The 70th anniversary of the discovery of DNA secondary structure

Godina 2023. je godina jubileja u kojoj se obeležava 70 godina od otkrića sekundarne strukture molekula DNK. U ovom radu preglednog tipa dat je istorijski osvrt na otkrića i eksperimente koji su doveli do razvoja molekularne biologije, a kasnije i genomike kao nove naučne discipline. Poseban osvrt dat je na događaje i ljude koji su doprineli postavljanju trodimenzionalnog modela molekula DNK.This year marks the 70th anniversary of the discovery of the secondary structure of DNA molecule. This review- type paper provides an overview of the most important discoveries and experiments that led first to the development of molecular biology, and later to the development of genomics as a new scientific discipline. A special review is given to the events and people who contributed to the establishment of the three-dimensional model of the DNA molecule

Genetic Association Studies on Prostate Cancer

The modern research on molecular basis of prostate cancer (PCa) development includes studies aiming to identify potential genetic markers which could be used in diagnostics and/or monitoring of PCa. Genome-wide association studies (GWASs) have identified over 75 variants associated with PCa risk. One of the major PCa-related regions identified through GWASs is found to be a segment of 8q24. Other important PCa-susceptibility regions are 17q12, 17q24, 10q11, and 19q13. Candidate-gene based approach has also provided evidence of association between PCa risk and genetic variants located in functionally significant genes (both protein-coding and noncoding RNA genes) involved in normal prostatic cell growth, malignant transformation, or in the development of metastases. Nevertheless, the success of these studies is questionable, since numerous candidates for PCa-susceptibility variants were identified, but these results failed to replicate. The main aim of both types of genetic association studies on PCa is the identification of potential PCa genetic markers which could be used for constructing reliable algorithms for evaluating the risk for PCa development and/or PCa progression

Incidence of pelvic lymph node metastasis in radical prostatectomy

Background/Aim. Radical prostatectomy (RP) provides the best cancer control in patients with clinically prostate gland confined cancer. Multiple models and nomograms combining preoperative prostate-specific antigen (PSA) serum level, clinical stage and Gleason score have been developed to predict the probability of metastatic disease. In prostate cancer (PC) the presence of metastases to the pelvic lymph nodes (PLNs) is recognized widely as an unfavorable prognostic factor. Currently, PLNs dissection is not done in a low-risk group of prostate cancer patients. The aim of this study was to analyze PLN metastases in PC patients, in clinically localized stages of PC. Methods. Radical prostatectomy specimens with pelvic lymphadenectomy specimens from 82 PC patients were reviewed. In this group of patients, serum preoperative PSA values ranged from 2 to 23 ng/ml. Results. We diagnosed 11/82 (13.4%) patients with PLN metastases. There were 8 (72%) patients with pT3c pathological stage, and 3 (28%) patients with pT4a stage. PSA below 4 ng/ml was detected in 2/5 (40%) patients with PLN metastases. There was no statistically significant difference between preoperative PSA values and postoperative T stage, and PLN metastases. A statistically significant correlation between PLN metastases and the stage was found in the patients with pT4 and the patients with pT3c PC stages (p < 0.05). Conclusion. Recent RP series indicate PLN metastases to be less than 10%. We demonstrated higher detection of PLN metastases (13.4%) in our RP series. Our results suggest that PLNs dissection should be performed even in patients with low-risk PC

Variability of the chloroplast dna of sessile oak (Quercus petraea agg. Ehrendorfer, 1967) in Serbia

Genetic variability of sessile oak (Quercus petraea agg. Ehrendorfer, 1967) in Serbia is estimated applying cpDNA universal primer pairs that were characterized by a high informative level for chloroplast genome variability assessment in previous investigations. Five different haplotypes were detected in the analyzed sample material from populations in Serbia

Efekat epistatičkih interakcija između varijanti u genima za mikrornk i genima za proteine utišavajućeg kompleksa na rizik za razvoj i progresiju karcinoma prostate

