Genotype-Phenotype Correlations in 293 Russian Patients with Causal Fabry Disease Variants.

BACKGROUND: Fabry disease (FD) is a rare hereditary multisystem disease caused by variants of the GLA gene. Determination of GLA gene variants and identification of genotype-phenotype correlations allow us to explain the features of FD associated with predominant damage of one or another system, both in the classical and atypical forms of FD, as well as in cases with late manifestation and involvement of one of the systems. METHODS: The study included 293 Russian patients with pathogenic variants of the GLA gene, which were identified as a result of various selective screening programs. Screening was carried out for 48,428 high-risk patients using a two-step diagnostic algorithm, including the determination of the concentration of the biomarker lyso-Gb3 as a first-tier test. Screening of atypical FD among patients with HCM was carried out via high-throughput sequencing in another 2427 patients. RESULTS: 102 (0.20%) cases of FD were identified among unrelated patients as a result of the study of 50,855 patients. Molecular genetic testing allowed us to reveal the spectrum and frequencies of 104 different pathogenic variants of the GLA gene in 293 examined patients from 133 families. The spectrum and frequencies of clinical manifestations in patients with FD, including 20 pediatric patients, were described. Correlations between the concentration of the lyso-Gb3 biomarker and the type of pathogenic variants of the GLA gene have been established. Variants identified in patients with early stroke were described, and the association of certain variants with the development of stroke was established. CONCLUSIONS: The results of a large-scale selective FD screening, as well as clinical and molecular genetic features, in a cohort of 293 Russian patients with FD are described.

Clinical features of pediatric Danon disease and the importance of early diagnosis.

Successful therapy in a cohort with early onset Danon disease (DD) highlights the potential importance of earlier disease recognition. We present experience from the largest National Pediatric Center in Russia for cardiomyopathy patients. This report focuses on identification of early clinical features of DD in the pediatric population by detailed pedigree analysis and review of medical records. RESULTS: Nine patients (3 females) were identified with DD at the Russian National Medical Research Center of Children's Health ("National Pediatric Center") aged birth to 16 years. At presentation/evaluation: all patients had left ventricular hypertrophy (LVH), ECG features of Wolff-Parkinson-White (WPW), and an increase in hepatic enzymes (particularly lactate dehydrogenase (LDH)); three had marked increase in NT-proBNP; two had HCM with impaired LV function; one had LVH with LV noncompaction; five had arrhythmia with paroxysmal supraventricular and/or ventricular tachycardia. Two teenagers died at ages 16-17 from refractory heart failure and two underwent heart transplantation. All patients were found to have a pathogenic/likely pathogenic variant in the LAMP2 gene, six patients had no family history and a de novo evolvement was documented in 4/6 of those available for genetic tested. Retrospective review related to family background and earlier clinical evaluations revealed a definitive or highly suspicious family history of DD in 3, early clinical presentation with cardiac abnormalities (ECG, echo) in 3, and cerebral, hepatic and/or neuromuscular symptoms in 5. Abnormalities were detected 9,5 months to 5,8 years, median 3,5 years prior to referral to the National Pediatric Center. CONCLUSION: The earliest clinical manifestations of Danon disease occur in the first 12 years of life with symptoms of skeletal muscle and cerebral disease, raised hepatic enzymes, and evidence of cardiac disease on ECG/echo.

Sliding tracheoplasty of complete tracheal cartilage rings in children.

INTRODUCTION: Complete tracheal rings are a rare malformation that occurs in 1 out of 100,000 live births. It is rare, isolated tracheal or tracheobronchial anomaly developed due to abnormal cartilage growth with formation of complete ring and often resulting in airway stenosis. Slide tracheoplasty, as it was originally described by Tsang et al. and popularized by Grillo et al., overlaps stenotic segments of trachea, shortening trachea itself, thus, doubling the circumference and diameter of the stenotic area. MATERIALS AND METHODS: We have performed slide tracheoplasty in 12 children during the period of 2019-2021 in thoracic surgery department of our center. Median age was 15 ± 21,1 months (2 months-6 years),median weight - 8,04 ± 4,75 kg (3-20,7 kg),tracheal lumen varied from 2.5 to 3.0 mm, stenosis length - from 40 to 70% of the trachea length. RESULTS: Slide tracheoplasty was performed using central veno-arterial extracorporeal membrane oxygenation in 7 cases and using cardiopulmonary bypass in 5 cases. Concomitant heart disease was revealed in 5 children (pulmonary artery sling in 3 cases, ventricular septal defects - 1, aberrant subclavian artery -1). 5 children underwent one-stage correction of VSD: plastic VSD -1; left pulmonary artery reimplantation - 3; subclavian artery reimplantation - 1. All patients were on mechanical ventilation for 4,3 ± 2,78 days at postoperative period. Patients were discharged 16,3 ± 5,14 days after surgery. Satisfactory result of treatment in the form of respiratory failure relief was achieved in 10 patients. It was possible to increase the trachea lumen from 1.5 to 2 times in all cases. There were 2 (16,6%) fatal cases due to sepsis and multi-organ failure development. CONCLUSIONS: Children with complete tracheal rings are very complicated patients with various comorbidities. Despite the advances in medicine, sometimes it is impossible to save lives of these children. The use of extracorporeal circulation (ECMO and bypass) allows us to safely perform reconstructive surgery on the trachea and save the child from respiratory failure manifestations. If needed, simultaneous correction of heart and tracheal defects is possible. Slide tracheoplasty allows to increase trachea lumen at least in 1.5-2 times. Mechanical ventilation is an unfavorable predictive factor for the outcomes of congenital tracheal stenosis management. LEVEL OF EVIDENCE: III.

