Quantitative Fluorescent PCR – A Rapid Approach to Prenatal Diagnostics of Common Autosomal Aneuploidies

Autosomal trisomies account for more than 80 % of significant chromosomal disorders and are routinely detected by the cytogenetic analysis of cultivated amniotic fluid cells. However, this approach is time-consuming and requires a significant level of training and expertise. The main aim of our work was to introduce QF-PCR to our lab, a quicker, simpler and cheaper method. We also aimed to evaluate the usefulness of the chosen marker set in the Croatian population and the reliability and accuracy of the obtained results. STR loci from chromosomes 13, 18 and 21 were co-amplified, separated by capillary electrophoresis and analysed. Characteristic triplets and/or 2:1 patterns were detected for trisomic samples while normal samples were either homozygous or heterozygous. The tested set of loci showed high heterozygosity and therefore a good potential for analyzing the Croatian population. The results of QF-PCR were in full compliance with the cytogenetic analysis which was also performed for cultivated amniotic fluid cell samples

Identification of human remains from the Second World War mass graves uncovered in Bosnia and Herzegovina

Aim To present the results obtained in the identification of human remains from World War II found in two mass graves in Ljubuški, Bosnia and Herzegovina. Methods Samples from 10 skeletal remains were collected. Teeth and femoral fragments were collected from 9 skeletons and only a femoral fragment from 1 skeleton. DNA was isolated from bone and teeth samples using an optimized phenol/chloroform DNA extraction procedure. All samples required a pre-extraction decalcification with EDTA and additional post-extraction DNA purification using filter columns. Additionally, DNA from 12 reference samples (buccal swabs from potential living relatives) was extracted using the Qiagen DNA extraction method. QuantifilerTM Human DNA Quantification Kit was used for DNA quantification. PowerPlex ESI kit was used to simultaneously amplify 15 autosomal short tandem repeat (STR) loci, and PowerPlex Y23 was used to amplify 23 Y chromosomal STR loci. Matching probabilities were estimated using a standard statistical approach. Results A total of 10 samples were processed, 9 teeth and 1 femoral fragment. Nine of 10 samples were profiled using autosomal STR loci, which resulted in useful DNA profiles for 9 skeletal remains. A comparison of established victims’ profiles against a reference sample database yielded 6 positive identifications. Conclusion DNA analysis may efficiently contribute to the identification of remains even seven decades after the end of the World War II. The significant percentage of positively identified remains (60%), even when the number of the examined possible living relatives was relatively small (only 12), proved the importance of cooperation with the members of the local community, who helped to identify the closest missing persons’ relatives and collect referent samples from them

Visoke vrijednosti omjera vjerojatnosti ne moraju biti dostatne u procesu identifikacije žrtava rata s pomo}u analize DNA

With the advance in typing tools and extraction procedures in recent years, DNA analysis has developed into an amazingly powerful method for forensic analysis. For a number of years, autosomal STR (Short Tandem Repeat) typing has been used as a tool in the process of identification of war victims in Croatia. Although DNA typing is very effective in detecting possible identities of exhumed skeletal remains, this approach bears some risk of false identification. The paper presents the case of a match between skeletal remains and the son and wife of a missing person in 13 STR loci. Even though these skeletal remains also matched in 13 loci the mother of the same missing person, additional genetic testing (Y-chromosome and mitochondrial DNA) unequivocally excluded the proposed identity. Although likelihood ratio is the best measure of the significance of a genetic match between exhumed skeletal remains and relatives of the missing person, the meaning of likelihood ratio is not as clear in database matching as in simple paternity cases and great care is needed to avoid wrong interpretation. To reduce the risk of possible false identifications, in addition to DNA evidence, other types of evidence (such as information about the time, place and other conditions of disappearance), as well as on anthropological and other »classical« forensic data are being used as a »control mechanism« in the DNA-lead process.Zahvaljujući stalnom napretku u metodama izolacije i karakterizacije, analiza DNA razvila se u vrlo moćnu metodu forenzičke analize. Već niz godina analizu autosomnih STR regija primjenjuje se kao temeljnu metodu u procesu identifikacije žrtava rata u Hrvatskoj. Iako je analiza DNA vrlo učinkovita u pronalaženju mogućega identiteta ekshumiranih posmrtnih ostataka, ovakav pristup nosi i određeni rizik pogrešne identifikacije. Ovdje je opisan slučaj poklapanja posmrtnih ostataka sa sinom i suprugom nestale osobe u 13 STR lokusa. Iako su se isti posmrtni ostaci također poklapali i s majkom iste nestale osobe u 13 lokusa, dodatnim genetičkim ispitivanjima (Y kromosom i mitohondrijska DNA) nedvojbeno je isključena mogućnost da se radi o toj osobi. Omjer vjerojatnosti najbolji je pokazatelj značajnosti genetičkoga podudaranja skeletnih ostataka i rodbine nestalih osoba, no kad je to podudaranje pronađeno nasumičnim pretraživanjem baze podataka, značaj omjera vjerojatnosti nije tako jasan kao u jednostavnome utvrđivanju očinstva i potreban je velik oprez kako ne bi došlo do njegove pogrešne interpretacije. Radi smanjivanja rizika pogrešne identifikacije, osim na rezultate analize DNA, pri utvrđivanju identiteta velik značaj potrebno je dati i drugim tipovima dokaza (poput vremena, lokacije i uvjeta nestanka) te antropološkim i ostalim »klasičnim« forenzičkim dokazima, kao kontrolnome mehanizmu u procesu identifikacije predvođenom analizom DNA

