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Traditional methods in cell proliferation studies are based on immunohistochemical detection of proliferating cells in the target tissue. Since they are time consuming, optimization of novel, more efficient methods is important for large scale proliferation studies. In this study, we aimed to optimize the isolation of single epithelial rat forestomach cells for flow cytometry. As a marker of cellular proliferation we used the Ki-67 antibody to detect this nuclear protein expressed in proliferating cells. We also performed immunohistochemical detection of Ki-67 positive cells and propidium iodide staining to validate the results. 3-tert- butyl -4-hydroxyanisole was used as the positive control to ensure cellular proliferation. The results showed that isolation of epithelial cells with collagenase, trypsin and cell strainer ensures great cell viability (>95%) and the purity of the samples. Flow cytometry and immunostaining with the Ki-67 antibody indicated that 3-tert- butyl-4-hydroxyanisole treatment leads to a significant increase in proliferation. A significant positive correlation was observed between the results obtained by immunohistochemistry and flow cytometry, but the flow cytometric data had a smaller measurement error, suggesting the equal sensitivity and greater accuracy of this method. Propidium iodide staining showed that the percentage of cells in the G2+S phase of the cell cycle correlated positively with the percentage of Ki-67 positive cells assessed by flow cytometry, indicating that Ki-67 positive cells reflect an active dividing cell pool. We conclude that the isolation of forestomach epithelial cells described is a simple and reliable method for obtaining viable cells for use in flow cytometry. Compared to immunohistochemistry, flow cytometric detection of the Ki-67 antigen is equally sensitive, but much faster and provides more accurate results., Tradicionalne metode u ispitivanju stanične proliferacije temelje se na imunohistokemijskom otkrivanju proliferacijskih stanica u ciljanom tkivu. Kako su dugotrajne, optimizacija novih i učinkovitijih metoda važna je za velika istraživanja o proliferaciji. U ovom smo radu željeli optimizirati izolaciju epitelnih stanica prednjeg želuca štakora za protočnu citometriju. Kao marker stanične proliferacije koristili smo Ki-67 protutijelo za otkrivanje ovoga nuklearnog proteina izraženog u proliferacijskim stanicama. Također smo učinili imunohistokemijsku detekciju Ki- 67 pozitivnih stanica i bojenje propidij-jodidom kako bismo potvrdili rezultate. Butil-hidroksianizol korišten je kao pozitivna kontrola da se osigura stanična proliferacija. Rezultati su pokazali da izolacija epitelnih stanica s kolagenazom, tripsinom i staničnim cjedilom osigurava veliku vijabilnost stanica (> 95 %) i čistoću uzoraka. Protočna citometrija i Ki-67 bojenje pokazali su da tretman butil-hidroksianizolom dovodi do znakovitog porasta proliferacije. Primijećena je znakovita pozitivna korelacija između rezultata dobivenih imunohistokemijom i protočnom citometrijom, dok su protočni citometrijski podaci imali manju pogrešku mjerenja, što upućuje na jednaku osjetljivost i veću točnost ove metode. Bojenje propidij-jodidom pokazalo je da postotak stanica u G2+S fazi staničnog ciklusa pozitivno korelira s postotkom Ki-67 pozitivnih stanica procijenjenih protočnom citometrijom, što upućuje na to da Ki-67 oslikava stanice u aktivnoj diobi. Zaključujemo da je opisana izolacija epitelnih stanica prednjeg želuca štakora jednostavna i pouzdana metoda za dobivanje održivih stanica za upotrebu u protočnoj citometriji. U usporedbi s imunohistokemijom, protočna citometrijska detekcija antigena Ki-67 jednako je osjetljiva, ali mnogo brža i daje točnije rezultate.

To preserve material for future genetic studies, human B-lymphocytes from whole blood samples are routinely transformed into lymphoblastoid cell lines (LCLs) by in vitro infection with Epstein–Barr virus. To determine the rate and frequency of chromosomal changes during long-term culture, we established 10 LCLs (from eight individuals). Before transformation, these cases showed a normal karyotype (three cases), a small supernumerary marker chromosome (three cases), or an aberrant karyotype (four cases). Chromosome analyses were performed at 8-week intervals over a period of at least 1 year, up to 3 years. Surprisingly, we demonstrate that chromosomal instability is the rule, rather than the exception, during long-term culture of LCLs. The most commonly observed acquired clonal aberration was trisomy 12, which emerged in all cell lines within 21 to 49 weeks after infection. Telomeric fusions indicating telomere shortening were found after ~21 weeks. After 1 year of cultivation, the proportion of cells with the original karyotype decreased to ≤10% in 7 of the 10 cell lines. To preserve cells with aberrant genomes, we conclude the cultivation time of LCLs must be restricted to the absolute minimum time required

