia (B-ALL) represents the most common form of ALL in children and the leading cause of pediatric cancer related mortality. Defining its distinct genetic profile is paramount for B-ALL classification, risk stratification and treatment evaluation. Recently, in a group of B-ALL patients with no prognostically significant, recurrent genetic rearrangements (such as BCR/ABL, MLL/AF4, TEL/AML, PBX/E2A,
hyper/hypodiploidy and other cytogenetic aberrations), new entity emerged, provisionally defined as BCR/ABL1-like ALL, associated with adverse outcome. Fusion of a member of the purine nucleotide G protein-coupled receptor gene family with cytokine receptor-like factor 2 gene (P2RY8/CRLF2) is defined as one of the most common hallmark of this entity, with reccurence of 5-8% in BCP-ALL cases. Aim of this study was to determine the presence of
P2RY8/CRLF2 rearrangement in a group of pediatric B-ALL patients with no recurrent molecular and cytogenetic alterations. From november 2009. to june 2021. year in Laboratory of Medical Genetics at Mother and Child Health Care Institute of Serbia 340 B-ALL pediatric patients at day of diagnosis were analyzed for BCR/ABL, MLL/AF4, TEL/AML, PBX/E2A rearrangements and cytogenetics. In total, 59 samples negative for aforementioned aberrations, were tested for P2RY8/CRLF2 fusion. Chromosomal analysis, detection of recurrent aberrations and P2RY8/CRLF2 fusion transcripts was done on bone marrow (BM) samples using GTG banding and RT-PCR protocols. Among 59 patients 24 (41%) were positive for P2RY8/CRLF2 fusion. This rearrangement, acorrding to the current WHO classification, defines these patients as B-ALL subgroup with translocation involving tyrosine kinase/cytokine receptors, which is described as BCR/ABL1-like. Aproximative incidence in whole cohort of patients of this rearrangement was 7% (24/340) and is in line with literature. According to current guidelines, P2RY8/CRLF2 rearrangement has potential prognostical and therapy related impact in a significant group of patients. BCR/ABL1-like ALL entity has defined genetic signature which is the hallmark for adverse prognosis. High prevalence of this alteration in BCP-ALL patients with no reccurent aberrations found, pose its significance in redefining risk stratification and implementation in routine testing.
Keywords: BCR/ABL1-like, P2RY8/CRLF2, B-ALL
Correspondence: jelenavuk@ymail.com

Milica Jaksic Karisik (University of Belgrade, School of Dental Medicine, Department of Human Genetics, Belgrade, Serbia):

OSTEOGENIC DIFFERENTIATION POTENTIAL OF ORAL CANCER STEM CELLS

Abstract

Milica Jaksic Karisik, Milos Lazarevic, Maja Milosevic Markovic, Jelena Milasin
University of Belgrade, School of Dental Medicine, Department of Human Genetics, Belgrade, Serbia

Cancer stem cells (CSCs) are responsible for cancer aggressiveness, drug resistance, and tumor relapse. CD44 has been identified as a CSC surface marker and has been used for the isolation and enrichment of cultures with CSC population in different types of cancers, including oral squamous cell carcinoma (OSCC). Some CSCs possess the potential to transdifferentiate into different cell lineages, as do normal adult stem cells. Such plasticity
of CSCs is considered to be a promising therapeutic target. The aim of this study was to examine osteogenic differentiation potencial of CD44⁺ cells isolated from comercial cell line SCC-25. CD44⁺ and CD44⁻ cells were magnetically separated using magnetic-activated cell sorting system. CD44⁺ and CD44⁻ cells were seeded into 24-well culture plates (8×10 4 per well) and cultured in osteogenic differentiation medium for 7 and 14 days. Cells were
cultivated under standard conditions in humidified atmosphere with 5% CO 2 at 37°C. After 14 days total RNA was extracted from the culture cells with TRIzol Reagent. The expression levels of CSC markers (Oct-4, Sox2, Nanog) and markers of osteo-differentiation (ALP, BMP4, Runx2) were analyzed by qPCR. Specific staining techniques and cell morphology were used for differentiation confirmation. Concomitantly with differentiation and the increase of
osteogenic markers, the levels of cancer stem cell markers decreased in the cultures. Osteogenic markers showed that CD44⁺ had significantlly higher osteo-differentiation potential in comparison with CD44⁻ cells. In conclusion, OSCC CD44+ cells exhibit the capacity to differentiate into osteoblastic lineage, a characteristic that may potentially be useful in the development of new strategies for OSCC differentiation therapy.
Keywords: cancer stem cells, oral squamous cell carcinoma, differentiation potential, osteogenic differentiation
Correspondence: milica.jaksic@stomf.bg.ac.rs

Svetlana Janceva (University Clinic for Rheumatology, Skopje, Republic of North Macedonia):

THE IMPORTANCE OF ANTI-dsDNA ANTIBODIES SCREENING AS A USEFUL TOOL FOR EVALUATION AND TREATMENT OF PATIENTS WITH SYSTEMIC RHEUMATIC DISEASE

Abstract

Svetlana Janceva 1 , Nevenka Velickova 2
1 University Clinic for Rheumatology, Skopje, Republic of North Macedonia
2 University “Goce Delcev”, Faculty of medical sciences, Stip, Republic of North Macedonia

Anti-Double Stranded DNA or anti-dsDNA antibodies are anti-nuclear antibodies (ANA) typically produced by the immune system, who target the antigens of the double-stranded DNA. These anti-nuclear antibodies target the essential parts of the cell’s nucleus especially the genetic material. In normal condition the antibodies protect the organism from different types of infections and inflammations, contrary to that these anti-antibodies don’t
differentiate their own cells. In those circumstances the anti-nuclear antibodies attack their own healthy cells, causing pathophysiological changes in the tissue and the organ itself. By direct binding to self-antigens or indirect formation of immune complexes, anti-dsDNA antibodies can be accumulated in the glomerular and tubular basal membranes. Systemic lupus erythematosus (SLE) is characterized by high-titer serological autoantibodies,
including double-stranded DNA molecule (dsDNA) antibodies. The main objective of this research is to define the importance of anti-dsDNA antibodies screening as a useful tool for the evaluation and treatment of patients with systemic rheumatic disease. Evaluation and screening of anti-dsDNA antibodies is done and confirmed with enzyme-linked immunosorbent assay (ELISA) on patients with SLE in the period from July to December
2020, monitored at the University Clinic for Rheumatology in Skopje, Republic of North Macedonia. The enzyme-linked immunosorbent assay (ELISA) for anti-dsDNA antibodies shows 98-100% specificity and 40-60% sensitivity for SLE. The prevalence for SLE is around 4/100.000 of the population, but more than 99% of the cases occur in young women, although there is no age range. If a patient has a positive ANA test results plus shows
symptoms like SLE, the laboratory diagnostics and anti-dsDNA antibodies screening is very useful tool for evaluation and treatment of patients with systemic rheumatic disease. Early diagnostic of SLE and lupus nephritis is very important for suitable therapy, it could stop the progression of the disease, decrease the mortality, and increase the degree’s quality of life on these patients.
Keywords: anti-dsDNA antibodies, anti-nuclear antibodies, systemic lupus erythematosus, enzyme-linked immunosorbent assay (ELISA)
Correspondence: nevenka.velickova@ugd.edu.mk

