Annotation of the results obtained in 2021
We carried out research in three Work Packages: creation and analysis of in vivo models of hereditary diseases accompanied by impaired intellectual development; creation and analysis of in vitro models; development of new diagnostic technologies. In addition, throughout this period, we recruited patients with idiopathic intellectual disability in order to search for new gene and chromosomal variants for modeling. WP1. Creation and analysis of in vivo models. To create in vivo (mouse) model of Nascimento syndrome (OMIM 300860), genetic constructs for overexpression, knockout and knockdown of the Ube2a gene were designed. The efficiency of overexpression and knockout of the Ube2a gene was confirmed by testing in mouse neuroblastoma cells N2A, followed by detection by Western blot analysis. Analysis of the expression of mRNA of the Ube2a gene at different embryonic stages, carried out using in situ hybridization, demonstrated that Ube2a mRNA is detected in neuronal precursors at stages E12.5, E14.5, and E15.5. At stages E16.5 and E18.5, expression of Ube2a is also detected in the cortical plate. Further, the Ube2a was inactivated in the cerebral cortex of mouse embryos by in utero electroporation of genetic constructs into the lateral ventricles of the embryonic brain. As a result, delayed migration of cortical neurons was found with a decreased expression of the Ube2a gene in comparison with the control. To simulate the effects of epigenetic silencing of chromosomal aberrations in carriers of small supernumerary marker chromosomes (sSMC), we began to create experimental mouse strains with deletions in the pericentromeric and peritelomeric regions of chromosomes using CRISPR/Cas9 genome editing. Chromosomal rearrangements were introduced by microinjection of components of the CRISPR/Cas9 system into the cytoplasm of mouse zygotes. Currently, we have received 384 zygotes from 35 female mice of the C57BL line; 167 zygotes were injected with components of the CRISPR/Cas9 system. Of these, 119 zygotes were transplanted, however, only 8 offspring were obtained out of 86 transplanted zygotes with termed gestation, for another 33 zygotes the results are still pending. WP2. Creation and analysis of in vitro models. We studied the case of carriage of the small supernumerary marker chromosome (sSMC) detected during prenatal diagnosis in the fetus. To determine the origin and structure of the marker chromosome, standard cytogenetic analysis, SNP-microarrays, quantitative real-time PCR, FISH analysis with centromere-specific, telomere-specific and locus-specific DNA probes; chromosomal microdissection with reverse in situ hybridization; whole genome amplification and sequencing of a microdissection DNA library from single copy of marker chromosome were performed. We found that sSMC is a ring-shape derivative of chromosome 10 and includes the regions 10p11.21-p11.1 and 10q11.21-q11.23 of 1.243 and 7.173 Mb, respectively. The presence in the karyotype of an additional chromosomal material with a size of about 9 Mb, containing 107 genes, including 7 genes indexed in OMIM, did not lead to any clinical manifestations at the time of the child's birth. This allowed us to formulate a hypothesis about the mechanisms of epigenetic silencing of genes localized in the pericentromeric region of the ring marker chromosome (Lebedev et al., 2021; PMID 34440234). Since this family refused to further participate in the study, we began in vitro modeling of the effects of epigenetic and transcriptional gene silencing in pericentromeric regions of chromosomes based on cell lines from another healthy carrier of sSMC - a derivative of chromosome 4. We established the patient's fibroblast line (TAF16) from which we obtained 12 iPSC lines. Standard cytogenetic analysis revealed that 72% of iPS cells contain sSMC. The chromosomal origin of sSMC was determined by FISH with pericentromeric DNA-probes, demonstrating that sSMC is a derivative of chromosome 4, and FISH with telomere-specific and locus-specific probes, indicating its ring shape in iPSCs. Using directed differentiation by overexpression of the NGN2 gene, we obtained lines of cortical neurons from iPSCs lines of the patient with microduplication of the CNTN6 gene, line with a corrected genotype, and control lines. To determine the reasons for the variability in the manifestation of microduplications at the 3p26.3 locus, we evaluated the distribution of epigenetic markers localized near the CNTN6 gene. ChIP-seq analysis showed that for patient-specific iPSCs with duplication, heterochromatinization of this region is characteristic, extending beyond it to an area up to 7 Mb. Heterochromatinization of the duplicated region in iPSCs indicates a decreased expression of the CNTN6 gene and/or genes of long noncoding RNAs located in this region. This is consistent with our previously published data on the imbalance in the allelic expression of CNTN6 in a patient with