Aim: This paper presents the latest surgical approaches for epispadias treatment in the pediatric population, as well as those for adolescent and adult populations after initial failed repair in childhood. Methods: The retrospective study was conducted between March 2005 and May 2020 and included 18 patients with the mean age of 21 months (range 11-48 months) (Group A), who underwent primary epispadias repair and 15 patients with the mean age of 18 years (range 13-29 years) (Group B), who underwent redo surgery after failed epispadias repair in childhood. In Group A, the surgery was performed as a one-stage procedure using complete penile disassembly technique, while, in Group B, the surgery was done as a two-stage procedure and included complete straightening and lengthening of the penis, followed by urethral reconstruction. Penile straightening and lengthening were achieved by tunica albuginea incision and grafting. In Group A, the urethral plate was mobilized, transposed ventrally, and tubularized and augmented with vascularized preputial skin flap where needed. In Group B, the urethra was reconstructed either using the buccal mucosa graft and genital skin flaps or with tubularization of genital skin flaps. Successful treatment was defined as a functional and esthetically acceptable penis without complications. Results: The mean follow-up was 88 months (range 15-197 months). Satisfactory results were achieved in 26/33 patients. Urethral fistula occurred in 4/18 patients from Group A and in 3/15 patients in Group B and was surgically repaired after four months. Skin dehiscence occurred in eight patients, five from Group A and three from Group B. Recurrent penile curvature was observed in 2/18 patients from Group A and required surgical correction and in 2/15 patients from Group B and was mild and did not need surgical repair. Eleven patients from Group B who filled out the International Index for Erectile Function reported satisfying erectile function, sexual desire, intercourse, and overall satisfaction. Conclusion: Primary or redo epispadias repair is challenging even for experienced reconstructive urologists. Only radical surgical approach can lead to complete correction of all deformities and provide successful outcome.

Preeclampsia (PE) is a leading cause of maternal and neonatal morbidity and mortality worldwide. Defects in trophoblast invasion, differentiation of extravillous trophoblasts and spiral artery remodeling are key factors in PE development. Currently there are no predictive biomarkers clinically available for PE. Recent technological advancements empowered transcriptome exploration and led to the discovery of numerous non-coding RNA species of which microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) are the most investigated. They are implicated in the regulation of numerous cellular functions, and as such are being extensively explored as potential biomarkers for various diseases. Altered expression of numerous lncRNAs and miRNAs in placenta has been related to pathophysiological processes that occur in preeclampsia. In the following text we offer summary of the latest knowledge of the molecular mechanism by which lnRNAs and miRNAs (focusing on the chromosome 19 miRNA cluster (C19MC)) contribute to pathophysiology of PE development and their potential utility as biomarkers of PE, with special focus on sample selection and techniques for the quantification of lncRNAs and miRNAs in maternal circulation.

Otopalatodigital spectrum disorder (OPDSD) is rare group of X-linked disorders caused by mutations in the filamin A (FLNA) gene. It is characterized by skeletal dysplasia of variable severity and different extra skeletal manifestations. Its presentation in the fetal period is quite unspecific, so diagnosis is usually made after birth. We present prenatal ultrasonography and postmortem findings that led us to a diagnosis of the mildest form of OPDSD (OPD type I) in two consecutive pregnancies. This is the first report on prenatal diagnosis (PND) of OPD type I. Affected fetuses showed facial dysmorphy (hypertelorism, micrognathia, cleft palate) and digital anomalies, features typical of OPD type I. In addition, microphtalmia and early neonatal death due to severe respiratory distress syndrome are described as a novel characteristics of the disorder. Clinical exome sequencing revealed a hemizygous missense pathogenic variant in the FLNA gene (NM_ 001110556.1: c.620C>T). We suggest that the presence of hypertelorism, micrognathia, digital anomalies on prenatal ultrasound examination should alert suspicion to OPDSD. Detailed clinical examination of mother and other female relatives is of great importance in establishing definitive diagnosis of OPD type I.

Introduction Management of severe penile trauma presents great challenges for reconstructive urologists since these injuries vary from abrasions to total emasculation. A review of our case experience with penile amputation is presented, emphasizing techniques used to salvage or reconstruct the most difficult of penile injury cases. Materials and methods A total of 13 patients with penile amputation injury referred to us between 2007 and 2016 were analyzed. Mean age at surgery was 16 years (ranged from 4 to 29 years). Etiology of penile amputation (partial or total) combined with management and outcomes were evaluated. Management included different surgical procedures with the aim to achieve good functional and esthetical outcomes. Postoperative questionnaire was used for assessment of patient's overall satisfaction. Results Follow-up ranged from 13 to 182 months (mean 53). Causes of penile injury were iatrogenic trauma (8), self-amputation (2), electrocution (1), intentional sexual assault (1) and mother's hair strangulation (1). Outcome criteria including aesthetic appearance, urinary function and ability to engage in satisfactory coitus, were noted in 11 cases (85%). Two cases with ensuing complications relating to the total phalloplasty required additional treatment due to urethral fistula. Conclusions Severe penile injuries should be treated on a case by case basis utilizing the most propitious techniques. We respectfully propose that the needs of such patients are best served by referral centers with extensive experience.

