3D organization of chicken genome demonstrates evolutionary conservation of topologically associated domains and highlights unique architecture of erythrocytes' chromatin.

How chromosomes are folded, spatially organized and regulated in three dimensions inside the cell nucleus are among the longest standing questions in cell biology. Genome-wide chromosome conformation capture (Hi-C) technique allowed identifying and characterizing spatial chromatin compartments in several mammalian species. Here, we present the first genome-wide analysis of chromatin interactions in chicken embryonic fibroblasts (CEF) and adult erythrocytes. We showed that genome of CEF is partitioned into topologically associated domains (TADs), distributed in accordance with gene density, transcriptional activity and CTCF-binding sites. In contrast to mammals, where all examined somatic cell types display relatively similar spatial organization of genome, chicken erythrocytes strongly differ from fibroblasts, showing pronounced A- and B- compartments, absence of typical TADs and formation of long-range chromatin interactions previously observed on mitotic chromosomes. Comparing mammalian and chicken genome architectures, we provide evidence highlighting evolutionary role of chicken TADs and their significance in genome activity and regulation.

Heterochromatic regions in Japanese quail chromosomes: comprehensive molecular-cytogenetic characterization and 3D mapping in interphase nucleus.

Chromosomes of Japanese quail (Coturnix coturnix japonica, 2n=78), a galliform domestic species closely related to chicken, possess multiple heterochromatic segments. Due to the difficulties in careful analysis of such heterochromatic regions, there is a lack of data on their DNA composition, epigenetic status, as well as spatial distribution in interphase nucleus. In the present study, we applied giant lampbrush chromosome (LBC) microdissection for high-resolution analysis of quail centromeric regions of macrochromosomes and polymorphic short arms of submetacentric microchromosomes. FISH with the dissected material on mitotic and meiotic chromosomes indicated that in contrast to centromeres of chicken macrochromosomes, which are known to harbor chromosome-specific and, in some cases, tandem repeat-free sequences, centromeres of quail macroautosomes (CCO1-CCO11) have canonical organization. CCO1-CCO11 centromeres possess massive blocks of common DNA repeats demonstrating transcriptional activity at LBC stage. These repeats seem to have been subjected to chromosome size-correlated homogenization previously described primarily for avian microchromosomes. In addition, comparative FISH on chicken chromosomes supported the previous data on centromere repositioning events during galliform karyotype evolution. In interphase nucleus of different cell types, repetitive elements specific for microchromosome short arms constitute the material of prominent centrally located chromocenters enriched with markers of constitutive heterochromatin and rimmed with clusters of microchromosomal centromeric BglII-repeat. Thus, clustering of such repeats is responsible for the peculiar architecture of quail interphase nucleus. In contrast, centromere repeats of the largest macrochromosomes (CCO1 and CCO2) are predominantly localized in perinuclear heterochromatin. The possible involvement of the isolated repeats in radial genome organization is discussed.

Microdissection of lampbrush chromosomes as an approach for generation of locus-specific FISH-probes and samples for high-throughput sequencing.

BACKGROUND: Over the past two decades, chromosome microdissection has been widely used in diagnostics and research enabling analysis of chromosomes and their regions through probe generation and establishing of chromosome- and chromosome region-specific DNA libraries. However, relatively small physical size of mitotic chromosomes limited the use of the conventional chromosome microdissection for investigation of tiny chromosomal regions. RESULTS: In the present study, we developed a workflow for mechanical microdissection of giant transcriptionally active lampbrush chromosomes followed by the preparation of whole-chromosome and locus-specific fluorescent in situ hybridization (FISH)-probes and high-throughput sequencing. In particular, chicken (Gallus g. domesticus) lampbrush chromosome regions as small as single chromomeres, individual lateral loops and marker structures were successfully microdissected. The dissected fragments were mapped with high resolution to target regions of the corresponding lampbrush chromosomes. For investigation of RNA-content of lampbrush chromosome structures, samples retrieved by microdissection were subjected to reverse transcription. Using high-throughput sequencing, the isolated regions were successfully assigned to chicken genome coordinates. As a result, we defined precisely the loci for marker structures formation on chicken lampbrush chromosomes 2 and 3. Additionally, our data suggest that large DAPI-positive chromomeres of chicken lampbrush chromosome arms are characterized by low gene density and high repeat content. CONCLUSIONS: The developed technical approach allows to obtain DNA and RNA samples from particular lampbrush chromosome loci, to define precisely the genomic position, extent and sequence content of the dissected regions. The data obtained demonstrate that lampbrush chromosome microdissection provides a unique opportunity to correlate a particular transcriptional domain or a cytological structure with a known DNA sequence. This approach offers great prospects for detailed exploration of functionally significant chromosomal regions.

