Mitochondrial DNA copy number changes, heteroplasmy, and mutations in plasma-derived exosomes and brain tissue of glioblastoma patients.

Glioblastoma is the most common malignant tumor of the central nervous system (CNS) in adults. Glioblastoma cells show increased glucose consumption associated with poor prognosis. Since mitochondria play a crucial role in energy metabolism, mutations and copy number changes of mitochondrial DNA may serve as biomarkers. As the brain is difficult to access, analysis of mitochondria directly from the brain tissue represents a challenge. Exosome analysis is an alternative (still poorly explored) approach to investigate molecular changes in CNS tumors. We analyzed brain tissue DNA and plasma-derived exosomal DNA (exoDNA) of 44 glioblastoma patients and 40 control individuals. Quantitative real-time PCR was performed to determine mtDNA copy numbers and the Kruskal-Wallis and Mann-Whitney U test were used for statistical analysis of data. Subsequently, sequencing libraries were prepared and sequenced on the MiSeq platform to identify mtDNA point mutations. Tissue mtDNA copy number was different among controls and patients in multiple comparisons. A similar tendency was detected in exosomes. Based on NGS analysis, several mtDNA point mutations showed slightly different frequencies between cases and controls, but the clinical relevance of these observations is difficult to assess and likely less than that of overall mtDNA copy number changes. Allele frequencies of variants were used to determine the level of heteroplasmy (found to be higher in exo-mtDNA of control individuals). Despite the suggested potential, the use of such biomarkers for the screening and/or diagnosis of glioblastomas is still limited, thus further studies are needed.

Repression of a large number of genes requires interplay between homologous recombination and HIRA.

During homologous recombination, Dbl2 protein is required for localisation of Fbh1, an F-box helicase that efficiently dismantles Rad51-DNA filaments. RNA-seq analysis of dbl2Δ transcriptome showed that the dbl2 deletion results in upregulation of more than 500 loci in Schizosaccharomyces pombe. Compared with the loci with no change in expression, the misregulated loci in dbl2Δ are closer to long terminal and long tandem repeats. Furthermore, the misregulated loci overlap with antisense transcripts, retrotransposons, meiotic genes and genes located in subtelomeric regions. A comparison of the expression profiles revealed that Dbl2 represses the same type of genes as the HIRA histone chaperone complex. Although dbl2 deletion does not alleviate centromeric or telomeric silencing, it suppresses the silencing defect at the outer centromere caused by deletion of hip1 and slm9 genes encoding subunits of the HIRA complex. Moreover, our analyses revealed that cells lacking dbl2 show a slight increase of nucleosomes at transcription start sites and increased levels of methylated histone H3 (H3K9me2) at centromeres, subtelomeres, rDNA regions and long terminal repeats. Finally, we show that other proteins involved in homologous recombination, such as Fbh1, Rad51, Mus81 and Rad54, participate in the same gene repression pathway.

Technical and Methodological Aspects of Cell-Free Nucleic Acids Analyzes.

Analyzes of cell-free nucleic acids (cfNAs) have shown huge potential in many biomedical applications, gradually entering several fields of research and everyday clinical care. Many biological properties of cfNAs can be informative to gain deeper insights into the function of the organism, such as their different types (DNA, RNAs) and subtypes (gDNA, mtDNA, bacterial DNA, miRNAs, etc.), forms (naked or vesicle bound NAs), fragmentation profiles, sequence composition, epigenetic modifications, and many others. On the other hand, the workflows of their analyzes comprise many important steps, from sample collection, storage and transportation, through extraction and laboratory analysis, up to bioinformatic analyzes and statistical evaluations, where each of these steps has the potential to affect the outcome and informational value of the performed analyzes. There are, however, no universal or standard protocols on how to exactly proceed when analyzing different cfNAs for different applications, at least according to our best knowledge. We decided therefore to prepare an overview of the available literature and products commercialized for cfNAs processing, in an attempt to summarize the benefits and limitations of the currently available approaches, devices, consumables, and protocols, together with various factors influencing the workflow, its processes, and outcomes.

Non-invasive prenatal testing (NIPT) by low coverage genomic sequencing: Detection limits of screened chromosomal microdeletions.

To study the detection limits of chromosomal microaberrations in non-invasive prenatal testing with aim for five target microdeletion syndromes, including DiGeorge, Prader-Willi/Angelman, 1p36, Cri-Du-Chat, and Wolf-Hirschhorn syndromes. We used known cases of pathogenic deletions from ISCA database to specifically define regions critical for the target syndromes. Our approach to detect microdeletions, from whole genome sequencing data, is based on sample normalization and read counting for individual bins. We performed both an in-silico study using artificially created data sets and a laboratory test on mixed DNA samples, with known microdeletions, to assess the sensitivity of prediction for varying fetal fractions, deletion lengths, and sequencing read counts. The in-silico study showed sensitivity of 79.3% for 10% fetal fraction with 20M read count, which further increased to 98.4% if we searched only for deletions longer than 3Mb. The test on laboratory-prepared mixed samples was in agreement with in-silico results, while we were able to correctly detect 24 out of 29 control samples. Our results suggest that it is possible to incorporate microaberration detection into basic NIPT as part of the offered screening/diagnostics procedure, however, accuracy and reliability depends on several specific factors.

Non-invasive prenatal testing as a valuable source of population specific allelic frequencies.