Objective The objective of this study was to analyze if the addition of simple cardiac scan in cases with increased nuchal translucency (NT) and/or abnormal ductus venosus (DV) blood flow, and/or tricuspid regurgitation (TCR) can improve detection of congenital heart defects (CHD) in chromosomally normal fetuses without non-cardiac defects at 11-13 + 6 gestational weeks in a population of singleton pregnancies. Methods During the 10 years period, all singleton pregnancies at 11-13 + 6 weeks were routinely scanned for NT, DV blood flow and TCR assessment and, if a single of these parameters was abnormal, simple cardiac scan with 2D gray scale and color and/or directional power Doppler in 4-chamber (4-CV) and 3 vessel and trachea views (3VTV) was performed. Results The sensitivity and specificity of NT ≥ 95th + DV R/A a-wave + TCR in detecting CHD were 77% and 97%, respectively, and of simple cardiac scan, 67% and 98%, respectively. Area under the curve of receiver operating characteristic curve of NT ≥ 95th + DV R/A a-wave + TCR was 0.838, and of NT ≥ 95th + DV R/A a-wave + TCR + simple cardiac scan was 0.915. Conclusions In chromosomally normal fetuses without non-cardiac anomalies, addition of simple cardiac scan to the combined first trimester screening parameters improves detection of major CHD during first trimester.

Background::Obesity, diabetes, and associated diseases are increasing all over the world, and pose a great burden on public health. According to the latest reports, 440 million people are suffering from diabetes. Diabetes is caused by impaired ability to produce or respond to the hormone insulin consequently resulting in hyperglycemia.Methods::Data used for this review was obtained by using PUBMED/MEDLINE (1987-2018). The main data search terms were: Gentiana lutea, Gentiana lutea extract, Gentiana lutea constituents, obesity, diabetes mellitus, diabetic complications.Results::In the present review, we describe the potential of root powder of yellow gentian (Gentiana lutea) for the prevention of obesity and diabetes including complications related to this disease.Conclusion::Reasonably effective, low-cost alternatives could fulfill an important role for a large part of the human population and could be of great value for the food market. Even a modest reduction of morbidity and mortality with respect to this disease translates into millions of lives saved.

We investigated whether gestational diabetes mellitus (GDM) and gestational arterial hypertension (GH) are associated with increased oxidative stress and DNA damage. Study included 3 groups of pregnant women (GDM, GH and control). DNA damage biomarkers (micronuclei MNi, nucleoplasmic bridges NPBs and nuclear buds NBUDs) were assessed by cytokinesis-block micronucleus cytome assay. Oxidative stress levels were evaluated by analyzing malondialdehyde equivalents (TBARS) and 8-hydroxy-2'-deoxyguanosine (8-OHdG). Genotoxic effect of methyldopa, drug used to treat GH, was evaluated in in vitro experiment. TBARS levels, MNi, NPBs and NBUDs frequencies were significantly increased in both GDM and GH group. Concentrations of 8-OHdG were significantly higher in GDM than in other groups. Since methyldopa did not affect MNi, NPBs and NBUDs frequencies, nor TBARS and 8-OHdG levels, we concluded that methyldopa has no genotoxic effect. Thus, even when hyperglycemia or hypertension are present only during pregnancy they induce oxidative stress, DNA damage and chromosomal aberrations.

Introduction. Partial trisomy of chromosome 12 long arm is rare condition with significant clinical impact and is usually diagnosed postnatally. Case report. We present prenatal sonographic findings and molecular cytogenetic characterization of partial trisomy 12q and partial monosomy 2q in two consecutive pregnancies of a healthy non-consanguineous couple. A 35-year-old pregnant woman G3P1A1 was referred to genetic counseling due to sonographic anomalies detected in the fetus. First trimester ultrasound examination revealed hyperechogenic focus in the left cardiac ventricle, single umbilical artery, hyperechogenic bowel and unilateral clubfoot with knee joint ankylosis. Previous pregnancy of the couple was terminated at 26th gestation weeks due to multiple fetal anomalies: bilateral ventriculomegaly, corpus callosum hypoplasia, single umbilical artery and clubfoot. In G3P1A1, amniocentesis was performed and cytogenetic analyses revealed a derivative chromosome 2. Subsequent cytogenetic analyses of parental lymphocytes showed that paternal karyotype was normal, while maternal karyotype showed a der(2). Metaphase fluorescence in situ hybridization (FISH) studies demonstrated partial trisomy 12q24.2?12qter and partial monosomy 2q37.3?2qter in the fetus, resulting from an unbalanced segregation of a maternal balanced translocation t(2;12)(q37.3;q24.2). To date, this is the first such prenatally detected case. Literature search revealed three more cases of prenatally detected partial trisomy 12q and anomalies described were consistent with ones detected in present case. Our findings contribute to further clinical delineation of partial trisomy 12q. Conclusion. Prenatal detection of single umbilical artery, clubfoot, arthogryposis and ventriculomegaly should alert suspicion to chromosome 12q aberrations.

