Analysis of microRNAs in Small Urinary Extracellular Vesicles and Their Potential Roles in Pathogenesis of Renal ANCA-Associated Vasculitis.

Antineutrophil cytoplasmic antibodies (ANCA)-associated vasculitis (AAV) represents an autoimmunity disease characterized by high mortality. For successful treatment, the detailed knowledge of its complex pathogenesis and the set of biomarkers for differential diagnostics are desired. Analysis of molecular content of small urinary extracellular vesicles (uEV) offers the possibility to find markers in the form of microRNAs (miRNAs) and study the pathways involved in pathogenesis. We used next-generation sequencing in the first preliminary study to detect the miRNAs with altered expression in uEVs of patients with AAV in comparison with age-matched controls. We confirmed the results using single-target quantitative polymerase chain reaction tests on different sets of samples and found five miRNAs (miR-30a-5p, miR-31-3p, miR-99a-5p, miR-106b-5p, miR-182-5p) with highly elevated levels in uEVs of patients. We performed the comparison of their targets with the differentially expressed proteins in uEVs of patients included in the first phase. We realized that upregulated miRNAs and proteins in uEVs in AAV patients target different biological pathways. The only overlap was detected in pathways regulating the actin cytoskeleton assembly and thus potentially affecting the glomerular functions. The associations of upregulated miRNAs with pathways that were neglected as components of complex AAV pathogenesis, e.g., the epidermal growth factor receptor signaling pathway, were found.

Improving structural variant clustering to reduce the negative effect of the breakpoint uncertainty problem.

BACKGROUND: Structural variants (SVs) represent an important source of genetic variation. One of the most critical problems in their detection is breakpoint uncertainty associated with the inability to determine their exact genomic position. Breakpoint uncertainty is a characteristic issue of structural variants detected via short-read sequencing methods and complicates subsequent population analyses. The commonly used heuristic strategy reduces this issue by clustering/merging nearby structural variants of the same type before the data from individual samples are merged. RESULTS: We compared the two most used dissimilarity measures for SV clustering in terms of Mendelian inheritance errors (MIE), kinship prediction, and deviation from Hardy-Weinberg equilibrium. We analyzed the occurrence of Mendelian-inconsistent SV clusters that can be collapsed into one Mendelian-consistent SV as a new measure of dataset consistency. We also developed a new method based on constrained clustering that explicitly identifies these types of clusters. CONCLUSIONS: We found that the dissimilarity measure based on the distance between SVs breakpoints produces slightly better results than the measure based on SVs overlap. This difference is evident in trivial and corrected clustering strategy, but not in constrained clustering strategy. However, constrained clustering strategy provided the best results in all aspects, regardless of the dissimilarity measure used.

Optimization of diagnostic strategy for non-invasive cell-free foetal RHD determination from maternal plasma.

BACKGROUND AND OBJECTIVES: The aim of the study was to optimize routine non-invasive prenatal detection of fetal RHD gene from plasma of RhD-negative pregnant women (the median of gestational age was 25 weeks, range 10-38) to detect RhD materno-fetal incompatibility and to avoid the redundant immunoprophylaxis. MATERIALS AND METHODS: Initially only one exon of RHD gene (exon 10) was investigated in 281 plasma samples (144 verified after delivery), in the second phase three RHD exons (5, 7, 10) were analyzed in 246 samples of plasma and maternal genomic DNA (204 verified) by real-time PCR method. Detection of Y-chromosomal sequence DYS-14 and five X-chromosomal insertion/deletion polymorphisms was used to confirm the fetal cfDNA detectability in plasma. Specific polymorphisms in RHD gene were detected by sequence-specific primer PCR in nine samples. RESULTS: When only the RHD exon 10 was tested, 2·8% of verified samples were false positive and 3·5% false negative. With three RHD exons (5, 7, 10) and maternal genomic DNA testing, only one case was false negative (0·5%). Nine samples were inconclusive due to RHD-positive results in maternal genomic DNA. These samples were analyzed for specific mutations in RHD gene. Combination of both methods for fetal cfDNA verification succeeded in 75% of tested group. CONCLUSION: Implementation of analysis of three RHD exons and maternal genomic DNA to routine practice lowers dramatically the ratio of false positive and negative results. This method enables more accurate determination of fetal RHD status with the reduction of unnecessary medical care and RhD immunoprophylaxis.

Detection of cell-free foetal DNA fraction in female-foetus bearing pregnancies using X-chromosomal insertion/deletion polymorphisms examined by digital droplet PCR.

In families with X-linked recessive diseases, foetal sex is determined prenatally by detection of Y-chromosomal sequences in cell-free foetal DNA (cffDNA) in maternal plasma. The same procedure is used to confirm the cffDNA presence during non-invasive prenatal RhD incompatibility testing but there are no generally accepted markers for the detection of cffDNA fraction in female-foetus bearing pregnancies. We present a methodology allowing the detection of paternal X-chromosomal alleles on maternal background and the confirmation of female sex of the foetus by positive amplification signals. Using digital droplet PCR (ddPCR) we examined X-chromosomal INDEL (insertion/deletion) polymorphisms: rs2307932, rs16397, rs16637, rs3048996, rs16680 in buccal swabs of 50 females to obtain the population data. For all INDELs, we determined the limits of detection for each ddPCR assay. We examined the cffDNA from 63 pregnant women bearing Y-chromosome negative foetuses. The analysis with this set of INDELs led to informative results in 66.67% of examined female-foetus bearing pregnancies. Although the population data predicted higher informativity (74%) we provided the proof of principle of this methodology. We successfully applied this methodology in prenatal diagnostics in a family with Wiscott-Aldrich syndrome and in pregnancies tested for the risk of RhD incompatibility.

Genome-wide miRNA profiling in plasma of pregnant women with down syndrome fetuses.

Down syndrome (DS) is one of the most common causes of intellectual disability and new approaches allowing its rapid and effective prenatal detection are being explored. In this study, we investigated the diagnostic potential of plasma microRNAs (miRNAs). This study builds upon our previous study in DS placentas, where seven miRNAs were found to be significantly up-regulated. A total of 70 first-trimester plasma samples from pregnant women were included in the present study (35 samples with DS fetuses; 35 with euploid fetuses). Genome-wide miRNA profiling was performed in the pilot study using Affymetrix GeneChip™ miRNA 4.1 Array Strips (18 samples). Selected miRNAs were then analysed in the validation study using quantitative reverse transcription PCR (RT-qPCR; 52 samples). Based on the current pilot study results (12 miRNAs), our previous research on chorionic villi samples (7 miRNAs) and the literature (4 miRNAs), a group of 23 miRNAs was selected for the validation study. Although the results of the pilot study were promising, the validation study using the more sensitive RT-qPCR technique and a larger group of samples revealed no significant differences in miRNA profiles between the compared groups. Our results suggest that testing of the first-trimester plasma miRNAs is probably not suitable for non-invasive prenatal testing (NIPT). Different results could be theoretically achieved at later gestational ages; however, such a result probably would have limited use in clinical practice.