EBS in Children with De Novo Pathogenic Variants Disturbing Krt14.

Epidermolysis bullosa simplex (EBS) is a dermatological condition marked by skin fragility and blister formation resulting from separation within the basal layer of the epidermis, which can be attributed to various genetic etiologies. This study presents three pathogenic de novo variants in young children, with clinical manifestations appearing as early as the neonatal period. The variants contribute to the EBS phenotype through two distinct mechanisms: direct keratin abnormalities due to pathogenic variants in the Krt14 gene, and indirect effects via pathogenic mutation in the KLHL24 gene, which interfere with the natural proteasome-mediated degradation pathway of KRT14. We report one severe case of EBS with mottled pigmentation arising from the Met119Thr pathogenic variant in KRT14, another case involving a pathogenic KLHL24 Met1Val variant, and a third case featuring the hot spot mutation Arg125His in KRT14, all manifesting within the first few weeks of life. This research underscores the complexity of genetic influences in EBS and highlights the importance of early genetic screening for accurate diagnosis and management.

Bridging Gaps in HDR Improvement: The Role of MAD2L2, SCAI, and SCR7.

This study aimed to enhance homology-directed repair (HDR) efficiency in CRISPR/Cas-mediated genome editing by targeting three key factors regulating the balance between HDR and non-homologous end joining (NHEJ): MAD2L2, SCAI, and Ligase IV. In order to achieve this, a cellular model using mutated eGFP was designed to monitor HDR events. Results showed that MAD2L2 knockdown and SCR7 treatment significantly improved HDR efficiency during Cas9-mediated HDR repair of the mutated eGFP gene in the HEK293T cell line. Fusion protein Cas9-SCAI did not improve HDR. This study is the first to demonstrate that MAD2L2 knockdown during CRISPR-mediated gene editing in HEK293T cells can increase precise correction by up to 10.2 times. The study also confirmed a moderate but consistent effect of SCR7, an inhibitor of Ligase IV, which increased HDR by 1.7 times. These findings provide valuable insights into improving HDR-based genome editing efficiency.

Rational Design of ssODN to Correct Mutations by Gene Editing.

Gene editing allows to make a variety of targeted changes in genome, which can potentially be used to treat hereditary human diseases. Despite numerous studies in this area, effectiveness of gene editing methods for correcting mutations is still low, so these methods are not allowed in routine practice. It has been shown that rational design of genome editing components can significantly increase efficiency of mutation correction. In this work, we propose design of single-stranded oligodeoxyribonucleotides (ssODNs) to increase efficiency of gene editing. Using a model system to repair knocked out EGFP that is integrated into the genome of HEK293T cell culture, we have shown that only a small part of ssODN (about 20 nucleotides: from the 15th nucleotide at 3'-end to the 4th nucleotide at 5'-end), a donor molecule for repairing double-stranded DNA breaks, is integrated into the site of the break. Based on the obtained data, it is possible to rationally approach the design of ssODNs to correct mutations using CRISPR-Cas9 method for the development of gene therapy for hereditary human diseases.

Influence of Copper Oxide Nanoparticles on Gene Expression of Birch Clones In Vitro under Stress Caused by Phytopathogens.

Recently, metal oxide nanoparticles (NPs) have attracted attention as promising components for the protection and stimulation of plant microclones in tissue culture in vitro. However, the effect of NPs on the genetic mechanisms underlying plant adaptive responses remains poorly understood. We studied the effect of column-shaped CuO NPs 50 nm in diameter and 70-100 nm in length at a concentration of 0.1-10 mg/L on the development of phytopathogenic fungi Alternaria alternata, Fusarium oxysporum, and Fusarium avenaceum in culture, as well as on the infection of downy birch micro-clones with phytopathogens and the level of genes expression associated with the formation of plant responses to stress induced by microorganisms. CuO NPs effectively suppressed the development of colonies of phytopathogenic fungi A. alternata and F. avenaceum (up to 68.42% inhibition at 10 mg/L CuO NPs) but not the development of a colony of F. oxysporum. Exposure to the NPs caused multidirectional responses at the level of plant genes transcription: 5 mg/L CuO NPs significantly increased the expression level of the LEA8 and MYB46 genes and decreased the expression of DREB2 and PAL. Infection with A. alternata significantly increased the level of MYB46, LEA8, PAL, PR-1, and PR-10 transcripts in birch micro-clones; however, upon exposure to a medium with NPs and simultaneous exposure to a phytopathogen, the expression of the MYB46, PR-1, and PR-10 genes decreased by 5.4 times, which is associated with a decrease in the pathogenic load caused by the effect of NPs and the simultaneous stimulation of clones in vitro. The results obtained can be used in the development of preparations based on copper oxide NPs for disinfection and stimulation of plant phytoimmunity during clonal micropropagation of tree crops.