Introduction: MicroRNAs have a significant role in the pathogenesis of preeclampsia. Circulating microRNAs could represent a potential biomarker for preeclampsia. The aim of this study was to evaluate plasma miR210-3p and miR518b in preeclampsia and healthy pregnancy for the first time by digital droplet PCR (ddPCR). Methods: Thirty-six pregnant women (seventeen healthy pregnancies, nineteen preeclampsia patients) were involved from the Clinic for Gynaecology and Obstetrics “Narodni front” in Belgrade, Serbia. Plasma miR210-3p, miR518b and cel-miR-39 as a spike-in control were measured by ddPCR. Results: MiR518b was significantly elevated in preeclampsia compared to a healthy pregnancy (P = 0.034; 0.302(0.217–0.421) vs. 0.171(0.110–0.266)). MiR210-3p showed no significant difference between the two groups (P = 0.951). The adjustment of miR518b was made for a gestational age and smoking status and the difference between the preeclampsia and healthy pregnancy group was more significant (P = 0.026; 0.300(0.216–0.419) vs. 0.172(0.121–0.245)). Plasma miR-518b was significantly higher in the group of preeclampsia patients with proteinuria above the 75th percentile for the group (P=0.033), in women who smoked (P=0.039), and was positively related to uric acid in preeclampsia (P = 0.018, r = 0.536). Plasma miR518b was able to significantly discriminate between preeclampsia and healthy pregnancy, yielding AUC of 0.712 (95%CI:0.539–0.891), P = 0.028. Conclusions: In this study plasma microRNA were measured for the first time in preeclampsia and healthy pregnancies with ddPCR. Placenta-specific miR-518b could serve as a potential biomarker for discriminating preeclampsia and healthy pregnancy, which should be confirmed on a larger study population. This study has failed to confirm the same potential for miR210-3p. This article has been corrected. Link to the correction: [https://farfar.pharmacy.bg.ac.rs/handle/123456789/3669]

Gender affirmation surgery for transmale patients is still challenging, as creation of the neophallus is one of the most demanding steps in surgical treatment. Metoidioplasty, as a one-stage procedure, can be considered in patients who desire gender affirmation surgery without undergoing a complex, multistage procedure with creation of an adult-sized neophallus. Metoidioplasty presents one of the variants of phalloplasty for patients in whom the clitoris is large enough under testosterone treatment. Advanced urethral reconstruction provides low complication rates with satisfying results of standing micturition.

Background:Recurrent pregnancy loss (RPL) is a heterogeneous condition affecting up to 5% of women of reproductive age. Inherited thrombophilia have been postulated as one of the causes of RPL. Here we examined the prevalence of nine thrombophilic gene polymorphisms among women with history of recurrent miscarriages and fertile controls.Methods:The study included 70 women with history of at least three early pregnancy losses and 31 fertile controls with no miscarriages. We investigated mutations in genes responsible for clotting and fibrinolysis, including factor V(FV) Leiden, FV H1299R, factor II (FII) G20210A, methyl-ene tetrahydrofolate reductase (MTHFR) C677T and A1298C, factor XIII (FXIII) V34L, plasminogen activator inhibitor-1 (PAI-1) 4G/5G and endothelial protein C receptor (EPCR) H1 and H3 haplotypes using reverse polymerase chain reaction ViennaLab cardiovascular disease StrippAssays. Results:Our results showed no significant increase inprevalence of tested polymorphisms in women with RPL. However, relative risk for PRL among women heterozygousfor FXIII V34L was 2.81 times increased (OR 2.81, 95% CI1.15–6.87, P=0.023). Haplotype analysis showed that combined presence of high-risk genotypes for FXIII andPAI-1 significantly increases risk for RPL (OR 13.98, CI95% 1.11–17.46, P=0.044).Conclusions:This is the first study in Serbian population that investigated prevalence of FVR2, A1298C, FXIII V34Land EPCR gene variants. Compound heterozygosity forFXIII V34L and PAI-1 4G is significant risk factor for recur-rent miscarriage. Our results should be viewed in context of small case-control study, so further large prospective studies are need for confirmation of our findings. Uvod: Ponavljani spontani pobačaji (PSP) su etiološki heterogeni i javljaju se kod 5% parova u reproduktivnom period. Jedan od mogućih uzroka PSP su i nasledne trombofilije. U okviru ove studije analizirali smo učestalost devet trombofilnih polimorfizama kod pacijentkinja sa ponavljanim spontanim pobačajima. Metode: Ispitanici su u studiji podeljeni u dve grupe na osnovu anamnestičkih podataka o broju spontanih pobačaja (70 u grupi sa PSP i 31 u kontrolnoj grupi). Ispitivani su sledeći genski polimorfizmi: faktor V Lajden (FVL), FVR2, faktor II (FII) G21210A, metilentetrahidrofolat reduktaza (MTHFR) C677T i A1298C polimorfizmi, inhibitor aktivatora plazminogena 1 (PAI-1) 4G/5G, faktor XIII (FXIII) V34L i endotelni protein C receptor (EPRC) H1, H2 i H3 haplotipovi. Za detekciju navedenih polimorfizama je korišćena metoda multipleks reakcije lančanog umnožavanja i reverzne hibridizacije na ViennaLab stripovima. Rezultati: Dobijeni rezultati nisu pokazali povećanu učestalost ispitivanih polimorfizama u grupi sa PSP. Posmatrajući uticaj pojedinačnih polimorfizama na ishod trudnoće pokazano je da polimorfizam FXIII V34L povećava rizik za ponavljane spontane pobačaje (OR 2,81, 95%CI 1,15-6,87, P=0,023). Analizom haplotipova ustanovljeno je da kombinovano prisustvo V34L i PAI-1 4G varijanti značajno povećava rizik za PSP (OR 13,98, CI 95% 1,11-17,46, P=0,044). Zaključak: Ovo je prva studija koja je ispitivala prevalencu FVR2, A1298C, FXIII V34L and EPCR polimorfizama u populaciji žena iz Srbije. Složeni heterozigoti za FXIII V34L i PAI-1 4G polimorfizme imaju značajno povišen rizik sa ponavljane gubitke trudnoće. Radi potvrde dobijenih rezultata potrebne su veće prospektivne studije.

