Jasenka Wagner

Stability of N-glycan profiles in human plasma

Glycan heterogeneity was shown to be associated with numerous diseases and glycan analysis has a great diagnostic potential. Recently, we reported high biological variability of human plasma N-glycome at the level of population. The observed variations were larger than changes reported to be associated with some diseases; thus, it was of great importance to examine the temporal constancy of human N-glycome before glycosylation changes could be routinely analyzed in diagnostic laboratories. Plasma samples were taken from 12 healthy individuals. The blood was drawn on seven occasions during 5 days. N-Linked glycans, released from plasma proteins, were separated using hydrophilic interaction high-performance liquid chromatography into 16 groups (GP1-GP16) and quantified. The results showed very small variation in all glycan groups, indicating very good temporal stability of N-glycome in a single individual. Coefficients of variation from 1.6% for GP8 to 11.4% for GP1 were observed. The average coefficient of variation was 5.6%. These variations were comparable to those observed when analytical procedure was tested for its precision. Good stability of plasma N-glycome in healthy individuals implies that glycosylation is under significant genetic control. Changes observed in glycan profiles are consequence of environmental influences and physiologic responses and therefore have a significant diagnostic potential.

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.

Free serum DNA is an early predictor of severity in acute pancreatitis

OBJECTIVES Cell-free DNA has been investigated as a diagnostic marker in many diseases, including acute conditions. Our hypothesis was that in acute pancreatitis free serum DNA correlates with the extent of pancreatic necrosis and that it may be an early marker of severity. DESIGN AND METHODS Free DNA was measured in sera from 30 patients with acute pancreatitis at admission, on the first, fourth and seventh day following admission. RESULTS On the first day following admission patients who would develop severe pancreatitis had significantly higher serum DNA levels than those with mild disease (median 0.271 ng/microL vs. 0.059 ng/microL respectively; P<0.001). This parameter showed very good characteristics as a potential severity predictor (area under ROC curve 0.97). Free serum DNA was in correlation with the extent of pancreatic necrosis. CONCLUSIONS Free serum DNA correlates with the extent of pancreatic necrosis and is a potential early marker of severe acute pancreatitis.

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.

Down syndrome: parental origin, recombination, and maternal age.

The aims of the present study were to assess (1) the parental origin of trisomy 21 and the stage in which nondisjunction occurs and (2) the relationship between altered genetic recombination and maternal age as risk factors for trisomy 21. The study included 102 cases with Down syndrome from the Croatian population. Genotyping analyses were performed by polymerase chain reaction using 11 short tandem repeat markers along chromosome 21q. The vast majority of trisomy 21 was of maternal origin (93%), followed by paternal (5%) and mitotic origin (2%). The frequencies of maternal meiotic I (MI) and meiotic II errors were 86% and 14%, respectively. The highest proportion of cases with zero recombination was observed among those with maternal MI derived trisomy 21. A higher proportion of telomeric exchanges were presented in cases with maternal MI errors and cases with young mothers, although these findings were not statistically significant. The present study is the first report examining parental origin and altered genetic recombination as a risk factor for trisomy 21 in a Croatian population. The results support that trisomy 21 has a universal genetic etiology across different human populations.

The relationship between interferon-γ gene polymorphism and acute kidney allograft rejection.

Cytokine gene polymorphisms have been associated with modified gene expression and cytokine production. Gamma interferon (IFN‐γ) plays an important role in the pathogenesis of kidney transplant rejection. This study evaluated the association between IFN‐γ gene polymorphisms and the history of acute allograft rejection in 53 adult first‐transplant recipients receiving cadaveric kidney grafts. They were followed up in a single centre until 2006, for a median time of 4 years after transplantation (1–22 years). IFN‐γ gene polymorphisms +874 T/A (rs2430561) were determined by polymerase chain reaction (PCR). T/T high IFN‐γ genotype was found in 12, intermediate T/A in 29 and low A/A in 12 patients. Twenty‐six acute kidney rejection episodes were evidenced in 20 patients, of which none occurred in the 12 patients with low IFN‐γ genotype A/A. Age, gender, number of HLA (human leukocyte antigen) mismatches, ABO blood groups, HLA, time after transplantation, creatinine clearance and immunosuppressive regimens were excluded as confounding factors associated with IFN‐γ genotype distribution between rejectors and non‐rejectors. IFN‐γ gene polymorphisms could be an important risk factor for acute kidney transplant rejection, whereas the low A/A IFN‐γ genotype could be protective against rejection.

