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Background: Methotrexate (MTX), a folate analogue, is the most commonly used disease-modifying drug in the treatment of rheumatoid arthritis (RA). However, high interindividual differences in drug response are present among RA patients.Research design and methods: In a group of 234 RA patients treated with MTX, we investigated whether rs1650697 polymorphism in DHFR gene may have an impact on MTX efficacy and/or adverse drug effects (ADEs). Relative DAS28 values (rDAS28) were used for the estimation of MTX therapy and all ADEs were recorded. Patients were genotyped for selected polymorphism by real-time PCR method. Results: According to the European League Against Rheumatism criteria after 6 months of MTX therapy, 196 patients (83.8%) were classified as responders (25 (10.7%) were good and 171 (73.1%) were moderate) and 38 patients (16.2%) as nonresponders. ADEs were observed in 55 patients (23.5%). Conclusions: Our results showed that the presence of T allele might be protective against MTX hepatotoxicity measured by transaminase levels (p = 0.05). Furthermore, among patients who also received low-dose corticosteroids, we have found a lower rDAS value in patients with CC genotype (p = 0.039).

Досадашња истраживања: Јонизујућа зрачења у контакту са живом материјом изазивају оштећења биомолекула. Биолошки маркери експозиције и ефекта озрачивања (хромозомске аберације и микронуклеуси) и репаративни капацитети (способност поправке зрачењем индукованих оштећења), испитивани су у досадашњим истраживањима у евалуацији индивидуалне радиосензитивности in vitro. Како је један од узрочника оштећења молекула ДНК дејством јонизујућег зрачења и настанак слободних радикала, циљ овог истраживања био је утврђивање утицаја параметара оксидативног стреса, антиоксидантног одговора и оксидативног статуса испитаника, на учесталост цитогенетичких биомаркера деловања зрачења у лимфоцитима периферне крви и испитивање корелације између формирања две врсте биомаркера. Метод: Истраживање је обухватило анализу узорака крви 56 испитаника-донора на постојање цитогенетички видљивих промена генетичког материјала и биохемијски потврђених промена у вредностима параметара оксидативног стреса, у одговору на деловање три различите апликоване дозе зрачења у односу на неозрачене узорке. Резултати: У истраживању је анализирано укупно 103 цитогенетичкa и биохемијскa параметра на 4 дозе зрачења (0, 0.75, 1.5 и 3 Gy). Резултати су показали постојање интер-индивидуалне варијабилности у свим анализираним параметрима, на свим дозама зрачења, као и дозно-зависно формирање свих параметара, уз издвајање две групе: прве-која обухвата скоро све анализиране параметре и која се карактерише повећањем вредности са порастом дозе и друге, која се карактерише смањењем вредности са порастом дозе, а обухвата само један параметар-тотални антиоксидантни статус. За појединачне параметре, корелациона анализа показала је, на свакој од апликованих доза зрачења, одсуство корелација дицентричних хромозома и микронуклеуса са биохемијским параметрима. Међутим, корелациона анализа параметара који говоре о свеукупном одговору донора , израженом кроз однос између броја „Н“ вредности и укупног броја мерења (H-response ratio - „, Ionizing radiation in living cells can directly act on atomic structures, producing damage to biomolecules. Earlier investigations evaluating individual radiosensitivity in vitro were focused on biomarkers of exposure and effect (chromosomal aberrations and micronuclei) as well as reparative capacity. Since ionizing radiation can also cause oxidative damage and produce reactive oxygen species, the main goal of this investigation was to establish the influence of parameters of oxidative status and oxidative damage on chromosomal aberrations and micronuclei frequency in peripheral blood lymphocytes from donors and to examine possible correlations. Method: Blood samples from fifty six health male and female donors were irradiated with four different doses (0, 0.75, 1.5 and 3 Gy) and then analyzed cytogenetically and biochemically. Results: A total of 103 cytogenetic and biochemical parameters were evaluated. The results showed significant inter-individual variability in all analyzed parameters, as well as dose-dependent increases of almost all of them, except for total antioxidant status, which exhibited a dose-dependent decrease. Correlation analysis indicated no association between cytogenetic radiation biomarkers (chromosomal aberrations and micronuclei) and biochemical oxidative stress parameters. However, findings for overall response („HRR“ parameters) confirmed the t-test analysis, i.e. donors with lower values for antioxidant status parameters had increased levels of cytogenetic damage and higher responses to irradiation. Unlike them, the group of donors with better antioxidative protection showed a lower response than expected. Conclusion: Besides well established cytogenetic biomarkers of radiation exposure, our results indicated a promising future use for biochemical oxidative status parameters in routine radiation protection practice, since together they can provide a complete radiation response profile in cases of continuous low dose exposure, as well as i