Three-dimensional architecture of tandem repeats in chicken interphase nucleus.

Tandem repeats belong to a class of genomic repetitive elements that form arrays of head-to-tail monomers. Due to technical difficulties in sequencing and assembly of large tandem repeat arrays, it remains largely unknown by which mechanisms tandem-repeat-containing regions aid in maintenance of ordered radial genome organization during interphase. Here we analyzed spatial distribution of several types of tandem repeats in interphase nuclei of chicken MDCC-MSB1 cells and somatic tissues relative to heterochromatin compartments and nuclear center. We showed that telomere and subtelomere repeats generally localize at the nuclear or chromocenters periphery. A tandem repeat known as CNM, typical for centromere regions of gene-dense microchromosomes, forms interchromosome clusters and occupies DAPI-positive chromocenters that appear predominantly within the nuclear interior. In contrast, centromere-specific tandem repeats of the majority of gene-poor macrochromosomes are embedded into the peripheral layer of heterochromatin. Chicken chromocenters rarely comprise centromere sequences of both macro- and microchromosomes, whose territories localize in different radial nuclear zones. Possible mechanisms of observed tandem repeats positioning and its implication in highly ordered arrangement of chromosome territories in chicken interphase nucleus are discussed.

Characterization of pathogenic genetic variants in Russian patients with primary ciliary dyskinesia using gene panel sequencing and transcript analysis.

BACKGROUND: Primary ciliary dyskinesia (PCD) is a group of rare genetically heterogeneous disorders caused by defective cilia and flagella motility. The clinical phenotype of PCD patients commonly includes chronic oto-sino-pulmonary disease, infertility, and, in about half of cases, laterality defects due to randomization of left-right body asymmetry. To date, pathogenic variants in more than 50 genes responsible for motile cilia structure and assembly have been reported in such patients. While multiple population-specific mutations have been described in PCD cohorts from different countries, the data on genetic spectrum of PCD in Russian population are still extremely limited. RESULTS: The present study provides a comprehensive clinical and genetic characterization of 21 Russian families with PCD living in various country regions. Anomalies of ciliary beating in patients` respiratory epithelial cells were confirmed by high-speed video microscopy. In the most cases, custom-designed panel sequencing allowed to uncover causative variants in well-known or rarely mentioned PCD-related genes, including DNAH5, DNAH11, CFAP300, LRRC6, ZMYND10, CCDC103, HYDIN, ODAD4, DNAL1, and OFD1. The variations comprised common mutations, as well as novel genetic variants, some of which probably specific for Russian patients. Additional targeted analysis of mRNA transcripts from ciliated cells enabled us to specify functional effects of newly identified genetic variants in DNAH5 (c.2052+3G>T, c.3599-2A>G), HYDIN (c.10949-2A>G, c.1797C>G), and ZMYND10 (c.510+1G>C) on splicing process. In particular, the splice site variant c.2052+3G>T, detected in four unrelated families, resulted in skipping of exon 14 in DNAH5 transcripts and, according to haplotype analysis of affected probands, was proposed as an ancestral founder mutation in Udmurt population. CONCLUSIONS: The reported data provide a vital insight into genetic background of primary ciliary dyskinesia in the Russian population. The findings clearly illustrate the utility of gene panel sequencing coupled with transcriptional analysis in identification and clinical interpretation of novel genetic variants.

High-resolution mapping and transcriptional activity analysis of chicken centromere sequences on giant lampbrush chromosomes.