Dobrijević Z., J. Karanović, D. Savić-Pavićević, G. Brajušković (2023). The effect of epistatic interactions between genetic variants located in microRNA and silencing complex genes on prostate cancer progression risk.- Genetika, Vol 55, No.1, 263-275. Previous studies conducted in Asian and European populations have provided evidence of the association between microRNA-related genetic variants and prostate cancer (PCa) risk and/or progression. Nevertheless, the results obtained in these studies are inconsistent, which could be explained by the limitations of single-locus main effect evaluations to detect joint effects of multiple genetic variants, reflected in statistical epistases. Therefore, we conducted the analysis of potential epistatic interactions between variants located in microRNA genes and in genes encoding the components of RNA-induced silencing complex (RISC) in relation with PCa risk/aggressiveness. Raw data on genotyping results from our previous studies involving four microRNA polymorphisms and five variants in RISC genes were subjected to the exclusion of samples based on missing data criterion, followed by the re-evaluation of Hardy-Weinberg equilibrium. Afterwards, these genotyping results were included in the Multifactor dimensionality reduction (MDR) analysis. Permutation testing was conducted in order to assess statistical significance of the best models from MDR tests. MDR tests on the risk of developing PCa yielded statistically insignificant results. Nevertheless, the MDR results for comparison of PCa patients with high and low cancer progression risk were statistically significant for the analysis that included rs11614913, with the 3-locus best model comprising this genetic variant, rs7813 and rs784567. We conclude that statistical epistasis between rs11614913 in hsa-miR-196a2, rs7813 in GEMIN4 and rs784567 in TARBP2 shows association with the invasiveness of PCa.Ranije studije u evropskim i azijskim populacijama ukazale su na značajnu asocijaciju genetičkih varijanti sa efektom na funkciju mikroRNK sa rizikom za razvoj i/ili progresiju karcinoma prostate (KP). Međutim, rezultati navedenih studija su nepodudarni, za šta je jedan od mogućih razloga nemogućnost pristupa baziranih na proceni pojedinačnih efekata genetičkih varijanti da detektuju značajne zajedničke efekte više varijanti, a koji se reflektuju u statističke epistaze. Iz tog razloga, cilj naše studije bila je analiza potencijalnih epistatičkih interakcija između varijanti lociranih u genima za mikroRNK molekule i u genima za komponente utišavajućeg kompleksa indukovanog sa RNK (RISC) kao faktora rizika za razvoj i/ili progresiju KP. Rezultati genotipizacije dobijeni tokom sprovođenja naših ranijih studija, a koji uključuju podatke za četiri varijante u genima za mikroRNK i pet u genima za komponente RICS, podvrgnuti su inicijalnoj obradi podataka u smislu isključivanja uzoraka sa nedostajućim rezultatima, nakon čega je procenjeno odstupanje od Hardi-Vajnbergove ravnoteže. Rezultati su zatim analizirani metodom redukcije dimenzionalnosti višestrukih faktora (Multifactor dimensionality reduction - MDR analysis). Permutacioni testovi su sprovedeni sa ciljem procene statističke značajnosti najboljih modela iz MDR analize. Dobijeni rezultati MDR testova koji su se odnosili na rizik za razvoj KP nisu bili statistički značajni. S druge strane, MDR rezultati koji se odnose na rizik za progresiju KP bili su značajni za model koji uključuje tri lokusa: rs11614913, rs7813 and rs784567. Stoga, zaključci naše studije ukazuju na značaj epistatičkih interakcija između varijanti rs11614913 u hsa-miR-196a2, rs7813 u GEMIN4 i rs784567 u TARBP2 kao faktora koji ispoljavaju efekat na invazivnost KP

Asocijacija varijanti u genima PRMT6, PEX10 I SOX5 sa idiopatskim muškim sterilitetom: dokazi iz populacije Severne Makedonije i ažurirana meta-analiza