Nika Puseljic (University of Osijek, Faculty of Medicine, Osijek, Croatia):

ANORECTAL ATRESIA AS PART OF MICRODUPLICATION SYNDROME 16p13.3

Abstract

Nika Puseljic 1 , Visnja Tomac 1,2 , Nora Puseljic 3 ; Ema Poznic 4 ; Silvija Puseljic 1,2
1 University of Osijek, Faculty of Medicine, Osijek, Croatia
2 University Hospital Center, Department of pediatrics, Division of neurology, genetic, metabolic disease and endocrinology, Osijek, Croatia
3 KBC Osijek, Emergency medicine, Osijek, Croatia
4 DZO Osijek, Family medicine, Osijek, Croatia

16p13.3 microduplication syndrome is a rare genetic condition mostly characterized by: typical facial dysmorphisms and variable intellectual disability. Other features include microcephaly, growth retardation, limb anomalies, and defects of the brain, heart, genitalia, palate, and eyes. The aim was to show anorectal atresia as part of microduplication syndrome 16p13.3 which hasn’t been found in the literature. A 2-year-old girl, born from the second pregnancy, of healthy non-consanguine parents. At 20 weeks of gestation, oligohydramnios was suspected. She has been monitored multidisciplinary since birth due to multiple difficulties as part of the malformation syndrome: anal atresia with rectoperinal fistula, open ductus arteriosus – spontaneous closure, atrial septal defect II, congenital laryngomalacia with inspiratory stridor, hypotonia, frenulum linguae breve. Phenotypic dysmorphia includes larger neurocranium compared to viscerocranium, poorly shaped and malpositioned ears, epicanthal folds, wider nasal root, clinodactyly. In the first year of life, anorectal atresia surgery was performed. Laryngomalacia is now less pronounced and less interferes with feeding, established coordination of breathing and swallowing, and no crisis of cyanosis. Chromosomopathy has been ruled out by karyotyping and due to the need for
further genetic evaluation, Chromosomal Microarray Analysis was performed – analysis revealed three copies of the 262 kb genome in the region 16p13.3, which includes the RBFOX1 gene. This gene is predominantly expressed in brain neurons and plays a key role in regulating neuronal excitation and influencing the susceptibility to the development of epilepsy. This presentation expanded the range of clinical manifestations for microduplication syndrome 16p13.3 with a unique clinical feature of anorectal atresia.
There is no specific treatment for this condition, affected individuals need multidisciplinary monitoring in the direction of improving the quality of life. The therapeutic measures should be on supporting the parents and also suggest genetic counseling for the next planned pregnancies.
Keywords: anorectal atresia, microduplication syndrome 16p13.3, chromosomal microarray analysis, RBFOX1 gene
Correspondence: nika.puseljic1@gmail.com

Bioengineering, Biotechnology and Bioinformatics

Anesa Ahatovic (University of Sarajevo, Institute for Genetic Engineering and Biotechnology, Sarajevo, Bosnia and Herzegovina):

IDENTIFICATION OF ACC DEAMINASE PRODUCING METAL TOLERANT BACTERIA INHABITING Medicago lupulina L. RHIZOSPHERE

Abstract

Anesa Ahatović, Mujo Hasanović, Adaleta Durmić-Pašić
University of Sarajevo – Institute for Genetic Engineering and Biotechnology

In natural environments plants are continuously exposed to different biotic and abiotic stressors which affect their growth. When confronted by various kinds of stressors, plants respond by readjusting levels of ethylene which reduces shoot and root growth, thus re-establishing homeostasis. The ACC (1-aminocyclopropane-1-carboxylic acid) is key intermediate in ethylene synthesis. Some plant-growth-promoting rhizobacteria produce
ACC deaminase which cleaves ACC and interrupt ethylene synthesis. Serpentine soils, derived from ultramafic rocks, with its unique physical and chemical properties are inhospitable environment for plant growth. As these soils are expected to be the source of metal tolerant bacteria we collected samples of rhizosphere associated with Medicago lupulina L. from five sites in Krivaja – Konjuh ophiolite complex (Bosnia and Herzegovina).
Heavy metals and nutrients concentrations were determined by flame atomic absorption spectroscopy. Heavy metal tolerance of isolated rhizobacteria was tested using tryptone yeast agar supplemented with Cu, Ni and Co. Metal-tolerant isolates were screened for ACC deaminase activity by inoculation of bacterial suspensions on DF (Dworkin &amp; Foster) salts solid medium supplemented with 1 mM ACC and medium without ACC (negative control).
Identification of metal tolerant isolates with ACC deaminase activity was performed using DNA sequencing of the 16S rRNA gene. Total of 124 tested bacterial isolates showed strong resistance to Cu and Ni and weaker resistance to Co. Isolates that showed multiple heavy metal resistance were screened for ACC deaminase activity. Out of 35 tested metal tolerant bacterial isolates, 24 showed intense growth on DF media supplemented with ACC. This indicates using ACC as a sole nitrogen source and implies ACC deaminase activity. Identification showed that most isolates belong to Phyla Proteobacteria with Pseudomonas as most abundant genus. Some representatives of Firmicutes were also identified. ACC utilizing bacterial strains which have the potential to curb stress induced ethylene production in plants will be further screened for other PGP traits and used in plant inoculation experiments in order to test their capacity to mitigate stress in plants.
Keywords: rhizobacteria, ethylene, ACC deaminase, serpentine soil, heavy metals
Correspondence: anesaahatovic@gmail.com