Čimbenici rizika i molekularne predispozicije za displaziju vrata maternice u žena iz istočne Hrvatske

Purpose: The aim of this study was to investigate possible association between high-risk Human papillomavirus (HR HPV) – induced cervical infection, HR HPV-related cervical dysplasia, HR HPV genotypes with two Toll-like receptor (TLR) 9 gene polymorphisms and other risk factors. Methods: During a three-year period, 100 women positive for cervical HR HPV infection (97 with cervical dysplasia and 3 positive women without dysplasia) were genotyped using the Linear Array HPV Genotyping assay (Roche Diagnostics). Furthermore, two polymorphisms of TLR9 (-1486T/C, rs187084 and 2848C/T rs352140) were determined using real-time Polymerase Chain Reaction; 50 HR HPV negative women of similar ethnicity were included as controls. Results: This study showed that infections with HPV 16 in women with cervical dysplasia were more frequently found compared with HPV 18 infections (p=0.0539). Comparison between HR HPV positive and negative women showed significant association between age >35 years (p=0.0058), being unmarried women (p=0.0001), nocondom usage (p=0.0304) and active tobacco smoking (p=0.0376) with HR HPV cervical infection. No significant associations between two TLR9 gene polymorphisms, HR HPV infection and cervical dysplasia were found. Conclusion: Our results indicated that: i) women with cervical dysplasia showed significant higher rate of HR HPV 16 infection compared to HR HPV 18, ii) HR HPV – infection was strongly correlated with social risk factors and iii) TLR9 gene polymorphisms (rs187084; rs352140) did not correlate with HR HPV infection and cervical dysplasia. Further genome-wide association studies could open new frontier in understanding the relationship between polymorphisms at TLR9 and immunological mechanisms in HPV-induced carcinogenesis.Cilj: Cilj ovog istraživanja bio je utvrditi povezanost između infekcije vrata maternice visokorizičnim humanim papilomavirusima (engl. high-risk Human papillomavirus - HR HPV), displazije vrata maternice uzrokovane visokorizičnim genotipovima HPV-a, visokorizičnih genotipova HPV-a te dva polimorfizma gena za Toll-u sličan receptor (TLR) 9 i drugih rizičnih čimbenika. Metode: Tijekom trogodišnjeg razdoblja, u 100 žena s cervikalnom infekcijom visokorizičnim genotipovima HPV-a (97 s displazijom vrata maternice i 3 s pozitivnim nalazom HPV-a ali bez displazije vrata maternice) određeni su genotipovi virusa primjenom molekularnog testa Linear Array HPV Genotyping assay (Roche Diagnostics). Također su analizirana i dva polimorfizma gena za TLR9 (-1486T/C, rs187084 i 2848C/T rs352140) koristeći metodu lančane reakcije polimerazom u stvarnom vremenu; te je 50 žena s negativnim hrHPV ili sličnog etniciteta uključeno kao kontrolna skupina. Rezultati: Istraživanje je pokazalo da su HR HPV infekcije genotipom 16 češće u žena sa displazijom vrata maternice u usporedbi sa ženama s infekcijom genotipa 18 (p=0.0539). Usporedbom žena s pozitivnim i negativnim nalazom na HR HPV, utvrđena je značajna povezanost između dobi >35 godina (p=0.0058), bračnog statusa (slobodne osobe, p=0.0001), nekorištenja kondoma (p=0.0304) i aktivnog pušenja (p=0.0376) i infekcije s HR HPV.“ Povezanost između dva polimorfizma gena za TLR9, infekcije HR HPV i displazije vrata maternice nije dokazana. Zaključak: Rezultati ovog istraživanja pokazali su da: i) je u žena sa displazijom vrata maternice značajno veća učestalost infekcije s genotipom 16 u usporedbi s genotipom 18, ii) infekcija s HR HPV povezana je sa socijalnim faktorima rizika i iii) polimorfizmi gena za TLR9 (rs187084; rs352140) nisu povezani s HR HPV infekcijom i displazijom vrata maternice. Daljnja genomska istraživanja mogu proširiti spoznaje u razumijevanju odnosa između polimorfizma gena za TLR9 i imunoloških mehanizama u HPV-om induciranoj karcinogenezi