Beckwith–Wiedemann syndrome (BWS; OMIM #130650) is an overgrowth syndrome caused by different genetic or epigenetic alterations affecting imprinted regions on chromosome 11p15.5. Here we report a family with multiple offspring affected with BWS including giant omphalocoeles in which maternal transmission of a chromosomal rearrangement including an inversion and two deletions leads to hypomethylation of the imprint control region 2 (ICR2). As the deletion includes the promoter and 5′ part of the KCNQ1 gene, we suggest that transcription of this gene may be involved in establishing the maternal methylation imprint of the ICR2, which is located in intron 10 of KCNQ1.

OBJECTIVE The aim of this study was to evaluate the screening performances of abnormal ductus venosus (DV) blood flow for the detection of heart defects in chromosomally normal fetuses with increased nuchal translucency (NT) thickness at 11-13 + 6 weeks' gestational in a population of singleton pregnancies. METHODS During an 8-year period, all singleton pregnancies from 11 + 0 to 13 + 6 weeks were scanned for NT and DV blood flow assessment. Two groups of cases with abnormal NT were evaluated: NT ≥ 95th and NT ≥ 99th centile. DV waveforms were considered to be abnormal if the a-wave was reversed or absent (R/A). RESULTS Addition of DV R/A a-wave to either NT ≥ 95th or NT ≥ 99th percentile increased specificity (p

Fanconi anemia (FA) is a rare genetically heterogeneous disorder associated with bone marrow failure, birth defects and cancer susceptibility. Apart from the disease- causing mutations in FANC genes, the identification of specific DNA variations, such as single nucleotide polymorphisms (SNPs), in other candidate genes may lead to a better clinical description of this condition enabling individualized treatment with improvement of the prognosis. In this study, we have assessed 95 SNPs located in 52 key genes involved in base excision repair (BER), nucleotide excision repair (NER), mismatch repair (MMR), double strand break (DSB) repair and cell cycle control using a DNA repair chip (Asper Biotech, Estonia) which includes most of the common variants for the candidate genes. The SNP genotyping was performed in five FA-D2 patients and in one FA-A patient. The polymorphisms studied were synonymous (n=10), nonsynonymous (missense) (n=52) and in non-coding regions of the genome (introns and 5 ‘and 3’ untranslated regions (UTR)) (n=33). Polymorphisms found at the homozygous state are selected for further analysis. Our results have shown a significant inter-individual variability among patients in the type and the frequency of SNPs and also elucidate the need for further studies of polymorphisms located in ATM, APEX APE 1, XRCC1, ERCC2, MSH3, PARP4, NBS1, BARD1, CDKN1B, TP53 and TP53BP1 which may be of great importance for better clinical description of FA. In addition, the present report recommends the use of SNPs as predictive and prognostic genetic markers to individualize therapy of FA patients. [Projekat Ministarstva nauke Republike Srbije, br. 173046]

Ciliopathies are genetically heterogeneous disorders characterized by variable expressivity and overlaps between different disease entities. This is exemplified by the short rib-polydactyly syndromes, Jeune, Sensenbrenner, and Mainzer-Saldino chondrodysplasia syndromes. These three syndromes are frequently caused by mutations in intraflagellar transport (IFT) genes affecting the primary cilia, which play a crucial role in skeletal and chondral development. Here, we identified mutations in IFT140, an IFT complex A gene, in five Jeune asphyxiating thoracic dystrophy (JATD) and two Mainzer-Saldino syndrome (MSS) families, by screening a cohort of 66 JATD/MSS patients using whole exome sequencing and targeted resequencing of a customized ciliopathy gene panel. We also found an enrichment of rare IFT140 alleles in JATD compared with nonciliopathy diseases, implying putative modifier effects for certain alleles. IFT140 patients presented with mild chest narrowing, but all had end-stage renal failure under 13 years of age and retinal dystrophy when examined for ocular dysfunction. This is consistent with the severe cystic phenotype of Ift140 conditional knockout mice, and the higher level of Ift140 expression in kidney and retina compared with the skeleton at E15.5 in the mouse. IFT140 is therefore a major cause of cono-renal syndromes (JATD and MSS). The present study strengthens the rationale for IFT140 screening in skeletal ciliopathy spectrum patients that have kidney disease and/or retinal dystrophy.