duplication (Gridina et al., 2018, PMID 29327201), and confirm the hypothesis about the influence of the epigenetic status of the duplicated region on the phenotypic manifestations of copy number alterations. To study the role of the UBE2A gene in human neurogenesis, we creating a cell model based on iPSCs. The model will include iPSCs from patients with neurogenesis abnormalities, intellectual disability and rearrangements in the Xq24 segment (microdeletion and microduplication), iPSCs from their healthy mothers - carriers of similar chromosomal rearrangements, iPSCs with knockout and overexpression of the UBE2A gene, and control iPSCs. To date, 10 iPSC lines have been obtained from two asymptomatic carriers of rearrangements and a patient with Xq24 microdeletion (a total of 30 lines with rearrangements affecting the UBE2A gene locus). We began the creation of knockout cell lines and lines with hyperexpression of the UBE2A gene by editing the iPSC genome. To knockout the UBE2A gene, we used CRISPR-Cas9 genomic editing of iPSC line FD5S from a healthy donor with karyotype 46,XX, followed by results evaluation using Sanger sequencing. Eighteen clones (44%) carried a knockout indel in the UBE2A gene in a homozygous state, 7 clones (18%) were compound heterozygous for knockout indels, one clone was heterozygous for a knockout single nucleotide insertion. Interestingly, the proliferative activity of the UBE2A knockout clones was higher than that of the initial line. We studied differentially expressed genes (DEG) in neurons obtained from iPSC lines from patients with microdeletions and microduplications of the CNTN6 gene and healthy individuals. It was shown predominantly a decrease in DEG expression for neurons with different CNTN6 copy numbers in comparison with control. We identified 4 categories of transcriptional effects of reciprocal CNVs affecting this gene: 1) unidirectional increase in expression (25 DEG); 2) unidirectional decrease in expression (402 DEG); 3) decreased expression in duplication and increased expression of these genes in CNTN6 deletion (51 DEG); 4) increased gene expression in duplication and decreased expression of these genes in CNTN6 deletion (51 DEG). The products of total DEG in neurons with microdeletion and microduplication of CNTN6 are involved in synaptic signal transmission and neurogenesis. WP3. Development of diagnostic technologies. We collected a sample of families to identify new, undescribed mutations that lead to brain malformations and intellectual disabilities. Previously, we excluded the patients with numerical and large structural chromosomal abnormalities, hereditary metabolic disorders and known monogenic hereditary syndromes. Whole exome sequencing was performed in 4 families with familial forms of intellectual disability. We revealed a de novo missense mutation in a heterozygous state on chromosome 1 in the MACF1 gene, affecting a conservative amino acid in patient M. (7 years old) with Dandy-Walker syndrome, agenesis of the corpus callosum, pachygiria of the frontal lobes, epileptic encephalopathy. The clinical significance of this variant is currently unknown; it was not described in the literature as of December 2021. The results of in silico algorithms predict the pathogenic effect of this substitution on the protein structure. To search for CNVs with pleiotropic effects in the prenatal and postnatal periods of development, we performed a chromosomal microarray analysis of 25 paired samples of placental tissues (chorion and extraembryonic mesoderm, which are derivatives of different germ layers) from spontaneous abortions with a normal karyotype previously determined by conventional G-banding analysis of metaphase chromosomes. CNVs were analyzed for type, gene content, clinical relevance, and origin. We detected several common CNVs for miscarriages and patients with intellectual disabilty. Interestingly, in addition to CNVs identified in two types of placental tissues, we found a large number of mosaic CNVs of mitotic origin. We began to study the characteristics of fetal extracellular DNA fractions associated with the cell surface and circulating in the mother's blood. Free-circulating extracellular DNA (plasma fraction) and extracellular DNA associated with the cell surface by weak ionic interactions (“weakly bound”) were isolated from peripheral blood of women carrying a fetus with chromosomal aneuploidy. Blood samples from 21 pregnant women was obtained and fractionated. The concentration and length distribution of extracellular DNA fragments were determined. We elaborate new approach for detecting chromosomal microdeletions/microduplications during preimplantation genetic testing (PGT) for families where the spouse is a healthy carrier of pathogenic CNV. This approach combines the principles of PGT-M for monogenic hereditary diseases with the haplotypes analysis marking the CNV-bearing chromosome and PGT for aneuploidy (PGT-A) to exclude embryos with numerical chromosomal