Low-coverage massively parallel genome sequencing for non-invasive prenatal testing (NIPT) of common aneuploidies is one of the most rapidly adopted and relatively low-cost DNA tests. Since aggregation of reads from a large number of samples allows overcoming the problems of extremely low coverage of individual samples, we describe the possible re-use of the data generated during NIPT testing for genome scale population specific frequency determination of small DNA variants, requiring no additional costs except of those for the NIPT test itself. We applied our method to a data set comprising of 1501 original NIPT test results and evaluated the findings on different levels, from in silico population frequency comparisons up to wet lab validation analyses using a gold-standard method based on Sanger sequencing. The revealed high reliability of variant calling and allelic frequency determinations suggest that these NIPT data could serve as valuable alternatives to large scale population studies even for smaller countries around the world.

Ultracentrifugation enrichment protocol followed by total RNA sequencing allows assembly of the complete mitochondrial genome.

The mitochondrial genome is an independent genetic system in each eukaryotic cell outside the nuclear genome. Mitochondrial DNA (mtDNA) appears in high copy number within one cell, unlike nuclear DNA, which exists in two copies. But nevertheless, mtDNA represent only small part of total cellular DNA what causes problematic analysis and identification of relevant mutations. While most researchers tend to overlook it because of its small size, the mitochondrial genome contains genes that are essential for cellular energetics and survival. Because of the increased awareness on the importance of metabolism and bioenergetics in a wide variety of human diseases, more and more mtDNA studies were performed. Mitochondrial genome research has established the connection between mtDNA and a wide variety of diseases such as cancer or neurodegenerative disorders. At the present time, several methods are known, that allow sequencing of mtDNA. However, genomic analysis is often complicated due to the low content of mtDNA compared to nuclear DNA. For this reason, we have designed a new approach to obtaining the genomic mitochondrial sequence. We chose RNA based sequencing. Since human mtDNA does not contain introns, the reconstruction of whole mitochondrial genome through RNA sequencing seems to be effective. Our method is based on total RNA sequencing coupled with simple ultracentrifugation protocol and de novo assembly. Following our protocol, we were able to assemble a complete mammalian mitochondrial genome with a length of 16,505 bp and an average coverage of 156. The method is a relatively simple and inexpensive which could help in the further research or diagnostics of mtDNA-based diseases.

Combining count- and length-based z-scores leads to improved predictions in non-invasive prenatal testing.

MOTIVATION: Non-invasive prenatal testing or NIPT is currently among the top researched topic in obstetric care. While the performance of the current state-of-the-art NIPT solutions achieve high sensitivity and specificity, they still struggle with a considerable number of samples that cannot be concluded with certainty. Such uninformative results are often subject to repeated blood sampling and re-analysis, usually after two weeks, and this period may cause a stress to the future mothers as well as increase the overall cost of the test. RESULTS: We propose a supplementary method to traditional z-scores to reduce the number of such uninformative calls. The method is based on a novel analysis of the length profile of circulating cell free DNA which compares the change in such profiles when random-based and length-based elimination of some fragments is performed. The proposed method is not as accurate as the standard z-score; however, our results suggest that combination of these two independent methods correctly resolves a substantial portion of healthy samples with an uninformative result. Additionally, we discuss how the proposed method can be used to identify maternal aberrations, thus reducing the risk of false positive and false negative calls. AVAILABILITY AND IMPLEMENTATION: The open-source code of the proposed methods, together with test data, is freely available for non-commercial users at github web page https://github.com/jbudis/lambda. SUPPLEMENTARY INFORMATION: Supplementary materials are available at Bioinformatics online.

A Novel Hantavirus of the European Mole, Bruges Virus, Is Involved in Frequent Nova Virus Coinfections.

Hantaviruses are zoonotic viruses with a complex evolutionary history of virus-host coevolution and cross-species transmission. Although hantaviruses have a broad reservoir host range, virus-host relationships were previously thought to be strict, with a single virus species infecting a single host species. Here, we describe Bruges virus, a novel hantavirus harbored by the European mole (Talpa europaea), which is the well-known host of Nova virus. Phylogenetic analyses of all three genomic segments showed tree topology inconsistencies, suggesting that Bruges virus has emerged from cross-species transmission and ancient reassortment events. A high number of coinfections with Bruges and Nova viruses was detected, but no evidence was found for reassortment between these two hantaviruses. These findings highlight the complexity of hantavirus evolution and the importance of further investigation of hantavirus-reservoir relationships.

The First Reported Case of Meckel-Gruber Syndrome Associated With Abnormal Karyotype Mosaic Trisomy 17.

Meckel-Gruber syndrome (MKS) is a rare lethal autosomal recessive disorder with typical anomalies including encephalocele, multicystic renal dysplasia, congenital liver fibrosis, and polydactyly. MKS is caused by mutations of genes localized on different chromosomes. Karyotypes of published Meckel-Gruber syndrome cases are without any aberrations. We present a male fetus with meningoencephalocele, multicystic renal dysplasia, congenital liver fibrosis, and other anomalies. Standard cytogenetic examination of cultured fetal skin and muscle fibroblasts showed mosaic trisomy 17. Homozygous deletion in CC2D2A gene was found by Sanger sequencing. This is to our knowledge the first case of genetically confirmed Meckel-Gruber syndrome with incidental cofinding of mosaic trisomy 17. Abnormal karyotype does not exclude diagnosis of MKS with risk of recurrence 25% in next pregnancy. In the case of anomalies typical for Meckel-Gruber syndrome, genetic analysis is indicated.