Uvod: Česta varijabilna imunodeficijencija (CVID) je bolest heterogene kliničke slike koju odlikuju poremećena diferencijacija i maturacija B-ćelija uz nedovoljnu produkciju antitela. Pripada grupi retkih bolesti sa procenjenom prevalencijom od 1:25000 do 1:50000, ali je ujedno najčešća klinički relevantna primarna imunodeficijencija. Početak i tok bolesti su veoma varijabilni. U kliničkoj slici dominiraju ponavljane bakterijske infekcije, uz brojne inflamatorne, limfoproliferativne i autoimunske poremećaje, kod približno polovine pacijenata. Sporadični oblici CVID se najverovatnije nasleđuju poligeno, ali je opisano više monogenskih uzročnih mutacija koje se javljaju u malom procentu prevashodno familijarnih oblika CVID. Opisani su brojni imunološki poremećaji, ali uzrok nastanka bolesti je još uvek nepoznat. Nekoliko istraživača je pretpostavilo da poremećaj u produkciji citokina, među kojima su i TNF, IL-10, IL-6 i IFN-gama, ima ulogu u etiopatogenezi poremećaja. Ciljevi istraživanja: Testirati hipotezu da genski polimorfizmi TNF (−308G/A), IFNG (+874T/A), IL10 (−1082G/A, −819T/C i −592A/C), i IL6 (−174G/C) doprinose sklonosti ka javljanju CVID ili posebnih kliničkih manifestacija bolesti. Ispitati distribucije alela i genotipova polimorfizama u genima IL-10, IL-6 u populaciji zdravih ljudi u Srbiji i uporediti ih sa objavljenim podacima za druge populacije. Ispitati produkciju TNF, IL-10, IL-6 i IFN-gama kod pacijenata sa CVID u odnosu na zdrave kontrole, kao i korelaciju genotipa pacijenata sa nivoima citokina. Pacijenti i metode: Trideset pet pacijenata sa CVID genotipizirani su pomoću Taqman eseja radi analize izabranih polimorfizama u TNF, IL6, IL10, i IFNG, kao i 250 zdravih kontrola za polimorfizme u genima IL6 i IL10 za koje nisu postojali podaci u zdravoj populaciji Srbije. Za ispitivanje slaganja distribucija dobijenih frekvencija genotipova u populaciji sa očekivanim vrednostima po Hardy-Weinberg ravnoteži, primenjivan je 2 test. Genotipsko-fenotipska, Introduction: Common Variable Immunodeficiency (CVID) is a heterogeneous disease characterized by impaired B cell differentiation and maturation accompanied with the defective antibody production. It is a rare disease, with estimated prevalence 1 in 25000 to 1 in 50000, but also the most common clinically relevant primary immunodeficiency. Age of onset and course of the disease are highly variable. Recurrent bacterial infections are dominant in clinical presentation, with inflammatory, lymphoproliferative and autoimmune disorders present in almost half of the patients. Sporadic CVID has putative polygenic inheritance, but several monogenic mutations are found in small percentage of patients, mostly in familiar cases of CVID. Plethora of immunological abnormalities is described, but the cause of the disorder is still unknown. Several investigators addressed the possibility that disturbed cytokine production of TNF, IL-6, IFN-gamma and IL-10, among a variety of others, may be implicated in CVID. Aims of the investigation: The aim of this study was to test the hypothesis that gene polymorphisms involving TNF (−308G/A), IFNG (+874T/A), IL10 (−1082G/A, −819T/C and −592A/C), and IL6 (−174G/C) cytokine genes might contribute to susceptibility to CVID or its clinical manifestations. In addition, we wanted to determine allele and genotype frequencies in the polymorphisms of the genes coding IL-10 and IL-6 in healthy Serbian population in order to compare them with the same data from other populations. Besides, the aim was to investigate TNF, IL-10, IL-6 and IFN-gamma cytokine production in the CVID patients related to healthy persons in control group, as well as to correlate the genotype of the patients with the cytokine production. Patients and methods: Thirty five patients with CVID were genotyped for indicated single nucleotide polymorphisms (SNP) in TNF, IL6, IFNG and IL10 using TaqMan-based assays, as well as 250 healthy controls for the SNPs in IL6 and IL10 which were