Fanconi anemia is rare inherited disease characterized by wide spectrum of congenital anomalies, progressive pancytopenia, and predisposition to hematological malignancies and solid tumors. Molecular genetic analysis of mutations in FANC genes is of a great importance for diagnosis confirmation, prenatal and carrier testing, as well as for prediction of chemotherapy outcome and disease complications. In this study we performed screening of frequently affected regions of FANCD2 gene for sequence variants in six unrelated FA-D2 patients in Serbia. This is the first molecular analysis of FANCD2 gene in Serbian FA-D2 patients. A total of 10 sequence variants were detected, one in homozygous, and nine in heterozygous state. Two variants were found within exons, and eight within introns, in deep intronic regions. In-silico analysis showed that among all detected variants one exon variant and three intron variants might have impact on splicing mechanism. Heterozygous variants found in intron 3, c.206-246delG; exon 26, c.2396 C>A and intron 28, c.2715+573 C>T were not previously reported. In-silico analysis revealed that among them, two (intron 3, c.206-246 delG and exon 26, c.2396 C>A) could be novel disease-causing mutations. Many variants were found in more than one patient, including those unreported, indicating their possible ethnic association. Great number of variants in some patients suggests their non-random emergence in Fanconi anemia pathway. [Project of the Serbian Ministry of Education, Science and Technological Development, Grant no. 173046]

Fanconi anemia (FA) is a rare genetically heterogeneous disease characterized by developmental abnormalities, progressive bone marrow failure, and cancer susceptibility. We examined spontaneous, diepoxybutane (DEB)- induced and radiation-induced sister chromatid exchanges (SCEs) in wholeblood lymphocyte cultures of bone marrow failure (BMF) patients including Fanconi anemia, mothers of affected individuals, and healthy controls. The baseline frequency of SCE in FA cells was similar to that observed in controls. However, in response to DEB SCE frequencies in FA patients and their mothers were significantly increased compared to both non-FA BMF families and healthy controls. In response to ionizing radiation, cells displayed increased frequency of SCE, but no differences between FA patients and non-FA BMF patients were seen. Our data confirm and expand previous findings by showing that SCE induced by DEB can be used as an adjunct diagnostic test not only for FA patients, but also for female heterozygous carriers, at least for complementation groups FANCA and FANCD2. [Projekat Ministarstva nauke Republike Srbije, br. 173046]

Objective The aim of this study was to examine the effectiveness of a combination of parameters at first-trimester screening for fetal aneuploidies, including ultrasound assessment of the nasal bone (NB), blood flow in the ductus venosus (DV) and flow across the tricuspid valve. Methods Screening for aneuploidy was carried out in 4172 singleton pregnancies between January 2006 and December 2010. Diagnostic accuracy of combined screening [inclusive of maternal age, fetal nuchal translucency (NT) thickness and maternal serum free beta-human chorionic gonadotropin and pregnancy-associated plasma protein A] and of secondary ultrasound markers [NB, tricuspid regurgitation (TR) and Doppler studies of the DV] obtained at the same visit was assessed using the receiver operating characteristic (ROC) curve analysis. Results The individual areas under the ROC curves of NT, NB, DV or TR ranged between 0.7 and 0.8, representing acceptable discrimination. The area under the ROC curve of combined first-trimester screening was 0.87, whereas the addition of secondary ultrasound markers increased the area under the curve to 0.92, which represents excellent discrimination. At a risk cutoff of 1 : 275, the detection rate for aneuploidy increased from 87% to 92% (z statistic = 1.78, P = 0.076), and the false positive rate decreased from 5.3% to 4.8%. Conclusion The addition of secondary ultrasound markers (NB, DV and TR) to combined first-trimester screening showed a tendency toward improved accuracy of the screening. © 2012 John Wiley & Sons, Ltd.

Among patients with bone marrow failure (BMF) syndrome, some are happened to have underlying Fanconi anemia (FA), a genetically heterogeneous disease, which is characterized by progressive pancytopenia and cancer susceptibility. Due to heterogeneous nature of the disease, a single genetic test, as in vitro response to DNA cross-linking agents, usually is not enough to make correct diagnosis. The aim of this study was to evaluate whether measuring repair kinetics of radiation-induced DNA double-strand breaks (DSBs) can distinguish Fanconi anemia from other BMF patients. An early step in repair of DSBs is phosphorylation of the histone H2AX, generating gamma-H2AX histone, which extends over mega base-pair regions of DNA from the break site and is visualised as foci (gamma-H2AX foci) with specific antibodies. The primary fibroblasts, established from FA patients, were exposed to gamma-rays, a dose of 2 Gy ((60)Co), incubated for up to 24 hours under repair-permissive conditions, and assayed for the level of gamma-H2AX foci and apoptosis at different recovery times after the treatment. Cell lines originating from FA patients displayed a significant delay in the repair of radiation-induced DNA DSBs relative to non-FA bone marrow failure (non-FA BMF) and control cell lines. The delay is especially evident at recovery time of 24 hours, and is seen as about 8-fold increase of residual gamma-H2AX foci compared to self-state before irradiation. The delay in repair kinetics of FA cells represents the unique feature of FA cellular phenotype, which should be exploited to distinguish FA cellular phenotype.

