Genomed (Poland)

Violacein is a natural indole-derived purple pigment of microbial origin that has attracted attention for its remarkable biological properties. Due to its poor solubility in aqueous media, most studies of this pigment use extracts of the compound obtained with common solvents. Violacein is also transported in bacterial extracellular vesicles (EVs) and transferred via this type of carrier remains stable in an aqueous environment. This paper is the first to present an in-depth study of Janthinobacterium lividum EVs as violacein carriers. J. lividum EVs were studied for their contribution to violacein translocation, size, morphology and protein composition. The production of violacein encapsulated in EVs was more efficient than the intracellular production of this compound. The average size of the violacein-containing EVs was 124.07 ± 3.74 nm. Liquid chromatography-tandem mass spectrometry analysis (LC–MS/MS) revealed 932 proteins common to three independent EVs isolations. The high proportion of proteins with intracellular localisation, which are involved in many fundamental cellular processes, suggests that J. lividum EVs could be generated in a cell lysis model, additionally stimulated by violacein production. Using human keratinocytes and melanoma cell lines, it was confirmed that J. lividum EVs are able to react with and deliver their cargo to mammalian cells. The EVs-delivered violacein was shown to retain its activity against melanoma cells, and the dose and timing of treatment can be selected to target only cancer cells. The characterisation of J. lividum EVs, described in the following paper, represents a milestone for their future potential anticancer application.Key points• This report focuses on the investigation of Janthinobacterium lividum EVs as a new delivery vehicle for violacein, a compound with a previously demonstrated broad spectrum of activity.• EVs were characterised for size, morphology and protein composition.• Studies on human keratinocytes and a melanoma cell model confirmed that the activity of violacein applied in the encapsulated form of EVs is similar to that of its organic solvent extract, but their production is much more environmentally friendly.

Age-related macular degeneration (AMD) is a complex eye disorder affecting millions worldwide. This article uses deep learning techniques to investigate the relationship between AMD, genetics and optical coherence tomography (OCT) scans. The cohort consisted of 332 patients, of which 235 were diagnosed with AMD and 97 were controls with no signs of AMD. The genome-wide association studies summary statistics utilized to establish the polygenic risk score (PRS) in relation to AMD were derived from the GERA European study. A PRS estimation based on OCT volumes for both eyes was performed using a proprietary convolutional neural network (CNN) model supported by machine learning models. The method's performance was assessed using numerical evaluation metrics, and the Grad-CAM technique was used to evaluate the results by visualizing the features learned by the model. The best results were obtained with the CNN and the Extra Tree regressor (MAE = 0.55, MSE = 0.49, RMSE = 0.70, R2 = 0.34). Extending the feature vector with additional information on AMD diagnosis, age and smoking history improved the results slightly, with mainly AMD diagnosis used by the model (MAE = 0.54, MSE = 0.44, RMSE = 0.66, R2 = 0.42). Grad-CAM heatmap evaluation showed that the model decisions rely on retinal morphology factors relevant to AMD diagnosis. The developed method allows an efficient PRS estimation from OCT images. A new technique for analysing the association of OCT images with PRS of AMD, using a deep learning approach, may provide an opportunity to discover new associations between genotype-based AMD risk and retinal morphology.

Coats disease (OMIM 300216) is a form of hereditary retinal dystrophy, which occurs due to congenital abnormality of retinal vessels and features unilateral exudative vitreoretinopathy. Coats disease mostly occurs sporadically; its genetic cause is still undetermined. Molecular genetic research including whole exome sequencing by the NGS method was used to define a genetic cause of the observed phenotype. Two heterozygous variants in different genomic loci associated with other forms of hereditary retinal dystrophy were detected, a rare variant in the HMCN1 gene c.9571C>T, p.(Arg3191Cys), and a known pathogenic variant in the NPHP4 gene c.2930C>T, p.(Thr977Met). The HMCN1 gene is responsible for dominant age-related macular degeneration (OMIM 603075), pathogenic variants in the NPHP4 gene cause recessive Senior-Løken syndrome 4 (OMIM 266900). These genes encode the proteins that are involved in the regulation of integrity of the blood-retinal barrier in the vascular endothelium (NPHP4) and retinal pigment epithelium (HMCN1). The identified mutation in the NPHP4 gene could lead to decreased function of the NPHP4 protein and contribute to the development of retinal degeneration, potentially of oligogenic nature.