abnormalities. The need for combined approach is due to the insufficient resolution of aCGH and NGS methods for the detection of CNVs after whole genome amplification of single cells in a trophectoderm biopsy specimen. The developed test system for preimplantation genetic diagnosis of microdeletions syndromes allowed to choose an embryo with a balanced karyotype for transfer in a family with delXq24, but the pregnancy didn’t achieved. In a family with del1q41, both embryos obtained had additional aneuploidies and were not recommended for transfer. A reference sample of 22 patients with known microdeletion/microduplication syndromes associated with intellectual disability and psychomotor developmental disorders was formed. Clinical characteristics of patients and molecular cytogenetic analysis using aCGH, followed by real-time PCR verification, were carried out. For each patient, we characterized the size and boundaries of rearrangements and its genetic content, and prioritized the genes, probably associated with the development of the disease symptoms. We develop a technology for the simultaneous analysis of 3D chromatin organization and its sequencing (Exo-C), which makes it possible to identify balanced chromosomal rearrangements with high accuracy. For this purpose 11 individuals with inversions and balanced translocations and 2 individuals with unbalanced rearrangements were recruited in our study. For the first time, the genomes of patients with inversions were analyzed using the Exo-C method. A characteristic change in the pattern of three-dimensional chromosomal contacts was shown, indicating the presence of large inversions. Visual analysis of the obtained Exo-C-maps made it possible to confirm the presence of inversions and establish the coordinates of their boundaries with an accuracy of 50 kb for samples of heterozygous inversions. This indicates that the sensitivity of the method is sufficient for detecting chromosomal rearrangements even against the background of contacts of the normal locus. The identified patterns will be used to develop an automatic algorithm for searching for balanced chromosomal rearrangements based on Exo-C data.

 

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Annotation of the results obtained in 2022
We study effects of mutations in several genes important for brain formation in mouse models. Transgenic mice with inactivation of the Ube2a gene, as well as mice with overexpression of the Ube2a cDNA were obtained. It has been shown that overexpression leads to a delay in neuronal differentiation, while inactivation causes an early exit from the mitotic cycle. The inactivating genetic construct was transfected into C57Bl6 mouse ESCs to simulate Ube3C gene deficiency. Chimeric animals were obtained after ESC injection into CD1 mouse blastocysts. The black progeny obtained from them proves the successful transfer of the inactivating mutation into germ cells. A strain of Ire1a knockout mice was created to study the role of this gene in the differentiation of neurons in the cerebral cortex. It was found that Ire1a inactivation leads to an increase in the number of neurons in the lower layers and a decrease in the number of neurons in the upper layers, while the opposite phenotype was revealed under Ire1a overexpression. We found that the loss of Ire1α causes a decrease in the rate of protein synthesis and leads to the arrest of ribosomes on RNA. The role of the multiadaptor protein Noma-GAP in the axonal transport of vesicles was studied. Rab11 has been shown to be required for dendritic-directed vesicular transport of postsynaptic proteins and for dendritic maturation. Vesicles containing Rab11 accumulate in the soma of NOMA-GAP-deficient neurons. Loss of NOMA-GAP is associated with faster retrograde vesicle movement. We used the NeuroD1/2/6 triple knockout to study the role of NeuroD1/2/6 in the regulation of cell death in cortical neurons. We studied the activity of caspase 3 during the development of the cortex. The number of apoptotic cells was increased with different intensity at all stages studied. We create experimental mouse strains with rearrangements on chromosomes 1 and 12 to study the effects of epigenetic silencing of chromosomal aberrations. Targeted chromosomal rearrangements were obtained using microinjections of CRISPR/Cas9 components into mouse zygotes. We found a low birth rate of mice - 10 mice out of 317 transferred zygotes - in experiments using Cas9 mRNA and sgRNA to chromosome 12, which indicates the toxicity of the components for zygotes and early-stage embryos. Analysis of experiments for creation chromosome rearrangements in blastocysts and early mouse embryos shows the presence of target rearrangements in chromosome 12. After birth, mice will be analyzed in a similar way. Analysis of mice born in the experiment on chromosome 1 did not reveal deletions and duplications, but revealed one animal with an inversion. Hi-C analysis of the fibroblast cell line from