Prevalence and Genotype Distribution of High-risk Human Papillomavirus (HR HPV) in Male Genital Samples of Osijek-Baranja County

This is a first cross-sectional study on the prevalence and distribution of HPV infection in asymptomatic, heterosex- ual men from Osijek-Baranja County, Croatia. Between 2009 and 2011, 330 men tested for sexually transmitted diseases (STDs) were recruited. Their genital swabs were tested for high-risk HPV (HR HPV) infection by the AMPLICOR HPV test and further genotyped by the Linear Array HPV Genotyping Test (both by Roche). Infection with a single HR HPV was detected in almost one third of men (39%) whereas multiple HPV types, in more than a half of HR HPV-positive men (61%). The highest HR HPV prevalence was detected in those younger than 20 (37.5%) and lowest in 31–35 year old men (27.8%). The most common genotypes were HPV 6 (24%), 16 (17.8%), 51 (9%), 52 (6%), 35, 55, 66, 84 (each 5%), 31, 62 (each 4%), 39, 58, 59, 83 (each 2.5%), and finally 56, 18, 53, and 54 (each 1.3%). Having more than one sexual partner per year was significantly associated with HR HPV infection in age group between 26 and 30 years (p=0.001). Due to the high prevalence of HR HPV infection in men of this County and its risk of transmission to women, we recommend more public awareness about this particular STD and initiating vaccination programs of young men and women

DNA isolation from Aspergillus flavus: Optimal method selection

The methods for fungal genomic DNA isolation for PCR amplification, including commercially available kits, must often be adapted in order to produce sufficient amounts of high-quality DNA from specific fungal species. The aim of this study was to select an optimal method for the isolation of DNA from Aspergillus flavus suitable for PCR reaction. Four different methods were compared according to their efficiency in isolating pure DNA, their price and time consumption. DNA quantification and purity estimation were performed using the NanoDropTM 1000 UV/VIS spectrophotometer and DNA integrity and PCR products were determined by gel-electrophoresis. DNA quantity ranged from 92.77 ± 11.52 to 5477.4 ± 22.03 ng/µL, with A260/280 from 1.14 ± 0.10 to 1.94 ± 0.16, and A260/230 0.37 ± 0.05 to 1.91 ± 0.17. There were also great differences in time consumption per sample, ranging from 1 hr 15 min to 7 hr 5 min. The determined costs per sample were ranging from 0.12 € to 2.29 € per sample. All tested methods were suitable for the isolation of A. flavus genomic DNA and subsequently for PCR reaction

The Association of Cardiovascular Disease with the T3111C Polymorphism in the CLOCK Gene

Background and Objectives: Cardiovascular diseases (CVDs) are among the leading causes of death worldwide, although CVD mortality has decreased in developed countries. Numerous pathophysiological processes lead to the development of CVDs. The circadian rhythm coordinates many physiological processes, and its disruption can lead to many pathophysiological changes. One of the significant circadian rhythm genes is the CLOCK gene, whose polymorphisms are associated with CVD risk factors. Research findings of the association between CLOCK gene polymorphism and CVDs and its comorbidities are not consistent. This meta-analysis was conducted to quantify the associations between T3111C polymorphism and the risk of CVDs. Materials and Methods: The PubMed and Scopus databases were searched for studies reporting onthe association between T3111C (rs1801260) in the circadian CLOCK gene and cardiovascular disease and its comorbidities such as obesity, hypertension, insulin resistance, and coronary artery disease. A fixed-effect model was used to calculate the pooled odds ratio and 95% confidence interval by comprehensive meta-analysis software. Results: Five independent studies, including case-control, cross-sectional, and cohort research methods, were analyzed with 3123 subjects in total. The meta-analysis revealed a significant association between T3111C polymorphism and cardiovascular disease (OR = 1.32, 95% CI: 1.16–1.50, p < 0.001) with significant heterogeneity (I2 = 91.1%, p < 0.001) and no publication bias. The subgroup analysis on comorbidity related to CVDs revealed that hypertension was associated with T3111C polymorphism (OR = 2.02, 95% CI: 1.60–2.54, p < 0.001). Conclusions: Our meta-analysis based on available studies using a fixed model shows that T3111C polymorphism in the CLOCK gene is associated with CVD susceptibility