PRMT6, PEX10 and SOX5 genetic variants were identified as male infertility-associated loci in a genome-wide association study and further validated in various populations. Still, the results of previous case-control studies varied, which could be due to differences in participants’ ethnic backgrounds. The main purpose of the present study was to evaluate the supposed association of these variants with idiopathic male infertility in North Macedonian population. Furthermore, we aimed to conduct the systematic quantitative data synthesis which includes the results of previous studies on the same issue in other European and non-European populations. A total of 137 men from North Macedonia diagnosed with idiopathic infertility and 130 age-matched fertile controls were included in the present case-control study. PCR-RFLP method was used for genotyping. Meta-analysis was performed by OpenMeta-analyst statistical software. Variants rs10842262 in SOX5, rs2477686 in PEX10 and rs12097821 in PRMT6 showed the lack of statistically significant differences in genotype distributions between men diagnosed with idiopathic infertility and the control group. Still, rs10842262 allele G frequency was significantly increased in men with poor sperm concentration (P= 0.024, OR = 2.10, 95%CI 1.08-4.06). Meta-analysis further showed the association of rs10842262 and rs12097821 with the risk of idiopathic male infertility. Our results obtained in North Macedonian population supported the previous reports on the involvement of rs10842262 in the genetic basis of male infertility. The meta-analysis confirmed the association of rs10842262 and rs12097821 with male infertility occurrence. Still, additional studies are needed to support the present findings.Asocijacija varijanti u genima PRMT6, PEX10 i SOX5 sa muškim sterilitetom identifikovana je u studiji genetičke asocijacije na čitavom genomu i kasnije analizirana u studijama slučajeva i kontrola u različitim populacijama. Rezultati prethodnih studija su pokazali značajnu varijabilnost, što može biti posledica razlika u etničkom poreklu studijskih grupa. Osnovni cilj ovog istraživanja je analiza asocijacije navedenih genetičkih varijanti sa rizikom za pojavu idiopatskog muškog steriliteta u populaciji Severne Makedonije. Takođe, naš cilj je bio i sprovođenje sistematske kvantitativne sinteze podataka iz studija sa istom ili sličnom temom istraživanja sprovedenim u drugim evropskim i neevropskim populacijama. Ukupno 137 muškaraca sa idiopatskim sterilitetom iz Severne Makedonije i 130 fertilnih kontrola slične starosti uključeno je u studiju slučajeva i kontrola. Genotipizacaija je vršena PCR-RFLP metodom, dok je za meta-analizu korišćen statistički softver OpenMeta-analyst. Za varijante rs10842262 u SOX5, rs2477686 u PEX10 i rs12097821 u PRMT6 nije utvrđena statistički značajna razlika u distribucijama genotipova između grupe ispitanika sa idiopatskim sterilitetom i kontrolne grupe. Međutim, učestalost alela G varijante rs10842262 bila je značajno povećana kod muškaraca sa niskom koncentracijom spermatozoida (P= 0.024, OR = 2.10, 95%CI 1.08– 4.06). Meta-analizom pokazana je asocijacija rs10842262, ali i rs12097821, sa rizikom za razvoj idiopatskog muškog steriliteta. Naši rezultati ustanovljeni u populaciji Severne Makedonije idu u prilog prethodnim navodima o učešću rs10842262 u genetičkoj osnovi muškog steriliteta. Ipak, dodatne studije su neophodne kako bi potvrdile značaj rezultata ovog istraživanja

Modern approaches in research of the molecular basis of prostate cancer

U ovom radu preglednog tipa prikazano je desetogodišnje iskustvo istraživačkog tima PROSTATSERBIA koji za temu svog istraživanja ima molekularnu osnovu karcinoma prostate (KP). Centar za humanu molekularnu genetiku Biološkog fakulteta Univerziteta u Beogradu poseduje kolekciju uzoraka i banku podataka za gotovo 1000 muškaraca sa bolestima prostate (KP i benigna hiperplazija prostate) i preko 350 muškaraca bez znakova bolesti prostate iz populacije Republike Srbije. Najveći broj studija bio je dizajniran kao studije asocijacije odabranih genetičkih varijanti sa rizikom za razvoj i progresiju KP. U ovim studijama slučajeva i kontola ispitivane su genetičke varijente kako u genima za proteine i nekodirajuće molekule RNK, tako i u nekodirajućim regionima genoma (tzv. „genskim pustinjama“). Pored studija slučajeva i kontrola, sprovođene su i meta-analize kao i analize statističkih epistatičkih interakcija analiziranih genetičkih varijanti. Krajnji cilj ovih studija je kreiranje algoritma za procenu rizika za progresiju bolesti koji bi se koristio u fazi aktivnog praćenja bolesnika sa ranodijagnostikovanim KP. U poslednje vreme, istraživanja su usmerena na egzozome i njihov makromolekulski sadržaj (proteine i nekodirajuće molekule RNK) kao potencijalne biološke markere tečne biopsije KP. Pored toga, biološki makromolekuli na površini egzozoma predstavljaju i ciljane molekule u novim strategijama lečenja KP.This review-type paper will present the ten-year experience of the PROSTATSERBIA research team, which has the molecular basis of prostate cancer (PCa) as its research topic. The Center for Human Molecular Genetics of the Faculty of Biology, University of Belgrade has a collection of samples and a data bank for almost 1000 men with prostate diseases (PCa and benign prostatic hyperplasia) and for over 350 men without signs of prostate diseases, all from Serbian population. Most of the studies were designed as studies of association of selected genetic variants with the risk for development and progression of PCa. In these case-control studies, genetic variants were examined both in genes for proteins and non-coding RNAs, as well as in non-coding regions of the genome (so-called gene deserts). In addition to case control studies, both meta-analyses and the analysis of statistical epistatic interactions of the analyzed genetic variants were conducted. The main goal of all these studies was to create an algorithm for risk assessment for disease progression that would be used in the phase of active monitoring of patients with early diagnosed PCa. Recently, we shifted our research focus on exosomes and their macromolecular content (proteins and microRNAs) as potential biological markers of liquid prostate cancer biopsy. In addition, macromolecules at the exosome surface represent target molecules for new PCa treatment strategies