Belmina Saric (University of Sarajevo, Institute for Genetic Engineering and Biotechnology, Sarajevo, Bosnia and Herzegovina):

GLU288 AND LYS137 OF SARS-CoV-2 MAIN PROTEASE AS TARGETS FOR INHIBITION BY SELECTED THYME PHYTOCONSTITUENTS

Abstract

Saric Belmina 1 , Tomic Nikolina 1 , Bajrovic Kasim 1 , Lojo-Kadric Naida 1 , Ramic Jasmin 1 , Pojskic Lejla 1 *
1 University of Sarajevo, Institute for genetic engineering and biotechnology, Sarajevo, Bosnia and Herzegovina

Since the outbreak of the Coronavirus Disease 19 (COVID-19) pandemic, researchers have been trying to investigate various active compounds found in plants that could have potential to inhibit the proliferation of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2).
Thymus vulgaris L., member of Lamiacae family, is an aromatic and medicinal plant that has been used in traditional medicine. It has showed antihelminthic, expectorant, antiseptic, antispasmodic, anti-microbial, antifungal, antioxidative, antivirotic, carminative, sedative, and diaphoretic effects due to the possession of a wide range of secondary metabolites. Therefore, the aim of this study was to evaluate bioactive compounds found in Thymus vulgaris L. as potential antiviral components and inhibitors of the SARS-CoV-2 main protease (Mpro), using a molecular docking approach. The main protease (3-chymotrypsin-like protease) plays a key role in viral gene expression and replication and serves as an important target in drug designing against COVID-19. Using in silico method we analyzed the binding affinity of thymol, carvacrol, rutin and thymoquinone, as phytoconstituents of Thyme, to SARS-CoV-2 main protease Mpro (PDB ID: 6Y84), using acetoside as a positive control of binding affinity. Obtained results by molecular docking showed highest affinities (rmsd l.b. 0.000; rmsd u.b. 0.000) for rutin (-10.3
kcal/mol), thymol (-6.1 kcal/mol), thymoquinone (-6.1 kcal/mol), carvacrol (-5.9 kcal/mol), and for positive control acetoside binding affinity was -10.0 kcal/mol. Visualization of interaction between SARS-CoV-2 main protease and ligands showed that carvacrol, rutin and thymol had interaction close to positions Glu288. Also, carvacrol and thymol showed similarity for binding modes to main protease close to position of Lys137 just as positive control acetoside. After analyzing the docking modes and docking scores we have found that phytoconstituents of Thyme have potential to be inhibitors of SARS-CoV-2 Mpro and also to be used as adjuvant in treatment of COVID-19, but greater studies and clinical researches are needed to confirm that and to elucidate mechanism of action.
Key words: Thyme, molecular docking study, SARS-CoV-2, main protease
Corresponding author’s e-mail: belmina.saric@ingeb.unsa.ba

Tarik Corbo (University of Sarajevo, Institute for Genetic Engineering and Biotechnology, Sarajevo, Bosnia and Herzegovina):

IMPORTANCY OF MACHINE LEARNING IN BIOLOGICAL STUDIES

Abstract

Tarik Corbo, Abdurahim Kalajdzic, Naris Pojskic

 University of Sarajevo, Institute for Genetic Engineering and Biotechnology, Sarajevo, Bosnia and Herzegovina

 In the fields of genetics and genomics, machine learning approaches have been used in a variety of applications. Algorithms of this technique use specific data in order to detect certain patterns, build certain models, as well as make predictions based on the best possible model. This method has been used to annotate a wide range of genomic sequence elements and is perhaps most useful for the interpretation of large genomic data sets. Also, this approach is used to predict variants of various biological data, in particular genetic, whether in terms of DNA sequence, the protein structure or other markers. Machine learning systems that can annotate genes can be built using models that each recognize a specific type of genomic element, as well as learnt logic about their relative positions. To better understand the mechanics underlying gene expression, a range of machine learning algorithms have been created. Some methods seek to predict a gene’s expression only based on its DNA sequence, while more advanced methods aim to model the expression of all of the genes in a cell by developing a network model. However, in recent years, artificial neural networks have become increasingly popular in the processing and prediction of biological data. These networks, which resemble the central nervous system, are made up of interconnected neural computing elements that can respond to input stimuli and adaptation processes. By using algorithms that imitate real processes in neurons, the network itself can learn to solve a particular problem. The learning process includes recognition and training patterns. Recognition patterns are data classification processes that aim to identify potential correlations between variables. Training patterns aid neural networks in the adaptation process by training the network to recognize input patterns and generate output data linked with the output pattern. The expanding usage of artificial neural networks in biology is clear, with the core purpose being the same: to generate a prediction based on existing data. Because neural networks cannot resolve all problems, a combination of classical and modern bioinformatics analysis, with the addition of an artificial neural network, would be the optimum strategy.

 

Keywords: machine learning, neural network, prediction, simulation

Correspondence: tarik.corbo@gmail.com

Mirsada Salihovic (University of Sarajevo, Faculty of Pharmacy, Faculty of Science, Department of Biology, Sarajevo, Bosnia and Herzegovina):

INVESTIGATION OF THE ANTIOXIDANT ACTIVITY OF PHYSALIS ALKEKENGI L. VARIOUS EXTRACTS

Abstract

Mirsada Salihovic1, Mirha Pazalja1, Irma Mahmutovic-Dizdarevic2, Bakir Avdic2

1University of Sarajevo, Faculty of Pharmacy, Sarajevo, Bosnia and Herzegovina

2University of Sarajevo, Faculty of Science, Department of Biology, Sarajevo, Bosnia and Herzegovina

 

Physalis alkekengi L. belongs to the genus Physalis of the family Solanaceae. Studies have shown that it has good anti-inflammatory, antioxidant, antimicrobial, diuretic, immunomodulatory activities and that some compounds are responsible for inhibiting the proliferation of tumor cells. P. alkekengi contains active metabolites such as flavonoids, alkaloids, phenylpropanoids, and physalins, which are responsible for many of the effects of this plant. P. alkekengi fruits were dried in an airy room. Dried fruits were used to prepare methanolic extract using the maceration method and ethanolic extract using the Soxhlet method. The antioxidant activity of methanolic and ethanolic extracts of P. alkekengi was measured by the DPPH method at the IC50. The obtained IC50 values for ethanolic and methanolic extracts are 1.97 ± 0.35 and 3.34 ± 0.26 mg/mL. These values were compared with the obtained IC50 values for ascorbic acid, which is used as a commercially available antioxidant and ranges from 0.13 ± 0.03 mg/mL. The concentrations of ethanolic and methanolic extract required to neutralize 50% of DPPH radicals are significantly higher than the required concentration of ascorbic acid. The results indicated that the dry fruits of P. alkekengi provide good antioxidants.

