Ales Horinek

Cell-Free Plasma DNA during Peritoneal Dialysis and Hemodialysis and in Patients with Chronic Kidney Disease

The mechanisms of clearance of circulating plasma DNA are not fully understood, and so we aimed to examine it in patients with impaired renal function compared with healthy individuals. We also assessed the effect of peritoneal dialysis and hemodialysis on circulating plasma cell‐free DNA (cfDNA) in our treated patients. Overall, 20 healthy volunteers, 20 patients with chronic kidney disease (CKD), 18 patients undergoing peritoneal dialysis (PD), and 17 patients on hemodialysis (HD; high‐flux polysulfone membrane) were examined. Cell‐free DNA levels were determined using real‐time GADPH gene sequence amplification. The levels of cfDNA in all groups of our patients did not differ significantly from those of healthy volunteers. In HD patients, cfDNA levels were significantly increased compared with those of CKD patients (P < 0.05) and PD‐treated patients (P < 0.01). In PD‐treated patients, cfDNA was detectable in overnight effluent, with its levels correlating inversely with the duration of PD treatment (r=−0.619, Spearmans coefficient, P= 0.008). Factors contributing to these differences may include changes in the quality and quantity of the cell population of the peritoneum, highlighting the need for additional studies clarifying the dynamics of cfDNA during PD. The plasma levels of cfDNA do not seem to be dramatically altered even in CKD patients or those on PD or HD (as long as they are measured prior to the procedure in the latter two). Our data suggest renal elimination is not the main mechanism of circulating cfDNA clearance.

Application of the new insertion–deletion polymorphism kit for forensic identification and parentage testing on the Czech population

Insertion-deletion polymorphisms (INDELs) are diallelic markers derived from a single mutation event. Their low mutation frequency makes them suitable for forensic and parentage testing. The examination of INDELs thus combines advantages of both short tandem repeats (STR) and single nucleotide polymorphisms (SNP). This type of polymorphisms may be examined using as small amplicon size as SNP (about 100xa0bp) but could be analyzed by techniques used for routine STR analysis. For our population study, we genotyped 55 unrelated Czech individuals. We also genotyped 11 trios to analyze DIPplex Kit (QIAGEN, Germany) suitability for parentage testing. DIPplex Kit contains 30 diallelic autosomal markers. INDELs in DIPplex Kit were tested with linkage disequilibrium test, which showed that they could be treated as independent markers. All 30 loci fulfill Hardy–Weinberg equilibrium. There were several significant differences between Czech and African populations, but no significant ones within European population. Probability of a match in the Czech population was 1 in 6.8u2009×u20091012; combined power of discrimination was 99.9999999999%. Average paternity index was 1.13–1.77 for each locus; combined paternity index reached about 27,000 for a set of 30 loci. We can conclude that DIPplex kit is useful as an additional panel of markers in paternity cases when mutations in STR polymorphisms are present. For application on degraded or inhibited samples, further optimization of buffer and primer concentrations is needed.

Investigator® Argus X‐12 study on the population of Czech Republic: Comparison of linked and unlinked X‐STRs for kinship analysis

DNA samples of 523 unrelated anonymized individuals (307 males and 216 females) born and living in the Czech Republic were genotyped using Investigator® Argus X‐12 system in the following loci localized in four linkage groups: DXS10148, DXS10135, DXS8378, DXS7132, DXS10079, DXS10074, DXS10103, HPRTB, DXS10101, DXS10146, DXS10134, DXS742. Haplotype frequencies were calculated for each LG (Linkage Group). The frequency of most common haplotype was 0.016, 0.036, 0.042, and 0.023 for LG1, LG2, LG3, and LG4, respectively. The combined power of discrimination was more than 0.999999999 both for female and male samples. The mean exclusion chance was 0.99999999 (trios) and 0.999999 (duos). Informativity and suitability of Investigator® Argus X‐12 for kinship determination was assessed by computing in several female–female duos using LR (Likelihood Ratio) determination for autosomal STR (PowerPlex ESI‐17), linked (Investigator® Argus X‐12 system), and unlinked (X‐STR Decaplex) X‐STR kits. Investigator® Argus X‐12 proved to be very useful for sibship determination, since its LR values were relatively similar to LR for autosomal STR kit. This work presents the first population data for Investigator® Argus X‐12 system in the Czech Republic.