Cilj: Rabdomiosarkom (RMS) je tumor visokog stepena maligniteta. Poslednje dve decenije prognoza pacijenata sa ovim tumorom je značajno poboljšana, osim kod pacijenata iz visokorizične grupe. U cilju utvrđivanja novih prognostičkih faktora ispitivali smo imunohistohemijsku ekspresiju proteina regulatora ćelijskog ciklusa i apoptoze, kao i obrasce metilacije promotora gena p16 u slučajevima RMS, i dobijene rezultate korelirati sa kliničkopatološkim parametrima. Metodologija: Ova studija je obuhvatila 67 slučajeva RMS dijagnostikovanih na Institutu za patologiju Medicinskog fakulteta, Univerziteta u Beogradu, u periodu od 1994-2014 godine Analizirani su sledeći kliničko-morfološki podaci: pol, starost, lokalizacija i veličina tumora, histološki tip i gradus, zahvaćenost regionalnih limfnih nodusa i postojanje metastatske bo lesti. Rezultati: Evaluacijom ekspresije ciklina A u odnosu na kliničko-patološke parametre pacijenata sa RMS, uočena je statistička značajnost samo u odnosu na histolški tip tumora (p=0.047). U ćelijama RMS, ciklin D1 je pokazivao jedarnu ekspresiju i aberantna ekspresija ovog markera nije pokazivala značajnu korelaciju sa ispitivanim parametrima. Rezultati različite ekspresija p57 u ovom tumoru su pokazivali statističku značajnost u odnosu na histološki tip (p=0,042),gradus tumora (p=0,026) i u odnosu na ishod bolesti (p=0,021). Ekspresija survivina u tumorskim ćelijama RMS bila je citoplazmatska i jedarna, Za razliku od citoplazmatske ekspresije survivina gde nije uočena njena povezanost sa prognostičkim faktorima, jedarna ekspresije ovog markera je bila statistički značajna u odnosu na starosne grupe, histološki tip i gradus tumora (p=0.009, p=0.008, p=0.010, redom). Analiza ekspresije nestina je pokazala statistički značaj u odnosu na starost pacijeanta (p = 0,028) i gradus tumora (p = 0,022)..., Objective: Rhabdomyosarcoma (RMS) is a tumor of high-grade malignancy. During the last two decades the prognosis of patients with this tumor has been significantly improved, except in patients from the high-risk group. In order to determine new prognostic factors, we investigated the immunohistochemical expression of proteins, the regulators of cell cycles and apoptosis as well as the patterns of methylation of the p16 gene promoter in cases of RMS, and the results obtained were to be correlated with clinicopathological parameters. Methodology: This study included 67 cases of RMS diagnosed at the Institute of Pathology, School of Medicine, University of Belgrade, in the period from 1994 to 2014. The following clinical and morphological data were analyzed: gender, age, tumor size and localization, histological type and grade, affected regional lymph nodes and the presence of metastatic disease. Results: By evaluation of the expression of cyclin A in relation to the clinicopathological parameters of patients with RMS, it was found a statistical significance only in relation to the histological type of tumor (p = 0.047). In the RMS cells, cyclin D1 showed a nuclear expression and the aberrant expression of this marker did not show any significant correlation with the studied parameters. Results of different expressions of p57 in this tumor showed a statistical significance in relation to the histological type (p = 0.042), tumor grade (p = 0.026) and in relation to the disease outcome (p = 0.021). The survivin expression in RMS tumor cells was cytoplasmic and nuclear. Unlike cytoplasmic survivin expression where no correlation with prognostic factors was found out, the nuclear expression of this marker was statistically significant in relation to the age groups, histological type of tumor and tumor grade (p = 0.009 , p = 0.008, p = 0.010, respectively).Analysis of the expression of nestin showed statistical significance in relation to the age of patients (p = 0.028) and

