Jan Radvanszky

Background and Aims: Iron overload causes symptoms, which can (if not treated) damage organs and systems of the body. We report a Slovakian female patient with chronic anemia and neuropathy due to iron overloading. Methods and Results: Clinical workup included laboratory investigations, body imaging, and genetic study. Patients’ iron parameters showed a low serum iron, low iron saturation, elevated serum ferritin, several neurological symptoms, and later discovered abnormal liver function tests, low serum copper and extremely low ceruloplasmin (CP) levels. Initial direct genetic testing showed homozygous variant p.(His63Asp) in HFE and indicated hereditary hemochromatosis (HHC) type 1. The following whole genome sequencing (WGS) revealed the digenic origin of iron overload, since also compound heterozygous variants p.(Tyr890*) and p.(Leu273Pro) in CP were identified, responsible for aceruloplasminemia (ACP). However, clinical significance of p.(His63Asp) is difficult to evaluate due to its low penetrance. Since the patient was intolerant to phlebotomy, chelation therapy with deferasirox was started, which appeared to prevent her liver damage. Based on ACP diagnosis, she has been recently switched to treatment with deferiprone, which has better brain penetrance. Conclusions: The presented case of unique digenic iron overload condition (simultaneous HHC and ACP) clarified by WGS, demonstrated multiple diagnostic and therapeutic challenges encountered in rare diseases. It highlights the limitations of targeted genetic testing, while stressing the diagnostic value of comprehensive genomic approaches in complex genotype–phenotype presentations, co-navigating the complicated patient’s and physician’s journey that necessitates multidisciplinary collaboration and use of non-trivial clinical and therapeutic strategies.

Clinical interpretation of copy number variants (CNVs) is a complex process that requires skilled clinical professionals. General recommendations have been recently released to guide the CNV interpretation based on predefined criteria to uniform the decision process. Several semiautomatic computational methods have been proposed to recommend appropriate choices, relieving the clinicians from the tedious search in vast genomic databases. We have developed and evaluated such a tool called MarCNV and tested it on CNV records collected from the ClinVar database. Alternatively, the emerging machine learning-based tools, such as the recently published ISV (Interpretation of Structural Variants), showed promising ways of even fully automated predictions using wider characterization of affected genomic elements. Such tools utilize features that are additional to ACMG criteria, thus, they have the potential to significantly improve and/or provide supportive evidence for accurate CNV classification. Since both approaches contribute to evaluation of CNVs clinical impact, we propose a combined solution in the form of a decision support tool based on automated ACMG guidelines (MarCNV) supplemented by a machine learning-based pathogenicity prediction (ISV) for classification of CNVs. We provide evidence that such a combined approach is able to reduce the number of uncertain classifications and reveal potentially incorrect classifications using automated guidelines. CNV interpretation using MarCNV, ISV, and combined approach is available for non-commercial use at https://predict.genovisio.com/ .

Motivation Short tandem repeats (STRs) are regions of a genome containing many consecutive copies of the same short motif, possibly with small variations. Analysis of STRs has many clinical uses, but is limited by technology mainly due to STRs surpassing the used read length. Nanopore sequencing, as one of long read sequencing technologies, produces very long reads, thus offering more possibilities to study and analyze STRs. Basecalling of nanopore reads is however particularly unreliable in repeating regions, and therefore direct analysis from raw nanopore data is required. Results Here we present WarpSTR, a novel method for characterizing both simple and complex tandem repeats directly from raw nanopore signals using a finite-state automaton and a search algorithm analogous to dynamic time warping. By applying this approach to determine the lengths of 241 STRs, we demonstrate that our approach decreases the mean absolute error of the STR length estimate compared to basecalling and STRique. Availability WarpSTR is freely available at https://github.com/fmfi-compbio/warpstr Contact jozef.sitarcik{at}uniba.sk

Myotonic dystrophy type 2 (DM2) is caused by expansion of a (CCTG)_n repeat in the cellular retroviral nucleic acid-binding protein (CNBP) gene. The sequence of the repeat is most commonly interrupted and is stably inherited in the general population. Although expanded alleles, premutation range and, in rare cases, also non-disease associated alleles containing uninterrupted CCTG tracts have been described, the threshold between these categories is poorly characterised. Here, we describe four families with members reporting neuromuscular complaints, in whom we identified altogether nine ambiguous CNBP alleles containing uninterrupted CCTG repeats in the range between 32 and 42 repeats. While these grey-zone alleles are most likely not pathogenic themselves, since other pathogenic mutations were identified and particular family structures did not support their pathogenic role, they were found to be unstable during intergenerational transmission. On the other hand, there was no observable general microsatellite instability in the genome of the carriers of these alleles. Our results further refine the division of CNBP CCTG repeat alleles into two major groups, i.e., interrupted and uninterrupted alleles. Both interrupted and uninterrupted alleles with up to approximately 30 CCTG repeats were shown to be generally stable during intergenerational transmission, while intergenerational as well as somatic instability seems to gradually increase in uninterrupted alleles with tract length growing above this threshold.

Gains and losses of large segments of genomic DNA, known as copy number variants (CNVs) gained considerable interest in clinical diagnostics lately, as particular forms may lead to inherited genetic diseases. In recent decades, researchers developed a wide variety of cytogenetic and molecular methods with different detection capabilities to detect clinically relevant CNVs. In this review, we summarize methodological progress from conventional approaches to current state of the art techniques capable of detecting CNVs from a few bases up to several megabases. Although the recent rapid progress of sequencing methods has enabled precise detection of CNVs, determining their functional effect on cellular and whole-body physiology remains a challenge. Here, we provide a comprehensive list of databases and bioinformatics tools that may serve as useful assets for researchers, laboratory diagnosticians, and clinical geneticists facing the challenge of CNV detection and interpretation.

Circulating extracellular DNA (ecDNA) is known to worsen the outcome of many diseases. ecDNA released from neutrophils during infection or inflammation is present in the form of neutrophil extracellular traps (NETs). It has been shown that higher ecDNA concentration occurs in a number of inflammatory diseases including inflammatory bowel disease (IBD). Enzymes such as peptidyl arginine deiminases (PADs) are crucial for NET formation. We sought to describe the dynamics of ecDNA concentrations and fragmentation, along with NETosis during a mouse model of chemically induced colitis. Plasma ecDNA concentration was highest on day seven of dextran sulfate sodium (DSS) intake and the increase was time-dependent. This increase correlated with the percentage of cells undergoing NETosis and other markers of disease activity. Relative proportion of nuclear ecDNA increased towards more severe colitis; however, absolute amount decreased. In colon explant medium, the highest concentration of ecDNA was on day three of DSS consumption. Early administration of PAD4 inhibitors did not alleviate disease activity, but lowered the ecDNA concentration. These results uncover the biological characteristics of ecDNA in IBD and support the role of ecDNA in intestinal inflammation. The therapeutic intervention aimed at NETs and/or nuclear ecDNA has yet to be fully investigated.

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.