Cell-Free Fetal DNA in Maternal Plasma during Physiological Single Male Pregnancies: Methodology Issues and Kinetics

Objective: To analyze methodological influences and characterize the concentrations of cell-free fetal DNA (cffDNA) circulating in maternal plasma at different gestational ages in physiological pregnancies. Methods: We investigated 238 independent samples from single male-bearing pregnancies of different gestation age. In the other 50 pregnancies, the samples were collected three times during pregnancy (at all trimesters) to evaluate the kinetics of cffDNA. The manual and automated DNA extraction methods (Roche) were compared. cffDNA was amplified using real-time PCR method and Y-specific sequences SRY and DYS14. Total cell-free DNA circulating in maternal plasma was determined by the use of the GADPH sequence. Results: The elevation in the concentration of cffDNA during pregnancy with the highest value in the third trimester was observed independently on the DNA extraction method and on the Y-specific amplified sequence. The same is documented for the percentage of fetal DNA in total cell-free DNA in maternal plasma. It increases also in successive trimesters (8.3, 10.7 and 23.2%). Conclusions: We discuss methodological problems and describe statistical parameters of cffDNA concentrations in maternal plasma during pregnancy as the basic information for comparison with pregnancies having a pathological outcome.

Identification of six novel mutations in ZEB1 and description of the associated phenotypes in patients with posterior polymorphous corneal dystrophy 3.

Posterior polymorphous corneal dystrophy 3 (PPCD3) is a rare autosomal dominant disorder caused by mutations in ZEB1. To date all identified disease‐causing variants were unique to the studied families, except for c.1576dup. We have detected six novel ZEB1 mutations; c.1749_1750del; p.(Pro584*) and c.1717_1718del; p.(Val573Phefs*12) in two Czech families, c.1176dup; p.(Ala393Serfs*19), c.1100C>A; p.(Ser367*), c.627del; p.(Phe209Leufs*11) in three British families and a splice site mutation, c.685–2A>G, in a patient of Sri Lankan origin. An additional British proband had the c.1576dup; p.(Val526Glyfs*3) mutation previously reported in other populations. Clinical findings were variable and included bilateral congenital corneal opacity in one proband, development of opacity before the age of 2 years in another individual and bilateral iris flocculi in yet another subject. The majority of eyes examined by corneal topography (10 out of 16) had an abnormally steep cornea (flat keratometry 46.5–52.7 diopters, steep keratometry 48.1–54.0 diopters). One proband underwent surgery for cryptorchidism. Our study further demonstrates that PPCD3 can present as corneal edema in early childhood, and that an abnormally steep keratometry is a common feature of this condition. As cryptorchidism has been previously observed in two other PPCD3 cases, its association with the disease warrants further investigation.

Lateralization of 17Beta-Hydroxysteroid Dehydrogenase Type 10 in Hippocampi of Demented and Psychotic People

Objective: The multifunctional mitochondrial enzyme 17β-hydroxysteroid dehydrogenase type 10 could play a role in the development of Alzheimer disease via its high-affinity binding to amyloid-β peptides and its overexpression. Methods: We evaluated the specificity of alterations in mRNA/enzyme expression levels in human right and left hippocampi. Results: We observed a trend towards right/left laterality in nondemented nonpsychotic controls; however, the degree of asymmetry was higher for mRNA when compared to enzyme expression levels. In Alzheimer disease and schizophrenia, significant shifts to left/right asymmetry were found and the changes were associated with more marked increases in mRNA/enzyme expression in the left hemisphere. On the other hand, no alterations were observed in people with multi-infarct dementia. Conclusion: Our results support studies reporting an impairment of mitochondria in Alzheimer disease or schizophrenia and a higher vulnerability of the dominant hemisphere to pathological processes. Overexpression of the enzyme could be used to distinguish Alzheimer disease from multi-infarct dementia.

