Snježana Džijan

Non-invasive prenatal paternity testing from maternal blood

Prenatal paternity analysis can be performed only after invasive sampling of chorionic villi or amnionic fluid. Aiming to enable noninvasive paternity testing, we attempted to amplify fetal alleles from maternal plasma. Cell-free DNA was isolated from plasma of 20 pregnant women and amplified with ampFLSTR Identifiler and ampFLSTR Yfiler kits. Unfortunately, autosomal fetal alleles were heavily suppressed by maternal DNA, and the only locus that was reliably amplified with AmpFLSTR Identifiler kit was amelogenin, which revealed only fetal gender. Much better success was obtained with AmpFLSTR Yfiler kit, which, in the case of male fetuses, successfully amplified between six and 16 fetal loci. All amplified fetal alleles matched the alleles of their putative fathers, confirming the tested paternity. To the best of our knowledge, this is a first report of noninvasive prenatal paternity testing.

Analysis of multiple loci can increase reliability of detection of fetal Y chromosome DNA in maternal plasma

Aiming to develop more reliable methods for determination of fetal gender from maternal plasma we compared three different systems of polymerase chain reaction (PCR) detection of Y‐chromosome DNA.

Population Substructure Can Significantly Affect Reliability of a DNA‐led Process of Identification of Mass Fatality Victims

Abstract:  Aiming to evaluate the effects of population substructure on the reliability of a DNA correspondence in the process of human identification, we used the model of “in silico” constructed populations with and without substructure. Effects of population substructure were evaluated at the level of locus heterozygosity, Hardy–Weinberg equilibrium and mini‐haplotype distribution. Inbreeding in a subpopulation of 100 individuals through 10 generations did not significantly alter the level of heterozygosity and Hardy–Weinberg equilibrium. However, analysis of mini‐haplotype distribution revealed a significant homogenization in separated subpopulations. Average observed mini‐haplotype frequency (fo) increased to threefold from expected values (fe), and the number of mini‐haplotypes with fo/fe above 10 increased over sixfold, suggesting that the effects of population substructure on calculated likelihood ratios (LR) might be larger than previously estimated. In most criminal cases, this would not represent a problem, whereas for identifications in large‐scale mass fatality events, population substructure might considerably increase the risk of false identification.

Evaluation of the Reliability of DNA Typing in the Process of Identification of War Victims in Croatia

Abstract:  Aiming to estimate the frequency of various types of errors that can occur in the large‐scale process of identification, we identified and compared genotypes of 911 parent–child pairs in the database of 3498 relatives of people that disappeared during the 1991/1992 war in Croatia. Genotypes of 891 pairs (97.8%) were matching, while 20 pairs did not match in one or more loci. Reanalysis of these samples revealed that out of 1822 analyzed genotypes, one genotype was completely wrong, and two genotypes had one wrong allele because of human errors. Five genotypes had a single wrong allele due to either polymerase chain reaction or electrophoresis errors. In five genotypes mutations were the cause of mismatch. Genetic inconsistencies with parentage were found in four “fathers” (4.2%) and three “mothers” (0.36%). As the majority of observed single‐locus errors were caused by nonhuman errors, all databases produced with similar technology would probably have comparable level of errors.

Most common HCV genotypes in patients from north-eastern Croatia.

OBJECTIVE The aims of this study were to determine the HCV-RNA viral load, genotype distribution, risk factors and symptoms of HCVRNA positive viral load in HCV antibody-positive patients from north-eastern Croatia. MATERIALS AND METHODS From January 2009 to December 2011, 203 HCV antibody- positive patients (130 men and 73 women; median age 44.5 years) were analyzed for HCV-RNA by the COBAS TaqMan HCV test and genotyped by the Linear Array HCV Genotyping test (both from Roche). All patients completed a structured questionnaire about risk factors and symptoms. RESULTS The HCV-RNA percentage was 61.1% and was similar for men and women. The HCV-RNA viral load increased with age: while 55% of 20-50 year old patients were HCV-RNA positive, 73% of patients >50 years were positive (p=0.021). Genotype 1 was the most prevalent genotype (79.8%), followed by 3 (12.9%), 4 (6.5%), and 2 (0.8%); genotypes 5 and 6 were not determined. Patients with genotype 1 (median, 50 years) were older than patients with 3 (median, 33.5 years) or 4 (median, 38 years). The blood transfusions performed in Croatian hospitals before 1993 was significantly associated with HCV-RNA positive viral load (p<0.05). CONCLUSION These data indicated an elevated prevalence of genotype 1 in elderly HCV-RNA positive patients and it may continue to rise. Using RNA-based detection in HCV positive-antibody patients would allow early detection of HCV in the acute stage of HCV disease and the increased risk of HCV genotyperelated treatment failure.

Distribution of Chlamydia trachomatis Serotypes in Clinical Urogenital Samples from North-Eastern Croatia

The purpose of this study was to determine prevalence of Chlamydia trachomatis (Ct) urogenital infection and its serotype distribution from clinical samples in north-eastern Croatia. During a 3-year period, 2,379 urogenital samples were analyzed by real-time polymerase chain reaction (A group), while 4,846 genital swabs were analyzed by direct fluorescent antibody test (B group). 132 Ct positive specimens were genotyped by omp1 gene sequencing. The prevalence rate of Ct was 3.2 % in A and 1 % in B group. The most prevalent chlamydial genotype was E (44 %), followed by F (33 %), K (11.5 %), G (8 %), J/UW (5.3 %), D-IC (4.4 %), D-B120 (1.8 %), and B/IU, J/IU, Ia/IU (0.9 % each) serotypes. Single-nucleotide polymorphisms (SNPs) of omp1 gene were detected in E, K, and G serotypes. Some of these SNPs (C/T at position 272 and G/A at position 813 in E strain; C/T at position 884 in D strain) might represent novel omp1 variants.