Evaluation of the Complex p.[Leu467Phe;Phe508del] CFTR Allele in the Intestinal Organoids Model: Implications for Therapy.

In the cohort of Russian patients with cystic fibrosis, the p.[Leu467Phe;Phe508del] complex allele (legacy name [L467F;F508del]) of the CFTR gene is understudied. In this research, we present the results of frequency evaluation of the [L467F;F508del] complex allele in the Russian Federation among patients with a F508del/F508del genotype, its effect on the clinical course of cystic fibrosis, the intestinal epithelium ionic channel function, and the effectiveness of target therapy. The frequency of the [L467F;F508del] complex allele among patients with homozygous F508del was determined with multiplex ligase-dependent probe amplification followed by polymerase chain reaction and fragment analysis. The function of ionic channels, including the residual CFTR function, and the effectiveness of CFTR modulators was analyzed using intestinal current measurements on rectal biopsy samples and the forskolin-induced swelling assay on organoids. The results showed that the F508del/[L467F;F508del] genotype is present in 8.2% of all Russian patients with F508del in a homozygous state. The clinical course of the disease in patients with the F508del/[L467F;F508del] genotype is severe and does not vary from the course in the cohort with homozygous F508del, although the CFTR channel function is significantly lower. For patients with the F508del/[L467F;F508del] genotype, we can recommend targeted therapy using a combined ivacaftor + tezacaftor + elexacaftor medication.

The Cytogenomic "Theory of Everything": Chromohelkosis May Underlie Chromosomal Instability and Mosaicism in Disease and Aging.

Mechanisms for somatic chromosomal mosaicism (SCM) and chromosomal instability (CIN) are not completely understood. During molecular karyotyping and bioinformatic analyses of children with neurodevelopmental disorders and congenital malformations (n = 612), we observed colocalization of regular chromosomal imbalances or copy number variations (CNV) with mosaic ones (n = 47 or 7.7%). Analyzing molecular karyotyping data and pathways affected by CNV burdens, we proposed a mechanism for SCM/CIN, which had been designated as "chromohelkosis" (from the Greek words chromosome ulceration/open wound). Briefly, structural chromosomal imbalances are likely to cause local instability ("wreckage") at the breakpoints, which results either in partial/whole chromosome loss (e.g., aneuploidy) or elongation of duplicated regions. Accordingly, a function for classical/alpha satellite DNA (protection from the wreckage towards the centromere) has been hypothesized. Since SCM and CIN are ubiquitously involved in development, homeostasis and disease (e.g., prenatal development, cancer, brain diseases, aging), we have metaphorically (ironically) designate the system explaining chromohelkosis contribution to SCM/CIN as the cytogenomic "theory of everything", similar to the homonymous theory in physics inasmuch as it might explain numerous phenomena in chromosome biology. Recognizing possible empirical and theoretical weaknesses of this "theory", we nevertheless believe that studies of chromohelkosis-like processes are required to understand structural variability and flexibility of the genome.

A Molecular Genetic Analysis of RPE65-Associated Forms of Inherited Retinal Degenerations in the Russian Federation.

Pathogenic variants in the RPE65 gene cause the only known form of inherited retinal degenerations (IRDs) that are prone to gene therapy. The current study is aimed at the evaluation of the prevalence of RPE65-associated retinopathy in the Russian Federation, the characterization of known variants in the RPE65 gene, and the establishment of the specificities of the mutation spectrum in Russian patients. METHODS: The analysis was carried out on blood samples obtained from 1053 non-related IRDs patients. The analysis, which consisted of 211 genes, was carried out based on the method of massive parallel sequencing (MPS) for all probands. Variant validation, as well as biallelic status verification, were carried out using direct automated Sanger sequencing. The number of copies of RPE65 exons 1-14 was analyzed with quantitative MLPA using an MRC-Holland SALSA MLPA probemix. RESULTS: Out of 1053 non-related patients, a molecular genetic diagnosis of IRDs has been confirmed in 474 cases, including 25 (5.3%) patients with RPE65-associated retinopathy. We detected 26 variants in the RPE65 gene, nine of which have not been previously described in the literature. The most common mutations in the Russian population were c.304G>T/p.(Glu102*), c.370C>T/p.(Arg124*), and c.272G>A/p.(Arg91Gln), which comprised 41.8% of all affected chromosomes. CONCLUSIONS: The current study shows that pathogenic variants in the RPE65 gene contribute significantly to the pathogenesis of IRDs and comprise 5.3% of all patients with a confirmed molecular genetic diagnosis. This study allowed for the formation of a cohort for target therapy of the disorder; such therapy has already been carried out for some patients.