The impact of standard chemotherapy on miRNA signature in plasma in AML patients

AIMnIn our pilot study, we used plasma samples as liquid biopsy to search for miRNA signatures in patients with acute myeloid leukemia (AML) at diagnosis and in remission achieved after standard chemotherapy before planned transplantation.nnnMATERIAL AND METHODSnWe examined 10 plasma samples from healthy volunteers and 8 paired samples from patients with AML at diagnosis and in remission using TaqMan MicroRNA Arrays. The results were validated using single-target qPCR reactions run in triplicates.nnnRESULTSnWe selected 6 miRNAs with expressions significantly sensitive to therapy: miR-199b-5p, miR-301b, miR-326, miR-361-5p, miR-625 and miR-655. All selected miRNAs were not or very weakly expressed in healthy individuals. They were abundant in plasma in patients at diagnosis but their levels decreased after chemotherapy.nnnCONCLUSIONnWe detected a therapy sensitive miRNA signature in plasma of patients with AML.

Cell-free nucleic acids as biomarkers in dialyzed patients

In this review, we discuss the origin, possible biological meaning, quantitative and qualitative changes in the concentrations of cell-free nucleic acids in human circulation with regard to renal failure and the process of dialysis. We focus on the inflammatory response and apoptosis known to be in close relationship not only with hemodialysis but also with different comorbidities frequently detected in hemodialyzed patients. u2029Hemodialysis itself is able to promote the changes in the quantity and quality of circulating nucleic acid pool, but large spectrum of comorbidities in hemodialyzed subjects can further complicate the interpretations of results of cell-free nucleic acid analysis. Such analysis can provide additional information about the patient prognosis and monitor some aspects of comorbidity development. Recently, it has been shown that the analysis of cell-free nucleic acids may also be worthy in patients on peritoneal dialysis because the cell-free nucleic acids may also be detected in overnight effluents and their examination can be informative with regard to the functional state of the patients peritoneum. u2029We summarize what is recently known about the use of cell-free nucleic acids as biomarkers in patients with renal failure not only in hemodialysis but also in peritoneal dialysis and we describe the future perspectives in this field.

Alterations in methylation status of immune response genes promoters in cell-free DNA during a hemodialysis procedure

Objective: Elevations of cell-free DNA (cfDNA) concentrations during hemodialysis (HD) sessions were reported in numerous studies regardless of an applied therapeutic protocol. It is generally thought that the elevated concentrations represent the consequence of apoptosis on the dialysis membranes. No data concerning the qualitative characteristics of cfDNAs in HD patients have been published till today; therefore, we focus on the promoter methylation status of genes involved in immune response. Research design and methods: We isolated cfDNA from randomly selected patients before and after a HD session and from healthy subjects examined two times per day with 4 h interval. The extent of promoter methylation of 24 genes involved in immune response was examined using the ‘EpiTect Methyl qPCR Array Inflammatory Response and Autoimmunity’. Results: In our pilot study, we discovered significant changes in methylation patterns of genes IL-7, IL-13, IL-17C and TYK2 between HD patients and healthy subjects. Conclusion: Methylation of immune response genes promoters may be detected using EpiTect Methyl qPCR Array Inflammatory Response and Autoimmunity at the level of cfDNA to provide the information about the actual state of immune response in HD patients.

Urinary Cell-Free DNA Quantification as Non-Invasive Biomarker in Patients with Bladder Cancer

Introduction: Concentration of urinary cell-free DNA (ucfDNA) belongs to potential bladder cancer markers, but the reported results are inconsistent due to the use of various non-standardised methodologies. The aim of the study was to standardise the methodology for ucfDNA quantification as a potential non-invasive tumour biomarker. Material and Methods: In total, 66 patients and 34 controls were enrolled into the study. Volumes of each urine portion (V) were recorded and ucfDNA concentrations (c) were measured using real-time PCR. Total amounts (TA) of ucfDNA were calculated and compared between patients and controls. Diagnostic accuracy of the TA of ucfDNA was determined. Results: The calculation of TA of ucfDNA in the second urine portion was the most appropriate approach to ucfDNA quantification, as there was logarithmic dependence between the volume and the concentration of a urine portion (p = 0.0001). Using this methodology, we were able to discriminate between bladder cancer patients and subjects without bladder tumours (p = 0.0002) with area under the ROC curve of 0.725. Positive and negative predictive value of the test was 90 and 45%, respectively. Conclusion: Quantification of ucf DNA according to our modified method could provide a potential non-invasive biomarker for diagnosis of patients with bladder cancer.