 

Keywords: Physalis alkekengi L., ethanol extract, methanol extract, antioxidant activity, DPPH

Correspondence: mirsada.salihovic@ffsa.unsa.ba

Mujo Hasanović (University of Sarajevo, Institute for Genetic Engineering and Biotechnology, Sarajevo, Bosnia and Herzegovina):

SIDEROPHORE PRODUCTION BY BACTERIA ISOLATED FROM ROBINIA PSEUDOACACIA L. ROOTS AND SERPENTINE SOIL

Abstract

Mujo Hasanović 1 , Anesa Ahatović 1 , Emir Hrelja 2 , Adaleta Durmić-Pašić 1
1 University of Sarajevo-Institute for Genetic engineering and biotechnology, Sarajevo, Bosnia and Herzegovina
2 University of Sarajevo, Faculty of Science, Sarajevo, Bosnia and Herzegovina

Heavy metal contamination of soil poses concerns regarding human health and a balanced ecosystem. Mounting evidence suggests that devastating anthropogenic activities endanger different types of soil across the world and influence plant growth and their survival. Therefore, creating different strategies to alleviate the detrimental impacts of heavy metal burdened soils and understanding the plant-microbe interactions is a promising solution.
These interactions are often bolstered with various activities and compounds produced by plant-growth-promoting bacteria (PGPB). As compounds produced by PGPB, siderophores are small molecules responsible for the chelation of dissolved iron and solubilization of iron aggregates. Since siderophores can have a crucial role for plants in situations of low iron availability or nutrient deficiency, the main goal of this study was to isolate soil bacteria and
assess their ability to produce siderophores. Since Robinia pseudoacacia L. (black locust) exhibits considerable adaptability to nutritiously poor soil, we opted to collect bacterial isolates from serpentine soil and roots of R. pseudoacacia (Donja Paklenica, Bosnia and Herzegovina). Bacterial colonies from collected samples were isolated on yeast mannitol agar (YMA) and subsequently tested for the heavy metal tolerance on Tripton Yeast Agar
(TYA) supplemented with Cu, Ni, Co in different concentrations. Siderophore production was tested using qualitative (CAS Agar assay) and quantitative (spectrophotometric) methods.
The results of spectrophotometric method showed that all 26 selected metal tolerant isolates produced siderophores in a range from 10.96% to 96.66% siderophore units (SU).
After seven days of cultivation on CAS Agar, siderophore producing isolates exhibited an orange halo ranging from 3 to 20mm in radius. One root isolate did not produce siderophores on CAS Agar but showed 32.86% SU of production performing the quantitative method. This discrepancy between methods can be explained by the HDTMA activity (component of CAS Agar) which can inhibit or completely stop the growth of gram-positive
bacteria. However, this is yet to be confirmed by 16S rRNA sequencing. In comparison to root isolates, isolates collected from the serpentine soil showed intensive siderophore production. The forthcoming study will include screening of other PGP strains and 16S rRNA sequencing for bacterial identification.
Keywords: siderophore production, Robinia pseudoacacia, PGP bacteria, heavy metals
Correspondence: mujohh@gmail.com

Bakir Avdic (University of Sarajevo, Faculty of Pharmacy, Sarajevo, Bosnia and Herzegovina):

INVESTIGATION OF THE ANTIOXIDANT ACTIVITY OF PHYSALIS ALKEKENGI L. VARIOUS EXTRACTS

Abstract

Genetics of Natural Resources

Jasna Hasanbegovic (University Dzemal Bijedic of Mostar, Agromediterranean Faculty, Mostar, Bosnia and Herzegovina):

GENETIC STRUCTURE AND DIVERSITY OF INTERNATIONAL CULTIVARS OF ALMONDS (PRUNUS AMYGDALUS L.) IN HERZEGOVINA

Abstract

Jasna Hasanbegović 1 , Semina Hadžiabulić 1 , Fuad Gaši 2 , Azra Skender 3
1 University Dzemal Bijedic of Mostar, Agromediterranean Faculty, Mostar, Bosnia and Herzegovina
2 University of Sarajevo, Faculty of Agricultural and Food Sciences, Sarajevo, Bosnia and Herzegovina
3 University of Bihac, Biotechnical Faculty, Bihac, Bosnia and Herzegovina

The research of genetic characterization of international almond varieties in the territory of Herzegovina includes seven international almond cultivars. The international group of almonds consisted of selections from Italy (Tuono, Genco, Supernova), two selections from France (Fereagnes and Ferraduel) and two originating from the USA (Texas and Nonpareil).
Genetic characterization was performed using 10 microsatellite markers, of which 9 microsatellite markers were derived from Prunus persicae and 1 from Prunus armeniaca.
Microsatellite primers, used in the preparation of this paper, showed high polymorphism in previous studies by a group of authors who analyzed almond populations. The results of genetic characterization show that the total number of alleles detected by 10 pairs of primers in seven international almond cultivars was 5.4 alleles per locus. The average number of effective alleles for the ten SSR loci of international cultivars was 3.924. The
Shannon Information Index averaged 1.413. The observed heterozygosity (Ho) averaged 0.529 and the expected heterozygosity (He) was 0.686. The results of these studies indicate that in the territory of Herzegovina there are international cultivars of almonds that can be used in breeding programs to improve the represented genotypes of the free population of almonds in the territory of Herzegovina.
Keywords: almond, international group, cultivars, microsatellites, genetic characterization
Correspondence: jasna.hasanbegovic@unmo.ba

Lejla Usanovic (University of Sarajevo, Institute for Genetic Engineering and Biotechnology, Sarajevo, Bosnia and Herzegovina):

THE IMPORTANCE OF MONITORING TICKSIN THE URBAN REGION CITY OF SARAJEVO, BOSNIA AND HERZEGOVINA

Abstract

Lejla Usanovic 1 , Lejla Lasic 1 , Alma Sejtarija-Memisevic 2 , Jasna Hanjalic 1 , Dalila Destanovic 1 , Belma Kalamujic Stroil 1
1 University of Sarajevo-Institute for Genetic Engineering and Biotechnology, Sarajevo, Bosnia and Herzegovina
2 Clinical Center of University of Sarajevo, Department of Infectology, Sarajevo, Bosnia and Herzegovina