the healthy carrier of sSMC(4) confirmed the ring shape of the marker chromosome, precisely determined its size and the content of 166 genes. It makes surprising the absence of clinical manifestations in the carrier’s phenotype. Bisulfite DNA sequencing identified a region on chromosome 4 with a pronounced increase in coverage, the coordinates of this region correspond to sSMC. CpG islands were demethylated both in sSMC- positive cells and in control lines. It can be concluded that in iPSCs with sSMC, the level of methylation in this region not significantly differs from isogenic iPSC without sSMC, which raises the question about the mechanisms of dosage compensation in the presence of additional chromosomal material in the karyotype. Experiments completed on directed differentiation of iPSC lines with duplication of the gene CNTN6 into cortical neurons in order to assess the expression and epigenetic status of genes localized near chromosomal rearrangements at the 3p26.3 locus. Chip-seq analysis was performed using antibodies to H3K9me3 histone. The results of sequencing of most of the libraries have been received, data analysis is in progress. Four iPSCs lines from a patient and a healthy carrier of duplications of the CNTN6 gene were differentiated into embryoid bodies with further analysis by the RNA-seq method. A total of 4645 differentially expressed genes (DEG) were found between all samples from a patient and a healthy duplication carrier. Enrichment analysis was carried out. We have constructed a cellular model to study the role of the UBE2A gene in the development of Nascimento syndrome, consisting of isogenic iPSCs with normal expression, with no expression, and with overexpression of the UBE2A gene, as well as iPSCs of the patient. In all knockout clones, we observed a significant decrease in the expression of the UBE2A gene and the absence of the UBE2A protein. Using lentiviral transfection, clones with inducible overexpression of the UBE2A gene were obtained, confirmed by quantitative RT-PCR. A functional characterization of the UBE2A gene is being carried out. We have shown that iPSCs with a knockout of the UBE2A and fibroblast-like cells differentiated from them attach to the surface faster than control. Faster adhesion is accompanied by an increased number of focal contacts per area. At the same time, knockout of the UBE2A gene does not affect the proliferation rate of iPSCs and neuronal precursors, and the growth rate of brain organoids. We found that knockout iPSCs and iPSCs with overexpression of the UBE2A gene differ in nuclear morphology and demonstrate a significant increase in the size of the nucleus compared to the original cells. The increase in nuclear size is not associated with changes in the nuclear lamina, in the distribution of constitutive heterochromatin within the nucleus, or differences in the distribution of cells through the cell cycle. An experiment to assess the role of the UBE2A gene in the repair of radiation-induced double-strand DNA breaks did not reveal a statistically significant difference between iPSCs with a knockout of the UBE2A gene and wild-type iPSCs. 110 patients with neurodevelopmental disorders were examined clinically and genetically. Microarray analysis was performed for 83 patients. Pathogenic/potentially pathogenic CNVs were found in 37% of the probands, all rearrangements were fully characterized. Whole exome sequencing was performed for 52 individuals from 17 families; clinically significant mutations were found in 15 probands. Those mutations that are significant for the formation of the brain are candidates for study in cell culture and animal models. A total of 13 pairs of DNA from placental tissues of abortuses (chorion and extraembryonic mesoderm) and their parents were analyzed on SNP microarrays. 130 CNVs have been identified, all of which have been fully characterized. 163 common CNVs for embryonic death and intellectual development disorders have been identified. CNVs with a pleiotropic effect are candidates for studying in cell culture models at the next stages of this work. Fifteen primary cell lines of human extraembryonic fibroblasts were established from dead embryos with a normal karyotype, the lines were cryopreserved. NIPT modifications were tested for screening microstructural chromosomal disorders on a model system - DNA of patients with microstructural rearrangements and their mothers. Two relative sample concentrations were used, 10% and 30%, for which 22% and 50% rearrangements were detected, respectively. The concentration of fetal DNA has a significant impact on the ability to detect CNV using NGS. We performed Exo-C analysis for 9 patients with intellectual disabilities and chromosomal rearrangements. The effectiveness of various bioinformatics tools for modeling 3D chromatin contacts has been evaluated. The 3DGenBench platform has been developed to predict changes in the 3D architecture of the genome caused by chromosomal rearrangements.