Pilot Program of Newborn Screening for 5q Spinal Muscular Atrophy in the Russian Federation.

5q spinal muscular atrophy (5q SMA) is one of the most common autosomal recessive disorders in the Russian Federation. The first medication to treat 5q SMA was registered in the Russian Federation for treatment of all 5q SMA types in 2019, and the last of the three currently available in December 2021. We launched the pilot newborn screening (NBS) program for 5q SMA in Moscow, the Russian Federation, starting in 2019. During the pilot program, 23,405 neonates were tested for the deletion of exon 7 of the SMN1 gene, the most common cause of 5q SMA. We used the SALSA® MC002 SMA Newborn Screen Kit (MRC Holland) to specifically detect homozygous deletions of SMN1 exon 7. We used the restriction fragment length polymorphism (RFLP) approach to validate detected homozygous deletions and the SALSA MLPA Probemix P060 SMA Carrier Kit (MRC Holland) to determine the SMN2 exon 7 copy number to prescribe gene therapy for 5q SMA. Three newborns with a homozygous deletion of the SMN1 gene were detected. The calculated birth prevalence of 1:7801 appears to be similar to the results in other European countries. The children did not show any signs of respiratory involvement or bulbar weakness immediately after birth. Until now, no 5q SMA case missed by NBS has been detected.

Satellite III (1q12) Copy Number Variation in Cultured Human Skin Fibroblasts from Schizophrenic Patients and Healthy Controls.

BACKGROUND: The chromosome 1q12 region harbors the genome's largest pericentromeric heterochromatin domain that includes tandemly repeated satellite III DNA [SatIII (1)]. Increased SatIII (1) copy numbers have been found in cultured human skin fibroblasts (HSFs) during replicative senescence. The aim of this study was to analyze the variation in SatIII (1) abundance in cultured HSFs at early passages depending on the levels of endogenous and exogenous stress. METHODS: We studied 10 HSF cell lines with either high (HSFs from schizophrenic cases, n = 5) or low (HSFs from healthy controls, n = 5) levels of oxidative stress. The levels of endogenous stress were estimated by the amounts of reactive oxygen species, DNA damage markers (8-hydroxy-2'-deoxyguanosine, gamma-H2A histone family member X), pro- and antioxidant proteins (NADPH oxidase 4, superoxide dismutase 1, nuclear factor erythroid 2-related factor 2), and proteins that regulate apoptosis and autophagy (B-cell lymphoma 2 [Bcl-2], Bcl-2-associated X protein, light chain 3). SatIII (1) copy numbers were measured using the nonradioactive quantitative hybridization technique. For comparison, the contents of telomeric and ribosomal RNA gene repeats were determined. RNASATIII (1 and 9) were quantified using quantitative Polymerase Chain Reaction (PCR). RESULTS: Increased SatIII (1) contents in DNA from confluent HSFs were positively correlated with increased oxidative stress. Confluent cell cultivation without medium replacement and heat shock induced a decrease of SatIII (1) in DNA in parallel with a decrease in RNASATIII (1) and an increase in RNASATIII (9). CONCLUSIONS: During HSF cultivation, cells with increased SatIII (1) content accumulated in the cell pool under conditions of exaggerated oxidative stress. This fraction of cells decreased after the additional impact of exogenous stress. The process seems to be oscillatory.

Somatic mosaicism in the diseased brain.

