A case report of Pallister-Killian syndrome with an unusual mosaic supernumerary marker chromosome 12 with interstitial 12p13.1-p12.1 duplication.

Pallister-Killian syndrome (PKS) is a rare inherited disease with multiple congenital anomalies, profound intellectual disability, and the presence in the karyotype of sSMC - i(12)(p10). The frequency of PKS may be underestimated due to problems with cytogenetic diagnosis caused by tissue-specific mosaicism and usually a low percentage of peripheral blood cells containing sSMC. Such tissue-specific mosaicism also complicates a detailed analysis of the sSMC, which, along with the assessment of mosaicism in different tissues, is an important part of cytogenetic diagnosis in PKS. Unfortunately, a full-fledged diagnosis in PKS is either practically impossible or complicated. On the one hand, this is due to problems with the biopsy of various tissues (skin biopsy with fibroblast culture is most often used in practice); on the other - a low percentage of dividing peripheral blood cells containing sSMC, which often significantly complicates the analysis of its composition and organization. In the present study, a detailed analysis of sSMC was carried out in a patient with a characteristic clinical picture of PKS. A relatively high percentage of peripheral blood cells with sSMC (50%) made it possible to perform a detailed molecular cytogenetic analysis of de novo sSMC using chromosomal in situ suppression hybridization (CISS-hybridization), multicolor FISH (mFISH), multicolor chromosome banding (MCB), array CGH (aCGH), and quantitative real-time PCR (qPCR), and short tandem repeat (STR) - analysis. As a result, it was found that the sSMC is not a typical PKS derivative of chromosome 12. In contrast to the classical i(12)(p10) for PKS, the patient's cells contained an acrocentric chromosome consisting of 12p material. Clusters of telomeric repeats were found at the both ends of the sSMC. Furthemore, the results of aCGH and qPCR indicate the presence of interstitial 8.9 Mb duplication at 12p13.1-p12.1 within the sSMC, which leads to different representations of DNA from different segments of 12p within cells containing sSMC. The obtained data raise the question of the instability of the sSMC and, as a consequence, the possible presence of additional rearrangements, which, in traditional cytogenetic analysis of patients with PKS, are usually described as i(12)(p10).

Generation of iPS cell line (ICGi040-A) from skin fibroblasts of a patient with ring small supernumerary marker chromosome 4.

Human induced pluripotent stem cell (iPSC) line, ICGi040-A, was obtained from skin fibroblasts derived from a male patient with mosaic ring small supernumerary marker chromosome 4 (sSMS(4)) and infertility. ICGi040-A cells have karyotype 47,XY,+r(4) in 97% of cells and express a set of pluripotent markers, as well as are able to differentiate in vitro into derivatives of all three embryonic germ layers.

Complex biology of constitutional ring chromosomes structure and (in)stability revealed by somatic cell reprogramming.

Human ring chromosomes are often unstable during mitosis, and daughter cells can be partially or completely aneuploid. We studied the mitotic stability of four ring chromosomes, 8, 13, 18, and 22, in long-term cultures of skin fibroblasts and induced pluripotent stem cells (iPSCs) by GTG karyotyping and aCGH. Ring chromosome loss and secondary aberrations were observed in all fibroblast cultures except for r(18). We found monosomy, fragmentation, and translocation of indexed chromosomes. In iPSCs, aCGH revealed striking differences in mitotic stability both between iPSC lines with different rings and, in some cases, between cell lines with the same ring chromosome. We registered the spontaneous rescue of karyotype 46,XY,r(8) to 46,XY in all six iPSC lines through ring chromosome loss and intact homologue duplication with isoUPD(8)pat occurrence, as proven by SNP genotype distribution analysis. In iPSCs with other ring chromosomes, karyotype correction was not observed. Our results suggest that spontaneous correction of the karyotype with ring chromosomes in iPSCs is not universal and that pluripotency is compatible with a wide range of derivative karyotypes. We conclude that marked variability in the frequency of secondary rearrangements exists in both fibroblast and iPSC cultures, expanding the clinical significance of the constitutional ring chromosome.

Generation of iPSC line ICGi024-A from human skin fibroblasts of a patient with ring chromosome 18.

Ring chromosome 18 is a rare chromosomal disorders that usually originate de novo and correlate with clinical manifestation: developmental delay as well as microcephaly, brain and ocular malformations, hypotonia and skeletal abnormalities. We generate iPSC clonal cell line ICGi024-A with pluripotency properties which were demonstrated in vitro by three germ layer differentiation capacity. ICGi024-A can be used for disease modeling and fundamental investigation of ring chromosome instability.