 

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Annotation of the results obtained in 2023
The role of the Ire1α gene in the regulation of Satb2 and Ctip2 translation was studied. It was shown that the level of Ctip2 expression does not depend on the presence of Ire1α. At the same time, a direct dependence of the translation efficiency of Satb2 on the presence of Ire1α has been established. Mass spectrometry analysis identified about 30 proteins whose expression levels differed in the cerebral cortex of Ire1α mutant mice compared to wild type mice. In particular, the expression levels of eEF-2 and eIF4A1 were reduced. Overexpression of eEF-2 by in utero electroporation (IUE) in wild-type embryos did not result in a change in the proportion of Satb2 in neuronal progeny cells, but was associated with a defect in the positioning of upper layer neurons similar to that observed with Ire1α loss. Inactivation of eIF4A1 resulted in a phenotype similar to that of Ire1α knockouts with respect to the types of neurons generated. Thus, it is eIF4A1 that is the target of Ire1α, which is responsible for the decrease in the level of Satb2 in the brain of Ire1α mutant mice. We studied the proliferation and fate of neuronal stem cells after mitotic cycle exit in mice with mutations in the Ube3C gene using BrdU labeling. It was found that Ube3C mutant cells differentiate into neurons faster than wild-type cells, and Ube3C inactivation leads to impaired neurogenesis and accelerated neuronal differentiation. We tested this hypothesis in experiments with overexpression of Ube3C in the embryonic cerebral cortex. We found that increased expression of Ube3C in progenitor cells leads to increased production of astrocytes, while only neurons appeared in the control. Analysis of the proteome of neurons with the Ube3C mutation and the wild type revealed that the Hakai protein showed the maximal difference in expression, which was also confirmed using Western blot. Thus, it has been established that Ube3C controls neurogenesis of the cerebral cortex. To study the role of a de novo point mutation in the SEMA4D gene, cDNA constructs were synthesized containing both wild-type human Sema4D and mutant Sema4D. In vivo experiments have shown that overexpression of mutant Sema4D in the embryonic cerebral cortex leads to impaired neuronal migration, as well as impaired axonal growth of the corpus callosum. We studied the effects of mutations in two isoforms of the HP1 gene (HP1beta and HP1gamma) on the development of the cytoarchitecture of the cerebral cortex and hippocampus in mice. It was found that the proliferation of neuronal stem cells in the hippocampus of the HP1beta/HP1 gamma double mutant was reduced compared to the wild type. Also, in mutants, neurons in the CA3 region of the hippocampus have less complex basal dendrites in comparison with wild-type animals. Measurements of total brain volume showed that young mutant animals had moderately reduced volume of both the cortex and the entire brain. HP1β/γ DKO animals exhibited impairments in spatial learning and memory. We performed microinjection experiments with components of the CRISPR/Cas9 system into mouse zygotes to produce animals with a 950-kb deletion in the pericentromeric region of chromosome 1. After transplantation of 406 embryos to surrogate mothers, 46 F0 offspring were obtained. Of these, genotyping revealed 9 animals with rearrangements of chromosome 1: two with deletions, four with inversions, one with duplication and two with a combination deletion/inversion, confirmed by Sanger sequencing. All F0 founders were fertile. Next, first generation descendants (F1) were obtained from crossing F0 with C57/Bl mice, and heterozygous individuals were crossed into F2 and F3. Genotyping identified homozygous rearrangement carriers in the offspring of seven F0 founders. The excess of heterozygotes and a marked deficiency of homozygotes for deletions suggest increased mortality of homozygous mice during the embryonic period. Similar experiments involving microinjection of CRISPR/Cas9 components into zygotes were performed to produce mice with another rearrangement, a 750-kb deletion in the peritelomeric region of the long arm of chromosome 12. More than 600 zygotes were