Changes in soil, vegetation, and climate, as well as the anthropogenic pressure, make living beings adapt, migrate or become extinct. Ticks are very dependent on biotic and abiotic conditions for their local survival and reproduction. Changes in the composition of ticks, as vectors of different pathogens, affect the dynamics of the development of vector-borne diseases in a particular region. Bosnia and Herzegovina does not have a continuous monitoring program on ticks, including the City of Sarajevo, the most crowded urban area.
Thus, we aimed to preliminary evaluate tick diversity and abundance in the City of Sarajevo and their impact on pathogen prevalence. Researches were conducted in 2013, 2019, and 2021 in the vegetation period between May and September. Samples from 2013 and 202 represent ticks collected by dragging method from vegetation in the urban areas, while the 2019 sample consisted of ticks removed from patients in the Emergency Medical Assistance.
After morphological identification, several samples could not be identified with certainty as Ixodes ricinus, so molecular confirmation was performed targeting the ITS region. The Ixodes ricinus was the most abundant species in all three periods with a tendency of growth (respectively 51.3%, 98.9%, and 100%), indicating the possibility of a high prevalence of Borrelia in this urban area. Additionally, we found Rhipicephalus sanguineus (48.7%) in
2013, and Hyalomma marginatum (0.53%) and Dermacentor reticulatus (0.53%) in 2019.
Although from sporadic and discontinuous researches, these results preliminary show a difference in distribution and the diversity of ticks in this timeline. The establishment of continuous monitoring of ticks at the same localities in Sarajevo, accompanied with data on temperature, humidity, potential hosts and main factors influencing the life cycle of ticks, should be a consequential result of this study.
Keywords: ticks, Ixodes, biomonitoring, pathogen
Correspondence: usanovic.lejla@gmail.com

Dalila Destanovic (University of Sarajevo, Institute for genetic engineering and biotechnology, Sarajevo, Bosnia and Herzegovina):

IS DNA BARCODING A FEASIBLE TOOL FOR BIODIVERSITY ASSESSMENT IN THE PROTECTED AREAS OF CANTON SARAJEVO?

Abstract

Dalila Destanovic 1,2 , Lejla Usanovic 1,2 , Jasna Hanjalic 1,2 , Lejla Lasic 1,2 , Adnan Cucukovic 2 , Semir Doric 2 , Jasmina Subara-Cehic 2 , Belma Kalamujic Stroil 1,2
1 University of Sarajevo, Institute for genetic engineering and biotechnology, Sarajevo, Bosnia and Herzegovina
2 Society for Genetic Conservation of Bosnia and Herzegovina’s Endemic and Autochthonous Resources – GENOFOND, Sarajevo, Bosnia and Herzegovina

There are five protected areas in Canton Sarajevo (Bentbaša, Bijambare, Skakavac, The Spring of Bosna River and Trebević), and each includes a form of a freshwater body. Therefore, special attention should be paid to the preservation of freshwater species. The traditional approach in species identification of benthic macroinvertebrates can be challenging for many reasons and requires efforts of several narrowly specialized taxonomists. Consequently, DNA barcoding has been widely applied as an auxiliary method in assessing and biomonitoring plankton, benthos and nekton. With protected areas of Canton Sarajevo as a model, the goal of our study was to test the applicability of DNA barcoding for species determination using available infrastructure and biodiversity data. The total macrozoobenthic community was sampled at the Natural monument “Skakavac” at four sub-localities. Caddisflies, the most researched group of benthic macroinvertebrates in Bosnia and Herzegovina, were sampled at all five protected areas. Sampling was done according to the AQEM methodology. Genomic DNA from specimens was isolated by a modified salting-out protocol. Both Folmer and JJ-primers were used to amplify the standardized DNA barcoding region of cytochrome c oxidase subunit I (COI). A subset of representative specimens was DNA barcoded and determined to the lowest taxonomic level possible. Results were as follows: Ephemeroptera-100% to genus level; Plecoptera-66.66% to species level, 33.33% to genus level, Diptera-83.33% to genus level, 16.66% to family level; Annelida-100% to genus level; Trichoptera- 54.05% to species level, 45.95% to genus level. At the current state of research and present records of species of Bosnia and Herzegovina in the BOLD database, DNA barcoding cannot be used as a sole tool in biomonitoring of protected areas in Canton Sarajevo. Further research in these areas, coupled with morphological species identification in collaboration with specialized
taxonomists and genetic characterization, is what academic institutions and non-governmental organizations should strive for.
Keywords: Macrozoobenthos, cytochrome c oxidase subunit I, freshwater bioassessment
Corresponding author’s e-mail: dalila.destanovic@gmail.com

Biomonitoring and Genetic Toxicology

Dea Luzha (University of Prishtina, Department of Biology, Xhorxh Bush, Kosovo):

GENOTOXIC AND ANTIGENOTOXIC PROPERTIES OF THE HYPERICUM PERFORATUM L. EXTRACTS ON ALLIUM CEPA L. ROOT TIP CELLS

Abstract

Dea Luzha, Fisnik H Asllani, Avni Hajdari, Avdulla J Alija
University of Prishtina, Department of Biology, Xhorxh Bush, Kosovo