Cell-free (cf) DNA screening is a noninvasive prenatal screening approach that is typically used to screen for common fetal trisomies, with optional screening for sex chromosomal aneuploidies and fetal sex. Genome-wide cfDNA screening can screen for a wide variety of additional anomalies, including rare autosomal aneuploidies (RAAs) and copy number variants. Here, we describe a multi-cohort, global retrospective study that looked at the clinical outcomes of cases with a high-risk cfDNA screening result for a RAA. Our study cohort included a total of 109 cases from five different sites, with diagnostic outcome information available for 68% (74/109) of patients. Based on confirmatory diagnostic testing, we found a concordance rate of 20.3% for presence of a RAA (15/74) in our study population. Pregnancy outcome was also available for 77% (84/109) of cases in our cohort. Many of the patients experienced adverse pregnancy outcomes, including intrauterine fetal demise, fetal growth restriction, and preterm birth. These adverse outcomes were observed both in patients with fetal or placental confirmation of the presence of a RAA, as well as patients that did not undergo fetal and/or placental diagnostic testing. In addition, we have proposed some suggestions for pregnancy management and counseling considerations for situations where a RAA is noted on a cfDNA screen. In conclusion, our study has shown that genome-wide cfDNA screening for the presence of rare autosomal aneuploidies can be beneficial for both patients and their healthcare practitioners. This can provide a possible explanation for an adverse pregnancy outcome or result in a change in pregnancy management, such as increased monitoring for adverse outcomes.

Neutrophils are a heterogenous population capable of both antimicrobial functions and suppressor ones, however, no specific pattern of transcription factors controlling this plasticity has been identified. We observed rapid changes in the neutrophil status after stimulation with LPS, pre-activating concentration of TNF-α, or IL-10. Chromatin immunoprecipitation sequencing (ChIP-Seq) analysis of histone H3K4me3 allowed us to identify various transcriptional start sites (TSSs) associated with plasticity and heterogeneity of human neutrophils. Gene Ontology analysis demonstrated great variation within target genes responsible for neutrophil activation, cytokine production, apoptosis, histone remodelling as well as NF-κB transcription factor pathways. These data allowed us to assign specific target genes positioned by H3K4me3-marked histone with a different pattern of gene expression related to NF-κB pathways, apoptosis, and a specific profile of cytokines/chemokines/growth factors realised by neutrophils stimulated by LPS, IL-10, or TNF-α. We discovered IL-10-induced apoptotic neutrophils being transcriptionally active cells capable of switching the profile of cytokines/chemokines/growth factors desired in resolving inflammation via non-canonical NF-κB pathway with simultaneous inhibition of canonical NF-κB pathway. As apoptotic/suppressive neutrophils induced by IL-10 via positioning genes within H3K4me3-marked histone were transcriptionally active, newly described DNA binding sites can be considered as potential targets for immunotherapy.Graphical H3K4me3 histone ChIP-Seq analysis reveals molecular drivers critical for switching neutrophils from their pro- to anti-inflammatory properties.

Introduction. The prevalence of congenital malformations (CMFs) in fetal central nervous system (CNS) ranges from 1.5 to 3 % and covers around 29 % among all malformations, whereas percentage in the structure of perinatal and infant mortality reaches 25–26 %.Aim: to estimate frequency of pathogenic copy number variations (CNVs) in fetuses with congenital malformations of CNS and normal karyotyping cytogenetic analysis.Materials and Methods. There were enrolled 42 pregnant women underwent invasive prenatal diagnostics in 2013–2019 due to ultrasound detection of congenital CNS defect in fetus. Fetal samples were studied by using chromosome microarray analysis (CMA).Results. Various pathogenic CNVs were detected in 7 (16.6 %) fetuses with prenatally diagnosed congenital CNS malformations. Non-syndrome pathogenic CNVs were detected in 85.7 %.Conclusion. Thus, performing chromosome microarray analysis as the first-line assay allows to diagnose not only aneuploidy, but also microdeletion/microduplication, the size of which below resolution threshold for standard cytogenetic karyotyping

Wolf – Hirschhorn syndrome is a rare genetic disease caused by the deletion of the end of the short arm of the 4th chromosome; it is manifested by numerous congenital malformations, delayed physical and psychomotor development. The article describes clinical experience of managing a patient with Wolff – Hirschhorn syndrome born to exposed parents who lived in a territory contaminated with radionuclides after the Chernobyl accident. The article describes pathogenetic aspects of the development of the disease and the need for timely diagnostics.