Establishment of an induced pluripotent stem cell line (ICGi025-A) from fibroblasts of a patient with 46,XY,r(8)/45,XY,-8 mosaicism.

Ring chromosomes are structural aberrations commonly associated with disease phenotype. We consider necessary to create the iPSCs with a ring chromosome 8, which can be used for disease modeling and related research. The ICGi025-A iPSCs line was obtained by the reprogramming of the skin fibroblasts from a 1-year-old boy with 46,XY,r(8)/45,XY,-8 mosaicism, developmental delay, microcephaly, dysmorphic features, diffuse muscle hypotonia, moderate proximal muscle weakness, feeding problems, and motor alalia. The iPSCs had expression of the pluripotency-associated markers. In vitro differentiated cells expressed the markers of the cells of three germ layers. That data allowed us to conclude that ICGi025-A cells were pluripotent.

Generation of four iPSC lines from two siblings with a microdeletion at the CNTN6 gene and intellectual disability.

The human induced pluripotent stem cell (iPSC) lines, ICGi009-A, ICGi009-B, ICGi013-A and ICGi013-B, were generated from skin fibroblasts of two siblings with intellectual disability. Both patients were carriers of CNTN6 gene microdeletion (Kashevarova et al., 2014). iPSC lines have normal karyotype, express pluripotency markers, are able to differentiate in vitro into derivatives of all three germ layers and represent a unique tool to study neurodevelopmental disorders.

Generation of the induced pluripotent stem cell line, ICAGi002-A, from unaffected carrier megabase scaled duplication involving the CNTN6 gene.

The 3p26.3 microduplication involving the CNTN6 gene cause developmental delay and the intellectual disability. However, the incomplete penetrance is described for this copy number variation (CNV). Here we describe ICAGi002-A line, which is supposed to use as a model for studying of the penetrance of the CNV in 3p26.3. The ICAGi002-A iPSCs line was obtained by the reprogramming of the skin fibroblasts from a healthy donor with 3p26.3 microduplication involving the CNTN6 gene. The ICAGi002-A cells was pluripotent as it was shown by the expression of the pluripotency-associated markers and in vitro differentiation into the cells of three germ layers.

Induced pluripotent stem cell line, ICAGi001-A, derived from human skin fibroblasts of a patient with 2p25.3 deletion and 2p25.3-p23.3 inverted duplication.

Skin fibroblasts from a patient with developmental delay and chromosome 2p25.3 deletion syndrome were reprogrammed into induced pluripotent stem cells (iPSCs) and the clonal stem cell line ICAGi001-A (iTAF9-11) was established. ICAGi001-A pluripotency was demonstrated in vitro by three germ layer differentiation capacity. This line is a good model for studying of the developmental delay and brain disorder.

Induced pluripotent stem cell line, IMGTi003-A, derived from skin fibroblasts of an intellectually disabled patient with ring chromosome 13.

Skin fibroblasts from a patient with neurodevelopmental and speech delay, anxiety disorder, macrocephaly, microorchidism, multiple anomalies of internal organs and ring chromosome 13 were reprogrammed into induced pluripotent stem cells (iPSCs) to generate a clonal stem cell line IMGTi003-A (iTAF6-6). IMGTi003-A pluripotency was demonstrated by three germ layer differentiation capacity in vitro, and this cell line had a mosaic karyotype with 46,XY,r(13) as a predominant cell subpopulation. IMGTi003-A line is a good model for studying of the mitotic instability of the ring chromosome 13.

Generation of two iPSC lines (IMGTi001-A and IMGTi001-B) from human skin fibroblasts with ring chromosome 22.

Skin fibroblasts from a patient with intellectual disability and ring chromosome 22 were reprogrammed into induced pluripotent stem cells (iPSCs) to establish a clonal stem cell lines, IMGTi001-A (iTAF5-29) and IMGTi001-B (iTAF5-32). Because of ring chromosome mitotic instability these cell lines show mosaic karyotypes with 46,XX,r(22) in >83% cells, 45,XX,-22 as minor class and sporadically cells with other karyotypes. Differentiation in derivatives of all three germ layers was shown in teratoma assay for IMGTi001-A, and in embryoid bodies for both cell lines. To our knowledge, human iPSC lines with ring chromosome are described for the first time.