Keratins as an Inflammation Trigger Point in Epidermolysis Bullosa Simplex.

Epidermolysis bullosa simplex (EBS) is a group of inherited keratinopathies that, in most cases, arise due to mutations in keratins and lead to intraepidermal ruptures. The cellular pathology of most EBS subtypes is associated with the fragility of the intermediate filament network, cytolysis of the basal layer of the epidermis, or attenuation of hemidesmosomal/desmosomal components. Mutations in keratins 5/14 or in other genes that encode associated proteins induce structural disarrangements of different strengths depending on their locations in the genes. Keratin aggregates display impaired dynamics of assembly and diminished solubility and appear to be the trigger for endoplasmic reticulum (ER) stress upon being phosphorylated by MAPKs. Global changes in cellular signaling mainly occur in cases of severe dominant EBS mutations. The spectrum of changes initiated by phosphorylation includes the inhibition of proteasome degradation, TNF-α signaling activation, deregulated proliferation, abnormal cell migration, and impaired adherence of keratinocytes. ER stress also leads to the release of proinflammatory danger-associated molecular pattern (DAMP) molecules, which enhance avalanche-like inflammation. Many instances of positive feedback in the course of cellular stress and the development of sterile inflammation led to systemic chronic inflammation in EBS. This highlights the role of keratin in the maintenance of epidermal and immune homeostasis.

hTERT-Driven Immortalization of RDEB Fibroblast and Keratinocyte Cell Lines Followed by Cre-Mediated Transgene Elimination.

The recessive form of dystrophic epidermolysis bullosa (RDEB) is a crippling disease caused by impairments in the junctions of the dermis and the basement membrane of the epidermis. Using ectopic expression of hTERT/hTERT + BMI-1 in primary cells, we developed expansible cultures of RDEB fibroblasts and keratinocytes. We showed that they display the properties of their founders, including morphology, contraction ability and expression of the respective specific markers including reduced secretion of type VII collagen (C7). The immortalized keratinocytes retained normal stratification in 3D skin equivalents. The comparison of secreted protein patterns from immortalized RDEB and healthy keratinocytes revealed the differences in the contents of the extracellular matrix that were earlier observed specifically for RDEB. We demonstrated the possibility to reverse the genotype of immortalized cells to the state closer to the progenitors by the Cre-dependent hTERT switch off. Increased ß-galactosidase activity and reduced proliferation of fibroblasts were shown after splitting out of transgenes. We anticipate our cell lines to be tractable models for studying RDEB from the level of single-cell changes to the evaluation of 3D skin equivalents. Our approach permits the creation of standardized and expandable models of RDEB that can be compared with the models based on primary cell cultures.

Signatures of Dermal Fibroblasts from RDEB Pediatric Patients.

The recessive form of dystrophic epidermolysis bullosa (RDEB) is a debilitating disease caused by impairments in the junctions of the dermis and the basement membrane of the epidermis. Mutations in the COL7A1 gene induce multiple abnormalities, including chronic inflammation and profibrotic changes in the skin. However, the correlations between the specific mutations in COL7A1 and their phenotypic output remain largely unexplored. The mutations in the COL7A1 gene, described here, were found in the DEB register. Among them, two homozygous mutations and two cases of compound heterozygous mutations were identified. We created the panel of primary patient-specific RDEB fibroblast lines (FEB) and compared it with control fibroblasts from healthy donors (FHC). The set of morphological features and the contraction capacity of the cells distinguished FEB from FHC. We also report the relationships between the mutations and several phenotypic traits of the FEB. Based on the analysis of the available RNA-seq data of RDEB fibroblasts, we performed an RT-qPCR gene expression analysis of our cell lines, confirming the differential status of multiple genes while uncovering the new ones. We anticipate that our panels of cell lines will be useful not only for studying RDEB signatures but also for investigating the overall mechanisms involved in disease progression.