Exploration into morphofunctional organisation of centromere DNA sequences is important for understanding the mechanisms of kinetochore specification and assembly. In-depth epigenetic analysis of DNA fragments associated with centromeric nucleosome proteins has demonstrated unique features of centromere organisation in chicken karyotype: there are both mature centromeres, which comprise chromosome-specific homogeneous arrays of tandem repeats, and recently evolved primitive centromeres, which consist of non-tandemly organised DNA sequences. In this work, we describe the arrangement and transcriptional activity of chicken centromere repeats for Cen1, Cen2, Cen3, Cen4, Cen7, Cen8, and Cen11 and non-repetitive centromere sequences of chromosomes 5, 27, and Z using highly elongated lampbrush chromosomes, which are characteristic of the diplotene stage of oogenesis. The degree of chromatin packaging and fine spatial organisations of tandemly repetitive and non-tandemly repetitive centromeric sequences significantly differ at the lampbrush stage. Using DNA/RNA FISH, we have demonstrated that during the lampbrush stage, DNA sequences are transcribed within the centromere regions of chromosomes that lack centromere-specific tandem repeats. In contrast, chromosome-specific centromeric repeats Cen1, Cen2, Cen3, Cen4, Cen7, Cen8, and Cen11 do not demonstrate any transcriptional activity during the lampbrush stage. In addition, we found that CNM repeat cluster localises adjacent to non-repetitive centromeric sequences in chicken microchromosome 27 indicating that centromere region in this chromosome is repeat-rich. Cross-species FISH allowed localisation of the sequences homologous to centromeric DNA of chicken chromosomes 5 and 27 in centromere regions of quail orthologous chromosomes.

Centromere positions in chicken and Japanese quail chromosomes: de novo centromere formation versus pericentric inversions.

Chicken (Gallus gallus domesticus, GGA) and Japanese quail (Coturnix coturnix japonica, CCO) karyotypes are very similar. They have identical chromosome number (2n = 78) and show a high degree of synteny. Centromere positions on the majority of orthologous chromosomes are different in these two species. To explore the nature of this divergence, we used high-resolution comparative fluorescent in situ hybridization mapping on giant lampbrush chromosomes (LBCs) from growing oocytes. We applied 41 BAC clones specific for GGA1, 2, 3, 11, 12, 13, 14, and 15 to chicken and quail LBCs. This approach allowed us to rule out a pericentric inversion earlier proposed to explain the difference between GGA1 and CCO1. In addition to a well-established large-scale pericentric inversion that discriminates GGA2 and CCO2, we identified another, smaller one in the large inverted region. For the first time, we described in detail inversions that distinguish GGA3 from CCO3 and GGA11 from CCO11. Despite the newly identified and confirmed inversions, our data suggest that, in chicken and Japanese quail, the difference in centromere positions is not mainly caused by pericentric inversions but is instead due to centromere repositioning events and the formation of new centromeres. We also consider the formation of short arms of quail microchromosomes by heterochromatin accumulation as a third scenario that could explain the discrepancy in centromeric indexes.

Generation of iPSC line FAMRCi010-A from patient with restrictive cardiomyopathy carrying genetic variant FLNC p.Gly2011Arg.

Human iPSC cell line FAMRCi010-A was generated from a patient with restrictive cardiomyopathy carrying FLNC p.Gly2011Arg genetic variant. Patient-specific peripheral blood mononuclear cells were reprogrammed using non-integrative Sendai viruses containing OCT4, SOX2, KLF4, and CMYC. FAMRCi010-A was generated and characterized through the study. The reported iPSC line could be useful tool for in vitro modeling of FLNC-associated cardiomyopathies.

Generation of iPSC line (FAMRCi009-A) from patient with familial progressive cardiac conduction disorder carrying genetic variant FLNC p.Val2264Met.

Human iPSC cell line FAMRCi009-A was generated from a patient with restrictive cardiomyopathy and congenital myopathy carrying FLNC p.Val2264Met genetic variant. Patient-specific peripheral blood mononuclear cells were reprogrammed using non-integrative Sendai viruses. Generated iPSC lines showed normal karyotype, expressed pluripotency markers and exhibited trilineage differentiation potential in vitro. The reported iPSC lines could be used for a deeper study of filaminopathies.

Case Report: A novel PHOX2B p.Ala248_Ala266dup variant causing congenital central hypoventilation syndrome.