[Genomic architecture of human chromosomal diseases].

The genomic architecture predisposed to the emergence of DNA copy number variation causing a new class of human chromosomal diseases­reciprocal microdeletion and microduplication syndromes­ is reviewed in the paper. The molecular mechanisms of such chromosomal abnormalities are described. The problems of the interpretation of their clinical significance and genotype-phenotype correlations are discussed. The classification of phenotypes due to reciprocal chromosomal microdeletions and microduplications is shown. Published by 2015, reciprocal mutations associated with inherited and congenital human pathology and involving 58 chromosomal regions are summarized.

[Estimation of association of CNTN6 copy number variation with idiopathic intellectual disability].

Analysis of the prevalence of copy number variations of the CNTN6 gene, recently selected as a new candidate gene for intellectual disorders, was performed. Real-time PCR did not detect any change in the number of CNTN6 gene copies in a group of 200 patients with impaired intellectual development. However, taking into account our data from the previous aCGH analysis and published data, the overall frequency of microdeletions and microduplications of CNTN6 was estimated as 1: 265 (0.4%). The common phenotypic features of 40 patients with microdeletions and microduplications of CNTN6 appeared to be the autism spectrum disorders, developmental delay, intellectual disability, seizures, cognitive impairment, cardiological defects, and behavioral problems.

[Methylation status of line-1 retrotransposon in chromosomal mosaicism during the early stages of human embryonic development].

Early stages of human embryonic development are characterized by spatio-temporal coincidence of events of total epigenetic genome reprogramming and elevated level of mosaic forms of numerical chromosome abnormalities. It is possible that the abnormal reprogramming of various regions of the genome can lead to violations of local epigenetic chromatin organization and gene expression, affecting the correct chromosome segregation during mitosis. In this study, a comparative analysis of the methylation index of LINE-1 retrotransposon, which is largely reflecting the methylation profile of the genome, is performed in placental tissues of spontaneous abortions with complete and mosaic forms of aneuploidy, and with a normal karyotype, as well as in the control group of induced abortions of the first trimester of pregnancy. It was shown that extraembryonic mesoderm and chorionic cytotrophoblast of spontaneous abortions with chromosomal mosaicism are characterized by the highest index of LINE-1 methylation among all groups studied. At the same time excessive hypomethylation of transposable genetic element recorded in spontaneous abortions with normal karyotype. It is suggested that violations of parental genomes demethylation during epigenetic reprogramming at preimplantation stages of development may be associated with an increased frequency of mitotic errors in chromosome segregation, leading to the formation of a mosaic karyotype.

[ROLE OF CONTACTINS IN NEUROGENESIS IN HUMAN AND ANIMALS].

Development of central and peripheral nervous system is one of the most complicated processes of embryogenesis. Dendritogenesis is an important component of this process because properties of dendritic branching define input and output signals received by neuron. Moreover, communications between neurons require transition of signal from dendrite of one neuron to axon of another, and this process of signal transduction underlies mechanisms of synaptic plasticity and memory formation. The neural cell adhesion molecules of the immunoglobulin superfamily involved in the control of dendritogenesis. In current review we focus our attention on 6 members of this adhesion molecules family: contactins 1-6. The contactins are proteins that control key events of neurogenesis: adhesion and migration of neuronal cells, orientation of growth of neurites and axons myelination. Functions of contactins are actively studied using model animals that express contactins in central and peripheral nervous system with almost similar to human pattern. Mutations of contactin-encoding genes result in abnormalities of neurogenesis process and development of multiple neurological disorders. Review is devoted to the role of contactin proteins in neurogenesis and nervous system disorders.

[Clinical and genetic analysis of idiopathic intellectual disability based on array comparative genomic hybridization].

In this study authors searched for chromosomal aberrations in 71 children with developmental delay or idiopathic mental retardation using Human Genome CGH Microarray Kits 4×44K and 8×60K (Agilent Technologies, USA). Microdeletions and microduplications, as well as CNV, which may be related to intellectual disability and associated with regions of known hereditary diseases or chromosomal syndromes were identified in 14 (20%) children (these patients are described in this article). During the analysis, candidate genes localized within the regions of aberrations and associated with development and functioning of nervous system were denoted.

[Epigenetic effects of trisomy 16 in the human placenta].