The aim of this research was to compare knowledge retention and course satisfaction among the students who attended a combined teaching course (traditional form and online course) and those who attended only the traditional course in human genetics. The study comprised 169 students divided in two groups. The first group included 88 students who attended the combined course, and the second group participated in the traditional course. A knowledge test was given after the end of the courses and six months later.. The results of both groups were compared. Further on, the students in the combined course were divided in three categories according to the levels of online activities: weak, moderate and very active. The categories were compared with each other, and with the results of the students who attended the traditional course. The students who attended the combined course achieved considerably higher results on both tests compared to the students who attended the traditional course. The average number of points on both tests was statistically considerably higher in the group of very active students compared to those moderately active or weak and, of course, better than the students who attended the traditional course The results show that the online course was marked highly, showed higher knowledge retention and that the satisfaction with the combined course was exceptional., Cilj ovog istraživanja bio je da se uporedi retencija znanja i zadovoljstvo kursom među studentima koji su pohađali kombinovanu nastavu (tradicionalan oblik i onlajn kurs) i onima koji su pohađali tradicionalni oblik nastave iz predmeta humana genetika. Studija je obuhvatila 169 studenata podeljenih u dve grupe. Prvu grupu je činilo 88 studenata koji su pohađali kombinovani kurs, a drugu grupu su činili studenti koji su pohađali tradicionalni kurs. Testiranje znanja je rađeno nakon završetka kursa i ponovo šest meseci kasnije. Rezultati testiranja su upoređeni između ove dve grupe. Takođe, studenti sa kombinovanog kursa su podeljeni u tri kategorije prema nivou aktivnosti na onlajn kursu: slabo aktivni, umereno aktivni i veoma aktivni. Kategorije su upoređene međusobno, kao i sa rezultatom studenata koji su pohađali tradicionalnu nastavu. Na oba testa su studenti koji su završili kombinovani kurs bili znatno bolji od studenata koji su pohađali tradicionalni oblik nastave. Prosečan broj poena na oba testa koje su postigli veoma aktivni studenti bio je statistički značajno veći u odnosu na umereno aktivne, slabo aktivne i studente koji su pohađali tradicionalnu nastavu. Rezultati ispitivanja procene studenata pokazuju da je onlajn kurs ocenjen visokim ocenama i da je zadovoljstvo kombinovanim oblikom nastave bilo izuzetno. Rezultati su pokazali veću efektivnost, tj. retenciju znanja kod studenata koji su pohađali kombinovani kurs.

Purpose Gamma-glutamyl hydrolase (GGH), cyclin D1 (CCND1) and thymidylate synthase (TS) genes encode enzymes that are involved in methotrexate (MTX) action. In a group of 184 RA patients treated with MTX, we have investigated whether selected polymorphisms in these genes modulate MTX efficacy and/or have impact on adverse drug effects (ADEs). Methods The efficacy of the MTX therapy has been estimated using the disease activity score in 28 joints (DAS28-ESR) based on EULAR criteria and relative DAS28 values (rDAS28). All adverse drug events were recorded. Patients were genotyped for selected polymorphisms of the GGH (-354 G>T and 452 C>T), CCND1 (870 A>G) and TYMS (variable number of tandem repeats, VNTR, and G to C substitution of triple repeat, 3R allele) gene. Association studies have been performed between obtained genotypes and the efficacy and toxicity of MTX. Results According to the EULAR response criteria, 146 RA patients (79.3 %) were classified as responders (good/moderate response) and 38 (20.7 %) as nonresponders (poor response). Higher frequency of the TYMS 3 G/3 G genotype has been found among non-responders as compared to individuals with remaining genotypes (p=0.02). ADEs were recorded in 53 patients. Among those patients eight experienced bone marrow toxicity, all of them carried GGH -354GG genotype (p=0.003). No other significant association were observed. Conclusion The 3 G/3 G genotype of the TYMS gene may indicate predisposition of poor response to MTX and GG genotype of GGH -354 T>G polymorphism may have high predictive value for myelosuppression in RA patients.