Introduction: Congenital central hypoventilation syndrome (CCHS) is a rare disease characterized by central alveolar hypoventilation and impaired autonomic regulation, caused by pathogenic variants of PHOX2B gene. More than 90% of patients have a polyalanine repeat mutation (PARM) in the heterozygous state, characterized by the expansion of GCN repeats and an increase in the number of alanine repeats, so that genotypes 20/24-20/33 are formed (the normal genotype is 20/20). The remaining 10% of patients harbor non-PARMs. Case description: We present a clinical case of a girl with a novel PHOX2B heterozygous genetic variant in the exon 3: NM_003924.4: c.735_791dup, p.Ala248_Ala266dup. The duplication includes 16 GCN (alanine) repeats and 3 adjacent amino acids. Both clinically healthy parents demonstrated a normal PHOX2B sequence. In addition, the girl has a variant of unknown significance in RYR1 gene and a variant of unknown significance in NKX2-5 gene. The child's phenotype is quite special. She needs ventilation during sleep, and has Hirschsprung's disease type I, arteriovenous malformation S4 of the left lung, ventricular and atrium septal defects, coronary right ventricular fistula, hemodynamically nonsignificant, episodes of sick sinus and atrioventricular dissociation with bradycardia, divergent alternating strabismus, and oculus uterque (both eyes) (OU) retinal angiopathy. Two episodes of hypoglycemic seizures were also registered. Severe pulmonary hypertension resolved after appropriate ventilation adjustment. Diagnostic odyssey was quite dramatic. Conclusion: Detection of a novel PHOX2B variant expands the understanding of molecular mechanisms of CCHS and genotype-phenotype correlations.

Case Report: COVID-19-Associated ROHHAD-Like Syndrome.

It is known that the SARS-CoV-2 virus may cause neurologic damage. Rapid-onset obesity, hypoventilation, hypothalamus dysfunction, and autonomic dysregulation (ROHHAD) syndrome is a disease of unknown etiology with a progressive course and unclear outcomes. The etiology of ROHHAD syndrome includes genetic, epigenetic, paraneoplastic, and immune-mediated theories, but to our knowledge, viral-associated cases of the disease have not been described yet. Here we present the case of a 4-year-old girl who developed a ROHHAD syndrome-like phenotype after a COVID-19 infection and the results of 5 months of therapy. She had COVID-19 pneumonia, followed by electrolyte disturbances (hypernatremia and hyperchloremia), hypocorticism and hypothyroidism, central hypoventilation-requiring prolonged assisted lung ventilation-bulimia, and progressive obesity with hypertriglyceridemia, dyslipidemia, hyperuricemia, and hyperinsulinemia. The repeated MRI of the brain and hypothalamic-pituitary region with contrast enhancement showed mild post-hypoxic changes. Prader-Willi/Angelman syndrome as well as PHOX2B-associated variants was ruled out. Treatment with non-steroidal anti-inflammatory drugs and monthly courses of intravenous immunoglobulin led to a dramatic improvement. Herein the first description of ROHHAD-like syndrome is timely associated with a previous COVID-19 infection with possible primarily viral or immune-mediated hypothalamic involvement.

Case Reports: Emery-Dreifuss Muscular Dystrophy Presenting as a Heart Rhythm Disorders in Children.

Emery-Dreifuss muscular dystrophy (EDMD) is inherited muscle dystrophy often accompanied by cardiac abnormalities in the form of supraventricular arrhythmias, conduction defects and sinus node dysfunction. Cardiac phenotype typically arises years after skeletal muscle presentation, though, could be severe and life-threatening. The defined clinical manifestation with joint contractures, progressive muscle weakness and atrophy, as well as cardiac symptoms are observed by the third decade of life. Still, clinical course and sequence of muscle and cardiac signs may be variable and depends on the genotype. Cardiac abnormalities in patients with EDMD in pediatric age are not commonly seen. Here we describe five patients with different forms of EDMD (X-linked and autosomal-dominant) caused by the mutations in EMD and LMNA genes, presented with early onset of cardiac abnormalities and no prominent skeletal muscle phenotype. The predominant forms of cardiac pathology were atrial arrhythmias and conduction disturbances that progress over time. The presented cases discussed in the light of therapeutic strategy, including radiofrequency ablation and antiarrhythmic devices implantation, and the importance of thorough neurological and genetic screening in pediatric patients presenting with complex heart rhythm disorders.