The methylation profiles of the placental tissues of human embryos with normal karyotype and trisomy 16 were compared using Infinium HumanMethylation27 BeadChip array (Illumina, United States). Numerous differences between the extraembryonic tissues with diploid and aneuploid karyotypes were observed. The extraembryonic mesoderm of embryos with trisomy 16 appeared to be less methylated compared to the diploid tissue, whereas the cytotrophoblast of aneuploid embryos was hypermethylated. The presence of the supernumerary chromosome was shown to influence the epigenetic profile of the genome changing the level of methylation of CpG sites of all chromosomes. However, the biggest number of differentially methylated loci was found on the chromosome 16. Besides, more often the epimutations were tissue-specific. The hypomethylated genes in both tissues belong to the groups of genes responsible for different metabolic processes, whereas the hypermethylated genes control the processes of development, cell adhesion, immune response, and response to stimulus.

[DNA methylation profile in human placental tissues].

For the first time using genome-wide Infinium HumanMethylation27 BeadChip Array (Illumina, USA) DNA methylation pattern was determined in the human cytotrophoblast and extraembryonic mesoderm. These tissues are the derivatives of trophectoderm and inner cell mass of blastocyst and have substantial differences in dynamics of epigenetic genome reprogramming during early stages of differentiation. The genome of the extraembryonic mesoderm cells has been shown to be more methylated compared to the cytotrophoblast similarly to other mammalian species. Differences in methylation pattern of single CpG-dinucleotides and dinucleotides localized within promoter CpG-islands have been found. It has been shown that the majority of single CpG-dinucleotides in both tissues were methylated whereas promoter CpG-island sites were not. Comparative analysis revealed 202 differentially methylated genes in extraembryonic mesoderm and 40 genes in cytotrophoblast. These genes are responsible for diverse biological processes. However, in the extraembryonic mesoderm the main functional groups included genes responsible for DNA binding and transcriptional factors' activity whereas in the cytotrophoblast--for transport and protein and cytokine secretion.

[Methylation profiling of the cell cycle regulating genes in placenta of human embryos with chromosomal mosaicism].

To date the investigation of epigenetic mechanisms of cell cycle regulation, ensuring genomic stability maintaining and accurate transfer of hereditary information to daughter cells, is of a considerable interest. Development of the up-to-date molecular technologies allows determining methylation pattern of the whole genome at a single round of analysis. In this work for the first time the epigenetic status of placental tissues of human embryos with mosaic karyotype was studied using the genome-wide Illumina Infinium HumanMethylation27 BeadChip Array (Illumina, USA). The groups of genes, related to the cell cycle and its regulation and containing differentially methylated CpG-sites in their promoter regions, are determined. The methylation level of oncogenes (ARHGEF1, RGF5), tumor-suppressors (APC2, BRACA2, DCC, GRLF1, RB1, TP73, TSPYL2, VHL) as well as genes, participating in chromosome segregation regulation (CNTROB, GMNN, PROCR, TACC1), was changed most frequently.

[Skewed X-chromosome inactivation in human embryos with mosaic trisomy 16].

The sex ratio and X-chromosome inactivation were analyzed in placental tissues of human spontaneous abortuses with pure and mosaic forms of chromosome 16 trisomy. The sex ratio value was found to decrease with an increase in the share of cells with the trisomic karyotype, which suggests differential survival of embryos belonging to different sexes. The pattern of X-chromosome inactivation in cells of extraembryonic mesoderm in the control group of embryos and in spontaneous abortuses with the level of trisomy 16 below 80% corresponded to random X-inactivation, whereas in most embryos with a frequency of trisomy 16 exceeding 80% skewed inactivation was observed. Our results support the hypothesis about the existence of an autosomal transfactor influencing the initiation of X-chromosome inactivation and suggest its possible localization on chromosome 16.

[Estimation of the methylation status of the promoter region of the cell cycle gene P14ARF in placental tissues of spontaneous abortuses with chromosomal mosaicism].

The methylation status of the promoter region of the cell cycle gene P14ARF was studied in the extraembryonic mesoderm and in the chorion cytotrophoblast of 46 human spontaneous abortuses with chromosomal mosaicism. Aberrant methylation of alleles of this gene was revealed for the first time in placental tissues of 9% of embryos. The identified epimutations were found to be characteristic of embryos with aneuploid cell clones of postzygotic origin. It is suggested that epigenetic inactivation of loci responsible for the regulation of cell division and for segregation of chromosomes is associated with the occurrence of mosaic forms of the karyotype at early stages of human embryonic development.