Our experience of using Losartan for esophageal stenosis in children with dystrophic form of congenital epidermolysis bullosa.

INTRODUCTION: Dystrophic epidermolysis bullosa (DEB) is one of the most severe forms of congenital epidermolysis bullosa and characterized by the formation of many surgical complications. Esophageal stenosis is a common complication of DEB and occurs in almost 76% of cases. Balloon dilatation (BD) under X-ray control is the main therapeutic technique, however conservative treatment is necessary to prevent restenosis. The use of the drug losartan is promising due to its antifibrotic effect through the suppression of transforming growth factor-ß1 (TGF-ß1). PURPOSE: To evaluate the efficacy of losartan in the prevention of restenosis after BD of esophageal stenosis in children with DEB. MATERIALS AND METHODS: The study included 19 children from 2 to 16 years old (mean age 9.2 ± 3.58 years) with DEB and X-ray confirmed esophageal stenosis. All children underwent BD. In the main group 9 children after BD have received losartan, in the control group of 10 children - only standard therapy. The observation period was 12 months. RESULTS: In the main group, 1 child (11.1%) required repeated dilatation, in the control group - 4 children (40%). Indicators of nutritional deficiency (THINC scale) and the disease severity index (EBDASI) were significantly lower in the group of children treated with losartan. No undesirable actions of the drug were recorded. CONCLUSIONS: In this study losartan showed its safety, contributed to a decrease in the restenosis frequency and an improvement in the nutritional status of children with DEB after BD. However, further studies are required to confirm its effectiveness. LEVEL OF EVIDENCE: IV.

Targeted NGS in Diagnostics of Genodermatosis Characterized by the Epidermolysis Bullosa Symptom Complex in 268 Russian Children.

The pathogenic variants of genes encoding proteins, participating in the formation and functioning of epidermis and dermo-epidermal junctions, create a large variety of clinical phenotypes from: small localized to severe generalized dermatitis, as well as early, or even, prenatal death due to extensive epidermis loss. The diagnostic panel in this study was developed for the purposes of identifying these pathogenic genetic variants in 268 Russian children, who possessed the epidermolysis bullosa symptom complex in a selection of 247 families. This panel included the targeted areas of 33 genes, which are genetic variants that can lead to the development of the phenotype mentioned above. The usage of next generation sequencing allowed the revelation of 192 various altered alleles (of which 109 alleles were novel, i.e., had not been described previously). In addition, it allowed the definition of the genetic variants that are both typical for most of the examined children and for the separate ethnic groups inhabiting modern Russia. We found that the most characteristic mutations for the Dargin and Chechen ethnic groups are the c.3577del deletion in the COL7A1 gene and the c.2488G>A missense mutation in the COL17A1 gene, respectively. In addition, the study of haplotypes of microsatellite markers, which we managed to conduct in the Dargin population, confirmed the presence of the founder effect.

Chromosome and Molecular Analyses Reveal Significant Karyotype Diversity and Provide New Evidence on the Origin of Aegilops columnaris.

Aegilops columnaris Zhuk. is tetraploid grass species (2n = 4x = 28, UcUcXcXc) closely related to Ae. neglecta and growing in Western Asia and a western part of the Fertile Crescent. Genetic diversity of Ae. columnaris was assessed using C-banding, FISH, nuclear and chloroplast (cp) DNA analyses, and gliadin electrophoresis. Cytogenetically Ae. columnaris was subdivided into two groups, C-I and C-II, showing different karyotype structure, C-banding, and FISH patterns. C-I group was more similar to Ae. neglecta. All types of markers revealed significant heterogeneity in C-II group, although group C-I was also polymorphic. Two chromosomal groups were consistent with plastogroups identified in a current study based on sequencing of three chloroplast intergenic spacer regions. The similarity of group C-I of Ae. columnaris with Ae. neglecta and their distinctness from C-II indicate that divergence of the C-I group was associated with minor genome modifications. Group C-II could emerge from C-I relatively recently, probably due to introgression from another Aegilops species followed by a reorganization of the parental genomes. Most C-II accessions were collected from a very narrow geographic region, and they might originate from a common ancestor. We suggest that the C-II group is at the initial stage of species divergence and undergoing an extensive speciation process.