For centuries, traditional medicine incorporates the use of plant extracts for the treatment of various diseases. Hypericum perforatum L. is among the plants that are widely used in traditional medicine and it is one of the ethnomedical plant species used frequently in Kosovo too. Due to its beneficial effects, Hypericum perforatum L. remains popular and it is used also in modern therapies. On the other hand, there is a growing interest in evaluating
the toxicological properties of the extracts from this plant. In this study, the genotoxic and antigenotoxic properties of the Hypericum perforatum L. extracts were evaluated. The extracts were prepared by infusing plant dry parts in boiling water and keeping in for a total of 10 minutes. The Allium cepa bulbs were exposed to concentrations of 0.5, 1, and 5% of plant extracts for 24h. The effects on the frequency of the micronucleated cells as well as on
the frequency of chromosomal aberrations in root tip cells were assessed. The mitotic index was also assessed. The protective effects of the plant extracts in the concentrations of 0.5, 1, and 5% against the paraquat-induced damages were evaluated too. The extracts in the concentration of 0.5% showed no effect on the mitotic index, micronucleus frequencies, and chromosomal aberrations. The concentration of 1% had an effect (p&lt;0.01) in increasing the
frequency of chromosomal aberrations whereas the highest concentration (5%) of the extract caused a decrease (p&lt;0.001) in the mitotic index. On the other hand, the concentrations 0.5% and 1% showed a protective effect with regard to chromosomal aberrations induced by paraquat as well as a protective trend against the paraquat-induced decrease in the mitotic index. In addition, all three concentrations of the plant extracts showed protective effects with regard to the micronucleus frequencies caused by paraquat.
The obtained data from this study indicate a concentration-dependent genotoxic activity of the Hypericum perforatum L. extracts as well as a protective potential of these extracts against paraquat-induced damages in Allium cepa L. root tip cells.
Keywords: Hypericum perforatum L., genotoxic, Kosovo
Correspondence: avdulla.alija@uni-pr.edu

Maida Hadzic (University of Sarajevo, Institute for genetic engineering and biotechnology, Sarajevo, Bosnia and Herzegovina):

SELECTIVE GENOTOXICITY OF HALOGENATED BOROXINE, K2(B3O3F4OH) IN UT-7 LEUKEMIA CELL LINE AND NORMAL PERIPHERAL BLOOD MONONUCLEAR CELLS 

Abstract

Maida Hadžić 1 , Tamara Ćetković 1 , Mirta Milić 2 , Anja Haverić 1 , Sanin Haverić 1
1 University of Sarajevo – Institute for Genetic Engineering and Biotechnology, Sarajevo, Bosnia and Herzegovina
2 Institute for Medical Research and Occupational Health, Zagreb, Croatia

Halogenated boroxine (HB), K2(B3O3F4OH), is a synthetic derivative of boronic acid with previously proven bioactive potential in different in vitro and in vivo model-systems. Its effects in hematological malignancies have not been tested yet. In this work, we aimed to evaluate the genotoxic potential of HB using alkaline comet assay in human acute myeloid leukemia cell line UT-7 (ACC 137) and normal peripheral blood mononuclear cells (PBMCs),
respectively. UT-7 leukemia cells and normal PBMCs isolated from healthy donor were incubated at 37° C for the cultivation period of 72 h. Cultures were treated with HB in concentrations of 0.1, 0.2 and 0.4 mg/mL. Negative and positive controls were set up as well. Cell damage was analyzed using Comet Assay IV software (Instem, UK) and measuring tail intensity (%). Obtained results showed significant increase in tail intensity (p&lt;0.001) in
all HB treatments of UT-7 cells when compared to negative control. The frequency of hedgehog cells was registered in HB treatments of UT-7 cells for 0.2 mg/mL (49.5%) and 0.4 mg/mL (83.3%), indicating high dose-dependent genotoxic damage. In PBMCs, tail intensity was significantly increased (p&lt;0.001) only in the highest tested concentration of HB in comparison to negative control. Hedgehog cells were not found in HB treated PBMCs. These
results revealed selective genotoxicity of HB in tumor compared to normal cells in vitro.
Acknowledgements: The helpful support of the hCOMET action (CA15132) is gratefully acknowledged.
Keywords: comet assay, genotoxic damage, UT-7 cell line, PBMCs
Correspondence: maida5hadzic@gmail.com

Selma Behmen (University of Sarajevo, Institute for genetic engineering and biotechnology, Sarajevo, Bosnia and Herzegovina):

EVALUATION OF DNA DAMAGE IN ORAL LYMPHOCYTES USING COMET ASSAY METHOD IN THE GROUP OF HEALTHY INDIVIDUALS FROM SARAJEVO

Abstract

Selma Behmen, Alen Džaferspahić, Mahira Mehanović, Irma Durmišević, Tamara Ćetković, Maida Hadžić, Anja Haverić, Sanin Haverić
University of Sarajevo – Institute for genetic engineering and biotechnology, Sarajevo, Bosnia and Herzegovina

Human biomonitoring studies are used in assessing exposure to environmental genotoxins. Comet assay, used for measuring the level of DNA damage in single cells, has applications in human biomonitoring and genetic ecotoxicology. The assay enables analysis of DNA strand breaks in different types of cells including oral leukocytes which are suitable samples as their collection is non-invasive and convenient, especially for vulnerable populations. The main objective of this study was to initiate comet assay based biomonitoring of the background level of DNA damage in healthy individuals from Sarajevo, analyse inter-individual variation and correlate it with the lifestyle habits and environmental pollution. Oral leukocytes were collected and isolated from 33 healthy individuals during the summer and winter of 2019/2020. The comets were analysed by Comet assay IV (Instem, UK) software and statistical analysis performed over log-transformed values of tail intensity.
Overall results revealed no significant differences between two sessions. Comparative analysis of smokers and non-smokers revealed significantly higher damage in smokers compared to non-smokers in the summer period. For the winter period no significant differences were found but the damage was higher in smokers. Independent t-test revealed lower DNA damage in females compared to males. In addition, significant increase in DNA damage was found for the individuals above 49 years of age compared to younger participants that is most likely related to the reduced capacities of the DNA repair system.
With the trends of continuous and increasing air pollution, continuous biomonitoring studies are a necessity.
Keywords: DNA damage, smokers, non-smokers, lifestyle, airpollution
Correspondence: selma_b96@hotmail.com

Armina Zatagic (University of Sarajevo, Faculty of Science, Sarajevo, Bosnia and Herzegovina):

SATUREJA HORVATII SILIC AQUEOUS EXTRACT AS A RADIOPROTECTIVE AGENT IN EXPOSED HUMAN LYMPHOCYTES

Abstract

Armina Zatagic 1 , Tamara Cetkovic 2 , Lejla Caluk Klacar 2 , Arnela Hrbat 1 , Anes Dzehverovic 1 , Nedim Mujic 3 , Jasmina Cakar 2 , Anja Haveric 2
1 University of Sarajevo, Faculty of Science, Sarajevo, Bosnia and Herzegovina
2 University of Sarajevo, Institute of Genetic Engineering and Biotechnology, Sarajevo, Bosnia and Herzegovina
3 University of Sarajevo, Faculty of Veterinary Medicine, Sarajevo, Bosnia and Herzegovina