Affinity-based isolation of extracellular vesicles by means of single-domain antibodies bound to macroporous methacrylate-based copolymer

Correct elucidation of physiological and pathological processes mediated by extracellular vesicles (EV) is highly dependent on the reliability of the method used for their purification. Currently available chemical/physical protocols for sample fractionation are time-consuming, often scarcely reproducible and their yields are low. Immuno-capture based approaches could represent an effective purification alternative to obtain homogeneous EV samples. An easy-to-operate chromatography system was set-up for the purification of intact EVs based on a single domain (VHH) antibodies-copolymer matrix suitable for biological samples as different as conditioned cell culture medium and human plasma. Methacrylate-based copolymer is a porous solid support, the chemical versatility of which enables its efficient functionalization with VHHs. The combined analyses of morphological features and biomarker (CD9, CD63 and CD81) presence indicated that the recovered EVs were exosomes. The lipoprotein markers APO-A1 and APO-B were both negative in tested samples. This is the first report demonstrating the successful application of spherical porous methacrylate-based copolymer coupled with VHHs for the exosome isolation from biological fluids. This inexpensive immunoaffinity method has the potential to be applied for the isolation of EVs belonging to different morphological and physiological classes.Supplementary material: [https://cherry.chem.bg.ac.rs/handle/123456789/5153

SMN1 copy number as a modifying factor of survival in Serbian patients with sporadic amyotrophic lateral sclerosis

Introduction/Objective. Amyotrophic lateral sclerosis (ALS) is a devastating motor neuron disease. The majority of cases are apparently sporadic ALS (SALS) with variants in susceptibility genes or sometimes in high-risk ALS genes. Two ALS susceptibility genes are SMN1, whose functional loss causes spinal muscular atrophy (SMA), and a nearly identical SMN2 gene, which modulates SMA severity. In this study we examined the association of copy number variations (CNVs) of SMN1 and SMN2 genes and two additional genes, SERF1 and NAIP, residing in the same genomic region (i.e. 5q13.2 segmental duplication), with SALS in patients from Serbia. Methods. Multiplex ligation-dependent probe amplification was used to determine CNVs of each gene in a clinically well-characterised group of 153 Serbian SALS patients and 153 controls. Results. Individual association between SMN1, SMN2, SERF1 or NAIP CNVs and SALS susceptibility or survival was not found. Survival curves based on the multivariable Cox regression analysis showed that three SMN1 copies, lower ALS Functional Rating Scale Revised (ALSFRS-R) score at the time of diagnosis, faster decline of the ALSFRS-R score over time, and shorter diagnostic delay result in shorter survival of Serbian SALS patients. Conclusion. Clinical variables might be complemented with the SMN1 copy number to improve prediction of survival in Serbian SALS patients

Repeat Interruptions Modify Age at Onset in Myotonic Dystrophy Type 1 by Stabilizing DMPK Expansions in Somatic Cells

CTG expansions in DMPK gene, causing myotonic dystrophy type 1 (DM1), are characterized by pronounced somatic instability. A large proportion of variability of somatic instability is explained by expansion size and patient’s age at sampling, while individual-specific differences are attributed to additional factors. The age at onset is extremely variable in DM1, and inversely correlates with the expansion size and individual-specific differences in somatic instability. Three to five percent of DM1 patients carry repeat interruptions and some appear with later age at onset than expected for corresponding expansion size. Herein, we characterized somatic instability of interrupted DMPK expansions and the effect on age at onset in our previously described patients. Repeat-primed PCR showed stable structures of different types and patterns of repeat interruptions in blood cells over time and buccal cells. Single-molecule small-pool PCR quantification of somatic instability and mathematical modeling showed that interrupted expansions were characterized by lower level of somatic instability accompanied by slower progression over time. Mathematical modeling demonstrated that individual-specific differences in somatic instability had greater influence on age at onset in patients with interrupted expansions. Therefore, repeat interruptions have clinical importance for disease course in DM1 patients due to stabilizing effect on DMPK expansions in somatic cells