It is hard to believe that all the cells of a human brain share identical genomes. Indeed, single cell genetic studies have demonstrated intercellular genomic variability in the normal and diseased brain. Moreover, there is a growing amount of evidence on the contribution of somatic mosaicism (the presence of genetically different cell populations in the same individual/tissue) to the etiology of brain diseases. However, brain-specific genomic variations are generally overlooked during the research of genetic defects associated with a brain disease. Accordingly, a review of brain-specific somatic mosaicism in disease context seems to be required. Here, we overview gene mutations, copy number variations and chromosome abnormalities (aneuploidy, deletions, duplications and supernumerary rearranged chromosomes) detected in the neural/neuronal cells of the diseased brain. Additionally, chromosome instability in non-cancerous brain diseases is addressed. Finally, theoretical analysis of possible mechanisms for neurodevelopmental and neurodegenerative disorders indicates that a genetic background for formation of somatic (chromosomal) mosaicism in the brain is likely to exist. In total, somatic mosaicism affecting the central nervous system seems to be a mechanism of brain diseases.

Pediatric chordoma associated with tuberous sclerosis complex: A rare case report with a thorough analysis of potential therapeutic molecular targets.

Chordoma associated with tuberous sclerosis complex (TSC) is an extremely rare tumor that was described only in 13 cases since 1975. Сhordoma itself is a malignant slow-growing bone tumor thought to arise from vestigial or ectopic notochordal tissue. Chordoma associated with TSC differs from chordoma in the general pediatric population in the median age, where the diagnosis of TSC-associated chordoma is 6.2 months, whereas for chordoma in the general pediatric population it is set to 12 years. The majority of TSC-associated chordomas are localized in skull-based and sacrum regions, and rare in the spine. Chordomas are genetically heterogeneous tumors characterized by chromosomal instability (CIN), and alterations involving PI3K-AKT signaling pathway genes and chromatin remodeling genes. Here we present the 14th case of chordoma associated with TSC in a 1-year-old pediatric patient. Alongside biallelic inactivation of the TSC1 gene, molecular genetic analysis revealed CIN and involvement of epigenetic regulation genes. In addition, we found the engagement of CBX7 and apolipoprotein B editing complex (APOBEC3) genes that were not yet seen in chordomas before. Amplification of CBX7 may epigenetically silence the CDKN2A gene, whereas amplification of APOBEC3 genes can explain the frequent occurrence of CIN in chordomas. We also found that KRAS gene is located in the region with gain status, which may suggest the ineffectiveness of potential EGFR monotherapy. Thus, molecular genetic analysis carried out in this study broadens the horizons of possible approaches for targeted therapies with potential applications for personalized medicine.

Vitamin D Status Among Children With Juvenile Idiopathic Arthritis: A Multicenter Prospective, Non-randomized, Comparative Study.

Background: Juvenile idiopathic arthritis (JIA) is a chronic autoimmune disease characterized by destructive and inflammatory damage to the joints. The aim in this study was to compare vitamin D levels between children and adolescents, 1-18 years of age, with juvenile idiopathic arthritis (JIA) and a health control group of peers. We considered effects of endogenous, exogenous, and genetic factors on measured differences in vitamin D levels among children with JIA. Methods: Our findings are based on a study sample of 150 patients with various variants of JIA and 277 healthy children. The blood level of vitamin D was assessed by calcidiol level. The following factors were included in our analysis: age and sex; level of insolation in three regions of country (center, south, north); assessment of dietary intake of vitamin D; effect of prophylactic doses of cholecalciferol; a relationship between the TaqI, FokI, and BsmI polymorphisms of the VDR gene and serum 25(OH)D concentration. Results: We identified a high frequency of low vitamin D among children with JIA, prevalence of 66%, with the medial level of vitamin D being within the range of "insufficient" vitamin D. We also show that the dietary intake of vitamin D by children with JIA is well below expected norms, and that prophylactic doses of vitamin D supplementation (cholecalciferol) at a dose of 500-1,000 IU/day and 1,500-2,000 IU/day do not meet the vitamin D needs of children with JIA. Of importance, we show that vitamin D levels among children with JIA are not affected by clinical therapies to manage the disease nor by the present of VDR genetic variants. Conclusion: Prophylactic administration of cholecalciferol and season of year play a determining role in the development of vitamin D deficiency and insufficiency.