Lamiaceae species are well known for thier beneficial and protective properties. Satureja horvatii Silic, an endemic species of a Lamiaceae family has shown antigenotoxic and antiapoptotic characteristics in human lymphocyte cultures. Therefore, we aimed to test radioprotective potential of Satureja horvatii Silic aqueous extract in the culture of ex vivo irradiated human peripheral blood lymphocytes. Isotopic radium was used to irradiate human lymphocyte cultures of a healthy donor. A dose of 100 μg was applied over a 90, and a 9 min period for the high-risk, and low-risk radiation type, respectively. Following irradiation, the cultures were treated with an aqueous extract at a final concentration of 0.1 mg/ml. Negative controls along with the control cultures were set up to monitor the independent effects of radiation and extracts of Satureja horvatii. An alkaline comet test was applied, and 100 comets per treatment were analysed. The level of DNA damage was expressed as the percentage of DNA in the comet&#39;s tail. Results were compared by one-way ANOVA, followed by Newman-Keuls test with the significance level of p&lt;0.05. The results showed a significant increase in DNA damage at the high-risk dose compared to the low-risk dose, but also a significant decrease in DNA damage after aqueous extract treatment indicating radioprotective effect of Satureje horvatii Silic aqueous extract. Given various radiation exposures, additional research should address radioprotective effects of widely distributed Lamiaceae species. The promotion of sustainable and controlled use of natural resources should be inevitable.
Keywords: radiation, comet assay, DNA damage
Correspondence: arminazatagic1003@gmail.com

Forensic Genetics

Mirela Dzehverovic (University of Sarajevo, Institute for Genetic Engineering and Biotechnology, Sarajevo, Bosnia and Herzegovina):

DNA ANALYSIS OF HUMAN SKELETAL REMAINS FROM THE MEDIEVAL NECROPOLES IN BOSNIA AND HERZEGOVINA – A GENETIC STRUCTURE OF OUR ANCESTORS AND PREDICTION OF THEIR MIGRATIONS

Abstract

Mirela Dzehverovic 1 , Edin Bujak 2 , Amela Pilav 1 , Belma Jusic 1 , Naris Pojskic 1 , Jasmina Cakar 1
1 University of Sarajevo, Institute for genetic engineering and biotechnology, Sarajevo, Bosnia and Herzegovina
2 University of Sarajevo, Faculty of philosophy, Department of archaeology, Sarajevo, Bosnia and Herzegovina

Archaeological sites represent a rich historical and cultural heritage of medieval Bosnia. Among the archaeological sites, the most of them are with stećak tombstone. Stećak is medieval tombstone that appear in the period between the 12th and 16th century AD primarily on the territory of today&#39;s Bosnia and Herzegovina (B&amp;H). Even though individual excavations of skeletal remains from archaeological sites have been carried out continuously from the period of medieval Bosnia, there is no data about the genetic structure of ancient B&amp;H populations. The aim of this study was to obtain usable aDNA (ancient DNA) profiles, then compare and potentially find kinship relations, determine haplogroup based on obtained Y-STR profiles and mDNA sequence. Research which includes molecular-genetics characteristics of a population that lived in the medieval Bosnia, would give a new insight into the genetic structure of our ancestors, their potential relationships and migration processes. Molecular genetic characterization of medieval Bosnia populations will be compared with genetic data of recent B&amp;H populations, thus assessing the genetic differentiation between recent and medieval B&amp;H populations.
Keywords: Ancient DNA, STR markers, tombstone stećak, medieval Bosnia, kinship, population
Correspondence: dzehverovic.mirela @ingeb.unsa.ba

Belma Jusic (University of Sarajevo, Institute for Genetic Engineering and Biotechnology, Sarajevo, Bosnia and Herzegovina):

PATERNITY TESTING WITH TWO AUTOSOMAL STR MISMATCHES: CASES REPORT

Abstract

Belma Jusic, Amela Pilav, Mirela Dzehverovic, Jasmina Cakar
University of Sarajevo – Institute for Genetic Engineering and Biotechnology, Sarajevo, Bosnia and Herzegovina

DNA profiling including short tandem repeat (STR) markers is a conventional procedure used in forensic genetics and all kinds of kinship testing, including paternity testing. Paternity testing is based on the comparison of STR profiles of standard trio including child, mother and the alleged father or duo, where mother is not included. General rule is to exclude paternity when three or more mismatches have been observed. With two mismatches
detected by autosomal STR profiling, paternity cannot be excluded but has to be confirmed by an additional analysis. Therefore, we report two cases of paternity testing where were detected two autosomal STR mismatches so it required an additional analysis of X-linked STRs to achieve conclusive results. Buccal swab samples were collected from the female child, mother and the alleged father. Genomic DNA was extracted using QiagenDneasy™
Tissue Kit and amplified using PowerPlex® Fusion System and Investigator® Argus X-12.
 Amplified products were analysed by capillary electrophoresis carried out in ABI PRISM® 310  Genetic Analyzer according to manufacturer′s recommendations. STR data were collected using 310 Data Collection Software and analysed using GeneMapper™ v.3.2 software. Comparing the autosomal genetic profiles, in the first case were detected two mismatches among the 22 analysed STR loci, at D16S539 and D18S51 loci. Likewise, in the
second case were detected two mismatches, at D8S1179 and FGA loci. In both cases, detected mutations were from paternal source and probabilities of paternity including mutations into account were over 99,999999%. Analysis of 12 X-linked STR loci yielded one mismatch between child′s and father′s profile at DXS10135 locus in the first case, while in the second case was found a complete match. Single-step mutations observed at autosomal
and X-linked STR loci in the first case can be atributted to the age of father at the child′s birth. Results of an additional analysis contributed to paternity confirmation in both cases, where the probability of paternity was higher in the case with complete match between child′s and father′s profile. This case report pointed up the importance of including the analysis of X-chromosome STR markers in cases unsolvable by analysis of autosomal STR markers.
Keywords: kinship testing, STR markers, X-chromosome, mutations
Corresponding author: belma.jusic@ingeb.unsa.ba

Tamara Lukic (University of Sarajevo, Faculty of Science, Sarajevo, Bosnia and Herzegovina):