Postoperative immunosuppression markers and the occurrence of sepsis in patients with benign and malignant disease

SummaryAimTo investigate associations between the postoperative immune response and the levels of extracellular circulating DNA (cDNA), C-reactive protein (CRP), neutrophil/lymphocyte (N/L) ratio, and regulatory T (Treg) cells in the peripheral blood and their role as potential predictors of postoperative septic complications.MethodsThis was a prospective observational study involving 115 adult patients who underwent elective surgery. Patients were divided into three groups: with benign disease, with malignant disease, and with malignant disease and administration of dexamethasone. Serum CRP levels, N/L ratio, monocyte human leukocyte antigen-DR (HLA-DR) expression, proportion of Treg cells, and cDNA levels were measured at different time points before and after surgery.ResultsAll patients had increased CRP levels after surgery. Septic patients had higher serum CRP levels at baseline. Compared with the other groups, the dexamethasone group had significantly higher CRP levels before and after surgery, a significantly higher N/L ratio before surgery, a significantly lower rise in the N/L ratio after surgery, and a significantly lower HLA-DR expression at baseline, which remained stable after surgery. In the malignant-disease group, we observed a significant postoperative decrease in the HLA-DR expression.ConclusionsOur results suggest that the immunosuppressive effect of surgery and the presence of a malignant disease may contribute to a higher risk of postoperative sepsis. Preoperative CRP levels may be a reliable predictor of sepsis in oncological patients.ZusammenfassungZielder Studie war es, Zusammenhänge zwischen der postoperativen Immunantwort und den Konzentrationen der zirkulierenden extrazellulären DNA (cDNA), des CRPs, des Quotienten Neutrophile/Lymphozyten (N/L) und der regulatorischen T (Treg) Zellen im peripheren Blut sowie deren Rolle als mögliche Vorhersager von postoperativen septischen Komplikationen zu untersuchen.MethodenIn diese prospektive Beobachtungsstudie haben wir 115 erwachsene Patienten, die einer elektiven Operation unterzogen wurden, eingeschlossen. Die Patienten wurden in 3 Gruppen eingeteilt: eine mit benigner Erkrankung, eine mit maligner Erkrankung und eine mit maligner Erkrankung und Gabe von Dexamethason. Die Serum-Konzentrationen des CRPs, der N/L Quotienten, der HLA-DR Expression der Monozyten, des Anteils der Treg Zellen und die Konzentrationen der cDNA wurden zu verschiedenen Zeitpunkten vor und nach der Operation gemessen.ErgebnisseAlle Patienten hatten nach der Operation erhöhte CRP Konzentrationen. Bei den Patienten mit postoperativer Sepsis waren die CRP Ausgangswerte höher. Im Vergleich mit den anderen Gruppen hatte die Gruppe der Patienten mit Dexamethason signifikant höhere CRP Werte vor und nach der Operation. Außerdem waren die N/L Quotienten präoperativ vergleichsweise signifikant erhöht, während postoperativ bei dieser Gruppe ein signifikant niedrigerer Anstieg der N/L Quotienten beobachtet wurde. Die Ausgangswerte der HLA-DR Expression waren bei diesen Patienten signifikant erniedrigt und blieben postoperativ stabil. Bei der Gruppe der Patienten mit maligner Erkrankung beobachteten wir postoperativ einen signifikanten Abfall der HLA-DR Expression.SchlussfolgerungenUnsere Ergebnisse lassen vermuten, dass der immunsuppressive Effekt einer Operation und das Vorliegen einer malignen Erkrankung zu einem erhöhten Risiko für das Auftreten einer postoperativen Sepsis beitragen können. Präoperative CRP Werte scheinen einen verlässlichen Vorhersagewert bezüglich Sepsis bei onkologischen Patienten zu haben.

Genetic variation in leptin and leptin receptor genes is a risk factor for idiopathic recurrent spontaneous abortion

Aim To determine whether maternal leptin (LEP) and leptin receptor (LEPR) gene polymorphisms are associated with idiopathic recurrent spontaneous abortion (IRSA). Methods This case-control association study conducted from 2010 to 2012 at the Department of Gynecology and Obstetrics, University Hospital Center Osijek and Clinical Institute of Medical Genetics Ljubljana included 178 women with a history of three or more IRSAs before the 22nd week of gestation and 145 women with at least two live births and no history of pathologic pregnancies during reproductive period. Polymorphisms of maternal LEP (rs7799039, rs2122627, rs11761556, rs10244329) and LEPR (rs1137101, rs7516341, rs1186403, rs12062820) were assessed by allele specific real-time polymerase chain reaction. Genotype distribution, allele frequencies, and frequency of haplotypes at LEP and LEPR genetic loci were determined. Results We observed more frequent genotype for rs7516341 (nominal P = 0.034, odds ratio [OR] 0.61, 95% confidence interval [CI] 0.38-0.97) and rs1137101 (nominal P = 0.048, OR 1.66, 95% CI 1.00-2.80) in the LEPR gene in patients than in controls, but these results did not remain significant after correction for multiple testing according to Bonferroni (adjusted P value threshold was set at 0.05). We did not observe differential distribution of genotype frequencies in the LEP gene between cases and controls. In patients with IRSA, GTCC haplotype in the LEPR gene locus was significantly less frequent than in controls (P = 0.00865, OR 0.45), contrary to ACTC haplotype (P = 0.0087, OR 1.98). Conclusions We showed that genetic variability in the LEPR gene was associated with IRSA, warranting confirmation on a greater number of patients and pathogenesis investigation.