Objectives Identifying genetic predictors of methotrexate (MTX) treatment response in patients with rheumatoid arthritis (RA) may have great importance for optimising drug doses required for clinical benefit without toxicity. In a group of 125 RA patients treated with MTX we investigated whether selected polymorphisms in genes relevant for MTX action (aminoimidazole-4-carboxiamide ribonucleotide transformylase, ATIC, and dihydrofolate reductase, DHFR) modulate disease activity and/or have impact on therapy side effects. Methods The efficacy of treatment was estimated both by the disease activity score in 28 joints (DAS28), based on EULAR criteria, and relative DAS28 (rDAS28) score. Adverse drug events (ADEs) were also recorded. RA patients were genotyped using the PCR-RFLP method, followed by an association study between ATIC -129T>G, DHFR -216T>C and DHFR -317A>G polymorphisms and the efficacy and toxicity of MTX. Results According to the EULAR response criteria, 96 RA patients (76.8%) were classified as responders (good/moderate response) and 29 (23.2%) as non-responders (poor response). rDAS28 values ranged from -0.01 to 0.80 (mean value 0.31 +/- 0.19). Among 125 patients enrolled in this study 39 experienced at least one side effect. The DHFR -317AA genotype was associated with the less favourable response (reduction in rDAS28 score, p=0.05). None of the analysed polymorphisms was associated with MTX toxicity. Conclusion RA patients with DHFR-317AA genotype had less favourable response to MTX Further studies in larger patient populations are necessary to confirm the relationship between the analysed polymorphisms and MTX treatment response.

Myelodysplastic syndromes (MDS) are rare in children and only a few studies have analyzed their molecular mechanisms. The NPM1 gene encodes for nucleophosmin (NPM) which regulates hematopoiesis. Mutations in exon 12 of the NPM1 cause the nucleophosmin cytoplasmic dislocation and disrupt its functions. We have analyzed mutations of the NPM1 gene in archival bone marrow samples from 17 children with MDS and detected, in one patient, transition C to T in codon 293. To the best of our knowledge, this is the first analysis of NPM1 mutations in childhood MDS and the very first missense mutation of the NPM1 gene reported so far.

Methylenetetrahydrofolate reductase (MTHFR) regulates the metabolism of folate and methionine, essential components of DNA synthesis and methylation. Polymorphisms in the MTHFR gene have been associated with susceptibility to some types of cancer. We investigated a possible association of MTHFR polymorphisms (677C > T and 1298A > C) and increased risk for acute lymphoblastic leukemia in 78 affected children. The frequencies of both MTHFR 677 genotypes and alleles were significantly different between patients and controls. A significant association between CT/TT individuals and reduced risk of acute lymphoblastic leukemia was found. The odds ratios were 0.53 (95% confidence interval, 032-0.89) and 0.30 (95% confidence interval, 0.12-0.81). Polymorphism 1298 did not show statistical difference between patients and controls.