Chromosome Instability, Aging and Brain Diseases.

Chromosome instability (CIN) has been repeatedly associated with aging and progeroid phenotypes. Moreover, brain-specific CIN seems to be an important element of pathogenic cascades leading to neurodegeneration in late adulthood. Alternatively, CIN and aneuploidy (chromosomal loss/gain) syndromes exhibit accelerated aging phenotypes. Molecularly, cellular senescence, which seems to be mediated by CIN and aneuploidy, is likely to contribute to brain aging in health and disease. However, there is no consensus about the occurrence of CIN in the aging brain. As a result, the role of CIN/somatic aneuploidy in normal and pathological brain aging is a matter of debate. Still, taking into account the effects of CIN on cellular homeostasis, the possibility of involvement in brain aging is highly likely. More importantly, the CIN contribution to neuronal cell death may be responsible for neurodegeneration and the aging-related deterioration of the brain. The loss of CIN-affected neurons probably underlies the contradiction between reports addressing ontogenetic changes of karyotypes within the aged brain. In future studies, the combination of single-cell visualization and whole-genome techniques with systems biology methods would certainly define the intrinsic role of CIN in the aging of the normal and diseased brain.

New Regions With Molecular Alterations in a Rare Case of Insulinomatosis: Case Report With Literature Review.

Insulinomatosis is characterized by monohormonality of multiple macro-tumors and micro-tumors that arise synchronously and metachronously in all regions of the pancreas, and often recurring hypoglycemia. One of the main characteristics of insulinomatosis is the presence of insulin-expressing monohormonal endocrine cell clusters that are exclusively composed of proliferating insulin-positive cells, are less than 1 mm in size, and show solid islet-like structure. It is presumed that insulinomatosis affects the entire population of ß-cells. With regards to molecular genetics, this phenomenon is not related to mutation in MEN1 gene and is more similar to sporadic benign insulinomas, however, at the moment molecular genetics of this disease remains poorly investigated. NGS sequencing was performed with a panel of 409 cancer-related genes. Results of sequencing were analyzed by bioinformatic algorithms for detecting point mutations and copy number variations. DNA copy number variations were detected that harbor a large number of genes in insulinoma and fewer genes in micro-tumors. qPCR was used to confirm copy number variations at ATRX, FOXL2, IRS2 and CEBPA genes. Copy number alterations involving FOXL2, IRS2, CEBPA and ATRX genes were observed in insulinoma as well as in micro-tumors samples, suggesting that alterations of these genes may promote malignization in the ß-cells population.

Health Characteristics of Patients with Cystic Fibrosis whose Genotype Includes a Variant of the Nucleotide Sequence c.3140-16T>A and Functional Analysis of this Variant.

Cystic fibrosis (CF) is the most common monogenic autosomal recessive disease, associated with pathogenic variants in the CFTR gene. The splicing variant c.3140-16T>A (3272-16T>A) has been described previously and, according to the Russian CF Patients Registry, occurs with a frequency of 0.34%. The phenotypic features of CF patients with the c.3140-16T>A variant were compared with those of patients with the genotype F508del/F508del. Patients with the allele c.3140-16T>A had higher average age and age at diagnosis, and the allele was present in a greater proportion of adults. Patients carrying the c.3140-16T>A allele were characterised by better physical development indicators, both in adults and in children, had preserved pancreatic function, as well as the absence of a number of complications, and required pancreatic enzyme replacement therapy less often than patients with the F508del/F508del genotype. Sweat test values also were lower in patients with the c.3140-16T>A genotype. According to the results of clinical and laboratory studies, the phenotype of patients with the genetic variant c.3140-16T>A can be considered "mild". Functional CFTR protein activity in the presence of c.3140-16T>A was evaluated using intestinal current measurements (ICM) and the forskolin-induced swelling assay on organoids obtained from patients' rectal biopsies. c.3140-16T>A had high residual CFTR channel activity and was amenable to effective pharmacological correction with thea VX-770 potentiator. To evaluate the effect of the variant on CFTR pre-mRNA splicing we performed a minigene assay, as well as RT-PCR analysis of RNA isolated from the nasal epithelium and rectal biopsy of patients. We showed that the c.3140-16T>A variant creates a novel acceptor AG dinucleotide within CFTR intron 19, resulting in a 14-nucleotide extension of exon 20. This frameshift produces a premature termination codon and triggers mRNA degradation by the nonsense-mediated decay (NMD) mechanism. Moreover, we observed that the c.3140-16T>A allele could produce a residual amount of normally spliced transcript, thus explaining the patient's mild phenotype.