Mapping epigenetic modifications on chicken lampbrush chromosomes.

BACKGROUND: The epigenetic regulation of genome is crucial for implementation of the genetic program of ontogenesis through establishing and maintaining differential gene expression. Thus mapping of various epigenetic modifications to the genome is relevant for studying the regulation of gene expression. Giant transcriptionally active lampbrush chromosomes are an established tool for high resolution physical mapping of the genome and its epigenetic modifications. This study is aimed at characterizing the epigenetic status of compact chromatin domains (chromomeres) of chicken lampbrush macrochromosomes. RESULTS: Distribution of three epigenetic modifications - 5-methylcytosine, histone H3 trimethylated at lysine 9 and hyperacetylated histone H4 - along the axes of chicken lampbrush chromosomes 1-4, Z and W was analyzed in details. Enrichment of chromatin domains with the investigated epigenetic modifications was indicated on the cytological chromomere-loop maps for corresponding chicken lampbrush chromosomes. Heterogeneity in the distribution of 5-methylcytosine and histone H3 trimethylated at lysine 9 along the chromosome axes was revealed. CONCLUSIONS: On examples of certain chromomeres of chicken lampbrush chromosomes 1, 3, 4 and W we demonstrated that a combination of immunofluorescent staining and fluorescence in situ hybridization allows to relate the epigenetic status and a DNA sequence context of individual chromomeres.

Desmin mutations result in mitochondrial dysfunction regardless of their aggregation properties.

Desmin, being a major intermediate filament of muscle cells, contributes to stabilization and positioning of mitochondria. Desmin mutations have been reported in conjunction with skeletal myopathies accompanied by mitochondrial dysfunction. Depending on the ability to promote intracellular aggregates formation, mutations can be considered aggregate-prone or non-aggregate-prone. The aim of the present study was to describe how expression of different desmin mutant isoforms effects mitochondria and contributes to the development of myocyte dysfunction. To achieve this goal, two non-aggregate-prone (Des S12F and Des A213V) and four aggregate-prone (Des L345P, Des A357P, Des L370P, Des D399Y) desmin mutations were expressed in skeletal muscle cells. We showed that all evaluated mutations affected the morphology of mitochondrial network, suppressed parameters of mitochondrial respiration, diminished mitochondrial membrane potential, increased ADP/ATP ratio, and enhanced mitochondrial DNA (mtDNA) release. mtDNA was partially secreted through exosomes as demonstrated by GW4869 treatment. Dysfunction of mitochondria was observed regardless the type of mutation: aggregate-prone or non-aggregate-prone. However, expression of aggregate-prone mutations resulted in more prominent phenotype. Thus, in this comparative study of six pathogenic desmin mutations that cause skeletal myopathy development, we confirmed a role of mitochondrial dysfunction and mtDNA release in the pathogenesis of desmin myopathies, regardless of the aggregation capacity of the mutated desmin.

New Insights Into Chromomere Organization Provided by Lampbrush Chromosome Microdissection and High-Throughput Sequencing.

Giant lampbrush chromosomes (LBCs) typical for growing oocytes of various animal species are characterized by a specific chromomere-loop appearance and massive transcription. Chromomeres represent universal units of chromatin packaging at LBC stage. While quite good progress has been made in investigation of LBCs structure and function, chromomere organization still remains poorly understood. To extend our knowledge on chromomere organization, we applied microdissection to chicken LBCs. In particular, 31 and 5 individual chromomeres were dissected one by one along the macrochromosome 4 and one microchromosome, respectively. The data on genomic context of individual chromomeres was obtained by high-throughput sequencing of the corresponding chromomere DNA. Alignment of adjacent chromomeres to chicken genome assembly provided information on chromomeres size and genomic boarders, indicating that prominent marker chromomeres are about 4-5 Mb in size, while common chromomeres of 1.5-3.5 Mb. Analysis of genomic features showed that the majority of chromomere-loop complexes combine gene-dense and gene-poor regions, while massive loopless DAPI-positive chromomeres lack genes and are remarkably enriched with different repetitive elements. Finally, dissected LBC chromomeres were compared with chromatin domains (topologically associated domains [TADs] and A/B-compartments), earlier identified by Hi-C technique in interphase nucleus of chicken embryonic fibroblasts. Generally, the results obtained suggest that chromomeres of LBCs do not correspond unambiguously to any type of well-established spatial domains of interphase nucleus in chicken somatic cells.