Uvod. Mikrodelecije Y hromozoma su, posle Klinefelterovog (Klinefelter) sindroma, najčešći genetski uzrok neplodnosti muškaraca. Cilj rada. Cilj istraživanja je bio da se utvrdi učestalost mikrodelecija Y hromozoma kod muškaraca s idiopatskom neplodnošću koji su kandidati za mikrofertilizaciju (Intracytoplasmic Sperm Injection - ICSI), kao i da se ispita korelacija genotipa i fenotipa kod bolesnika sa delecijama Y hromozoma. Metod rada. U studiju je prvobitno uključeno 160 odabranih muškaraca s malim brojem spermatozoida (manje od 5×106 spermatozoida po mililitru ejakulata), međutim, 40 muškaraca je isključeno iz daljeg ispitivanja (kod 10 ispitanika su utvrđeni citogenetski poremećaji, a kod 30 je dijagnostikovan jedan od poznatih uzroka neplodnosti). Kontrolnu grupu ispitanika činilo je 150 muškaraca koji su ostvarili očinstvo bar jednom u poslednje dve godine. Genomska DNK je izolovana iz periferne krvi 120 odabranih ispitanika, a postojanje ili izostanak delecija Y hromozoma analizirano je u dve multipleks reakcije lančanog umnožavanja (Polimerase Chain Reaction - PCR) korišćenjem odgovarajućih prajmera. Rezultati. Izostanak amplifikacije korišćenjem bar jednog para prajmera dokazan je kod 12 ispitanika (10%), dok u kontrolnoj grupi ispitanika nije dokazana nijedna delecija Y hromozoma. Od ukupno 12 otkrivenih delecija, kod devet ispitanika delecija je lokalizovana u regionu AZFc (75%), kod jednog ispitanika je otkrivena u regionu AZFa (8%), dok su kod dva ispitanika delecije zabeležene u regionu AZFbc (17%). Zaključak. Ispitivanje mikrodelecija Y hromozoma treba razmotriti kao važan elemenat genetskog savetovanja infertilnih parova u Srbiji. Odluka o primeni tehnika asistirane reprodukcije treba da bude zasnovana na rezultatima kliničkog pregleda, spermograma, kariotipa, endokrinih ispitivanja, kao i na rezultatima analize mikrodelecija Y hromozoma., Introduction Y chromosome microdeletions are the second most frequent genetic cause of male infertility after Klinefelter's syndrome. Objective The aim of the study was to determine the frequency of Y chromosome microdeletions in a group of infertile men with an idiophatic cause of infertility, candidates for microfertilization (Intra-cytoplasmic Sperm Injection - ICSI) in Serbia and to correlate genotype-phenotype in patients with Y chromosome microdeletions. METHOD One hundred and sixty patients with low sperm count (less than 5x106 spermatozoa/ml) were enrolled in the study. Forty patients were excluded from the study: ten because they were diagnosed with cytogenetic abnormality and thirty patients were diagnosed with other known causes of infertility. The control group consisted of 150 men who fathered at least one child in the last two years. Genomic DNA was extracted from peripheral blood samples and two multiplex polymerase chain reactions (PCR) analyses were performed using specific primers to confirm the presence or absence of Y chromosome microdeletions. Results Microdeletions were detected in 12 of 120 (10%) cases, while no deletions were detected in the control group. Of total number of 12 deletions, nine were detected in AZFc region (75%), one in AZFa (8%), and two in AZFbc (17%). Conclusion Testing for Y chromosome microdeletions should be considered as an important element in diagnosis and genetic counselling of infertile couples in Serbia. Decisions regarding the assisted reproduction should be made based on the detailed clinical, endocrinological and cytogenetic examinations, spermogram, presence or absence and type of AZF microdeletions and CFTR gene mutations.

Prevalence of Y chromosome microdeletions in infertile men with severe oligozoospermia in Serbia: Aim: The aim of this study was to determine the prevalence and type of microdeletions of the Y chromosome of men with severe oligozoospermia-ICSI candidates in the Serbian population and to compare our findings with those from other parts of the world. Methods: In all patients spermiogram has been performed in order to determine the sperm concentration. Patients were subjected to detailed clinical, endocrinological and cytogenetic examinations. Microdeletion analysis was performed by polymerase chain reaction (PCR) on 203 patients with normal cytogenetic findings. The STS markers tested in each case were sY84, sY86 (AZFa); sY127, sY134 (AZFb); sY254, sY255 (AZFc). Results: at least one of the STS markers was deleted in I I of the 203 cases (5.4 %). Conclusion: AZFc microdeletions were identified with a rather high prevalence in men with severe oligozoospermia ICSI candidates in Serbian population.

Myelodysplastic syndromes (MDS) are rare disorders in children. Molecular mechanisims underlying MDS in children are not yet completely understood. Considering the role of FMS and TP53 gene mutations in adult MDS patients, we analyzed nnutations of these genes in a cohort of 35 children with MDS. Single-strand conformation polymorphism polymerase chain reaction analysis performed on FMS codon 969 and TP53 exons 5-9 showed no mutations in the analyzed sequences. Our results Suggest that molecular mechanisms of MDS evolution in children are different from those in adults.

In children, myelodysplastic syndromes (MDS) represent less then 10% of all hematological malignancies; consequently, molecular genetic studies dealing with this group of patients are scarce. We have analyzed 35 archival bone marrow samples of children with MDS for the presence of mutations in the first and second exons of the NRAS and KRAS2 genes. Mutations were detected with single-strand conformation polymorphism analysis in three patients. One patient harbored a mutation in the second exon of NRAS and two patients in the second exon of KRAS2. Sequencing was performed in two samples and novel mutations were found in both. One patient had a missense mutation in codon 45 of NRAS; the other had a silent mutation in codon 53 and a missense mutation in codon 55 of KRAS2.