Multiple Chromoanasynthesis in a Rare Case of Sporadic Renal Leiomyosarcoma: A Case Report.

We present the genetic profile of kidney giant leiomyosarcoma characterized by sequencing of 409 cancer related genes and chromosomal microarray analysis. Renal leiomyosarcomas are extremely rare neoplasms with aggressive behavior and poor survival prognosis. Most frequent somatic events in leiomyosarcomas are mutations in the TP53, RB1, ATRX, and PTEN genes, chromosomal instability (CIN) and chromoanagenesis. 67-year-old woman presented with a right kidney completely replaced by tumor. Immunohistochemical reaction on surgical material was positive to desmin and smooth muscle actin. Molecular genetic analysis revealed that tumor harbored monosomy of chromosomes 3 and 11, gain of Xp (ATRX) arm and three chromoanasynthesis regions (6q21-q27, 7p22.3-p12.1, and 12q13.11-q21.2), with MDM2 and CDK4 oncogenes copy number gains, whereas no copy number variations (CNVs) or tumor specific single nucleotide variants (SNVs) in TP53, RB1, and PTEN genes were present. We hypothesize that chromoanasynthesis in 12q13.11-q21.2 could be a trigger of observed CIN in this tumor.

Abnormal Hypermethylation of CpG Dinucleotides in Promoter Regions of Matrix Metalloproteinases Genes in Breast Cancer and Its Relation to Epigenomic Subtypes and HER2 Overexpression.

Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) substantially contribute to the regulation of intercellular interactions and thereby play a role in maintaining the tissue structure and function. We examined methylation of a subset of 5'-cytosine-phosphate-guanine-3' (CpG) dinucleotides in promoter regions of the MMP2, MMP11, MMP14, MMP15, MMP16, MMP17, MMP21, MMP23B, MMP24, MMP25, MMP28, TIMP1, TIMP2, TIMP3, and TIMP4 genes by methylation-sensitive restriction enzyme digestion PCR. In our collection of 183 breast cancer samples, abnormal hypermethylation was observed for CpGs in MMP2, MMP23B, MMP24, MMP25, and MMP28 promoter regions. The non-methylated status of the examined CpGs in promoter regions of MMP2, MMP23B, MMP24, MMP25, and MMP28 in tumors was associated with low HER2 expression, while the group of samples with abnormal hypermethylation of at least two of these MMP genes was significantly enriched with HER2-positive tumors. Abnormal methylation of MMP24 and MMP25 was significantly associated with a CpG island hypermethylated breast cancer subtype discovered by genome-wide DNA bisulfite sequencing. Our results indicate that abnormal hypermethylation of at least several MMP genes promoters is a secondary event not directly functional in breast cancer (BC) pathogenesis. We suggest that it is elevated and/or ectopic expression, rather than methylation-driven silencing, that might link MMPs to tumorigenesis.

Ontogenetic and Pathogenetic Views on Somatic Chromosomal Mosaicism.

Intercellular karyotypic variability has been a focus of genetic research for more than 50 years. It has been repeatedly shown that chromosome heterogeneity manifesting as chromosomal mosaicism is associated with a variety of human diseases. Due to the ability of changing dynamically throughout the ontogeny, chromosomal mosaicism may mediate genome/chromosome instability and intercellular diversity in health and disease in a bottleneck fashion. However, the ubiquity of negligibly small populations of cells with abnormal karyotypes results in difficulties of the interpretation and detection, which may be nonetheless solved by post-genomic cytogenomic technologies. In the post-genomic era, it has become possible to uncover molecular and cellular pathways to genome/chromosome instability (chromosomal mosaicism or heterogeneity) using advanced whole-genome scanning technologies and bioinformatic tools. Furthermore, the opportunities to determine the effect of chromosomal abnormalities on the cellular phenotype seem to be useful for uncovering the intrinsic consequences of chromosomal mosaicism. Accordingly, a post-genomic review of chromosomal mosaicism in the ontogenetic and pathogenetic contexts appears to be required. Here, we review chromosomal mosaicism in its widest sense and discuss further directions of cyto(post)genomic research dedicated to chromosomal heterogeneity.