Generation of two iPSC lines (FAMRCi004-A and FAMRCi004-B) from patient with familial progressive cardiac conduction disorder carrying genetic variant DSP p.His1684Arg.

Human iPSC cell lines (FAMRCi004-A and FAMRCi004-B) were generated from patient with progressive cardiac conduction disease and sick sinus syndrome carrying DSP p.His1684Arg genetic variant. Patient-specific adipose tissue-derived mesenchymal multipotent stromal cells were reprogrammed using non-integrative Sendai viruses. Established iPSC lines showed normal karyotype, expressed pluripotent markers and were able to differentiate toward three germ layers in vitro. The reported iPSC lines could be useful tool for in vitro modeling of progressive cardiac conduction disease associated with mutations in desmosomal genes.

Generation of two induced pluripotent stem cell lines (FAMRCi005-A and FAMRCi005-B) from patient carrying genetic variant LMNA p.Asp357Val.

LMNA mutations are often linked to laminopathies characterized by tissue-specific disorders. We generated two induced pluripotent stem cells lines from patient carrying genetic variant LMNA p.Asp357Val associated with paroxysmal ventricular tachycardia and myopathy. Reprogramming of patient's peripheral blood mononuclear cells was performed using Sendai viruses. Characterization of the FAMRCi005-A and FAMRCi005-B lines revealed that generated iPSC lines expressed pluripotent stem cell markers, had normal karyotype and demonstrated triliniage differentiation ability. Generated cell lines can be used to investigate the molecular links between LMNA genetic variants and cardiac disorders.

Generation of two iPSC lines (FAMRCi006-A and FAMRCi006-B) from patient with dilated cardiomyopathy and Emery-Dreifuss muscular dystrophy associated with genetic variant LMNAp.Arg527Pro.

Mutations in LMNA gene are known to cause a broad range of diseases called laminopathies. We have generated two induced pluripotent stem cell lines FAMRCi006-A and FAMRCi006-B from a patient carrying LMNA p. p.Arg527Pro mutation associated with Emery-Dreifuss muscular dystrophy and dilated cardiomyopathy. Patient-specific peripheral blood mononuclear cells were reprogrammed to iPSCs using Sendai virus reprogramming system. Characterization of iPSCs had revealed pluripotency marker expression, normal karyotype, ability to differentiate into three embryonic germ layers. The reported iPSC lines could be a useful tool for in vitro modeling of laminopathies associated with LMNA genetic variants.

Generation of two iPSC lines (FAMRCi007-A and FAMRCi007-B) from patient with Emery-Dreifuss muscular dystrophy and heart rhythm abnormalities carrying genetic variant LMNA p.Arg249Gln.

Human iPSC lines were generated from peripheral blood mononuclear cells of patient carrying LMNA mutation associated with Emery-Dreifuss muscular dystrophy accompanied by atrioventricular block and paroxysmal atrial fibrillation. Reprogramming factors OCT4, KLF4, SOX2, CMYC were delivered using Sendai virus transduction. iPSCs were characterized in order to prove the pluripotency markers expression, normal karyotype, ability to differentiate into three embryonic germ layers. Generated iPSC lines would be useful model to investigate disease development associated with genetic variants in LMNA gene.

Two New Cases of Hypertrophic Cardiomyopathy and Skeletal Muscle Features Associated with ALPK3 Homozygous and Compound Heterozygous Variants.

Hypertrophic cardiomyopathy associated with damaging variants in the ALPK3 gene is a fairly recent discovery, and only a small number of patients have been described thus far. Here we present two additional patients with hypertrophic cardiomyopathy caused by biallelic variants in ALPK3. Genetic investigation was performed using a targeted gene panel consisting of known cardiomyopathy-associated genes and whole exome sequencing. The patients showed a large difference in the age of onset, and both presented with extracardiac features that are often seen in ALPK3 patients. The patient with the later onset showed milder extracardiac symptoms, such as decreased muscle tone and distal muscular dystrophy, but had fast progression of cardiac complications leading to the need of heart transplantation. This study further elucidates the variability of both symptoms and age of onset among these patients.