The complete form of X-linked congenital stationary night blindness (CSNB) is a rare genetic disease caused by a mutation in the NYX gene. CSNB is associated with the mutations taking place in 17 genes, whilst its CSNB1A form is caused by the mutations in the NYX gene, which were characterized earlier, although nothing had been reported so far about the Russian founder principle. The paper analyzes the pathogenetic mechanisms in a family with diagnosed CSNB1A and a new genetically confirmed mutation in the NYX gene in four members of one Russian family. Two brothers of the four siblings (two boys, two girls) with congenital stationary night blindness, diagnosed in early childhood, and high myopia underwent a standard ophthalmic examination, supplemented with OCT, electroretinography and color blind test with tables by Rabkin and Farnsworth test, whereupon they were sent to molecular genetics confirmation of the diagnosis by whole exome sequencing with subsequent Sanger sequencing confirmation of the detected mutation in the proband and proband’s relatives. In members of the family with clinical features of CSNB1A the reading frame shift mutation was genetically confirmed in the NYX gene (c.283delC, p.His95fs, NM_022567.2). This mutation is inherited in X-linked form. This is the first report of a case with a novel and probable founder mutation from Russia associated with CSNB1A. Since the mRNA of a NYX gene consists of only 2696 base pairs, a gene replacement therapy, or CRISPR-based gene editing, or a similar approach may be envisaged for the correction of frameshift in His95fs position.

Background. A large number of single gene disorders with seizures in clinical picture has been described. Among them, a special place is held by early-onset epileptic encephalopathies (EEE) – a genetically diverse group of disorders characterized by manifestation of seizures in the first 2 years of life and severe progressing course. Currently, 58 genetic variants of EEE has been identified.The objective is to analyze the incidence, clinical and genetic characteristic of type II EEE in a sample of patients from Russia identified by whole exome sequencing using next generation sequencing.Materials and methods. The patient sample included 67 children (35 boys and 32 girls) with ages varying from 4 months to 10 years. All patients underwent neurological examination using the standard techniques. Monitoring of electroencephalography (EEG) was performed in accordance with the International 10–20 system. For magnetic resonance imaging, tomographs with magnetic induction of 1.5 and 3.0 T were used. The identified changes were confirmed by Sanger sequencing using DNA from the patients and their parents.Results. In total, 67 patients with EEE were diagnosed, 8 of which had causational mutations in the SCN2A gene: p.Leu1611Pro (c.4832T>C), p.Cys728* (c.2184C>A), p.Arg607* (c.1819C>T), p.Val1325Ile (c.623T>C), p.Leu419Met (c.1255T>A), p.Asp1487Glu (c.4461C>A), p.Val208Ala (c.3973G>A), p.Gln1211Lys (c.3631G>A). Analysis of parent DNA had shown that all mutations appeared de novo. At the time of disease onset, all patients had multiregional epileptiform activity per EEG. Focal seizures, myoclonic seizures, and epileptic spasms were the most common types of seizures in the patients. The majority of patients (5/8) had diffuse muscular hypotonia. All patients had pronounced mental deficiency. All patients received more than 2 therapy regimens (including hormonal therapy and ketogenic diet) but full remission wasn’t obtained. In some patients, partial positive dynamic was registered with hormonal therapy and sodium channel blockers. Various mutations in the SCN2A gene caused a similar clinical picture but had different functional significance which determined the effectiveness of anti-epileptic therapy.Conclusion. Identification of the mutated genes causing EEE and further study of their function are important for selection of therapeutic approach to epilepsy treatment.