injected, of which 90% survived. However, transplantation of more than 500 embryos into surrogate mothers did not result in the birth of F0 offspring. An additional in vitro study confirmed the absence of toxic factors in the injection mixture. The resulting embryos tended to have a reduced number of trophoblast cells and a significantly reduced number of inner cell mass cells. We hypothesized that reduced survival is the result of chromothripsis and possible elimination of chromosome 12 following zygote genome editing. We completed the creation of the model cellular system to study the functions of the UBE2A gene. It has been shown that in glial cells differentiated from iPSCs with abnormal doses of the UBE2A gene due to deletion, knockout, or inducible overexpression, a significant increasing of the cell nucleus size is observed, along with an increasing amount of the main nuclear lamina protein, lamin B1. Thus, we showed that the abnormal increase in nuclear size previously identified in iPSCs remains in differentiated cells. It was shown that deletion and knockout of UBE2A lead to a significant increase in the speed of glial cells migration. The transcriptome and proteome of neuronal progenitors obtained from iPSCs of the isogenic system and iPSCs from the patient were analyzed. In precursors differentiated from patient iPSCs, disturbances in the expression of genes associated with the development of axons, dendrites and the regulation of synapses were detected. The expression of genes responsible for binding of the myosin heavy chain was also reduced, which confirms our observations about impaired cell migration in the absence of the UBE2A protein. Proteome analysis demonstrated a paradoxical feature of UBE2A regulation, which makes neuronal progenitors with radical differences in UBE2A gene dosage similar to each other. Microarray analysis was performed for 159 patients with intellectual disability. Pathogenic and likely pathogenic CNVs were identified in 37% of patients. Whole exome sequencing was performed in 7 families with familial forms of intellectual disability; no pathogenic variants were identified. Previously identified pathogenic and likely pathogenic variants were confirmed by Sanger sequencing: in 6 families we found de novo variants, and variants in 2 families were inherited from mothers and localized on the X chromosome. Guidelines of the Russian Society of Medical Geneticists for chromosomal microarray analysis have been developed and published. The structure of chromosomal imbalance in spontaneous abortions has been clarified. 204 CNVs were detected in 21 embryos. Interestingly, 28% of CNVs were detected only in the chorionic villi cytotrophoblast (CV) and 25% in the extraembryonic mesoderm (EM), including inherited variants. In addition, tissue-specific mosaic regions of homozygosity (ROH), also confined by either CV or EM (18% each), were also registered for the first time. A possible mechanism for the occurrence of tissue-specific ROH and CNV may be the correction of meiotic trisomy, resulting to the restoration of the normal chromosome complement with the loss of different homologues in lines of embryonic and extra- embryonic origin. This means that from 36 to 53% of spontaneous abortions with a normal karyotype carry molecular markers of the process of self-correction of abnormal karyotype during early development. Analysis of the structure of chromosomal imbalance in 94 abortions with a previously established normal karyotype using molecular karyotyping and haplotyping technics revealed chromosomal abnormalities in 35% of abortions. For the first time, it was established that human pregnancy loss is accompanied by the accumulation of mosaic chromosomal abnormalities in the derivatives of the inner cell mass, compared to placental cells. We clarified the boundaries of chromosomal rearrangements detected by the Exo-C method using Oxford Nanopore long-read whole-genome sequencing (WGS) and targeted sequencing using nCATS and adaptive selection (AS) methods. An algorithm for searching for inversions has been developed, which makes it possible to automatize the search for chromosomal rearrangements on Hi-C maps with exome enrichment.

 

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