The Association between Circadian Clock Gene Polymorphisms and Metabolic Syndrome: A Systematic Review and Meta-Analysis

Metabolic syndrome (MetS) is a combination of cardiovascular risk factors associated with type 2 diabetes, obesity, and cardiovascular diseases. The circadian clock gene polymorphisms are very likely to participate in metabolic syndrome genesis and development. However, research findings of the association between circadian rhythm gene polymorphisms and MetS and its comorbidities are not consistent. In this study, a review of the association of circadian clock gene polymorphisms with overall MetS risk was performed. In addition, a meta-analysis was performed to clarify the association between circadian clock gene polymorphisms and MetS susceptibility based on available data. The PubMed and Scopus databases were searched for studies reporting the association between circadian rhythm gene polymorphisms (ARNTL, BMAL1, CLOCK, CRY, PER, NPAS2, REV-ERBα, REV-ERBβ, and RORα) and MetS, and its comorbidities diabetes, obesity, and hypertension. Thirteen independent studies were analyzed with 17,381 subjects in total. The results revealed that the BMAL1 rs7950226 polymorphism was associated with an increased risk of MetS in the overall population. In contrast, the CLOCK rs1801260 and rs6850524 polymorphisms were not associated with MetS. This study suggests that some circadian rhythm gene polymorphisms might be associated with MetS in different populations and potentially used as predictive biomarkers for MetS

Visoke vrijednosti omjera vjerojatnosti ne moraju biti dostatne u procesu identifikacije žrtava rata s pomo}u analize DNA

With the advance in typing tools and extraction procedures in recent years, DNA analysis has developed into an amazingly powerful method for forensic analysis. For a number of years, autosomal STR (Short Tandem Repeat) typing has been used as a tool in the process of identification of war victims in Croatia. Although DNA typing is very effective in detecting possible identities of exhumed skeletal remains, this approach bears some risk of false identification. The paper presents the case of a match between skeletal remains and the son and wife of a missing person in 13 STR loci. Even though these skeletal remains also matched in 13 loci the mother of the same missing person, additional genetic testing (Y-chromosome and mitochondrial DNA) unequivocally excluded the proposed identity. Although likelihood ratio is the best measure of the significance of a genetic match between exhumed skeletal remains and relatives of the missing person, the meaning of likelihood ratio is not as clear in database matching as in simple paternity cases and great care is needed to avoid wrong interpretation. To reduce the risk of possible false identifications, in addition to DNA evidence, other types of evidence (such as information about the time, place and other conditions of disappearance), as well as on anthropological and other »classical« forensic data are being used as a »control mechanism« in the DNA-lead process.Zahvaljujući stalnom napretku u metodama izolacije i karakterizacije, analiza DNA razvila se u vrlo moćnu metodu forenzičke analize. Već niz godina analizu autosomnih STR regija primjenjuje se kao temeljnu metodu u procesu identifikacije žrtava rata u Hrvatskoj. Iako je analiza DNA vrlo učinkovita u pronalaženju mogućega identiteta ekshumiranih posmrtnih ostataka, ovakav pristup nosi i određeni rizik pogrešne identifikacije. Ovdje je opisan slučaj poklapanja posmrtnih ostataka sa sinom i suprugom nestale osobe u 13 STR lokusa. Iako su se isti posmrtni ostaci također poklapali i s majkom iste nestale osobe u 13 lokusa, dodatnim genetičkim ispitivanjima (Y kromosom i mitohondrijska DNA) nedvojbeno je isključena mogućnost da se radi o toj osobi. Omjer vjerojatnosti najbolji je pokazatelj značajnosti genetičkoga podudaranja skeletnih ostataka i rodbine nestalih osoba, no kad je to podudaranje pronađeno nasumičnim pretraživanjem baze podataka, značaj omjera vjerojatnosti nije tako jasan kao u jednostavnome utvrđivanju očinstva i potreban je velik oprez kako ne bi došlo do njegove pogrešne interpretacije. Radi smanjivanja rizika pogrešne identifikacije, osim na rezultate analize DNA, pri utvrđivanju identiteta velik značaj potrebno je dati i drugim tipovima dokaza (poput vremena, lokacije i uvjeta nestanka) te antropološkim i ostalim »klasičnim« forenzičkim dokazima, kao kontrolnome mehanizmu u procesu identifikacije predvođenom analizom DNA