Antioxidant Properties of Fullerene Derivatives Depend on Their Chemical Structure: A Study of Two Fullerene Derivatives on HELFs.

Oxidative stress is a major issue in a wide number of pathologies (neurodegenerative, cardiovascular, immune diseases, and cancer). Because of this, the search for new antioxidants is an important issue. One of the potential antioxidants that has been enthusiastically discussed in the past twenty years is fullerene and its derivatives. Although in aqueous solutions fullerene derivatives have shown to be antioxidants, their properties in this regard within the cells are controversially discussed. We have studied two different water-soluble fullerene C60 and C70 derivatives on human embryonic lung fibroblasts at a wide range of concentrations. Both of them cause a decrease in cellular ROS at short times of incubation (1 hour). Their prolonged action, however, is fundamentally different: derivative GI-761 causes secondary oxidative stress whereas derivative VI-419-P3K keeps ROS levels under control values. To gain a better understanding of this effect, we assessed factors that could play a role in the response of cells to fullerene derivatives. Increased ROS production occurred due to NOX4 upregulation by GI-761. Derivative VI-419-P3K activated the transcription of antioxidant master regulator NRF2 and caused its translocation to the nucleus. This data suggests that the antioxidant effect of fullerene derivatives depends on their chemical structure.

QF-PCR examination of parental and meiotic origin of trisomy 21 in Central and Eastern Europe.

Study of parental/meiotic origin of free trisomy 21 in nuclear families from Russia (70 cases), Ukraine (32 cases), and 22 from Germany revealed maternal nondisjunction in 77.3% (Germany), 93.8% (Ukraine), and 91.4% (Russia), paternal origin in 13.6%, 6.2%, and 8.6%, respectively. Maternal meiosis I errors were found in 84.4% (Ukraine), 77.1% (Russia), paternal origin in 3.1% (Ukraine), 2.9% (Russia). Maternal meiosis II errors occurred in 9.4% and 14.3% and paternal in 3.1% and 5.7% in Ukraine and Russia, respectively. No significant differences were found in maternal/paternal origin among Ukraine, Russia, Germany, and published data from other European regions.

Aneuploidy and confined chromosomal mosaicism in the developing human brain.

BACKGROUND: Understanding the mechanisms underlying generation of neuronal variability and complexity remains the central challenge for neuroscience. Structural variation in the neuronal genome is likely to be one important mechanism for neuronal diversity and brain diseases. Large-scale genomic variations due to loss or gain of whole chromosomes (aneuploidy) have been described in cells of the normal and diseased human brain, which are generated from neural stem cells during intrauterine period of life. However, the incidence of aneuploidy in the developing human brain and its impact on the brain development and function are obscure. METHODOLOGY/PRINCIPAL FINDINGS: To address genomic variation during development we surveyed aneuploidy/polyploidy in the human fetal tissues by advanced molecular-cytogenetic techniques at the single-cell level. Here we show that the human developing brain has mosaic nature, being composed of euploid and aneuploid neural cells. Studying over 600,000 neural cells, we have determined the average aneuploidy frequency as 1.25-1.45% per chromosome, with the overall percentage of aneuploidy tending to approach 30-35%. Furthermore, we found that mosaic aneuploidy can be exclusively confined to the brain. CONCLUSIONS/SIGNIFICANCE: Our data indicates aneuploidization to be an additional pathological mechanism for neuronal genome diversification. These findings highlight the involvement of aneuploidy in the human brain development and suggest an unexpected link between developmental chromosomal instability, intercellural/intertissular genome diversity and human brain diseases.