The aim of this study was to evaluate the screening performances of abnormal ductus venosus (DV) blood flow for the detection of heart defects in chromosomally normal fetuses with increased nuchal translucency (NT) thickness at 11-13 + 6 weeks' gestational in a population of singleton pregnancies.During an 8-year period, all singleton pregnancies from 11 + 0 to 13 + 6 weeks were scanned for NT and DV blood flow assessment. Two groups of cases with abnormal NT were evaluated: NT ≥ 95th and NT ≥ 99th centile. DV waveforms were considered to be abnormal if the a-wave was reversed or absent (R/A).Addition of DV R/A a-wave to either NT ≥ 95th or NT ≥ 99th percentile increased specificity (p 0.001 and p 0.001, respectively), but not screening performances in detection of major heart defects (p = 0.73 and p = 0.91, respectively). Combination of DV R/A a-wave with NT ≥ 95th or NT ≥ 99th centile correlated with right heart defects (p = 0.024 and p = 0.013, respectively).In chromosomally normal fetuses, addition of abnormal DV a-wave to increased NT does not improve screening performances of NT in detection of major hearts defects in first trimester. However, there is correlation of such parameter with right heart defects and AV septal defects.

JOKSIC I., LESKOVAC, A., PETROVIC, S. and JOKSI, G. Vitamin B12 Reduces Ribavirin-Induced Genotoxicity in Phytohemaglutinin-Stimulated Human Lymphocytes. Toboku J. Exp. Med., 2006, 209 (4), 347-354 - Ribavirin, an N-glycosyl nucleoside (1-,6-D-ribofuranosyl-1, 2, 4-triazole-3 carboxamide), is a synthetic purine nucleoside analogue with a broad spectrum of antiviral activity, however, its high toxicity poses a major disadvantage of its use as a therapeutic. Various studies have shown that vitamin B 12 plays a significant role in maintaining the stability of the human genome. We therefore investigated the potential beneficial effect of vitamin B 12 in reducing ribavirin-induced genotoxicity. To test this, we used the cytokinesis-block micronucleus (CBMN) assay. Human blood cells were treated in vitro with increasing doses of ribavirin (0.05, 0.17, 0.32, 0.47 and 0.65,umol/ml) for three different periods of time (2, 4 and 17 hrs). Duplicate cultures were supplemented with 50 mu l of vitamin B 12 during the drug treatment (final concentration of 13.5 mu g/ml). Micronuclei formation and cell proliferation potential were then scored in both sets of samples and the corresponding controls. The results showed that supplementation with vitamin B 12 lowered the frequency of micronuclei (Z = 2.02, p LT 0.04) and recovered the proliferation potential of the treated cells for each treatment period, except for the conditions with the highest concentration of ribavirin and the shortest time. These observations underscore the unique beneficial effects of vitamin B12 in reducing genotoxicity, particularly by recovering the proliferation potential of treated cells, as demonstrated by the decrease in mononucleated cells and enhancement of binucleated and polynucleated cells. The mechanism by which vitamin B 12 reduces ribavirin-induced genotoxicity is related to de novo synthesis of nucleotides, and is worthy of further investigation. ribavirin; reduced genotoxicity; vitamin B 12; human lymphocytes (c) 2006 Tohoku University Medical Press.

Genomic instability tends to occur at specific genomic regions known as common fragile sites (FS). FS are evolutionarily conserved and generally involve late replicating regions with AT-rich sequences. The possible correlation between some FS and cancer-related breakpoints emphasizes on the importance of understanding the mechanisms of chromosomal instability at these sites. Although about 230 FS have already been mapped cytogenetically, only a few of them have been characterized on a molecular level. In this chapter, we provide a protocol for mapping of common FS using bacterial artificial chromosome (BAC) probes in fluorescence in situ hybridization (FISH) and suggest the usage of lymphocytes from Fanconi anemia patients as a model system. In the latter, rare FS are expressed much more frequently than in, for example, aphidicolin-induced blood lymphocyte preparations. Knowing the exact location of FS enables the molecular comparison of their location and breakpoints that appear during evolution, cancer development and inherited disorders.

Genomic instability tends to occur at specific genomic regions known as common fragile sites (FS). FS are evolutionarily conserved and generally involve late replicating regions with AT-rich sequences. The possible correlation between some FS and cancer-related breakpoints emphasizes on the importance of understanding the mechanisms of chromosomal instability at these sites. Although about 230 FS have already been mapped cytogenetically, only a few of them have been characterized on a molecular level. In this chapter, we provide a protocol for mapping of common FS using bacterial artificial chromosome (BAC) probes in fluorescence in situ hybridization (FISH) and suggest the usage of lymphocytes from Fanconi anemia patients as a model system. In the latter, rare FS are expressed much more frequently than in, for example, aphidicolin-induced blood lymphocyte preparations. Knowing the exact location of FS enables the molecular comparison of their location and breakpoints that appear during evolution, cancer development and inherited disorders.