MOLECULAR-GENETIC ANALYSIS OF SKELETAL REMAINS FROM MEDIEVAL ARCHAEOLOGICAL SITES FROM THE TRAVNIK AREA

Abstract

Tamara Lukic 1 , Mirela Dzehverovic 2 , Amela Pilav 2 , Jasmina Cakar 1,2
1 University of Sarajevo, Faculty of Science, Sarajevo, Bosnia and Herzegovina
2 University of Sarajevo, Institute for Genetic Engineering and Biotechnology, Sarajevo, Bosnia and Herzegovina

Molecular genetic analysis of archaeological skeletal remains is important for gaining insight into the origin and genetic structure of our ancestors. The analysis of ancient DNA (aDNA) is extremely challenging since DNA is frequently found in very low concentrations and is often highly degraded. Regarding mentioned, the main goal of aDNA studies is to successfully isolate DNA from skeletal remains with as little loss of biological material and generate usable electropherograms (DNA profiles) with as many amplified STR loci as possible.
Although there is not much information about the people who inhabited the Travnik region during the medieval Bosnia, it is believed that this area was one of the most populated in that period, as evidenced by the numerous archaeological necropolises found in this area. A total of 11 tooth samples from four localities from the Travnik area: Glavica – Han Bila, Fazlići, Alihodže and Klisa were analyzed. Amplification of the isolated aDNA was performed
with a commercial multiplex kit PowerPlex® Fusion System and PowerPlex® Y23 System in samples in which the presence of Y chromosomes was detected. Statistical recalculation of the probability of kinship was determined using IngebKinship software. For Y-haplogroup prediction, digital software Whit Athe&#39;s Haplogroup Predictor and NEVGEN were used.
Isolation of aDNA and amplification of STR loci from analysed samples was extremely successful, generating full profiles from eight out of 11 samples. The highest degree of kinship was found between the two samples with a kinship probability for relationship brother-brother 99.99996%. Analysis of Y haplotypes showed that all male individuals were related by the male line and that they belonged to the J2a haplogroup.
Keywords: ancient DNA, archeology, skelet remains, STR markers
Correspondence: lukic-tamara@outlook.com

Lejla Kovacevic (University of Sarajevo, Faculty of Science, Sarajevo, Bosnia and Herzegovina):

THE EFFECT OF STORAGE CONDITIONS OF BLOOD TRACES ON THE DNA ANALYSIS IN FORENSIC CASES

Abstract

Lejla Kovacevic 1 , Mirela Dzehverovic 2 , Amela Pilav 2 , Jasmina Cakar 2
1 University of Sarajevo, Faculty of Science, Sarajevo, Bosnia and Herzegovina
2 University of Sarajevo, Institute for Genetic Engineering and Biotechnology, Sarajevo, Bosnia and Herzegovina

Analysis of DNA from blood traces and other biological samples from crime scenes is an essential part of work in forensic laboratories, and represents a key step in the criminal investigation that forensic investigators have relied on for decades. Since biological traces found at the crime scene are exposed to numerous factors that can affect the integrity of the trace, the aim of this study was to analyze the impact of certain storage conditions on blood traces left on appropriate surfaces so that they can be used after some time. Blood traces on selected surfaces (glass, metal, fabric and paper) were left in different conditions (light and warm; dark and warm, dark and cold) packed in paper and plastic packaging. After 19 days, dry and wet swabs were sampled and the DNA molecule was isolated using modified Miller protocol. For amplification of isolated DNA PowerPlex® Fusion multiplex system was used. DNA profiles were generated using an ABIPRISM® 310 Genetic Analyzer and GeneMapper®ID 3.2 software. The results of the study indicate significant differences in obtained DNA profiles of individuals whose samples were stored in different conditions. As expected the success of the DNA analysis was highly dependent on type of the surface on which the biological trace was located, and light and temperature exposure. In our study the best DNA profiles were generated from blood traces collected from glass and fabric in all applied storage conditions, except two profiles from glass in light-warm and dark-cold conditions. Our results confirmed the importance of proper sampling and storage conditions of biological traces for successful DNA analysis in forensic cases.
Keywords: forensic traces, DNA profile, criminal investigation
Correspondence: lkovacevic2706@gmail.com

Semra Burkic (University of Sarajevo, Faculty of Science, Sarajevo, Bosnia and Herzegovina):

FREQUENCY ANALYSIS OF 12 SELECTED X-STR MARKERS IN POPULATION OF BOSNIA AND HERZEGOVINA

Abstract

Semra Burkic 1 , Abdrurahim Kalajdzic 2 , Amela Pilav 2 , Mirela Dzehverovic 2 , Jasmina Cakar 2
1 University of Sarajevo, Faculty of Science, Sarajevo, Bosnia and Herzegovina
2 University of Sarajevo, Institute for Genetic Engineering and Biotechnology, Sarajevo, Bosnia and Herzegovina

X-STR markers are routinely used in forensic practice mainly as a complementary analysis of autosomal STRs analysis in complex cases. However, in certain kinship scenarios, these markers can be the only means to obtain information needed to solve the case. In order to use X-STRs, it is necessary to determine the frequency of those markers in certain population and to establish population databases to be used for comparison in forensic analyses. In this study, a total of 195 individuals from Bosnia and Herzegovina (82 females and 113 males) were included in analysis of 12 X-STR markers. The Investigator® Argus X-12 QS kit was used to obtain allele frequencies for 12 polymorphic STR loci including DXS10103, DXS8378, DXS10101, DXS10134, DXS10074, DXS7132, DXS10135, DXS7423, DXS10146, DXS10079, HPRTB and DXS10148. The main heterogenity parameters for both female and male individuals were calculated in PowerMarker v 3.25 software. There was no statistically significant difference in measured values between males and females in Bosnian population as well as from those observed in neighbouring Balkan populations. DXS10135 locus was found to be the most discriminating locus among all loci included in study in both males and females with highest values of polymorphic information content as well. Expected heterozygosity values among all the studied loci ranged from 65.35% to 92.12%. The least polymorphic locus was DXS8378, with PIC values of 0.581515 in males and 0.63741 in females, respectively. Since only one population study using X-STR markers has been reported previously on the population of Bosnia and Herzegovina, these results expand Bosnian population database with information on eight new X-STR loci. Overall, the studied markers of the Argus 12 X-STR kit proved to be highly polymorphic tool for use in analysis of forensic issues such as missing person identification, incest, immigration disputes, paternity and kinship analysis as well as genealogical studies.
Keywords: X-STR, forensic parameters, Investigator Argus X-12 QS Kit
Correspondence: semra.burkic@gmail.com