Brain Biomarkers in Children After Mild and Severe Traumatic Brain Injury.

Brain biomarkers (protein S100b and neuron-specific enolase (NSE)), antibodies (aAb) to the NR2 subunit of N-methyl-D-aspartate (NR2(NMDA)) and to the GluR1 subunit of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (GluR1(AMPA)) subtype of glutamate receptors (GluR), NR2 and AMPA peptides, nitrogen oxides (NOx; "nitrites and nitrates"), and 3-nitrotyrosine (NT) were measured in blood from 159 children after mild traumatic brain injury (mTBI), moderate traumatic brain injury (mdTBI), or severe traumatic brain injury (sTBI) within 1-2 days and at intervals during the first 15 days after brain trauma. S100b and NSE levels on the first day were not a strict criterion for injury outcomes. Children with mTBI had the most significant elevations in antibodies to NR2(NMDA) and AMPA peptides, a slight increase in NOx, and, in 25% of cases, appearance of NT in the blood right after TBI. The lowest level of antibodies to NR2(NMDA) GluR detected shortly after the initial TBI was found in children with sTBI, with a negative outcome. The opposite characters of antibodies to NR2(NMDA) on the first day in children with mild and moderate versus severe TBI may be associated with an important mechanism aimed at protecting neurons from Glu excitotoxicity. We hypothesized that a slight increase in NOx after the onset of TBI rapidly activates the innate immune system and contributes to an increase in antibodies to NR2(NMDA). An increase in the AMPA peptide level in mTBI may be early signs of diffuse axonal injury.

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.

Smell Status in Children Infected with SARS-CoV-2.

OBJECTIVES/HYPOTHESIS: This study aimed to evaluate the olfactory status in children with laboratory confirmed SARS-CoV-2 using subjective and psychophysical methods. STUDY DESIGN: Prospective clinical cross-sectional study. METHODS: This is a prospective clinical cross-sectional study of 79 children with COVID-19. The 21st item of SNOT-22 questionnaire and odor identification test were used for smell assessment. Children were examined twice during the hospitalization, and a telephone survey was conducted 60 days after hospital discharge. RESULTS: Immediately after confirmation of COVID-19, smell impairment was detected in 86.1% of children by means of the Identification test and in 68.4% of children by means of the survey (P = .010). After 5 days survey revealed a statistically significant decrease in the number of patients with hyposmia (41 out of 79, 51.9%). On the first visit, the mean Identification test score corresponded to "hyposmia" (9.5 ± 2.7), while on the second visit, the average value was 13.1 ± 1.9, which corresponded to "normosmia." According to the telephone survey, recovery of the olfactory function occurred within 10 days in 37 of 52 patients (71.2%), 11 to 29 days - in 12 children (23.1%), and later than 30 days - in three cases (5.7%). CONCLUSIONS: In the pediatric population, olfactory dysfunction is an early and common symptom of COVID-19. There is a trend to quick recovery of olfactory function in children with COVID-19. The overwhelming majority of patients (94.3%) had no subjective olfactory complaints by the end of the first month. LEVEL OF EVIDENCE: 4 Laryngoscope, 131:E2475-E2480, 2021.

Lipopolysaccharide From E. coli Increases Glutamate-Induced Disturbances of Calcium Homeostasis, the Functional State of Mitochondria, and the Death of Cultured Cortical Neurons.

Lipopolysaccharide (LPS), a fragment of the bacterial cell wall, specifically interacting with protein complexes on the cell surface, can induce the production of pro-inflammatory and apoptotic signaling molecules, leading to the damage and death of brain cells. Similar effects have been noted in stroke and traumatic brain injury, when the leading factor of death is glutamate (Glu) excitotoxicity too. But being an amphiphilic molecule with a significant hydrophobic moiety and a large hydrophilic region, LPS can also non-specifically bind to the plasma membrane, altering its properties. In the present work, we studied the effect of LPS from Escherichia coli alone and in combination with the hyperstimulation of Glu-receptors on the functional state of mitochondria and Ca2+ homeostasis, oxygen consumption and the cell survival in primary cultures from the rats brain cerebellum and cortex. In both types of cultures, LPS (0.1-10 µg/ml) did not change the intracellular free Ca2+ concentration ([Ca2+]i) in resting neurons but slowed down the median of the decrease in [Ca2+]i on 14% and recovery of the mitochondrial potential (ΔΨm) after Glu removal. LPS did not affect the basal oxygen consumption rate (OCR) of cortical neurons; however, it did decrease the acute OCR during Glu and LPS coapplication. Evaluation of the cell culture survival using vital dyes and the MTT assay showed that LPS (10 µg/ml) and Glu (33 µM) reduced jointly and separately the proportion of live cortical neurons, but there was no synergism or additive action. LPS-effects was dependent on the type of culture, that may be related to both the properties of neurons and the different ratio between neurons and glial cells in cultures. The rapid manifestation of these effects may be the consequence of the direct effect of LPS on the rheological properties of the cell membrane.