Introduction. Aneuploidies are the major cause of perinatal death and early psychophysical disorders. Objective. In this study, we analyzed detection and false-positive rates of screening for aneuploidies in the first trimester by the combination of maternal age, fetal nuchal translucency (NT) thickness and maternal serum free beta-human chorionic gonadotrophin (β-hCG), and pregnancy-associated plasma protein-A (PAPP-A) at 11-13+6 weeks of gestation, using the appropriate software developed by the Fetal Medicine Foundation. Methods. Our screening study for aneuploidies analyzed 4172 singleton pregnancies from January 2006 to December 2010. The sensitivities and false-positive rates using the combined aneuploidies determination for the risk cut-off of 1:275 were evaluated. Results. In the trisomy 21 pregnancies, the fetal NT was higher than 95th centile, in 72.8%, serum free b-hCG concentration it was above the 95th centile in 55% and serum PAPP-A was below the 5th centile in 47% of the cases. In the trisomy 18 and 13, the fetal NT was above 95th centile in 66.6% and 44.4% of the cases, respectively. The serum free b-hCG concentration was above the 95th centile in 0 and 10%, but serum PAPP-A was below 5th centile in 80.9% and 88.8% of pregnancies. In the trisomy 21 pregnancies the median free beta-hCG was 2.3 MoM and the median PAPP-A was 0.45 MoM. Chromosomal abnormalities were detected in 169 fetuses: trisomy 21 (97), Turner syndrome (19), trisomy 18 (28), trisomy 13 (11) and others (14). Detection rate of combined screening for aneuploides were 86.0% with false positive rate of 5.3% (mean age 33±4.9 years, >35 years in 35% of pregnancies). Conclusion. Our study suggests that the strategy of first-trimester combined screening of biochemical values and ultrasonographic parameters at 12 gestational weeks identifies higher percentage of aneuploidies with a lower false-positive rate than a single parameter strategy.

In this paper we present the data of lymphocyte radiosensitivity testing used for characterization of radiosensitive cellular phenotype and diagnostics of ataxia-telangiectasia disease. We point out the advantage of lymphocyte micronucleus test (CBMN) over other cellular tests for assessment of radiosensitivity: the first advantage of CBMN is that primary patient cells are used (less than 1 ml), the second one is that the results of testing are obtained within 3 days and there is no need for establishing a patient-derived cell line, which requires additional time and application of more expensive methods. The third advantage of CBMN method is that it gives information about proliferative ability of cells, which can recognize dysfunctional ataxia-telangiectasia mutated protein. The results are fast and accurate in diagnostics of ataxia-telagiectasia diseases.

The aim of this study was to examine the effectiveness of a combination of parameters at first-trimester screening for fetal aneuploidies, including ultrasound assessment of the nasal bone (NB), blood flow in the ductus venosus (DV) and flow across the tricuspid valve.Screening for aneuploidy was carried out in 4172 singleton pregnancies between January 2006 and December 2010. Diagnostic accuracy of combined screening [inclusive of maternal age, fetal nuchal translucency (NT) thickness and maternal serum free beta-human chorionic gonadotropin and pregnancy-associated plasma protein A] and of secondary ultrasound markers [NB, tricuspid regurgitation (TR) and Doppler studies of the DV] obtained at the same visit was assessed using the receiver operating characteristic (ROC) curve analysis.The individual areas under the ROC curves of NT, NB, DV or TR ranged between 0.7 and 0.8, representing acceptable discrimination. The area under the ROC curve of combined first-trimester screening was 0.87, whereas the addition of secondary ultrasound markers increased the area under the curve to 0.92, which represents excellent discrimination. At a risk cutoff of 1 : 275, the detection rate for aneuploidy increased from 87% to 92% (z statistic = 1.78, P = 0.076), and the false positive rate decreased from 5.3% to 4.8%.The addition of secondary ultrasound markers (NB, DV and TR) to combined first-trimester screening showed a tendency toward improved accuracy of the screening.

Biological Effects ofEchinacea Purpureaon Human Blood CellsThe aim of this study was to investigate radioprotective properties ofEchinacea purpureatabletsin vivo. We analysed lymphocyte chromosome aberrations (CA), micronuclei (MN), apoptosis of leukocytes and haematological parameters in a group of radiation workers who were identified as carrying dicentric chromosomes in their lymphocytes. All radiation workers were taking two 275 mg Echinacea tabletsb.i.d., according to a pharmacist's recommendation. All parameters were analysed before and after the two-week treatment. At the end of the treatment lymphocyte CA frequency dropped significantly, and the number of apoptotic cells increased. The inverse lymphocyte-to-granulocyte ratio at the beginning of the study changed to normal at its end. In conclusion, biological effects observed after administration ofEchinacea purpureapreparation suggest that it may be beneficial for the prevention of adverse health effects in workers exposed to ionising radiation.