Čimbenici rizika i molekularne predispozicije za displaziju vrata maternice u žena iz istočne Hrvatske

Purpose: The aim of this study was to investigate possible association between high-risk Human papillomavirus (HR HPV) – induced cervical infection, HR HPV-related cervical dysplasia, HR HPV genotypes with two Toll-like receptor (TLR) 9 gene polymorphisms and other risk factors. Methods: During a three-year period, 100 women positive for cervical HR HPV infection (97 with cervical dysplasia and 3 positive women without dysplasia) were genotyped using the Linear Array HPV Genotyping assay (Roche Diagnostics). Furthermore, two polymorphisms of TLR9 (-1486T/C, rs187084 and 2848C/T rs352140) were determined using real-time Polymerase Chain Reaction; 50 HR HPV negative women of similar ethnicity were included as controls. Results: This study showed that infections with HPV 16 in women with cervical dysplasia were more frequently found compared with HPV 18 infections (p=0.0539). Comparison between HR HPV positive and negative women showed significant association between age >35 years (p=0.0058), being unmarried women (p=0.0001), nocondom usage (p=0.0304) and active tobacco smoking (p=0.0376) with HR HPV cervical infection. No significant associations between two TLR9 gene polymorphisms, HR HPV infection and cervical dysplasia were found. Conclusion: Our results indicated that: i) women with cervical dysplasia showed significant higher rate of HR HPV 16 infection compared to HR HPV 18, ii) HR HPV – infection was strongly correlated with social risk factors and iii) TLR9 gene polymorphisms (rs187084; rs352140) did not correlate with HR HPV infection and cervical dysplasia. Further genome-wide association studies could open new frontier in understanding the relationship between polymorphisms at TLR9 and immunological mechanisms in HPV-induced carcinogenesis.Cilj: Cilj ovog istraživanja bio je utvrditi povezanost između infekcije vrata maternice visokorizičnim humanim papilomavirusima (engl. high-risk Human papillomavirus - HR HPV), displazije vrata maternice uzrokovane visokorizičnim genotipovima HPV-a, visokorizičnih genotipova HPV-a te dva polimorfizma gena za Toll-u sličan receptor (TLR) 9 i drugih rizičnih čimbenika. Metode: Tijekom trogodišnjeg razdoblja, u 100 žena s cervikalnom infekcijom visokorizičnim genotipovima HPV-a (97 s displazijom vrata maternice i 3 s pozitivnim nalazom HPV-a ali bez displazije vrata maternice) određeni su genotipovi virusa primjenom molekularnog testa Linear Array HPV Genotyping assay (Roche Diagnostics). Također su analizirana i dva polimorfizma gena za TLR9 (-1486T/C, rs187084 i 2848C/T rs352140) koristeći metodu lančane reakcije polimerazom u stvarnom vremenu; te je 50 žena s negativnim hrHPV ili sličnog etniciteta uključeno kao kontrolna skupina. Rezultati: Istraživanje je pokazalo da su HR HPV infekcije genotipom 16 češće u žena sa displazijom vrata maternice u usporedbi sa ženama s infekcijom genotipa 18 (p=0.0539). Usporedbom žena s pozitivnim i negativnim nalazom na HR HPV, utvrđena je značajna povezanost između dobi >35 godina (p=0.0058), bračnog statusa (slobodne osobe, p=0.0001), nekorištenja kondoma (p=0.0304) i aktivnog pušenja (p=0.0376) i infekcije s HR HPV.“ Povezanost između dva polimorfizma gena za TLR9, infekcije HR HPV i displazije vrata maternice nije dokazana. Zaključak: Rezultati ovog istraživanja pokazali su da: i) je u žena sa displazijom vrata maternice značajno veća učestalost infekcije s genotipom 16 u usporedbi s genotipom 18, ii) infekcija s HR HPV povezana je sa socijalnim faktorima rizika i iii) polimorfizmi gena za TLR9 (rs187084; rs352140) nisu povezani s HR HPV infekcijom i displazijom vrata maternice. Daljnja genomska istraživanja mogu proširiti spoznaje u razumijevanju odnosa između polimorfizma gena za TLR9 i imunoloških mehanizama u HPV-om induciranoj karcinogenezi